(Dov - Prusky - Maria Benh Sau Thu Hoach PDF

Postharvest Pathology
Plant Pathology in the 21st Century:

Contributions to the 9th International Congress
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Dov Prusky M. Lodovica Gullino
●
Editors
Postharvest Pathology
Editors
Dov Prusky Maria Lodovica Gullino
Department of Postharvest Science Università di Torino
of Fresh Produce Grugliasco TO
Agricultural Research Organization Italy
Bet-Dagan marialodovica.gullino@unito.it
Israel
dovprusk@volcani.agri.gov.il
ISBN 978-1-4020-8929-9 e-ISBN 978-1-4020-8930-5

DOI 10.1007/978-1-4020-8930-5
Springer Dordrecht Heidelberg London New York
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Contents
1 The Role of Pre-formed Antifungal Substances

in the Resistance of Fruits to Postharvest Pathogens............................. 1
Nimal Adikaram, Chathurika Karunanayake,
and Charmalie Abayasekara
2 Mechanisms of Induced Resistance Against B. cinerea.......................... 13

Tesfaye Mengiste, Kristin Laluk, and Synan AbuQamar
3 Induced Resistance in Melons by Elicitors for the Control

of Postharvest Diseases.............................................................................. 31
Bi Yang, Li Yongcai, Ge Yonghong, and Wang Yi
4 Mechanisms Modulating Postharvest Pathogen

Colonization of Decaying Fruits............................................................... 43
Dov Prusky, Noam Alkan, Itay Miyara, Shiri Barad,
Maayan Davidzon, Ilana Kobiler, Sigal Brown-Horowitz,
Amnon Lichter, Amir Sherman, and Robert Fluhr
5 Global Regulation of Genes in Citrus Fruit in Response

to the Postharvest Pathogen Penicillium digitatum................................. 57
L. González-Candelas, S. Alamar, A.R. Ballester,
P. Sánchez-Torres, J. Forment, J. Gadea, M.T. Lafuente,
L. Zacarías, and J.F. Marcos
6 Epidemiological Assessments and Postharvest

Disease Incidence....................................................................................... 69
Themis J. Michailides, David P. Morgan, and Yong Luo
7 Preharvest Strategies to Control Postharvest

Diseases in Fruits........................................................................................ 89
N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias, and I. Viñas
v
vi Contents
8 New Developments in Postharvest Fungicide Registrations

for Edible Horticultural Crops and Use Strategies
in the United States.................................................................................. 107
J.E. Adaskaveg and H. Förster
9 New Approaches for Postharvest Disease Control in Europe.............. 119

M. Mari, F. Neri, and P. Bertolini
10 Quo Vadis of Biological Control of Postharvest Diseases..................... 137

Wojciech J. Janisiewicz
11 Improving Formulation of Biocontrol Agents Manipulating

Production Process................................................................................... 149
J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas, and I. Viñas
12 Host Responses to Biological Control Agents........................................ 171

Raffaello Castoria and Sandra A.I. Wright
13 Non-fungicidal Control of Botrytis Storage Rot in New Zealand

Kiwifruit Through Pre- and Postharvest Crop Management.............. 183
M.A. Manning, H.A. Pak, and R.M. Beresford
14 The Peach Story....................................................................................... 197

Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena,
Iray Gell, and Belen Guijarro
Index.................................................................................................................. 209
Recent Developments in Postharvest Pathology
This collection of papers includes some of the presentations given at the

International Congress for Plant Pathology held in Turin in 2008 in the session with
the above title. Fruit production for human consumption is an important part of the
market economy. Any waste due to spoilage and pest infestation, in the field but
mostly during the postharvest phase, results in significant economic losses which
are more pronounced as the losses occur closer to the time of produce sale. Careful
handling of perishable produce is needed for the prevention of postharvest diseases
at different stages during harvesting, handling, transport and storage in order to
preserve the produce high quality. The extent of postharvest losses varies markedly
depending on the commodities and country and are estimated to range between 4%
and 8% in countries where postharvest refrigeration facilities are well developed to
50% where these facilities are minimal. Microbial decay is one of the main factors
that determine losses compromising the quality of the fresh produce. For the devel-
opment of an integrated approach for decay management, cultural, preharvest,
harvest, and postharvest practices should be regarded as essential components that
influence the complex interaction between host, pathogen, and environmental con-
ditions. Orchard practices including preharvest fungicide applications can also
directly reduce the development of postharvest fruit decay. Among postharvest
practices, postharvest fruit treatments with fungicides are the most effective means
to reduce decay. Ideally, these fungicides protect the fruit from infections that occur
before treatment, including quiescent infections, as well from infections that are
initiated after treatment during postharvest handling, shipment, and marketing.
However the wide consumption in human diet of high-quality fresh fruits and veg-
etables and the increased concerns for the possible toxicity of fungicide residues
have lead to the development of new alternative approaches for disease control. One
of the alternatives is the use of antagonist applications, either alone or in combina-
tion with physical treatments and substances generally regarded as safe. The imple-
mentation of these alternatives techniques often requires modifying currently used
postharvest practices and development of new formulation for their applications.
Three chapters in this book deal with the mechanisms of host fruit and vegetable
resistance. Adikaram and co-workers referred to preformed antifungal substances
affecting the resistance of unripe fruits and changes in their level during fruit ripen-
ing. Mengiste and co-workers suggested that active processes related to the regulation
vii
viii Recent Developments in Postharvest Pathology
of cell death, plant hormone signalling and synthesis are implicated in disease
resistance to necrotrophic pathogens during storage. Interestingly Yang and co-
workers indicated that a variety of chemical, physical and biological elicitors may
modulate inducible mechanisms of resistance.
Two chapters in this book deal with fungal pathogenicity factors and their
relationship with the host response. Prusky and co-workers described an interesting
mechanism used by postharvest pathogens to modulate host environment (alkalini-
zation and acidification) leading to enhanced pathogenicity, while Gonzales-Candelas
and co-workers presented the first wide transcriptome analysis of citrus fruit response
to Penicillium digitatum infection.
Four chapters in this book deal with subjects related to disease assessments
before harvest and their relation to the postharvest treatment of fruits and vegetables.
Michailidis and co-workers emphasized the importance of weather and environmen-
tal conditions to pathogen infection and suggested different approaches for disease
assessment which could be used to predict the incidence of postharvest diseases.
Teixido and co-workers suggested the importance of preharvest applications of
biocontrol treatment efficacy in combination with nutrients and conclude on the
importance of preharvest treatment in postharvest disease control. The other two
chapters dealt specifically on the new development of postharvest edible crop in the
United States by Adaskaveg and Förster, and in Europe by Mari and co-workers.
Both suggested that integrations of combined technologies such as sanitation and
use of fungicides, physical and biological agents are of high importance.
Three chapters in this book are dealing with biological control of postharvest
diseases and host responses to the biocontrol agents. Janisiewicz, presented a sum-
mary of the biocontrol developments in his “Quo Vadis of Biological Control …”
chapter and their impact on the industry, while Usall and co-workers described the
different technological changes made during the development of new formulations
which allow the improvement of biocontrol efficiency. Castoria and Wright referred
in their chapter to the different mechanisms of perception and activation of host
resistance by the biocontrol agents.
The remaining chapters of the book are focused in specific study cases of crops
such as kiwifruit, peaches and grapes, where the integrations of different approaches
at the pre and postharvest levels are combined. These represent new types of pre-
sentations which were presented in the evening workshops of the ISPP program
with excellent attendance. Manning and Beresford described how management and
assessment of rot-risk-factors in the vine and storage conditions may allow preven-
tion of botrytis problems. Melgarejo and co-workers described the importance of
orchard management in combination with epidemiological assessment to predict
risk and optimal handling of fruits.
In summary the Postharvest Pathology sessions included excellent presentations
of new and exciting progress at the leading edge of Postharvest Pathology.
Dov Prusky
Department of Postharvest Science of Fresh Produce
Agricultural Research Organization
Bet Dagan, Israel
Chapter name Author(s) Chapter No.
The Role of Pre-formed Antifungal Substances in the Resistance Nimal Adikaram, Chathurika Karunanayake and Charmalie 1
of Fruits to Postharvest Pathogens Abayasekara
Mechanisms of Induced Resistance Against B. cinerea Tesfaye Mengiste, Kristin Laluk and Synan AbuQamar 2
Induced Resistance in Melons by Elicitors for the Control Bi Yang, Li Yongcai, Ge Yonghong and Wang Yi 3
of Postharvest Diseases
Mechanisms Modulating Postharvest Pathogen Colonization Dov Prusky, Itay Miyara, Noam Alkan, Shiri Barad, Maayan 4
of Decayed Fruits Davidzon, Ilana Kobiler, Sigal Brown-Horowitz, Amnon Lichter,
Amir Sherman, and Robert Fluhr
Global Regulation of Genes in Citrus Fruit in Response to the L. González-Candelas, S. Alamar, A.R. Ballester, P. Sánchez-Torres, 5
Postharvest Pathogen Penicillium digitatum J. Forment, J. Gadea, M.T. Lafuente,
L. Zacarías and J.F. Marcos
Recent Developments in Postharvest Pathology
Epidemiological Assessments and Postharvest Disease Incidence Themis J. Michailides; David P. Morgan and Yong Luo 6
Preharvest Strategies to Control Postharvest Diseases in Fruits N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias and I. Viñas 7
New Developments in Postharvest Fungicide Registrations J. E. Adaskaveg and H. Förster 8
for Edible Horticultural Crops and Use Strategies
in the United States
New Approaches for Postharvest Disease Control in Europe M. Mari, F., Neri and P. Bertolini 9
Quo Vadis of Biological Control of Postharvest Diseases Wojciech J. Janisiewicz 10
Improving Formulation of Biocontrol Agents Manipulating J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas and I. Viñas 11
Production Process
Host Responses to Biological Control Agents Raffaello Castoria and Sandra A. I. Wright 12
Non-fungicidal Control of Botrytis Storage Rot in New Zealand M.A. Manning, H.A. Pak and R.M. Beresford 13
Kiwifruit Through Pre- and Postharvest Crop Management
The Peach Story Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena, Iray Gell 14
and Belen Guijarro
ix
Chapter 1
The Role of Pre-formed Antifungal Substances
in the Resistance of Fruits to Postharvest
Pathogens
Nimal Adikaram, Chathurika Karunanayake, and Charmalie Abayasekara
Abstract Plants contain secondary metabolites with antifungal properties. In fruits

they are mostly concentrated in the peel at immature stage and decline during
ripening in coincidence with fungal rot development. The information on antifungal
systems in immature avocado and mango, reviewed here, suggests that they play
a role in natural disease resistance. Immature mangoes have evolved a formidable
antifungal system comprising several resorcinols, gallotannins and chitinases.
Resorcinols and gallotannins are inhibitory to major postharvest pathogens,
Colletotrichum gloeosporioides causing anthracnose and Botryodiplodia theobromae
causing stem-end rot. Their levels are generally higher in resistant cultivars than in
susceptible ones. Mango latex, distributed in a fine network of canals in the fruit
peel, contains chitinases which have the ability to rapidly digest conidia of
C. gloeosporioides. Gallotannins and resorcinols decline progressively during
ripening and the latex disappears when ripe rot development begins. Retention
of latex in the harvested fruit reduces anthracnose and stem-end rot development
during ripening. Treatment of harvested fruit with CO2 or inoculation with certain
non-pathogenic fungi increased antifungal resorcinol concentration. Immature
avocado fruits possess a pre-formed antifungal system comprising at least five
antifungal compounds. The quiescence of C. gloeosporioides in the immature fruit
has been attributed to the pre-formed antifungal activity of the peel. Lipoxygenase
activity increases during fruit ripening, while epicatechin levels decline, suggesting
that these events are linked to the decrease in di-ene concentrations. Inhibition
of lipoxygenase activity results in retention of antifungal di-ene during ripening
increasing fruit resistance. In freshly harvested avocados, the di-ene concentration
can be further enhanced by treatment with biotic and abiotic agents.
N. Adikaram (*), C. Karunanayake, and C. Abayasekara

Department of Botany, University of Peradeniya, Peradeniya, Sri Lanka
e-mail: nkba@pdn.ac.lk
D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology 1

in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_1,
© Springer Science + Business Media B.V. 2010
2 N. Adikaram et al.
1.1 Pre-formed Antifungal Substances
Plants produce a range of chemically diverse secondary metabolites with antifungal

activity. Some of these exist in healthy plants in their biologically active forms and
others occur as inactive precursors and are activated in response to tissue damage
or pathogen attack (Schonbeck and Schlosser 1976; Osbourn 1996). VanEtten et al.
(1994) have proposed the term phytoanticipin to distinguish these preformed
antimicrobial compounds from phytoalexins, which are synthesized from remote
precursors in response to pathogen attack, probably as a result of de novo synthesis
of enzymes. Phytoanticipins are low molecular weight, antimicrobial compounds
that are present in plants before challenge by microorganisms or are produced after
infection solely from preexisting constituents (VanEtten et al. 1994). Certain plants
contain high molecular weight constitutive hydrolytic enzymes such as chitinases
with considerable antifungal activity and these cannot be accommodated within the
umbrella of phytoanticipins, although they seem to play a role in plant defence.
Pathogens infecting fruits have to face challenges that the pathogens infecting
vegetative organs do not normally encounter. Fruits are generally protected by
mechanical and chemical barriers in the peel and their physiology changes
markedly during development, particularly when ripening occurs. Pathogens, when
confront unripe fruits, often cause quiescent infections or minor damage. Such
quiescent infections have been observed in tropical (Muirhead and Deverall 1981),
subtropical (Droby et al. 1987), and deciduous fruits (Hall 1971). The resistance of
unripe fruits to fungal decay has been shown to be associated with induced
(Adikaram et al. 1982) or preformed (Prusky and Keen 1993) antifungal substances
in the peel. The onset of decay coincides with fruit ripening and concurrent
decrease in the antifungal compounds to sub-toxic levels. Thus, quiescence may
therefore represent a mechanism for avoiding toxic levels of antifungal plant
compounds. There is considerable interest in determining mechanisms underlying
the natural resistance of unripe fruits to fungal pathogens and extending fruit
resistance to postharvest ripening phase.
In certain fruits, the preformed antifungal substances appear to play a supportive
role to their arsenal of inducible defences by excluding saprophytic and epiphytic
microorganisms. In other fruits such as avocado and mango where the inducible
defence system is weaker, the preformed antifungal compounds appear to perform
a definitive protective role. This Chapter reviews the preformed antifungal systems
in mango and avocado.
1.2 Preformed Antifungal Compounds in Mango

(Mangifera indica) fruit
Unripe mango fruit peel contains three classes of preformed antifungal substances,
gallotannins and resorcinols in the peel tissue, hydrolytic enzyme, chitinase, in the latex.
1 The Role of Pre-formed Antifungal Substances in the Resistance of Fruits 3
1.2.1 Resorcinols
Constitutive resorcinol type antifungal compounds were first isolated from the peel
of unripe mango fruit cultivars Tommy Atkins and Haden and identified as a mixture
of 5-substituted resorcinols, 5-(12-cis-heptadecenyl) resorcinol and 5-pentadecyl
resorcinol (Fig. 1.1) (Droby et al. 1986; Cojocaru et al. 1986). Identical resorcinols
were also shown in the peel of several other cultivars (Droby et al. 1986). High
Pressure Liquid Chromatography of the dichloromethane phase of peel extracts of
two Sri Lankan cultivars, Karutha Colomban and Willard, showed peeks represent-
ing 5-(12-cis-heptadecenyl) resorcinol, 5-pentadecyl resorcinol and an additional
peak due probably to a new resorcinol (Karunanayake 2008). Two other resorci-
nols, 5(7, 12-heptadecadienyl) resorcinol from the fruit peel (Prusky et al. 1996)
and 5-(9, 12-heptadecadienyl) resorcinol from the latex (Oka et al. 2004), have
been reported. The concentration of the former did not change significantly during
fruit ripening, therefore it did not appear to play a role in fruit resistance. Knodler
et al. (2007) in a more recent study identified 3 major and 12 minor C15, C17 and C19
substituted resorcinols and related analogues, however, their antifungal properties
are not yet known.
Resorcinols in mango varieties have been studied in relation to black spot
development by Alternaria alternata during ripening. In unripe fruit, the fungus
Fig. 1.1 Structures of three resorcinols from mango peel, 5-(7, 12-heptadecadienyl) resorcinol
(a), 5-pentadecyl resorcinol (b), and 5-(12-heptadecenyl) resorcinol (c) (Kobiler et al. 1998)
forms quiescent infections. The concentration of resorcinols in the unripe mango

fruit peel ranged between 154–232 mg g−1 fresh weight and this declined during
ripening to about 74–125 mg g−1 fresh weight. At the time of Alternaria rot devel-
opment, the concentration also ranged between 74–125 mg g−1 FW (Droby et al.
1986). 5-substituted resorcinols were present at fungitoxic levels in the unripe fruit
peel and decreased to non-toxic levels, with ripening, in uninoculated fruits at the
same time that disease symptoms appeared in inoculated fruits (Prusky and Keen
1993). Eight mango cultivars, Maya, Erwin, Palmer, Pairi, Mabroka, Tommy
Atkins, Haden and Keitt tested gave similar results.
Concentration of resorcinols in the mango fruit was also studied in relation to
development of anthracnose disease by Colletotrichum gloeosporioides during
ripening. In the unripe fruit where the fungus forms quiescent infections, the
resorcinols are present at higher concentrations and declined during fruit ripening
to very low levels when anthracnose rot development commenced (Fig. 1.2).
Recent studies have showed the presence of significantly higher amounts of anti-
fungal resorcinols in mango latex (Bandyopadhyay et al. 1985; Oka et al. 2004)
than in the fruit peel (Hassan 2006). The constitutive resorcinols identified in peel
extracts could actually be those present in the latex canals of the peel.
The concentration of three resorcinols varied among different cultivars.
In cultivars that are resistant to anthracnose disease, there was a higher concentra-
tion of 5-(12- cis-heptadecenyl), 5-pentadecyl and AR 21 resorcinol (Table 1.1), than
in susceptible ones (Karunanayake 2008). A strong positive correlation was present
between the level of resorcinols and the degree of resistance to C. gloeosporioides.
Mango cultivars, Kensington Pride and Keitt that are more resistant to anthracnose,
had the highest concentrations of 5-n- heptadecenyl resorcinol while Nam Doc Mai
and Honey gold susceptible to anthracnose had the lowest (Hassan et al. 2007). As
in mango peel, the concentration of 5-substituted resorcinols in mango latex also
varied significantly among cultivars (Hassan 2006). A high correlation exists
Fig. 1.2 Resorcinol concentration in the unripe and ripe fruit peel of cultivar Willard
Table 1.1 The concentration of resorcinols in five Sri Lankan mango cultivars (average of two
samples) determined by HPLC
Concentration of resorcinols mg/g fresh weight
5 – (12- cis-heptadecenyl) 5 – pentadecyl Resorcinol ar 21
Cultivar resorcinol resorcinol derivative Resorcinol
Karutha Colomban (R) 59.9 27.1 532.1 43.2
Rata (R) 91.8 26.8 2,306.3 79.0
Kohu (M) 38.1 8.2 911.4 139.2
Petti (S) 20.7 8.6 47.5 15.3
Willard (S) 34.4 58.3 14.8 25.7
(R) cultivars more resistant, (M) moderately susceptible, and (S) susceptible to anthracnose
(Karunanayake 2008)
between the concentration of resorcinols in mango latex and the percentage (w/w)
of the non-aqueous phase of mango latex (Hassan 2006).
The concentration of the 5-substituted resorcinols decreased faster during
ripening in cultivars like Tommy Atkins susceptible to Alternaria spot, than in less
susceptible cultivars, such as Haden (Droby et al. 1986). A similar trend was
observed in two Sri Lankan cultivars where the resorcinols in the cultivar Willard
susceptible to anthracnose declined faster during ripening than in the resistant
Karutha Colomban (Karunanayake 2008).
1.2.2 Gallotannins
Methanol extract of the mango fruit peel when bioassayed with Cladosporium
cladosporioides or C. gloeosporioides produced a prominent inhibition zone at
Rf. 0.00 (Fig. 1.3). The compounds responsible for inhibition were purified and
identified as a mixture of three closely related gallotannins (Fig. 1.3). The three
compounds vary in the number and the points of attachment of sugar molecules.
Earlier 18 different gallotannins have been reported from the mango fruit peel and
eight in the pulp (Berardini et al. 2004). The phenolic compounds which were
accounted for antibacterial activity, observed in mango seed (Kabuki et al. 2000),
showed almost an identical elution profile to gallotannins (Berardini et al.
2004). Young and mature leaves and florets of mango also contain gallotannins
(Karunanayake 2008).
The gallotannins are directly inhibitory to the anthracnose pathogen, C. gloeo-
sporioides and the stem-end rot pathogen, Botryodiplodia theobromae. Antifungal
activity due to gallotannins was higher in the peel of unripe fruit at harvesting
maturity and declined gradually during ripening. By colour break stage, the antifungal
activity had declined by about 20% from what it was at harvest. At the ripe stage,
when anthracnose development occurred in cultivar Karutha Colomban, the anti-
fungal activity had declined to about 50% of the initial level.
Fig. 1.3 (a) Inhibition areas on thin layer chromatography bioassay plates produced by gallotan-
nins in the methanol (left) and resorcinols in the dicholomethane extracts (right) of mango fruit
peel. (b) Structure of the antifungal gallotannins
Mango cultivars more resistant to anthracnose such as Gira and Rata show
greater gallotannin activity in their peel than more susceptible cultivars such as
Kohu and Willard. The decline of gallotannin activity during ripening was greater
in the more susceptible cultivars.
1.2.3 Chitinase Activity
The peel of unripe mango fruit consists of a network of fine canals with latex which
extends to the pedicel. At the abscission point of mango pedicel, the small canals
tend to coalesce to form three or four large canals. It is from these that the latex
flow spurts out when the fruit is removed from the tree. When the mango latex is
removed from the fruit and allowed to settle, it separates into an aqueous and oily
phase. The latex is toxic to the conidia of C. gloeosporioides, the causal agent of
mango anthracnose. When exposed to the undiluted aqueous phase of mango latex,
the conidia were gradually digested (Fig. 1.4). During early hours, a slight granulation
was visible in the conidia and later the conidial wall was gradually dissolved.
A gel diffusion assay carried out on glycol chitin-enriched agarose confirmed
the presence of chitinase enzyme in the aqueous phase of the mango latex. Under
UV light (365 nm), the areas hydrolyzed by chitinase appeared dark against blue
fluoresced areas containing undigested glycol chitin (Fig. 1.5). Three chitinases
with molecular weights 47, 87, and 97 KDa were present in the mango latex
(Karunanayake 2008). The level of chitinase activity in the aqueous phase varied
with the mango cultivar.
Development of anthracnose (Fig. 1.6) and stem-end rot (Fig. 1.6) during ripening
was significantly lesser in fruits from which latex was not drained off after harvest
B
&
Fig. 1.4 The state of conidia of C. gloeosporioides after different periods of exposure to the
undiluted aqueous phase of mango latex
Fig. 1.5 Chitinase activity of the aqueous phase of mango latex, papaya latex (positive control),
dilution buffer (negative control) and a commercial enzyme standard from S. marcescence
Fig. 1.6 Anthracnose (left) and natural stem-end rot (right) development in fruits of cultivar ‘Willard’
(anthracnose) and ‘Karutha Colomban’ (SER) from which latex was drained and not drained
than in fruits from which the latex was drained off. It was subsequently shown that
the peel of unripe mango fruits from which latex was not removed after harvest had
greater chitinase activity (Table 1.2) which could be the reason for greater fruit
resistance in latex-retained fruit.
Table 1.2 Chitinase activity in the fruit peel of cultivar ‘Willard’ when the latex was drained after
harvest
Chitinase activity in units/gram fresh weight tissue
Treatment Day 1 after harvest Day 3 after harvest
Latex retained fruits 0.48 ± 0.03 0.29 ± 0.02
Latex drained fruits 0.32 ± 0.08 0.22 ± 0.01
The values given in the table are the average of two independent trials.
1.2.4 Pre- and Postharvest Treatments Enhance Fruit

Resistance and Antifungal Activity
Exposure of harvested mangoes to CO2 at a flow rate of 100 mL of CO2/min for 24 h

significantly reduced anthracnose development, however, the optimum effective
dosage of CO2 varied according to cultivar, 20% CO2 for Keitt, 60% CO2 for Tommy
Atkins. A concomitant significant increase of 5-(7,12-heptadecadienyl) resorcinol
was also detected in Keitt fruits in response to the 30% CO2 treatment (Kobiler et al.
1998). Dipping fruit in hot water (55° C) for 5 min also resulted in an increase in
5-(7,12-heptadecadienyl) resorcinol.
Dipping the unripe mangoes in a suspension of conidia of Colletotrichum
magna, not pathogenic on mango, before inoculation with C. gloeosporioides,
significantly delayed the anthracnose development. A significant increase in
5-(7,12-heptadecadienyl)resorcinol was also detected in mango fruits dipped in a
conidia suspension of C. magna (Kobiler et al. 1998). Peeling increased five
substituted resorcinols in the flesh of peeled mango and the fruits became resistant
to A. alternata infection (Droby et al. 1987).
Inoculation of unripe fruits with C. gloeosporioides resulted in enhanced gallotan-
nins (Sinnaih, Unpublished data) and the total soluble phenol content (Karunanayake
2008). Concurrent histo-chemical tests carried out on inoculated fruit peel at different
time intervals supported the findings of chemical tests that tissue phenolics increased
following infection. Free phenols can directly act as antimicrobial substances and be
oxidized to form quinines which can inhibit extracellular enzymes of the pathogen
(Mayer 1987). Chitinase activity increased in the peel following inoculation with C.
gloeosporioides, however, whether the increased activity is due the same chi-
tinases found in the latex could not be ascertained (Karunanayake 2008).
A field trial was conducted to investigate the influence of soil potassium on
postharvest rot development and natural disease resistance. Stem-end rot development
was significantly less in mangoes from trees which received three times the annual
recommended dose of potassium (2,055 g × 3) compared to those which received the
annual recommended level of potassium (2,055 g) or no potassium (Table 1.3).
The fact that the tissue potassium levels were higher in the peel tissues
from fruits harvested from potassium-treated trees (16.24 and 12.38 ppm) than
the control (11.68 ppm) may indicate that potassium enhances fruit resistance.
Table 1.3 Stem-end rot in fruits harvested from trees which received different levels of potassium
Lesion area (mm2) at different days after inoculation
K level (g) Day 1 Day 2 Day 3 Day 4 Day 5
0 0 0 558.7a 3,873.7a 10,572a
2,055 0 0 458.8 a
4,140.1 a
9,109a
2,055 × 3 0 0 248.7 a
2,394.7 b
6,834b
Values followed by the same letter do not differ significantly at the 5% probability level
a, b
(Duncan’s Multiple Range Test)
Gallotannin activity was in fact higher in peel of fruits which received greater
potassium, the differences in gallotannin activity were, however, not significant.
1.3 Avocado (Persea americana) Fruit
Anthracnose disease caused by Colletotrichum gloeosporioides (Penz.) Penz. &

Sacc. and the stem-end rot caused by Phoma spp., Botryodiplodia theobromae and
Phomopsis spp. are recognized as major diseases in ripe avocado fruit. Young fruit
are usually free from visible symptoms and characteristic decay lesions develop
during fruit ripening. The anthracnose disease originates from quiescent infections
in the immature fruit long before harvest (Binyamini and Schiffmann-Nadel 1972).
In unripe fruit the fungus produces an appressorium, then an infection peg which
ceases the growth in the cuticle (Coates et al. 1993) and becomes quiescent.
The quiescence of C. gloeosporioides was attributed to the presence of substantial
preformed antifungal activity in the immature fruit peel (Prusky et al. 1982; Sivanathan
and Adikaram 1989). Avocado peel contains antifungal monoene, 1-acetoxy-2,4-
dihydroxy-n-heptadeca-16-ene (2) and diene, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-
12,15-diene (1) (Prusky et al. 1982; Prusky et al. 1991) and three other compounds
1,2,4-trihydroxyheptadec-16-yne (3), 1,2,4-trihydroxyheptadex-16-ene (4) and
1-acetoxy-2,4-dihydroxyheptadec-16-yne (5) (Adikaram et al. 1992):
CH3(CH2)4CH=CHCH2CH=CH(CH2)7COCH2CHOHCH2OCOCH3 (1)
CH2=CH(CH2)11CHOHCH2CHOCH2OCOCH3 (2)
CH2=CH(CH2)11CHOHCH2CHOHCH2OH (3)
CH º C(CH2)11CHOHCH2CHOHCH2OH (4)
CH º C(CH2)11CHOHCH2CHOHCH2OCOMe (5)
The most striking structural feature among the five antifungal compounds is the
presence of a trihydroxy fragment which could be a precursor to these compounds
(Adikaram et al. 1992).
The levels of antifungal di-ene in peel of unripe avocados are subject to complex
regulation and may be modulated by lipoxygenase, for which the di-ene is a
substrate (Prusky et al. 1983), and also by the flavan-3-ol epicatechin, an inhibitor
of lipoxygenase (Ardi et al. 1998; Prusky et al. 1982). Lipoxygenase activity
increases during fruit ripening, while epicatechin levels decline, suggesting that
these events are linked to the decrease in di-ene concentrations. In freshly harvested
unripe avocado fruits, di-ene concentrations can be further enhanced by a variety of
biotic and abiotic treatments including challenge with C. gloeosporioides, wounding,
irradiation, exposure to ethylene (at levels that do not induce ripening) or carbon
dioxide, and treatment with lipoxygenase inhibitors (Prusky et al. 1990; Prusky and
Keen 1995; Prusky et al. 1985; Prusky et al. 1991; Prusky et al. 1996). Treatment
with lipoxygenase inhibitors results in increased disease resistance (Prusky et al.
1985, 1991), offering potential strategies for the manipulation of fruit physiology for
control of postharvest diseases. Interestingly, inoculation of freshly harvested avocado
fruit with a non-pathogenic mutant strain of Colletotrichum magna also confers
protection against C. gloeosporioides, possibly by the induction of epicatechin and
modulation of the level of the antifungal di-ene (Prusky et al. 1994). Taken together,
this evidence suggests that for the avocado-C. gloeosporioides interaction, preformed
antifungal compounds may contribute to the resistance of unripe fruits to decay.
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Chapter 2
Mechanisms of Induced Resistance Against
B. cinerea
Tesfaye Mengiste, Kristin Laluk, and Synan AbuQamar
Abstract Botrytis cinerea is a widespread pre-and postharvest pathogen of diverse

crops. Current crop protection methods rely on fungicide application and on hor-
ticultural practices. Variation for genetic resistance is documented in many crop
plant species but has not been utilized. Studies in model and crop plant species are
revealing the biological processes that underlie plant responses to infection to
B. cinerea. The genetic control of pathogen recognition and activation of defense
to restrict pathogen ingress and colonization is likely to emerge from such studies.
Deeper understanding of resistance mechanisms and their genetic control will aid
produce cultivars with genetic resistance to B. cinerea. The genetic components of
induced resistance in different plant species and future implications are discussed.
Keywords Botrytis cinerea • genetic resistance • necrotrpohic pathogens • induced

resistance
2.1 Introduction
B. cinerea is a ubiquitious fungal pathogen with relative host unspecificity primarily

attacking dicot plants but also some monocot species. The fungus causes the gray
mold disease resulting in significant crop losses under different production
conditions. Gray mold occurs over a wide geographical area, in the open field, in
greenhouses and even in storages at 0–10°C. B. cinerea is the principal cause of
pre- and postharvest disease in grapes, berries, tomatoes and many other crops
(Coley-Smith et al. 1980; Williamson et al. 1995; Elad 1997). In grapes, where
B. cinerea causes “bunch rot”, the estimated loss to the vineyard can amount to
T. Mengiste (*), K. Laluk, and S. AbuQamar

Department of Botany and Plant Pathology, Purdue University, 915 W. State street,
West Lafayette, IN 47906, USA
e-mail: mengiste@purdue.edu

14 T. Mengiste et al.
15–40% of the harvest depending upon the season. The losses to strawberries and cut
flowers have been estimated at 10–20% (Legard et al. 2000). B. cinerea is regarded
as an expensive pathogen because of the qualitative and quantitative crop losses it
causes and because of its demand for high fungicide treatment. B. cinerea also
develops fungicide resistance, limiting chemical crop protection options. Chemical
protection is also discouraged due to public safety associated with fungicide residues
on fresh produce. Consequently, there is an increased effort to identify genetic
resistance. Genetic resistance provides cost-effective and sustainable plant protection.
However, no robust genetic resistance against B. cinerea has been identified in
crop plants. There is also a very limited understanding of the biological processes
underlying plant responses to necrotrophic pathogens in general and B. cinerea in
particular. The biology of B. cinerea has been studied extensively and the genome
of the fungus has been sequenced (Elad et al. 2004; van Kan 2006). The critical
factors in B. cinerea pathogenesis, pathogen derived effectors, the molecular events
associated with infection processes, infection related morphogenesis, and factors
that confer the relative host unspecificity are still not fully understood. Equally
unknown are the critical factors in plant disease resistance mechanisms in different
plant species. In this chapter we will highlight recent progress made towards
understanding of plant induced resistance to B. cinerea.
2.2 Plant Resistance to B. cinerea
Plant resistance to diseases is controlled by a multitude of environmental as well as

host and pathogen genetic factors that vary depending on pathogen-host combinations.
Figure 2.1 summarizes B. cinerea derived disease and/or defense elicitors and the
corresponding plant defense mechanisms. Necrotrophic pathogens in general and
B. cinerea in particular have evolved infection strategies to breach plant defenses
(Prins et al. 2000a). These infection strategies involve the secretion of diverse
chemical compounds before and during colonization. Some necrotrophic fungi are
host specific, producing toxins that promote chlorosis and host cell death only in
their hosts (host specific toxins, HSTs) (Wolpert et al. 2002). Many necrotrophic
fungi including B. cinerea are host unspecific and produce host non-sepecific toxins.
There is no HST identified from B. cinerea consistent with the host unspecificity of
the pathogen. B. cinerea produces botrydial, a non-host-specific toxin implicated in the
intitation and severity of disease (Colmenares et al. 2002). In addition, B. cinerea
produces cell wall degrading enzymes, other extracelluar enzymes, oxalic acid, and
reactive oxygen intermediates to promote disease and macerate plant tissues (Prins
et al. 2000b). These infection strategies differ from obligate pathogens that suppress
plant defenses through subtle mechanisms. B. cinerea promotes or benefits from
host cell death during pathogenesis. Excellent reviews have recently been published
on the pathogenesis of B. cinerea (van Kan 2006; Williamson et al. 2007).
Plants also have counter defense mechanisms that are built as layers of constitutive
and inducible resistance strategies. Broadly, plant defense is composed of primary
2 Mechanisms of Induced Resistance Against B. cinerea 15
Fig. 2.1 A generalized scheme showing major factors involved in the development of the gray
mold disease by B. cinerea and corresponding components of plant defense. Prior to recognition
of B. cinerea derived PAMPs and the activation of defense, the cuticle and the plant cell wall fend
off infection. These are also targets of B. cinerea virulence by lipases, cutinases and the plant cell
wall degrading enzymes. Plant defense is depicted as layers starting from the leaf epidermal cells
protected by the cuticle, cell wall, membrane localized PRRs that trigger gene expression and the
accumulation of molecules that confer resistance through diverse activities. Receptor mediated
recognition is not defined in the case of B. cinerea and plant interactions. OG-R, oligogalacturonide
receptors; PGIP, polygalacturonase-inhibiting protein; PRRs, pattern recognition receptors; ROIs,
reactive oxygen species; PAMPs, pathogen associated molecular patterns
line of defense at the cuticular membrane, plant cell wall based defenses and those
activated upon recognition of pathogen derived molecules by membrane localized
pattern recognition receptors. It is also possible that plants recognize pathogen
infection because of the stress, toxins and other pathogenesis events. Recognition
mediated activation of defense controls the production of defense compounds. Plant
survival under pathogen assault is a result of the combined effect of the various
layers on the pathogen making the plant environment inhospitable to the pathogen.
These include diverse phytoalexins, phytoanticipins, and antimicrobial peptides
and antibiotics. Resistance to necrotrophs producing HSTs can be conditioned by
single genes that neutralize the toxin or encode altered proteins not to be recognized
by the toxin (Wolpert et al. 2002). Thus, host resistance mechanisms to biotrophic
and necrotrophic pathogens differ significantly and resistance to host unspecific
necrotrophs is predicted to be complex, requiring the involvement of many genes
and pathways for full resistance. Genetic studies in model and crop plant species
have defined some of the major plant defense pathways and their components that
regulate resistance to B. cinerea at the various layers of defense (Table 2.1). Some
of these defense components are discussed throughout this chapter.
Table 2.1 Plant genes implicated in the regulation of responses to B. cinerea based on increased
or decreased diseases resistance of mutants or transgenic plants
Mutant Nature of protein Reference
Bodyguard, bdg Epidermis-specific extracellular (Kurdyukov et al. 2006)
a/ß-hydrolase fold protein
Botrytis Resistant1 (bre1) Long-chain acyl-CoA synthetase 2 (Kurdyukov et al. 2006)
(LACS2)
PGIP1/2 Polygalacturonase-inhibiting proteins (Ferrari et al. 2003b)
Pad2 Gamma-glutamylcysteine synthetase (Ferrari et al. 2003a) )
Pad3 Cytochrome P450 monooxygenase (Ferrari et al. 2003a);
Coi1/Jai1 Coronatine insesitive1, JA receptor (Thomma et al. 1999;
Li et al. 2004)
Jasmonic acid insensitive1, MYC2 transcription factor (NICKSTADT et al. 2004)
JIN1
Jasmonic acid resistant1, Adenylation of jasmonic acid (Ferrari et al. 2003a)
JAR1
JMT Jasmonic acid carboxyl (Seo et al. 2001)
methyltransferase
Fatty acid oxygenation Ca2+-permeant non-selective cation (Bonaventure et al. 2007a)
upregulated2, FOU2 channel
AOS Alene oxide synthase (Bonaventure et al. 2007a)
EIN2 ET signaling, Ethylene insensitivity (Thomma et al. 1999)
Ethylene-response-factor1 GCC-box-binding protein (Berrocal-Lobo et al. 2002)
Ethylene receptor1 Ethylene receptor (Ferrari et al. 2003a)
Weak ethylene-insensitive5, EIN3-related transcription factor (Alonso et al. 2003)
wei5
Suppressor of SA Stearoyl-ACP desaturase (Kachroo et al. 2001)
insensitivity2
Botyrtis induced kinase 1 Ser/Thr receptor-like kinase (Veronese et al. 2006b)
WRKY33, 70 WRKY transcription factors (AbuQamar et al. 2006;
Zheng et al. 2006)
Botrytis R2R3MYB transcription factor (Mengiste et al. 2003)
susceptible1(BOS1)
Botrytis susceptible 2,3,4 BOS2-BOS4 genes not identified (Veronese et al. 2004)
Defense no death, DND1 Cyclic nucleotide gated ion channel (Govrin and Levine 2000)
Enhanced disease EDR3 encode dynamin-related (Tang et al. 2006)
resistance protein 1E
Overexpressor of cationic Homeodomain transcription factor (Coego et al. 2005)
peroxidase3, OCP3
TPK1b Ser/Thr receptor-like kinase (Abuqamar et al. 2008)
SPR2 Tomato fatty acid desaturase (Li et al. 2003)
Acx1 JA deficient, b-oxidation (Li et al. 2005)
Sitens Abscisic acid-deficient (Audenaert et al. 2002)
Cel1, cel2 Tomato endo b-1,4-glucanase (Flors et al. 2007)
Notes: Arabidopsis OCP3, FOU2, JIN1, LACS2 and tomato Cel1, Cel2 mutations confer increased
resistance whereas the other mutations cause susceptibility. Lesion mimic mutations that result in
B. cinerea susceptibility due to the precocious cell death are excluded from this table. This table
includes data from mutants for which B. cinerea disease have been performed and excludes many
mutants that are susceptible to other necrotrophs and may also show susceptibility to B. cinerea
2.3 The First Line of Defense Against B. cinerea
Recent studies illuminate differences that underpin host responses to pathogens

depending on pathogen life style. The first line of defense against pathogen attack,
regardless of the type of the invader, are the barriers provided by the plant cuticle
and cell wall. Initiation of infection and pathogen ingress may be halted by such
structural defenses. The susceptibility of the plant cell wall to degradation by cell
wall-hydrolyzing enzymes can affect the severity of disease caused by B. cinerea.
Fungal polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall
pectin and are important virulence factors for some necrotrophic fungi, whereas the
plant polygalacturonase-inhibiting proteins (PGIPs) contribute to resistance by
counteracting fungal PGs (Powell et al. 2000; Ferrari et al. 2003b). Furthermore,
attacked plants build up papillae on the inner side of epidermal cell walls at the
point of attempted pathogen entry. Papillae are largely composed of callose (a
b-1,3-glucan) but also contain polysaccharides, phenolic compounds and reactive
oxygen intermediates (Flors et al. 2005). Contrary to intuitive expectations, defects
in plant secondary cell wall caused by a mutation in cellulose synthesis resulted in
resistance to necrotrophic pathogens (Hernandez-Blanco et al. 2007). Necrotrophic
fungi target the plant cell wall for degradation, and the strength of the wall to such
attack directly contributes to resistance. Recent data suggest that enzymes involved
in cell wall metabolism play a role in susceptibility to pathogens (Flors et al. 2007;
Cantu et al. 2008). In tomato, inhibition of expansin and polygalacturonases (PGs)
involved in the disassembly of the plant cell wall during fruit ripening resulted
in reduced cell wall thickness and decreased the susceptibility of the fruits to
B. cinerea, supporting the cell wall being an important virulence target for B.
cinerea (Cantu et al. 2008). Absence of the endo-b-1,4-glucanases Cel1 and Cel2
in tomato reduces susceptibility to B. cinerea. Thus, cell wall-based defense
mechanisms can decrease or enhance pathogen resistance.
Traditionally, the cuticle is considered to serve as protection against abiotic
stresses and a barrier to fungal infection. The plant cuticle protects against patho-
gen penetration and hence prevents the pathogen from establishing infection.
Unexpectedly, Arabidopsis mutants defective in components of the cuticle were
resistant to B. cinerea attributed to loss of virulence in the fungus in the absence
of cuticle-derived signals (Kurdyukov et al. 2006; Chassot et al. 2007). Mutation in
LACS2, a gene required for cutin biosynthesis, leads to altered cuticle development
in Arabidopsis and, interestingly, also leads to resistance to B. cinerea infection
(Bessire et al. 2007; Tang et al. 2007). Cutinase expressing as well as the body
guard Arabidopsis mutant plants, which is unable to form a continuous cuticular
membrane, is also more resistant to infection with this fungus (Chassot et al. 2007;
Bessire et al. 2007). It was argued that increased cuticle permeability facilitates
resistance to necrotrophic infection by allowing diffusion of defense signals and
effectors across the plant surface leading to an altered perception of fungal elicitors
of defense (Bessire et al. 2007; Chassot et al. 2007). This perception leads to accu-
mulation of antifungal compounds on the leaf surface. The degree of permeability
directly correlates with the amount of antifungal compounds released at the site of
infection and hence the degree of resistance.
2.4 Active Defense Against Botrytis cinerea Contrasts

with Resistance to Biotrophic Pathogens
Pathogen invasion activates diverse defense responses that have varying efficiencies
and specificities in disease resistance. Race-specific resistance is predominantly
effective against biotrophic pathogens (Jones and Dangl 2006). Pathogen produced
effector proteins are directly or indirectly recognized by the corresponding plant
resistance proteins to trigger the hypersensitive response (HR) which restricts further
pathogen ingress. There is no precedence for such recognition based resistance to B.
cinerea. The fungus causes necrotic cell death in plant cells that are remarkably simi-
lar to the HR cell death (Govrin and Levine 2000). Thus, cell death is a hall mark of
race specific resistance but can be a typical disease symptom in plants infected with
B. cinerea. R-gene mediated HR facilitates infection by B. cinerea (Govrin and
Levine 2000, 2002). Cell death resulting from resistance responses or other physio-
logical functions facilitates B. cinerea infection (Kachroo et al. 2001; Veronese et al.
2004b). Dead cells and necrotic tissues are sources of leaked nutrients and provide
saprophytic growth base from which B. cinerea further colonizes healthy tissue.
Accordingly, the expression of animal antiapoptotic genes in tobacco inhibits HR cell
death and enhances resistance to B. cinerea (Dickman et al. 2001). No R-gene has
been implicated in resistance to B. cinerea or other necrotrophic pathogens.
Interestingly, an Arabidopsis R-gene that mediates susceptibility to the necrotrophic
fungal pathogen Cochliobolus victoriae has been described (Lorang et al. 2007).
Mutations in genes mediating signaling downstream of Arabidopsis R genes, eds1
or ndr1, show no altered resistance to B. cinerea (Ferrari et al. 2003a).
Systemic acquired resistance (SAR) is an active defense intiated by infection
with certain necrotizing pathogens and confers resistance to secondary infection.
SAR is effective against a broad-spectrum of pathogens including viruses, bacteria,
fungi and oomycetes (Ryals et al. 1996; Sticher et al. 1997). Inhibition of salicylic
acid (SA) accumulation or biosynthesis impairs SAR (Gaffney et al. 1993; Nawrath
and Metraux 1999). In Arabidopsis, the biological and chemical induction of SAR
failed to inhibit B. cinerea growth (Govrin and Levine 2002). Mutants impaired in
the induction of SAR are not altered in B. cinerea resistance. Similarly, the HR
inhibits a secondary infection by biotrophic pathogens by causing SAR in systemic
tissues but facilitates infection by B. cinerea (Govrin and Levine 2002). Arabidopsis
mutants that form spontaneous lesions and constitutively express SAR show
increased susceptibility to B. cinerea (Kachroo et al. 2001). SA was associated with
resistance at the point of infection in Arabidopsis tissue (Ferrari et al. 2003a). In
tomato, benzothiadiazole (BTH), the chemical inducer of SAR, induced restance to
B. cinerea (Audenaert et al. 2002). Multiple pre-harvest treatments of grapevine
with BTH enhanced trans-resveratrol content (by about 40-fold) and induced SAR
in grapevine berries. The percentage of infected berries per cluster was significantly
reduced in grapes from BTH-treated plants suggesting that BTH treatments could
be exploited in vineyards to protect grapes against gray mold (Iriti et al. 2004). In
strawberry plants treated with 0.25–2 mg/mL BTH, the development of gray mold
was delayed by about 2 days on the harvested strawberry fruit at 5°C. This delay
was equivalent to a 15–20% increase in storage life of the fruit suggesting that
chemical plant activators could control grey mold on strawberry fruit and grapes
(Leon and Joyce 2000). Thus, the available data point to the potential of SAR
against B. cinerea in various crop plants despite evidence from Arabidopsis that
show the failure of SAR in restricting B. cinerea.
Induced systemic resistance (ISR) resembles SAR but is induced by root
colonization of specific strains of nonpathogenic plant growth-promoting rhizobac-
teria in contrast to SAR that is induced by necrotizing pathogens. Unlike SAR, ISR is
dependent on jasmonate and ethylene, independent on SA and not associated with
PR gene expression (Pieterse et al. 1996); (van Loon et al. 1998). Molecularly, both
SAR and ISR are intertwined through the Arabidopsis NPR1 gene. In Arabidopsis, the
root-colonizing bacteria Pseudomonas fluorscens establishes ISR against B. cinerea
(De Meyer et al. 1998; Ton et al. 2002).
2.5 The Plant Hormones Jasmonate and Ethylene Play

a Central Role in Defense Against B. cinerea
Most studies implicate the plant hormones jasmonic acid (JA) and ethylene (ET) as
the key regulators of defense against necrotrophic pathogens such as B. cinerea
(Glazebrook 2005). In Arabidopsis, pathogen infection and exogenous application
of JA and/or ET induce defense gene expression including those encoding plant
defensins and thionins (Epple et al. 1995, 1997); (Penninckx et al. 1996; Penninckx
et al. 1998). The plant defensin PDF1.2 and the tomato protease inhibitor 2 (PI-2)
genes are induced by B. cinerea and MeJA. These inductions require intact JA/ET
or JA pathways in these plants (Thomma et al. 1998, ref). In addition, in both plant
species, the JA receptor mutants coi1/jai1 fail to induce PDF1.2/PI-2 gene expression
and exhibit enhanced susceptibility to B. cinerea (Penninckx et al., 1998; Li et al.,
2004. Arabidopsis mutations blocking JA signaling and biosynthesis including
allene oxide synthase (aos), jasmonic acid resistant (jar1), jasmonate insensitive4
(jin4), fatty acid desaturase (fad3/fad7/fad8) display increased susceptibility to B.
cinerea and other necrotrophs (Thomma et al. 1998; Stintzi et al. 2001; Ferrari et al.
2003a). Additionally, over-expression of a jasmonic acid carboxyl methyltrans-
ferase (JMT) in Arabidopsis results in enhanced and constitutive JA responses as well
as resistance against B. cinerea (Seo et al. 2001). JMT catalyzes the production of
methyl-jasmonate (MeJA) from JA, demonstrating that elevated levels of MeJA can
confer resistance to necrotrophic pathogens possibly aiding in defense signal trans-
duction (Seo et al. 2001). Two Arabidopsis gain of function mutants, jasmonate
insensitive1 (jin1) and fatty acid oxygenation upregulated2 (fou2), exhibit enhanced
resistance to B. cinerea (Lorenzo et al. 2004; Bonaventure et al. 2007b). JIN1
encodes a transcription factor involved in the regulation of JA-inducible genes that
are involved in pathogen as well as wounding defense responses (Lorenzo et al.
2004). Mutation in FOU2 alters cation fluxes involved either directly or indirectly
in the feed-back regulation of JA synthesis (Bonaventure et al. 2007b). Overall,
phenotypic analysis of mutants altered in JA accumulation and signaling clearly
establishes JA as an important regulator of plant defense responses to B. cinerea.
Similarly, genetic data from various plant species show ET to be a regulator of
defense to B. cinerea infection. In Arabidopsis, ET acts in concert with jasmonate.
ET insensitive mutants, etr1 and etr2, in soybean are severely susceptible to the
necrotrophic pathogens Septoria glycines and Rhizoctonia solani (Hoffman et al.
1999). Arabidopsis ethylene insensitive (ein2) mutant plants which are blocked ET
signaling show increased susceptibility to B. cinerea (Thomma et al. 1998; Norman-
Setterblad et al. 2000; Ferrari et al. 2003a). Some evidence also suggests that ET
promotes disease caused by some fungal pathogens indicating that its role varies
among different pathosystems (Lund et al. 1998; Ciardi et al. 2000; van Loon et al.
2006). ET induces fruit ripening as well as plant senescence, developmental pro-
cesses linked to increased colonization by B. cinerea. Thus, ET may increase plant
susceptibility or resistance to B. cinerea depending on the infected plant species
and the kind of infected tissue (van Loon et al. 2006; Elad 1993; Hoffman et al.
1988). Interestingly, exposure of B. cinerea to ET in vitro causes a reduction in
growth and numerous transcriptional changes (Chagué et al. 2006). However,
expression of a putative pathogenicity gene was enhanced in B. cinerea growing on
ET-producing plants compared to expression during infection on ET non-producing
plants (Chagué et al. 2006). Additionally, in vitro and during infection, the fungus
produces ET that is required for growth and sporulation through its own ET synthe-
sis pathway. However, the amount of ET produced by B. cinerea during plant colo-
nization is significantly reduced compared to the levels produced in vitro suggesting
a suppression of biosynthesis either by the fungus or the host. Overall, the timing
of plant inoculation plays a major role in determining whether ET may increase or
decrease plant resistance to necrotrophic infection. Although it has been clearly
established that ET generally aids in resistance, exactly what function ET produc-
tion serves on the side of the pathogen during host interaction remains unclear.
2.6 The Emerging Role of Absciscic Acid as a Regulator Plant

Response to B. cinerea
The plant stress hormone, absciscic acid (ABA), regulates plant responses to patho-
gens. ABA regulated closure of stomata leads to decreased bacterial entry (Melotto
et al. 2006). There is no evidence for a defense based on the control of stomatal
opening for B. cinerea or other necrotrophic fungi. Recently, ABA has emerged as
a positive or negative regulator of disease resistance depending on the nature of the
host-pathogen interaction (Anderson et al. 2004; Lorenzo et al. 2004; Mauch-Mani
and Mauch 2005). Exogenous application of ABA caused susceptibility to B.

cinerea (Audenaert et al. 2002). ABA-deficiency in tomato and impaired ABA
responses in Arabidopsis result in increased resistance to B. cinerea and other
necrotrophic pathogens due to reduced ABA-signaling but increased JA- or
ET-responsive gene expression (Audenaert et al. 2002). The enhanced response to
ABA3 (ERA3) gene is allelic to EIN2 (Ghassemian et al. 2000), which is required for
resistance to B. cinerea (Thomma et al. 1999). In addition, resistance to the
necrotrophic oomycete Pythium irregulare and to the bacterial necrotroph Ralstonia
solanacearum requires ABA synthesis and responses indicating a positive role for
ABA in disease resistance (Adie et al. 2007b; Hernandez-Blanco et al. 2007).
Interestingly, ABA is also implicated as a positive signal required for plant resis-
tance to Pythium irregulare and initiates callose biosynthesis, a defense response to
certain pathogens (Flors et al. 2005; Adie et al. 2007a). On the other hand, mutations
in ZFAR1, encoding a putative zinc-finger protein with ankyrin-repeat domains,
cause sensitivity to ABA and local susceptibility to B. cinerea (AbuQamar et al.
2006). Additionally, B. cinerea contains a gene cluster involved in the synthesis of
ABA; however the involvement and impact of fungal produced ABA on virulence
remains to be elucidated (Siewers et al. 2006).
2.7 Resistance to B. cinerea and Cross-Talk

with Other Pathways
Arabidopsis exhibits basal resistance to B. cinerea. This basal resistance was used
as the basis for forward and reverse genetic screens to identify genes regulating basal
resistance to B. cinerea (Mengiste et al. 2003). The BOS1 (Botrytis Susceptible1)
gene is required to restrict the growth of B. cinerea in inoculated plants. Strikingly,
bos1 plants have impaired tolerance to water deficit, increased salinity, and oxidative
stress. BOS1 encodes an R2R3MYB transcription factor protein, and our results
suggest that it mediates responses to signals, possibly mediated by reactive oxygen
intermediates, from both biotic and abiotic stress agents. In addition, three B. cinerea
susceptible mutants bos2, bos3, and bos4 defining independent genetic loci required
for Arabidopsis resistance to B. cinerea were described (Veronese et al. 2004a).
The bos2 mutant is susceptible to B. cinerea but retains wild-type levels of
resistance to other pathogens tested, indicative of a defect in a response pathway
more specific to B. cinerea. The bos3 and bos4 mutants also show increased
susceptibility to A. brassicicola, another necrotrophic pathogen, suggesting a broader
role for these loci in resistance. PR-1, a molecular marker of the SA-dependent
resistance pathway, shows a wild-type pattern of expression in bos2, while in bos3
this gene was expressed at elevated levels, both constitutively and in response to
pathogen challenge. In bos3, the mutant most susceptible to B. cinerea and with the
highest expression of PR-1, removal of SA resulted in reduced PR-1 expression but
no change in response to B. cinerea. Expression of the plant defensin gene PDF1-2
was generally lower in bos2, bos3 and bos4 mutants compared to wild-type plants,
with a particularly strong reduction in bos3. Production of the phytoalexin camalexin

is another well-characterized plant defense response. The bos2 mutant accumulates
reduced levels of camalexin whereas bos3 accumulates significantly higher levels
of camalexin than wild-type plants in response to B. cinerea. These three mutants
mediate disease responses through mechanisms independent of the BOS1. Based on
the differences in the phenotypes of the bos mutants, it appears that they affect
different points in defense response pathways.
Similarly, through reverse genetic searches for genes affecting resistance to
B. cinerea, we isolated other Arabidopsis mutants showing increased susceptibility to
B. cinerea. Among these, the B. cinerea Induced Kinase 1 (BIK1) and the WRKY33
transcription factor genes have been described recently (Veronese et al. 2006;
Zheng et al. 2006). BIK1 is a positive regulator of resistance to B. cinerea (Veronese
et al. 2006). Inactivation of BIK1 caused severe susceptibility to B. cinerea and
A. brassicicola. In contrast, bik1 plants display enhanced resistance to the virulent
strain of the bacterial pathogen P. syringae, with a significant reduction of bacterial
growth and total absence of disease symptoms. JA- and ET-regulated defense
response pathways, generally associated with resistance to necrotrophic fungi, are
attenuated in bik1 as measured by the expression of the plant defensin PDF1-2 gene
transcript. Similarly, the Arabidopsis WRKY33 gene, encoding transcription factor,
regulates the antagonistic interactions between defense pathways (Zheng et al.
2006). Mutations of the Arabidopsis WRKY33 gene cause enhanced susceptibility
to B. cinerea, concomitant with reduced expression of PDF1-2. Over-expression of
WRKY33, on the other hand, increases resistance to the two necrotrophic fungal
pathogens. The wrky33 mutants do not show altered responses to a virulent strain
of the bacterial pathogen P. syringae, although the ectopic expression of WRKY33
results in enhanced susceptibility to this pathogen. The susceptibility of plants
expressing WRKY33 to P. syringae is associated with reduced expression of the
salicylate-regulated PR-1 gene. Thus, WRKY33 is an important transcription fac-
tor that regulates the antagonistic relationship between defense pathways mediat-
ing responses to P. syringae and B. cinerea.
2.8 Mechanisms of B. cinerea Resistance Are Conserved

Between Tomato and Arabidopsis
The predominant mechanism of B. cinerea resistance appears to be conserved between

tomato and Arabidopsis. Tomato mutants impaired in ET, JA, ABA responses or
synthesis pathways mirror the B. cinerea susceptibility of the Arabidopsis mutants.
This is encouraging for the possible transfer of resistance between the two species.
However, there is also not clear data elaborating why Arabidopsis and tomato show
significant differences in the rate of disease development. The rate of B. cinerea dis-
ease development is significantly faster in tomato as compared to Arabidopsis
(unpublished observations).
Recently, from searches for regulatory genes that control B. cinerea resistance,
we isolated the tomato protein kinase 1 (TPK1b) gene encoding a receptor-like
cytoplasmic kinase that is localized to the plasma membrane. Pathogen infection,

mechanical wounding and oxidative stress induce expression of TPK1b, indicative
of a role in mediating responses to diverse signals. Reduction of TPK1b gene
expression through RNA interference (RNAi) increases the susceptibility of tomato
plants to colonization by the necrotrophic fungus B. cinerea. Interestingly, TPK1b
RNAi plants were also susceptible to feeding by larvae of tobacco hornworm
(Manduca sexta). Notably, susceptibility to B. cinerea and insect feeding is
correlated with reduced expression of the proteinase inhibitor II (PI-II) gene
in response to B. cinerea and 1-aminocyclopropane-carboxylic acid (ACC),
the natural precursor of ET, but wild type expression in response to mechanical
wounding and methyl-jasmonate (MeJA). TPK1b RNAi seedlings are impaired in
ET responses suggesting that B. cinerea induces PI-II gene expression through a
TPK1b-dependent ET signaling pathway. TPK1b is a functional kinase with
autophosphorylation and MBP (Myelin Basis Protein) phosphorylation activities.
Thus, TPK1b is a key signaling component in defense response to necrotrophic
fungi and herbivorous insects as well as ET mediated defense against pathogens.
TPK1b is a functional kinase that localizes to the plasma membrane suggesting
that TPK1b acts early in pathogen and insect response pathway and functions in
recognition and/or signaling. The impaired disease and insect resistance in TPK1b
RNAi plants is accompanied by altered ET and B. cinerea induced defense gene
expression, supporting the role of TPK1b in ET-mediated defenses. ET integrates
plant responses to developmental and environmental signals, including responses to
pathogens (Klee 2004). The function of TPK1b suggest that plants deploy multiple
and overlapping mechanisms of defense against necrotrophic pathogens and chewing
insects, despite the disparate strategies of these organisms in deriving nutrition
from plants. Interestingly, ectopic expression of TPK1b rescues the phenotype of
the Arabidopsis bik1 mutant. TPK1b and BIK1 are not orthologs but perform
partially redundant functions. The susceptibility of TPK1b RNAi plants to tobacco
hornworm coupled with the lack of ET responses suggest that TPK1b mediated ET
responses are required for defense against pathogens and insect pests. The attenuated
PI-II gene expression in TPK1b RNAi plants is also consistent with the action
of ET in parallel with the octadecanoid/wounding pathway for full PI-II gene
expression (O’Donnell et al. 1996). Mutations causing ET insensitivity in soybean
(Glycine max L.) and Arabidopsis, and inhibition of ET perception in tomato result
in increased susceptibility to necrotrophic pathogens (Hoffman et al. 1999;
Thomma et al. 1999; Diaz et al. 2002).
Multiple lines of evidence suggest that responses to insects and necrotrophic
pathogens are mechanistically linked in tomato (McCormick 1991). B. cinerea
induces wound-like responses including the expression of the wound response gene
PI-II, possibly through the actions of oligogalacturonides released from plants cell
walls by the action of fungal endopolygalacturonases. The tomato JA and wound
response mutants show susceptibility to B. cinereaand their impaired resistance to
tobacco hornworm (M. sexta) or spider mites (Tetranychus urticae) (Schilmiller
and Howe 2005) demonstrate a clear overlap in the mechanisms of resistance to
necrotrophic fungi and chewing insect pests. These overlapping responses are
mediated in part by JA levels and signaling. JAI1, SPR2, ACX1, and DEF1 are
required for basal resistance of tomato to B. cinerea. The acx1 mutant is JA-deficient
and lacks local and systemic expression of defensive PI genes in response to
wounding due to a defect in the first step of ß-oxidation in the octadecanoid
pathway (Li et al. 2005). SPR2 is a fatty acid desaturase required for the synthesis
of JA and the generation of a systemic wound signal mediating defense gene
expression in tomato (Li et al. 2003). The def1 and jai1 mutants are impaired in JA
responses and both show susceptibility to B. cinerea (Li et al. 2002; Abuqamar et al.
2008). Thus, these genes in the JA pathway are part of defense against B. cinerea
and our findings suggest that B. cinerea resistance mechanisms regulated by JA are
generally conserved between Arabidopsis and tomato.
2.9 Phyotalexins in B. cinerea Resistance
The role of induced chemical compounds in relation to B. cinerea resistance has

been studied in various plant species (Elad 1997). The accumulation of phyotalexin
has been linked to B. cinerea resistance (Prins et al. 2000b). Grape plants accumulate
phyotalexins, mainly stilbenes such as trans-and cis-resveratrol, a-and e-viniferin,
and pterostilbene in response to infection by B. cinerea (Thomzik et al. 1997).
Over-expression of the phyotalexin biosynthesis enzymes stilbene synthase involved
in the synthesis of the phytolalexins stilbene and resveratol results in resistance
to B. cinerea in tobacco and tomato (Hain et al. 1993; Thomzik et al. 1997).
In Arabidopsis, the phytoalexin camalexin accumulates in response to infection by
B. cinerea, and reduced or delayed induction of camalexin results in increased
susceptibility to the pathogen (Tierens et al. 2002; Ferrari et al. 2003a; van Wees
et al. 2003). The synthesis of camalexin, the major phytoalexin in Arabidopsis, is
regulated by the MPK3/MPK6 cascade (Ren et al. 2008). Induction of camalexin
by B. cinerea was preceded by MPK3/MPK6 activation, and compromised in mpk3
and mpk6 mutants. MPK3/MPK6 controls the Phytoalexin Deficient 2 (PAD2) and
PAD3 genes indicating that the MPK3/MPK6 cascade regulates camalexin synthesis
through transcriptional regulation of the biosynthetic genes.
2.10 Changes in Genome Wide Gene Expression

During B. cinerea Infection
To determine the nature of the B. cinerea induced defense transcriptome and identify
genes involved in host responses against the pathogen infection, the expression
profiles of B. cinerea inoculated Arabidopsis plants were studied (AbuQamar et al.
2006). Wild type Arabidopsis plants showing basal resistance were compared to
coi1, ein2, and nahG plants that represent defects in various defense responses and/
or show increased susceptibility to B. cinerea. In wild type plants, the expression
of an average of 621 genes representing roughly 0.48% of the Arabidopsis

transcriptome was induced by >twofold. B. cinerea induced genes (BIGs) encode
diverse regulatory and structural proteins implicated in defense, abiotic and oxidative
stress responses. The coi1 mutation affected the B. cinerea induced expression of
the largest set of these BIGs consistent with its enhanced B. cinerea susceptibility.
The ein2 and nahG plants, impaired in ET signaling and SA accumulation, respec-
tively, affected the expression of 63 and 80 BIGs, respectively. Forty BIGs encode
putative DNA binding proteins that belong to ET response, Zinc finger, MYB,
WRKY and HD-ZIP family transcription factor proteins. The transcriptional
activation of genes involved in cell death, removal of reactive oxygen species,
plant hormone signaling and synthesis during B. cinerea infection coupled with the
susceptibility of mutants of some B. cinerea induced genes indicates the multiplicity
of pathways required for resistance to B. cinerea consistent with the complex
inheritance of B. cinerea resistance.
2.11 Conclusion and Perspective
Genetic approaches in Arabidopsis and tomato defined some components of the

basal B. cinerea resistance. Generally, processes related to the regulation of cell
death, plant hormone signaling and synthesis are implicated in disease resistance to
necrotrophic pathogens. Future research is likely to unravel other processes that
affect plant resistance to necrotrophs and the regulatory factors involved. Future
focus will be on the biochemical and molecular mechanisms underlying host
resistance to necrotrophs and its relationship with plant responses to other classes
of pathogens. The tissue specificity of B. cinerea resistance and the extent of
environmental control of defense responses need further investigation. In addition,
transfer of knowledge gained in model plant systems to crop plants under greenhouse,
field crop production and postharvest situation is very important.
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Chapter 3
Induced Resistance in Melons by Elicitors
for the Control of Postharvest Diseases
Bi Yang, Li Yongcai, Ge Yonghong, and Wang Yi
Abstract Melons fruit can be induced to develop enhanced resistance to pathogen

infection by pre- or postharvest treatment with a variety of chemical, physical and
biological elicitors. The elicitors include acibenzolar, soluble silicon, oxalic acid,
chitosan, b-aminobutyric acid, 2,6-dichloroisonicotinic acid, heat treatment and
harpin. Resistance induced is broad spectrum and long lasting, but rarely provides
complete control of infection. The mechanism of induced resistance is involved in
the accumulation of defense enzymes, antifungal compounds, increasing of reactive
oxygen species and lignification of epidermal cells. In order to maximize the efficacy
of resistance elicitors, it is required to understand of the mechanism of induced
resistance and the effect factors of pre- or postharvest. There also needs to evaluate
quality change in induced fruit. It is concluded that control of melons postharvest
disease by induced resistance would be the use of integrated approach combining
chemical, physical and biological control methods, and culture practices.
Keywords postharvest diseases • induced resistance • fruit • elicitors
3.1 Introduction
Melons, Cucumis melo L., are well known members of the Cucurbitaceae family,
has been divided into a number of botanical subspecies (Sykes 1990). The major
ones are cantaloupes (Cucumis melo var. cantalupensis), muskmelons (C. melo
var. reticulatus), oriental melons (C. melo var. chinensis) and winter melons
(C. melo var. inodorus). China is the biggest producer of melons in the world (Bi
et al. 2007b).
Bi Y.,(), Li Y.C., Ge Y.H., and Wang Y.

College of Food Science and Engineering, Gansu Agricultural University,
Lanzhou, 730070, China
e-mail: beyang62@163.com

32 B. Yang et al.
The melons are quite perishable after harvest. A various types of pathogens are
involved in fruit decay (Snowdon 1990; Barkai-Golan 2001). In China, melons are
infected by Alternaria rot (Alternaria alternata), blue mold rot (Penicillium spp.),
Fusarium rot (Fusarium spp.), Green mold rot (Cladosporium sp.), Mucor rot (Mucor
mucedo), pink mold rot (Trichothecium roseum), Rhizopus rot (Rhizopus spp.),
sour rot (Geotrichum candidum) (Bi et al. 2003). Both Alternaria and Fusarium
rots are related to latent infection (Ge et al. 2005b). Postharvest losses are estimated
to be as 35–50% because of rough handling after harvest, inadequate packaging and
temperature management (Bi et al. 2007b).
Postharvest disease of melons is often controlled by the application of synthetic
fungicides, such as benomyl and guazatine (Morris and Wade 1983), imazalil
(Aharoni et al. 1992), iprodione and azoxystrobin (Ma et al. 2004). However, due
to problems related to fungicide residues, development of fungicide resistance by
pathogens, and potential harmful effects on the environment, as well as the neces-
sity to reduce losses with minimal use of fungicides, new strategies for controlling
postharvest diseases have been proposed (Wilson et al. 1994).
Induced resistance is a natural defense response triggered by pathogen or elicitors
(Kuc 2001). Induced resistance includes local induced resistance and systemic
acquired resistance (SAR). The former is directly producing disease resistance at
the guided position; the latter is that untreated part produced disease resistance after
treated the other part (Ryals et al. 1996). Induction disease resistance in harvested
horticultural crops using physical, biological and chemical elicitors has received
increasing attention over recent years, it being considered a preferred strategy for
disease management (Terry and Joyce 2004). Fruit and vegetables can be induced
to develop enhanced resistance to pathogen infection by pre- or postharvest treatment
with a variety of chemical elicitors (Bi et al. 2007c). In this chapter, we provide a
description of research that indicates that induced resistance by elicitors can be part
of postharvest disease control, and provide a brief knowledge on the mechanism of
induced resistance in melons.
3.2 Chemically Induced Resistance
3.2.1 Acibenzolar
Acibenzolar (benzo-(1, 2, 3)-thiadiazole-7-carbo-thioicacidS-methylester; ASM;

BTH; Bion™; Actigard™) is perhaps the most potent synthetic SAR activator
discovered to date (Terry and Joyce 2004; Kessmann et al. 1994; Friedrich et al.
1996). The chemical is not phytotoxic and has proven an effective SAR elicitor in
both monocotyledons (Gorlach et al. 1996) and dicotyledons (Tally et al. 2000).
In recent years, ASM has been extensively studied to control postharvest diseases
in apple (Spadaro et al. 2004), pear (Cao and Jiang 2006), peach (Liu et al. 2005),
strawberry (Terry and Joyce 2000), passionfruit (Willingham et al. 2002), mango
(Dann and Zainunuri 2008) and potato (Bokshi et al. 2003).
3 Induced Resistance in Melons by Elicitors for the Control of Postharvest Diseases 33
Huang et al. (2000) found a pre-flowering foliar spray of ASM at 50 mg/L com-
bined with a fruit dip in guazatine at 500 mg/L at harvest substantially decreased
disease in stored rockmelons and Hami melons. Preharvest treatment with ASM at 50
mg/L four times from anthesis and one time (3 weeks before harvest) also reduced
Alternaria and Fusarium rots in harvested rockmelons (Bokshi et al. 2006). A foliar
spraying with ASM at 100 mg/L 1 week or 1 day before harvest decreased the lesion
area of harvested muskmelons inoculated with F. semitectum and T. roseum (Ge et al.
2008). Field spraying with ASM at 100 mg/L significantly reduced the latent infec-
tion rate of muskmelons. Four sprays from anthesis reduced latent infection rate in
fruit caused by A. alternata and Fusarium spp, by 66.7 and 60% (Zhang et al. 2006b).
Postharvest treatment with ASM at 200 mg/L noticeably reduced decay severity
caused by A. alternata, F. semitectum and T. roseum in Hami melon (Bi et al. 2006).
Postharvest ASM treatment at 100 mg/L suppressed lesion diameter in treated and
untreated halves of the same fruit inoculated with T. roseum, indicating that the
chemical induced local and systemic resistance (Wang et al. 2008).
As a functional analogue of SA, ASM acts downstream of SA and elicits accu-
mulation of the same SAR genes and pathogenesis-related proteins (PRs) as SA
(Friedrich et al. 1996). Bi et al. (2006b) reported that activity of peroxidase (POD),
chitinase (CHT) and phenylalanine ammonia lyase (PAL) was significantly enhanced
in harvested Hami melons treated with ASM. Similar results were also observed in
rockmelons (Bokshi et al. 2006) and muskmelons (Wang et al. 2008). ASM treat-
ment increased production of reactive oxygen species, augmented the content of the
preformed antifungal compounds, total phenolics and lignin, and accumulated
lignin, suberin and callose in muskmelon (Bi et al., unpublished data).
3.2.2 Silicon
Silicon (Si) is the second most abundant element in the lithosphere (27.70%) and it
is as important as phosphorus and magnesium (0.03%) in the biota (Exley 1998).
Si is also considered to be biologically active and to trigger a faster and more extensive
deployment of plant natural defenses (Fauteux et al. 2005). Si has been shown to
reduce postharvest diseases in pear (Spotts and Cervantes 1989), cherry (Qin and
Tian 2005) and jujube (Tian et al. 2005).
Postharvest application of silicon oxide and sodium silicate tended to suppress
postharvest pink rot severity caused by T. roseum in muskmelons (Guo et al. 2007).
Sodium silicate at 100 mM significantly reduced decay incidence and severity of
Hami melons inoculated with A. alternata, F. semitectum, and T. roseum. Si treat-
ments at 100 mM were also effective in reducing natural infections of Hami mel-
ons. Si treatments applied at 100 mM pre-inoculation with T. roseum had lower
decay incidence and severity than treatments applied post-inoculation, indicated
resistance induction of occurred in melon fruit (Bi et al. 2006a).
Postharvest treatment with Si proved effective for inhibiting pathogen growth as
well as inducing disease resistance in melons. Sodium silicate has been shown to
have direct inhibition in vitro antifungal activity against postharvest pathogens of
34 B. Yang et al.
melons (Bi et al. 2006a). Si treatment resulted in enhanced activity of POD and
PAL (Guo et al. 2007). Si also caused a more progressive and significant increase
in POD and CHT activities in Hami melons challenged by T. roseum (Bi et al.
2006a). Si treatment enhanced the production of reactive oxygen species and main-
tained fruit firmness. The accumulation of antifungal compounds was observed in
Si treated muskmelons (Bi et al., unpublished data).
3.2.3 Other Chemicals
The application of oxalic acid has already been shown to induce systemic resistance
against field diseases in cucumber (Mucharroman and Kuc 1991). The chemical has
been confirmed to reduce postharvest diseases in mango (Zheng et al. 2007) and
longan (Whangchai et al. 2006). Postharvest oxalic acid dipping at 50 mM reduced
significantly decay severity of muskmelon fruit inoculated with T. roseum. A systemic
resistance was observed in untreated halves of the same fruit (Deng et al. 2008).
Chitosan has been considered a promising means for enhancing disease resis-
tance in harvested horticultural commodities (Wilson et al. 1994; El Ghaouth
1994). A significant reduction of rots has been recorded in chitosan-treated apple
(Bautista-Banos et al. 2004), pear and kiwifruit (Du et al. 1997), table grape (Meng
et al. 2008), strawberries (Zhang and Quantick 1998), bell pepper (El Ghaouth et al.
1997), tomato (Liu et al. 2007), carrots (Cheah et al. 1997). Preharvest treatment with
chitosan at 1 mg/mL significantly reduced the latent infection rate of muskmelons.
Three and four sprays from anthesis significantly reduced latent infection rate in fruit
caused by A. alternata and Fusarium spp. Four sprays before harvest also decreased
the lesion area of harvested muskmelons inoculated with A. alternata, F. semitectum
and T. roseum, indicated resistance induced in melon fruit (Xie et al. 2008).
Although b-aminobutyric acid (BABA) is only rarely found naturally in plants,
it has proved to be a potent inducer of acquired resistance and has a broad spectrum
of activity against many disease-causing organisms (Cohen 2002; Jakab et al.
2001). Earlier foliage application of BABA at 2000 mg/L reduced the total storage
rots, Alternaria and Fusarium rots of fruit. The chemical caused activation of CHT
and POD activities in treated leaves of rockmelons (Bokshi et al. 2006).
2,6-dichloroisonicotinic acid (INA) protect many crops against their pathogens.
INA is weakly fungistatic in vitro, but effectively elicits SAR genes in tobacco
prior to TMV challenge inoculation (Ward et al. 1991). The INA-mediated resis-
tance has been reported to be against a broad spectrum of pathogens (Uknes et al.
1992) and the induced resistance has been suggested to have a long-lasting effect
(Lucas 1999). Bokshi et al. (2006) found that earlier foliage application of INA at
50 mg/L significantly reduces the postharvest diseases of rockmelons. Activity of
CHT and POD in treated leaves were stimulated and maintained at a higher level
three days after a first spray with INA rather than BABA, and second spray of INA
increased CHT and POD activities further and the increase lasted several weeks
until harvest.
3.3 Physically Induced Resistance
Heat treatment is considered a promising method for reducing postharvest disease

(Lurie 1998). It may be effective either by directly inhibiting pathogen develop-
ment, or by inducing natural resistance in the fruit (Klein and Lurie 1991; Schirra
et al. 2000). Induction of resistance against decay due to hot water treatment of
muskmelons for 3 min at 55°C before inoculation was reported by Zhang et al.
(2005). The resistance was found to be most effective when inoculation was carried
out 24 h after hot water treatment. Dipping melons in hot water not only reduced
pathogens causing storage disease but also significantly improved the life and mar-
ketability of fruit (Fallik et al. 2000). Teitel et al. (1989) found that with a longer
immersion time, a hot water dip may have provided effective protection for melon
fruit against storage rots. They observed that a reduced temperature of 52°C and a
longer dip time of 2 min controlled decay from Alternaria spp., Fusarium spp.,
Rhizopus spp. and Mucor spp. without causing external heat injury. Similar obser-
vation made by Mayberry and Hartz (1992), and Barkai-Golan et al. (1994), who
reported that hot water treatment of ‘Galia’ melon at 52–55°C effectively prevented
storage losses caused by A. alternata, Fusarium spp. and T. roseum. A mixture of
fungicides and hot water could result in an effective decay control of melons. Zhang
et al. (2005) observed that hot water treatment at 55°C for 1 min combined with 50
mg/ L azoxystrobin or 250 mg/L imazalil significantly controlled the rots of musk-
melons caused by R. stolonifer, Fusarium spp. and T. roseum.
3.4 Biologically Induced Resistance
Harpin (Messenge™) is an acidic, heat-stable, glycine-rich, 44-kDa protein, encoded

by the hrpN gene of the bacterium Erwinia amylovora (Wei et al. 1992). It is the
first known bacterial product able to elicit the hypersensitive response (HR) and to
induce systemic acquired resistance in plants (Baker et al. 1993; Dong et al. 1999;
Mullin et al. 1998). Postharvest treatment with harpin has been shown to induce
resistance in apple (de Capdeville et al. 2003) and pear (Wang et al. 2006).
Field spraying with harpin at 50 mg/L reduced the latent infection rate of musk-
melons. Three and four sprays from anthesis significantly decreased latent infection
rate and relative surface of fruit (Wang and Bi, unpublished data). Postharvest
harpin treatment at 90 mg/L decreased decay severity caused by A. alternata,
F. semitectum and T. roseum in Hami melons (Bi et al. 2007a). Similar results were
observed by Ge et al. (2005a) in muskmelons inoculated by F. semitectum and
T. roseum. A higher concentration over 90 mg/L failed to promote resistance and
did not cause phytotoxicity to melons. Harpin did not demonstrate any fungicide
effect in vitro, suppressed lesion diameter of fruit inoculated with T. roseum in
treated and untreated halves of the same melon, indicating that the chemical
induced local and systemic resistance. Besides, harpin provided greater level of
36 B. Yang et al.
decay control in long-storage-life cultivars (cv. 8601) than in short-storage-life

ones (cv. New Queen). The time between initial treatment with harpin and subse-
quent inoculation with T. roseum significantly affected efficacy of the induction
(Bi et al. 2005). Harpin treatment applied at early stage of maturity was more effective
than at later stage (Bi et al., unpublished data).
Studies have shown that harpin triggers a variety of cellular responses, such as
activation of reactive oxygen species and cell membrane depolarization (Dong et al.
1999). Harpin induced a significant and progressively increasing activity of POD
and CHT in muskmelon and Hami melons (Ge et al. 2005a; Bi et al. 2005; Wang
et al. 2008). Postharvest application of harpin also increased activity of defense
enzymes, and resulted in a production of activated oxygen species, an increase of the
preformed antifungal compounds and total phenolics, and an accumulation of lignin,
suberin and callose in epidermal cell of muskmelons (Bi, unpublished data).
3.5 Conclusions
Induced resistance may be an important part of postharvest disease control strategies

of the melons in the future. Because of the apparent safety and the broad-spectrum
nature of the induced resistance, research is underway to identify and develop elicitors
that can be used to induce resistance in melons and thus make this resistance
directly applicable to postharvest disease control. However, despite some studies as
mentioned above, only a few cultivars have been studied in any detail. Having more
concrete information on the biological spectrum of induced resistance on a cultivar-
by-cultivar basis will be of use in determining which cultivar may be suitable for
this type of control.
Having a better understanding of induced resistance response in more than one
cultivar is important in developing any generalizations about this type of resistance.
Information on the relative importance of putative defense mechanisms utilized in
induced resistance is also limited. We know that induction of resistance is accom-
panied by the accumulation of PR proteins, defense enzymes and antifungal
compounds, increasing of reactive oxygen and lignification of epidermal cells.
There are other likely defense reactions and compounds that are also probably
involved in the observed resistance. They could be more rapidly induced than the
markers, and could be more effective against the pathogen (Hammerschmidt 1999).
Each of the defense-related phenomena reported above have characteristics and
correlations that strongly suggest that each is associated with the resistance state.
However, the contribution of each to resistance appears to be small. This strongly
suggests that the expression of full resistance requires the expression of several
mechanisms, many of which are known to be coordinately regulated (Ward et al.
1991). Furthermore, understanding the defenses and the signals that regulate these
defenses will also provide new avenues for implementation of induced resistance
by genetic engineering or manipulation of signal pathway (Hammerschmidt and
Becker 1997).
In our haste to realize the potential offered by induced resistance for postharvest
disease control, we have paid too little attention to the factors that are likely to
influence its effectiveness, largely using it inappropriately as simply a fungicide
replacement. Therefore, there is an urgent need for understanding of the various
factors (such as cultivars, maturity, timing of treatment, field environment, posthar-
vest handling and storage condition) that will influence the expressing of induced
resistance in melon fruit, in order to maximize the efficacy of resistance elicitors
(Walters et al. 2005). A more realistic scenario to combat decay of harvested melons
would be the use of integrated control approach combining biological and physical
control strategies, with or without limited quantities of fungicides pre-harvest, and
with efficient management and handling practices.
As the releasing of volatile compounds could be inhibited in ASM treated
muskmelons (Jiang et al. 2007), there needs to evaluate quality change in melon
fruit treated with elicitors and be assurance that induced natural defense compounds
effective against pathogens are not present in consumed tissues at levels toxic to
mammals (Paiva 2000; Dann 2003).
The implementation of induced resistance by chemicals in melons should be
approached with cautious optimism. The enormous potential for reducing postharvest
diseases via their natural disease resistance mechanisms has been demonstrated.
However, more information is required to ensure that this type of resistance offers
a safe, effective and reliable complement to the existing methods.
Acknowledgements We are grateful to Dr. Dov Prusky (Department of Postharvest Science,

ARO, Volcani Center, Israel) for offering the opportunity to write this chapter. This work was
financially supported by National Natural Science Foundation of China (30671465), Ministry of
Science and Technology of China (2001BA501A09) and Australia Center of International
Agricultural Research (ACIAR, PHT/1998/140).
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Chapter 4
Mechanisms Modulating Postharvest Pathogen
Colonization of Decaying Fruits
Dov Prusky, Noam Alkan, Itay Miyara, Shiri Barad, Maayan Davidzon,
Ilana Kobiler, Sigal Brown-Horowitz, Amnon Lichter, Amir Sherman,
and Robert Fluhr
Abstract As biotrophs, insidious fungal infections of postharvest pathogens remain

quiescent during fruit growth while at a particular phase during fruit ripening and
senescence the pathogens transform to necrotrophs causing typical decay symptoms.
Exposure of unripe hosts to pathogens (hemi-biotroph or necrotrophs), initiates defen-
sive signal-transduction cascades that limit fungal growth and development. Exposure
to the same pathogens during ripening and storage activates a substantially different
signalling cascade which facilitates fungal colonization. This chapter will focus on
modulation of postharvest host-pathogen interactions by pH and the consequences of
these changes. Host pH can be raised or lowered in response to host signals, including
alkalization by ammonification of the host tissue as observed in Colletotrichum and
Alternaria, or acidification by secretion of organic acids as observed in Penicillium,
Botrytis and Sclerotinia. These changes sensitize the host and activate transcription and
secretion of fungal hydrolases that promote maceration of the host tissue. Several par-
ticular examples of coordinated responses which follow this scheme are described.
4.1 Introduction
Postharvest fungal pathogens exploit three main routes to penetrate the host tissue:
(i) through wounds caused by biotic and/or abiotic agents during growth and storage;
(ii) through natural openings such as lenticels, stem ends and pedicel-fruit inter-
phase, and (iii) by direct breaching of the host cuticle, which can occur throughout
D. Prusky (*), N. Alkan, I. Miyara, S. Barad, M. Davidzon,

I. Kobiler, S. Brown-Horowitz, and A. Lichter
Department of Postharvest Science of Fresh Produce, Agricultural Research Organization,
the Volcani Center, Bet Dagan, 50250
e-mail: dovprusk@agri.gov.il
A. Sherman
Department of Genomics, Agricultural Research Organization, the Volcani Center,
Bet Dagan, 50250
R. Fluhr
Department of Plant Genetics, The Weizmann Institute of Science Rehovot 76100, Israel

44 D. Prusky et al.
the fruit growth period. An active pathogenic process can start immediately after
spores land on the wounded tissue, whereas other pathogens can breach the unripe
fruit cuticle and then remain inactive for months until the harvested fruit ripens.
The penetration process may go unnoticed by the host, or it may result in rapid
defense signaling that results in the induction of defense molecules that will limit
fungal development (Prusky and Lichter 2007). The period from host infection to
the activation of fungal development and symptom expression is designated the
quiescent stage (Prusky 1996). After harvest, during ripening and storage, the
mechanism that protects the fruit from fungal attack becomes insufficient. This
transition from a resistant to susceptible state parallels physiological changes that
occur during ripening to which the pathogen senses and responds. In the present
chapter, we will focus on modulation of the host environment by the pathogen as a
mean for fungal colonization. We also touch upon the signals that may activate the
transition from quiescent to necrotrophic mode during fruit ripening.
4.2 The Quiescent Stage
During the colonization of plant hosts, postharvest fungal pathogens exploit two
main modes of nutrition: biotrophy, in which the nutrients are obtained from the
living host cells, and necrotrophy, in which nutrients are obtained from dead host
cells killed by the fungus (Perfect et al. 1999). Both of these nutritional modes are
exhibited by postharvest pathogens. Opportunistic postharvest pathogens may also
be located within the fruit in an inactive mode, awaiting fruit wounding, ripening
or senescence. The length of each period may vary among pathogens, hosts, and
host developmental stages. Pathogens such as Colletotrichum, Monilinia, Botrytis
and Alternaria may remain quiescent for long periods in developing fruit tissues,
but initiate immediate necrotrophic development on ripening and senescing fruits.
Colletotrichum is one of the major postharvest pathogens in which quiescence has
been studied. Colletotrichum spores adhere to and germinate on the plant surface,
produce germ tubes, and the tip of the germ tube developing from the appressorium
sends an infection peg through the cuticle. Following penetration, Colletotrichum
initiates subcuticular intramural colonization (Perfect et al. 1999) and spreads rapidly
throughout the tissue with both inter- and intracellular hyphae. After colonizing one
or more host cells, the infecting hyphae, which can be described as biotrophic
(Kramer-Haimovich et al. 2006), subsequently give rise to secondary necrotrophic
hyphae (Bailey and Jeger 1992; Coates et al. 1993; Latunde-Dada et al. 1996;
Mendgen and Hahn 2001; O’Connell et al. 1985). Botrytis and Monilinia can
penetrate through wounds and also breach the fruit cuticle by using an infection peg
from an appressorium that then remains quiescent for long periods of time (Fourie
and Holz 1995; Pezet et al. 2003). Depending on the physiological status of the
organ, these hyphae may continue the infective process or remain quiescent.
Based on published reports, three major hypothetical modes for the activation of
quiescent biotrophic pathogens have been suggested (Prusky 1996): (i) deficiency
4 Mechanisms Modulating Postharvest Pathogen Colonization of Decaying Fruits 45
in the host nutritional resources required for pathogen development; (ii) the presence
of preformed or inducible fungistatic antifungal compounds in resistant unripe
fruits, and (iii) an unsuitable environment for the activation of fungal pathogenicity
factors. In the present work we will concentrate in the modulation of host environ-
ment by the fungus as mechanism of fungal colonization.
4.3 pH Modulation of the Environment by Postharvest Fungal

Pathogens Is a Virulence Potentiation
The ability to modify pH may be expressed in either direction, and fungi that raise
or reduce it are described as “alkalizing fungi” or “acidifying fungi”, respectively.
The mechanisms regulating ambient pH modulation are tied to the levels of ambient
pH. Fine-tuning of enzyme expression in response to the ambient pH in the host by
the fungus further highlights the importance of the specific regulatory system that
is activated under a changing environmental pH which can lead to the activation of
quiescent infections (Prusky and Yakoby 2003).
4.3.1 Alkalizing Fungi
In normal ripening and senescing fruits, pH levels change as part of the ripening
process: for instance, the pH of avocado fruit increases from 5.2 to 6.0 during ripening
(Yakoby et al. 2000) (Fig. 4.1). Alkalinization of the fruit host environment during
Fig. 4.1 Changes in pH values of the pericarp (◊) of cultivar Fuerte avocado fruits during posthar-
vest ripening. Firmness (¨) is presented as a parameter of ripening. Arrow indicates the time of
decay initiation (DI) and of decay symptoms (DS) of C. gloeosporioides, following inoculation of
freshly harvested fruits. Bars represent the standard deviations of the mean from one representative
experiment. Cultivar Fuerte fruits were harvested at midseason
46 D. Prusky et al.
Fig. 4.2 Measurement of the local pH environment in tomato fruit at the leading edge of the
decay caused by C. coccodes infection. (a): pH values as a function of distance from the leading
edge of the decay in infected (¨) and uninfected () tissue. (b): Fluorescence micrograph with
BCECF at different distances from the region of decay. The changes in pH were detected
microscopically in 10 mm long by 1 mm thick strips of tissue stained by BCECF fluorescence dye
after correlating fluorescent values with direct pH determinations obtained by a piercing pH
electrode. Fluorescence was detected by Leica fluorescence binocular the average value of 50
evaluated cells is reported. A representative picture of cells at 1, 5 and 10 mm distance for the
hyphal front is presented in inset (b)
colonization by postharvest pathogens such as Colletotrichum and Alternaria is

associated with fungal secretion of ammonia (Eshel et al. 2002; Prusky et al. 2001a).
In avocado, the fruits pericarp showed a pH of 5.2 that increased up 7.5 and 8.0,
during C. gloeosporioides attack (Yakoby et al. 2000). In tomato fruits, the initial
pH ranged between 4.1 and 4.5 and decay development at the infection site increased
the pH to 8.0 and the ammonia accumulated to 3.6 mM compared to 0.2 mM in
the healthy tissue (Alkan et al. 2008; Prusky et al. 2001) (Fig. 4.2). In the case of
polyphage pathogens such as A. alternata, a threefold to tenfold increase in ammonia
concentration, and a pH elevation of 0.2 to 2.4 pH units were detected in several
hosts: tomato, pepper, melon, cherry and persimmon (Eshel et al. 2002). Ambient
alkalization by fungi is achieved by active secretion of ammonia, which is produced
as a result of protease activity and deamination of amino acids (Prusky and Yakoby
2003). Interestingly, the initial acidic pH of the fruit has been shown to be conducive
to the enhanced ammonium secretion and host-tissue alkalization that facilitate
fungal virulence (Alkan et al. 2008; Kramer-Haimovich et al. 2006) (Fig. 4.3).
The isolation of C. coccodes mutants with reduced ammonium production has
provided direct evidence for the necessity of ammonium accumulation during
Fig. 4.3 Effect of pH on ammonia secretion by C. gloeosporioides. Samples of medium were

quantified for ammonia accumulation at different times after transfer to secondary medium
containing KNO3, buffered with phthalate buffer to a pH range of 4.0 to 7.0. Symbols: ◊ pH 7,
pH 6.5, ▲pH 6, □ pH 5,  pH 4. Significance was calculated by comparing the concentrations
of ammonia at the different initial pHs on each sampling day and was analyzed statistically by
analysis of variance of three replications. Values for points on the same day labeled with the same
letter were not statistically different (P < 0.05)
Colletotrichum pathogenicity (Alkan et al. 2008), indicating that ammonium

accumulation is a critical factor contributing to Colletotrichum necrotrophic devel-
opment in ripening fruits (Prusky and Yakoby 2003). Pathogens alter the local pH
at the infection site to suit the increased expression of pathogenicity factors and the
enzymatic arsenal (Denison 2000; Yakoby et al. 2000; Prusky et al. 2001; Eshel
et al. 2002; Prusky and Yakoby 2003). Expression of the endoglucanase gene AaK1
by Alternaria alternata was found to be maximal at pH levels above 6.0, values that
are characteristic of decayed tissue. AaK1 was not expressed at the lower pH
values in which the pathogen was quiescent (Eshel et al. 2002). In the pathogen
C. gloeosporioides, the gene pelB was expressed when the pH was above 5.7 and
was positively correlated with ambient alkalinization (Yakoby et al. 2000, 2001).
The transcription factor pac1, which is involved in pH regulation, follows a pattern
similar to that of pelB, which suggests that they are co-regulated or that pac1
regulates the expression of pelB (Drori et al. 2003) (Fig. 4.4). Dpac1 mutants of
C. gloeosporioides showed 85% reduction of PELB transcript, delayed PL secretion
(Fig. 4.5) and dramatically reduced virulence, as demonstrated by infection assays
with avocado fruits (Miyara et al. 2008).
4.3.2 Acidifying Fungi
Other postharvest pathogens, such as P. expansum, P. digitatum, P. italicum (Prusky

et al. 2003), B. cinerea (Manteau et al. 2003) and S. sclerotiorum (Bateman and
Beer 1965; Ruijter et al. 1999), utilize tissue acidification to support their attack via
Fig. 4.4 Transcriptional activation of pelB and pac1 by C. gloeosporioides as a function of pH
levels. Expression as a function of pH: Northern analysis of total RNA isolated from C. gloeospo-
rioides mycelia 16 h after transfer to fresh secondary cultures buffered with phthalate. Blots were
probed with pelB (pelB panel) and then sequentially stripped and reprobed with pac1 (pac1 panel)
and rDNA probes
Fig. 4.5 Relative expression of PAC1, PELB and PL secretion by the wild-type and Dpac1 mutant
strains of C. gloeosporioides at pH 6.0. (a) Expression of PAC1. (b) Expression of PELB and PL
secretion. Relative expression was detected by RT-PCR 7 and 15 h after transfer of the growing
hyphae from the primary medium to the inductive secondary medium. The relative expression
values are the averages of three replications of the treatment ± standard deviations. Western blot
analyses were repeated three times, and results from a representative experiment are presented
the secretion of organic acids. Penicillium spp. acidifies the ambient environments
of apple and citrus fruit during decay development. P. expansum caused decay of
apple fruits of several cultivars, in which it caused pH decreases from values ranging
from 3.95 to 4.31 in the healthy mesocarp to values ranging from 3.64 to 3.88 in
the decaying tissue. P. digitatum and P. italicum caused similar pH declines in citrus
fruits (Prusky et al. 2004). In the necrotrophic fungus Sclerotinia sclerotiorum, during
plant infection, a pH gradient was established in relation to oxalic acid secretion
and the pH of the host reached as low as pH 4.0 (Rollins and Dickman 2001).
This production of oxalic acid during plant infection has been implicated as a
primary determinant of pathogenicity in this and other phytopathogenic fungi.
The size of lesions induced by different strains of B. cinerea on grapevine and
bean leaves correlated with the amount of OA that these strain in vitro (Germeier
et al. 1994). However it remains unclear whether the level of OA produced in planta
is sufficient to cause host cells to directly collapse. It is suggested that OA may
in fact be a co factor in pathogenesis rather than the primary phytotoxic agent
(Manteau et al. 2003).
Several mechanisms for tissue acidification have been proposed on the basis of
such studies. Penicillium spp. use two mechanisms for ambient acidification: the
production of organic acids, mainly citric and gluconic, and NH4+ utilization associ-
ated with H+ efflux. In decayed fruit by P. expansum and P. digitatum, both pathogens
produced significant amounts of citric and gluconic acids in the decayed tissue and
reduced the host pH by 0.5 to 1.0 units (Table 1). Ammonium depletion from the
growth medium or from the fruit tissue was directly related to ambient pH reduction.
The organic acids that accumulated during tissue acidification by P. expansum were
mainly gluconic (GA) and citric acids (Prusky et al. 2003) the same as for Aspergillus
(Ruijter et al. 1999) which secrete as well, gluconic and citric acids. The contribution
of gluconic acid secretion to the colonization of apple tissue by P. expansum was
Table 4.1 pH levels in healthy and Penicillium-decayed fruits

pH value±SE
Healthy Decayed
Penicillium sp. Host Cultivars
P. expansum Apple Granny Smith 3.95±0.06 3.64±0.01
Gala 4.31±0.06 3.88±0.03
Red Delicious 4.44±0.03 4.07±0.02
Fuji 4.44±0.06 3.96±0.02
Golden Delicious 4.54±0.06 3.88±0.03
P. digitatum Oranges Naval 4.77±0.45 3.12±0.07
Grapefruit Oro Blanco 4.74±0.05 3.10±0.14
P. italicum Oranges Naval 4.77±0.07 3.02±0.13
Grapefruit Oro Blanco 4.55±0.13 3.23±0.17
* pH changes induced by P. expansum, P. digitatum and P. italicum on different cultivars of apple
and citrus fruits. pH was measured directly with a micro-combination pH electrode Model
9810BN (Orion, Beverly, MA). Measurements were taken 7 days after inoculation.
50 D. Prusky et al.
analyzed by modulation (increase or decrease) of gluconic acid accumulation at the

infection court. P. expansum isolates that express higher gox2 transcripts, glucose
oxidase (GOX) activity and that secrete the highest amount of gluconic acid, caused
disease of apple at the fastest rate (Fig. 4.6). Furthermore, the detection of signifi-
cantly high levels of transcripts of gox2 and GOX activity at the edge of the decaying
tissue emphasize the involvement of GOX in tissue acidification of the decaying tis-
sue. Taken together, these results emphasize the importance of GOX in the production
of the gluconic acid that leads, in turn, to host tissue acidification (Hadas et al. 2007).
Organic acid accumulation findings, demonstrate that there is a close relation between
the accumulation of gluconic acid and virulence of P. expansum, S. sclerotiorum and
Fig. 4.6 Effect of the aggressiveness of Penicillium expansum isolates on the pH and on the
production of organic acids in infected Golden Delicious apples. (a) Decayed area 5 days after
inoculation with P. expansum; (b) pH of the infected tissue. Average values of 10 replicates (± SD)
are presented and (c) Amounts of organic acids (dark column indicates citric acid and bright column
indicates gluconic acid)
B. cinerea (Rollins and Dickman 2001; Manteau et al. 2003). The production of
oxalic acid by S. sclerotiorum is regulated by the ambient pH environment that met
the pathogen. This regulation of pH responsive genes is mediated in part by the zinc
finger transcription factor encoded by pac1, an orthologue of the Aspergillus nidulans
pacC gene. Accumulation of pac1 transcripts paralleled increases in ambient pH, and
oxalic acid production increases with the ambient pH of the growth medium, as does
oxaloacetase activity, the enzyme proposed to catalyze oxalic acid production by
hydrolysis of oxaloacetate (Rollins and Dickman 2001). Oxalic acid secretion is a
pH-regulated process and, in turn, its accumulation, by virtue of environment acidi-
fication, may serve as a regulator of acid pH-regulated processes. Functionally
characterization of the pacC gene homolog, pac1, from S. sclerotiorum' have demon-
strated that although oxalic acid production is alkaline pH-responsive, the kinetics
and magnitude of oxalate accumulation are dramatically altered and virulence, in
loss-of-function pac1 mutants, was dramatically reduced in infection assays with
tomato. Based on these results, pac1 appears to be necessary for the appropriate regu-
lation of physiological processes (Rollins 2003).
The accumulating findings suggested that pH modulation of the environment
enables the pathogen the “selection” of specific virulence factors needed for the par-
ticular host. Several studies on fungal pathogen demonstrate that the ambient pH
conditions in each host induce the expression of a specific subset of fungal genes,
selected from large gene families that encode cell-wall-degrading enzymes (Eshel
et al. 2002; Prusky and Yakoby 2003). In the case of P. expansum, the secretion of
gluconic acid and, to a lesser extent, citric acid enhanced the expression of pectolytic
enzymes and the establishment of conditions for necrotrophic development of P.
expansum (Hadas et al. 2007; Torres and Candelas 2003; Prusky et al. 2003). Analysis
of transcripts encoding the endopolygalacturonase gene, pepgl, from P expansum
accumulated under acidic culture conditions from pH 3.5 to 5.0, suggesting that the
acidification process is a pathogenicity enhancing factor of Penicillium spp. This
hypothesis was supported by the findings that cultivars with lower pH as well as citric
acid treatments that reduced tissue pH, increased P. expansum development. However,
organic acid treatment could not enhance decay development in naturally acidic
apples. Conversely, local alkalinization with NaHCO3 reduced decay development
(Prusky et al. 2003). The accumulated findings so far demonstrate that ambient pH is
a regulatory cue for processes linked to pathogenicity, and that specific genes are
expressed as a result of the modified host pH created by the pathogen.
4.4 Effectors That Activate the Transition from Quiescent

to Necrotrophic Infection
The transition from biotrophism to a necrotrophic-saprophytic stage appears to be

related to factors that are modulated at the intracellular level, and that are affected
by nutrients and by ambient pH. Each of the secreted compounds (organic acid or
52 D. Prusky et al.
ammonia) plays a critical physiological role in the initiation of necrotrophic devel-

opment. Speculation regarding the mechanisms by which secretion of organic acids
enhances virulence centers on three modes of action. First, oxalate may be directly
toxic to host plants and may weaken them, thereby facilitating invasion. Second, it
has been hypothesized that chelation of cell-wall Ca2+ by oxalic and gluconic acids
loosens the plant cell wall (Bateman and Beer 1965; Hadas et al. 2007). Finally,
oxalate secretion may suppress and activate ROS generation in a pH dependent
manner and the associated plant defense responses, thereby contributing to activation
of the necrotrophic mode of development (Cessna et al. 2000; Kim et al. 2008).
Ammonification of the tissue also has significant physiological and biochemical
effects both on the host and on the pathogen. Healthy plant cells have an electro-
chemical proton gradient across the plasma membrane that is important for ion
uptake, solute transport, and cell-wall growth. Several studies have demonstrated
that transient extracellular alkalization is essential for the induction of defense
responses by fungal elicitors (Schaller and Oecking 1999). Ammonium-toxicity
symptoms can lead to host stress responses expressing elevated ethylene synthesis
and various changes in membrane flux (Ingermarsson et al. 1987). Alkan et al. (2009)
demonstrate third role for ammonium secretion in the induction of host ROS
accumulation through the NADPH oxidase mechanism in a pH dependent manner.
ROS activation by ammonium can act as a factor promoting cell death in tomato
fruit at the leading edge of the colonizing hyphae (Alkan et al. 2009). Kramer-
Haimovich et al. (2006) suggested a fourth way to affect colonization. Ammonium
can directly affect the fungus pathogenicity by activating the expression of genes
encoding pathogenicity factors, such as pelB expression and PL secretion in
C. gloeosporioides. Finally ammonium can also indirectly affect the pathogen-host
interaction by elevating the local pH of the infection site to create an optimal
environment for production of pathogenicity factors as PL and endoglucanase
(Yakoby et al. 2001; Eshel et al. 2002). This suggests that ammonification by
Colletotrichum at the leading edge of the infection site may activate host responses
leading to cell death and activate fungal pathogenicity factors. This way ammonium
may act as an effector for activation of the transition from quiescent to necrotrophic
mode in the alkalizing pathogen life cycle.
4.5 Summary
It has become clear in recent years that the activation of quiescent infections is
not a simple process whereby a decline in host resistance results in the activation
of fungal attack. Activation of quiescent biotrophic infections seems to involve a
coordinated series of events. The complexity of this process can be attributed to
the significant physiological and biochemical changes that occur in the host dur-
ing ripening and senescence, and that lead to decreased host response and
increased susceptibility. In parallel, activation of quiescent fungal infections con-
sists of processes that compromise host defenses directly or indirectly by detoxi-
fication of antifungal agents. The physiological changes that accompany fruit

ripening and host senescence, e.g. host pH, sugar content, cell-wall components
and oxidation of wounded tissue, trigger fungal host modulation. The acidifica-
tion of the tissue by organic acids (oxalic and gluconic) or its alkalization by
ammonia and the possible modulation of ROS, may contribute to rapid necrotiza-
tion of the tissue. Further amplification of the decay can result from activation of
gene expression and release of cell-wall-degrading enzymes. The transition from
the biotrophic stage to the necrotrophic one could then be resolved by character-
izing the temporal changes in gene expression and protein during the transition.
Further clarification of the role of putative signals (pH, nitrogen and sugar) in
postharvest pathogenesis during fruit ripening is clearly needed. Nevertheless,
the current state of knowledge of fungal modulation of host pH has already
opened new avenues to the control of postharvest pathogens thus studies of viru-
lence as a result of pH-conditioning by the pathogen support the developing of
new strategies of postharvest fungal pathogen control involve local pH changes
(Prusky et al. 2006).
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Chapter 5
Global Regulation of Genes in Citrus Fruit
in Response to the Postharvest Pathogen
Penicillium digitatum
L. González-Candelas, S. Alamar, A.R. Ballester, P. Sánchez-Torres,

J. Forment, J. Gadea, M.T. Lafuente, L. Zacarías, and J.F. Marcos
Abstract Large-scale EST sequencing projects and microarray hybridization

nowadays constitute two major approaches to analyse biological systems at a molecular
level. Although the use of these methodologies is becoming commonplace in plant
pathology, their application to postharvest pathology has not yet been reported. In
this chapter we analyze the overall response of citrus fruit to Penicillium digitatum
infection from a genomic perspective. We have constructed both subtracted and
regular cDNA libraries from infected fruits. Analysis of the non-subtracted library
gives us a picture of what genes are being transcribed, whereas the subtracted
library provides more direct information on what citrus genes are induced upon
P. digitatum infection. A cDNA macroarray generated from the subtracted library
has been used to identify what genes are up-regulated in response to pathogen
infection, and to determine the influence of ethylene on their expression. Under the
framework of the Spanish ‘Citrus Functional Genomic Project, CFGP’ more than
fifty cDNA libraries have been generated encompassing more than 90,000 ESTs.
These clones have been used to develop a cDNA microarray representing roughly
7,000 unigenes. With this tool we have analyzed the differences and commonalities of
the citrus fruit responses to ethylene, wounding, P. digitatum infection and induced
resistance. We will present the results of such analyses emphasizing not only what
genes are affected to a larger extent but also describing what are the major changes
in biological processes and metabolic pathways.
Citrus is the major fruit crop worldwide with an estimated annual production
above 100 million metric tons in 2007 (FAO, http://faostat.fao.org). Although most
L. González-Candelas (*), S. Alamar, A.R.Ballester, P. Sánchez-Torres,

M.T. Lafuente, L. Zacarías, and J.F. Marcos
Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), PO Box 73
Burjassot, 46100, Valencia, Spain
e-mail: lgonzalez@iata.csic.es
J. Forment and J. Gadea
Instituto de Biología Molecular y Celular de Plantas (IBMCP), UPV-CSIC,
Avenida de los Naranjos s/n, 46 022, Valencia, Spain

58 L. González-Candelas et al.
of the produce is devoted to juice production, the primary destination in Spain is for
the fresh consumption market. During postharvest handling and storage, citrus fruit
are prone to fungal decay. Penicillium digitatum and Penicillium italicum are the
two major pathogens of citrus fruit being the causal agents of green and blue mould
diseases, respectively. Control of these pathogens has classically been conducted
with chemical fungicides, but the actual problems they face from a variety of points
of view makes it necessary to undertake new approaches for disease control that
minimize health and environmental risks.
Different alternative control methods are being developed by numerous research
groups worldwide. Most of these methods, either physical, chemical or biological,
do not rely on the knowledge of the pathosystem (Palou et al. 2008). However, a
deeper understanding of the fruit’s defence capabilities and of the pathogen’s patho-
genicity mechanisms may lead us to more rational approaches for disease control.
There are few studies focusing on the defence mechanisms of citrus fruit to
postharvest pathogens. Only some b-1,3-glucanases (Porat et al. 2002), chitinases
(Porat et al. 2001) and some phenylpropanoid (Arcas et al. 2000; Ballester et al.
2006; Droby et al. 2002; Kim et al. 1991, Lers et al. 1998; Ortuño et al. 2006) and
oxidative stress (Ballester et al. 2006) genes/compounds have been identified as
components of the fruit’ defence arsenal that are induced either in response to
fungal attack or to elicitor treatment. We have also recently shown the relevance of
ethylene metabolism in the response of citrus fruit to P. digitatum infection (Marcos
et al. 2005). However, all these studies are narrow-focused ones. Nowadays we
have the possibility of addressing a biological problem from a wider perspective by
using non-biased approaches and taking advantage of the new ‘-omics’ tools that
are being implemented in other fields of plant pathology.
In this context we have initiated a large scale characterization of the citrus
transcriptome with the aim of deciphering at the molecular level the defence
responses of citrus fruit to pathogens and to compare these responses with those
elicited by other treatments, such as wounding, exogenous ethylene application or
with treatments that increase the resistance of the fruit to a subsequent pathogen
attack. This initiative has been possible within the frame of the Spanish Citrus
Functional Genomics Project (CFGP; http://bioinfo.ibmcp.upv.es/genomics/
cfgpDB/), a joint effort devoted to the development of genomic tools for the genetic
improvement of citrus. To this end CFGP groups have generated a large collection of
ESTs from numerous cDNA libraries that have then been used for the construction
of a citrus cDNA microarray (Forment et al. 2005).
We have conducted a multifaced approach, as depicted schematically in Fig. 5.1.
On one hand we have obtained a subtracted cDNA library enriched in citrus genes
up-regulated at 24 h after inoculation (hai) with P. digitatum spores (RindPdigS
in Fig. 5.1). This library was prepared following the Suppression Substractive
Hybridization technique (Diatchenko et al. 1996) using mRNA derived from
the rind of ‘Navelina’ oranges as ‘tester’ RNA and mRNA from wounded and mock-
inoculated oranges as ‘driver’. By using wounded tissue as a control we expected
to enrich the cDNA library in genes that are induced specifically, or at least to a
higher level, in response to fungal infection. More than 1,400 cDNA inserts from
5 Global Regulation of Genes in Citrus Fruit 59
Fig. 5.1 Scheme representing the approaches undertaken to characterize the response of citrus
fruit to P. digitatum infection. Dashed lines indicate experiments conducted with ‘Navelina’
oranges, dotted lines indicate experiments conducted with ‘Clemenules’ mandarins
this library were spotted onto nylon membranes in order to conduct hybridization
analyses with RNA samples derived from orange fruits subjected to different
treatments: (i) control fruits stored in air at 20ºC and high relative humidity for 24 h,
(ii) fruits that have been wounded and mock-inoculated with sterile water and stored
at the same conditions for 24 h, (iii) fruits inoculated with P. digitatum spores at 106
conidia/mL and stored for 24 h, and (iv) fruits that have been incubated with 10 ppm
of ethylene in closed jars. Triplicate hybridization experiments were conducted and
the resulting images were analyzed to find genes showing differential expression,
either up- or down-regulation, among treatments. A first analysis between infected
and wounded orange tissues was done in order to confirm whether the library was
effectively enriched in fruit genes showing a higher expression level in response to
Penicillium infection. This comparison revealed that the number of genes showing
statistically higher expression in response to infection in comparison to wounding

represented more than 80% of the genes showing differential expression between both
treatments. An illustrative example of the macroarray hybridization with ‘wounding’
and ‘infection’ RNA samples is shown in Fig. 5.2. The overall results from the
macroarray hybridization experiments showed that ‘infection’ was the treatment
that triggered the largest number of changes in gene expression, both induction and
repression, in comparison to control, non-treated fruit. It is interesting to note that
roughly half of the genes that were up-regulated in response to P. digitatum infection
were also up-regulated either in response to exogenous ethylene treatment or in
response to wounding, a fact that reflects the important role of ethylene mediating
the fruit responses to pathogen attack. However, the overlap between ethylene and
infection treatments is not so extensive with the repressed genes. We have sequenced
more than 350 clones from the RindPdigS library encompassing representatives of
all different expression patterns obtained after macroarray hybridization.
On the other hand, we have constructed a regular cDNA library (neither
subtracted nor normalized) from the rind of ‘Clemenules’ mandarins that had been
Wounding (24 hpi)
Infection (24 hpi)
Fig. 5.2 Hybridizations of the macroarray derived from the RindPdigS cDNA library.
(a) Membranes were hybridized with RNA derived from wounded (W) or P. digitatum-infected
(I) orange fruits 24 h after inoculation. (b) Detailed area of the membranes showing within circles
several spots that show differential expression in both treatments
inoculated 24 h earlier with P. digitatum spores (RindPdig24 in Fig. 5.1) and

have sequenced more than 1100 randomly selected clones. Although the number of
sequenced clones was lower in the RindPdigS library, the redundancy, which reflects
the number of repeated sequences within the library, doubles that of RindPdig24.
This difference reflects the complementary nature of both cDNA libraries. The number
of ESTs in the subtracted cDNA library correlates with its induction level in the citrus
peel in response to P. digitatum infection with respect to the wound response, even
tough its absolute expression level is low. However, a highly represented gene in the
regular cDNA library corresponds to a gene with a high level of expression whether it
is induced or not. As a matter of fact many of the genes with a higher representation
in RindPdigS are not present in RindPdig 24. One notable exception is the CsACO
gene, which codes for an ACC oxidase and is highly abundant in both cDNA libraries,
confirming previous results that highlighted the important role of ethylene in the
defence response of citrus fruit to P. digitatum attack (Marcos et al. 2005).
A global analysis that integrates all the nucleotide sequences obtained from both
libraries can uncover the biological processes that are switched on or off in the peel
of citrus fruit when challenged with this important pathogen. Gene ontology (GO)
categorization is one of the tools that has become more popular for this purpose.
As most of the citrus cDNA sequences have a homolog counterpart in Arabidopsis
thaliana we have thus categorized citrus cDNA sequences according to the assignment
of their closest A. thaliana homologs. Table 5.1 summarizes the distribution of the
different ‘Biological Processes’ in RindPdigS and RindPdig24 cDNA libraries. For
comparison purposes the distribution of the whole CFGP dataset is also included.
The processes ‘response to stimulus’, ‘amino acid and derivative metabolism’ and
‘electron transport’ are over-represented in both cDNA libraries with respect to
the overall CFGP dataset, but to a much higher extent in the subtracted library.
Interestingly, ‘metabolism’ and ‘cell death’ processes are only more abundant in the
RindPdigS library. Although some of the categories are too broad to withdraw
conclusions from them, we can further analyze to a deeper level the annotation of the
members of each process. For example, the analysis of the subcategories included
in ‘metabolism’ indicates that secondary metabolism, and more specifically the
metabolism of phenylpropanoids, is the most represented process in RindPdigS.
Another interesting observation that highlights the importance of preparing
specific cDNA libraries is the high amount of genes that are only present in the
infection-derived libraries that otherwise would have not been isolated. Thus, one of
the two most abundant contigs in the subtracted cDNA library, with 11 ESTs and that
does not have homologous sequences in databases, is not present in any other of the
CFGP cDNA libraries that actually comprise more than 85,000 good quality ESTs.
The last approach we have undertaken to characterize the early response of
citrus fruit to P. digitatum infection is to perform a global analysis of citrus gene
expression by using the citrus ‘7k’ cDNA microarray containing 12,672 probes
corresponding to 6,875 unigenes that has been developed in the CFGP (Forment
et al., 2005). RNA from three biological replicates of ‘Clemenules’ mandarins
subjected to the same treatments described before were used to conduct hybridizations.
We employed the common reference strategy by which each sample was labelled
Table 5.1 ‘Biological process’ gene ontology annotation of RindPdigS and RindPdig24 cDNA
libraries in comparison with the overall CFGP database
GO biological process classification RindPdigS RindPdig24 CFGP
No biological process GO annotation 45.7 46.4 54.8
Cellular physiological process 21.7 21.3 14.0
Biological process unknown 12.0 13.2 14.5
Macromolecule metabolism 12.5 18.9 12.8
Biosynthesis 11.4 12.5 6.3
Metabolism 8.7 4.4 4.2
Nucleobase- nucleoside- nucleotide and nucleic acid 3.3 4.3 5.0
metabolism
Response to stimulus 8.2 4.8 3.0
Amino acid and derivative metabolism 6.0 2.7 1.7
Physiological process 0.7 0.7 0.5
Catabolism 2.7 3.6 1.5
Transport 3.8 4.9 4.3
Regulation of biological process 2.2 1.5 2.9
Electron transport 6.5 3.3 1.8
Cell communication 0.5 1.1 1.5
Development 1.1 0.7 0.6
Cell death 1.1 0.3 0.3
Numbers indicate the percentage of sequences from each dataset belonging to each process. A single
sequence can be assigned to more than one process.
with Cy5, visualized as red spots in the images, and hybridized with a reference
labelled with Cy3 that was made up with equal amounts of RNA from all analyzed
samples. Two technical replications were conducted for each sample. Thus, a total of
six hybridizations were conducted for each treatment. Differentially expressed genes
among the four conditions were identified by SAM with a FDR < 0.01 (Tusher
et al. 2001). When the results were compared against control fruit, infection was the
condition that induced the largest number of changes in gene expression, followed by
ethylene and wounding, being the difference more pronounced in down-regulated
genes. The two genes showing the largest induction in response to P. digitatum
infection showed homology to the cowpea CPRD2 gene, a gene that is induced in
response to dehydration and with no known function yet. This result also confirms
the validity of the RindPdigS library, as one of the most abundant contigs in this
library also shows homology to the same gene. CPRD2 codes for an oxidoreductase
that contains both a FAD-binding domain and a berberine bridge enzyme (BBE)
domain. BBE converts the N-methyl group of (S)-reticuline into the methylene
bridge moiety of (S)-scoulerine, a conversion that is unique in nature (Facchini et al.
2004). BBE is a key branchpoint enzyme in the biosynthesis of certain benzyliso-
quinoline alkaloids (Ziegler and Facchini 2008). Besides these two genes, among
the top positions of genes showing the highest induction in response to the pathogen
there are other genes coding for enzymes involved in secondary metabolism, such
as PAL, O-methyl transferases or a cytochrome P450, as well as enzymes involved
in the synthesis of ethylene, such ACC oxidase, two processes that were also
highlighted in the analysis of the subtracted cDNA library.
Although the overall results derived from both the subtracted cDNA library and
the microarray analyses are qualitatively similar, surveying thousand of genes at the
same time gives us the opportunity to take a deeper look inside the biology of the
fruit tissue. GO classification of those genes that showed differential expression
between conditions, either induction or repression, is one of the ways we can gather
more information beyond the identification of genes showing the strongest up or
down-regulation. Table 5.2 shows the GO classification of the genes that are
Table 5.2 Biological processes differentially represented (p < 0.05) among treatments

Infection Infection vs Infection vs
Biological process vs. air wounding ethylene Resistance
Level 6 Amine biosynthetic process + +
Amino acid derivative biosynthetic + +
process
Amino acid metabolic process + + +
Cellular response to water −
deprivation
Defense response, incompatible +
interaction
Phenylpropanoid metabolic process +
Translation + +
Regulation of nucleobase, −
nucleoside, nucleotide and
nucleic acid metabolic process
Nucleotide biosynthetic process −
Cell wall modification −
Level 7 Amino acid biosynthetic process + +
Aromatic amino acid family +
metabolic process
Aspartate family amino acid +
metabolic process
Methionine methabolic process +
Jasmonic acid and ethylene- +
dependent systemic resistance
Pyridine nucleotide metabolic +
process
Pyruvate metabolic process +
Level 8 Methionine metabolic process +
Sulfur amino acid biosynthetic + +
process
Aromatic amino acid family + +
biosynthetic process
Isopentenyl diphosphate +
biosynthetic process
The ‘+’ value denotes that the process is over-represented in the first condition, whereas the ‘−’
value indicates that it is under-represented. Only genes that showed statistically differential
expression in each comparison where consider for the analysis.
differentially expressed in the comparison between ‘infection’ and the other three
treatments. Lower levels of GO classification reflect broader processes and higher
levels show more specific ones. Several over-represented processes in the infected
tissue are related to secondary metabolism, either directly, as the phenylpropanoid
metabolism, or indirectly through the synthesis of the major precursors of the
different pathways, such as the aromatic amino acids tyrosine, tryptophan and
phenylalanine, or the terpenoid’s precursor isopentenyl diphosphate. Synthesis of
ethylene is included in processes involving the sulphur amino acid methionine.
Defence responses typical of incompatible interactions together with the activa-
tion of systemic resistance dependent of the hormones ethylene and jasmonic
acid are also processes triggered by P. digitatum, in agreement with the observed
activation of cell death that was observed in the subtracted cDNA library.
Classically, induction of cell death processes in the plant has been considered a
landmark of the hypersensitive response triggered in an incompatible interaction
by the recognition of an avirulence (avr) gene product from the pathogen by the
resistant (R) gene product in the plant. Although host cell death is effective in
deterring the progress of biotrophic pathogens, its role with necrotrophic fungi
might be completely different (Glazebrook 2005). Host cell death favours infec-
tion progress of the necrotroph fungus Botrytis cinerea. Moreover, B. cinerea is
able of triggering the death of host cells as a means to obtain nutrients (Govrin
and Levine 2000; Govrin et al. 2006). According to our results the same situation
may holds true for P. digitatum infection of citrus fruit.
Another important source of information derived from the microarray analysis
is the possibility of studying simultaneously the regulation of the genes belong-
ing to the same metabolic pathway. As an example, the synthesis of phenylala-
nine from the shikimate-chorismate pathway is presented in Fig. 5.3. In the citrus
7 k microarray there are 12 genes whose homologous A. thaliana counterparts
code for enzymes involved in the different reactions. Many of them are induced
in the citrus peel in response to the presence of the pathogen, mainly at entry of
the pathway.
In summary, the results obtained give us a global view on how the flavedo and
albedo cells of the fruit rind respond to the pathogen modifying their metabolism
towards secondary metabolism with ethylene being a major player in the process.
At least part of the response resembles the characteristics of a hypersensitive response
that characterize the incompatible interaction. However, despite this deployment of
responses by the fruit, the pathogen is able to successfully invade the cortical tissue.
What we do not know yet is whether this is achieved by down-regulating later on
the defences triggered by the fruit or by overcoming the different set of barriers
deployed by the host.
D-eritrose-4-phosphate Phosphoenolpyruvate
E W I
(1/1) DHS1 −0.1 0.7 2.2
3-deoxy-D-arabino-heptulosonate 7-phosphate
E W I
(1/1) At5g66120 −0.0 0.3 0.7
3-dehydroquinate
E W I
(1/1) At3g06350 0.1 0.1 0.8
3-dehydro-shikimate
E W I
(1/1) At3g06350 0.1 0.1 0.8
Shikimate
(0/6)
Shikimate-3-phosphate
E W I
(1/2) EPSPs 0.5 - 0.6
5-enolpyruvyl-shikimate-3-phosphate
(0/1)
Chorismate
E W I
(1/4) At3g29200 0.4 0.4 0.8
Prephenate
E W I
(1/6) At1g08250 0.0 0.0 −0.1
Arogenate Phenylpyruvate
E W I
ACS1 0.0 − −0.1
E W I (1/6) (4/7)
At1g08250 0.0 0.0 −0.1 At5g53970 −0.1 −0.6 −0.7
At4g34200 + + +
AtAAT −0.2 −0.0 −0.1
L-phenylalanine
Fig. 5.3 Representation of the pathway leading to the synthesis of phenylalanine. The first
number in parenthesis indicates the number of citrus genes present in the 7 k microarray that have
a homologous gene in A. thaliana and the second number indicates the number genes coding for
the corresponding enzyme in A. thaliana. For each gene, the E, W, and I values are the Log2 ratios
of the expression level for each condition (ethylene, wounding and infection, respectively) with
respect to control fruits stored in air. The ‘+’sign denotes lack of expression in air, whereas the ‘-’
sign indicates lack of expression in the particular treatment
Acknowledgments We acknowledge A. Izquierdo and M.J. Pascual for their excellent technical
assistance. This work was supported by grants from the Spanish Ministry of Science and
Education (AGL2000-1443, GEN2001-4885-C05-04, AGL2002-01727, AGL2005-04921-C02-01)
and the Generalitat Valenciana (AVCiT-GRUPOS03/008).
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Chapter 6
Epidemiological Assessments and Postharvest
Disease Incidence
Themis J. Michailides, David P. Morgan, and Yong Luo
Abstract Postharvest plant disease can be measured by “incidence,” by record-

ing the presence or absence of symptoms, and “severity,” the degree to which
the symptoms are expressed. Weather and other environmental conditions play
a significant role by causing stress in plants and lowering natural defenses, and
by creating conditions suitable for pathogens to infect the plants. Specifically for
postharvest diseases of fruit, infections can start as early as fruit set and continue
until harvest. Although weather conditions influence the epidemiology of a dis-
ease in the field, the incidence of postharvest disease depends on the incidence
of latent infections that initiate in the field during the season, contamination with
fungal propagules during harvest, the effectiveness of postharvest treatments, and
storage and marketing conditions. True latent infection, defined as a parasitic
relationship that eventually induces macroscopic symptoms (Verhoeff, K., Annu.
Rev. Phytopathol. 12:99–107, 1974) plays a major role in both the incidence and
severity of postharvest disease. If conditions are favorable, incidence and severity
of latent infections will be higher and the risk for postharvest disease development
will increase and vice versa. For example, in California kiwifruit there is a posi-
tive relationship between the incidence of latent infection of sepals or stem ends,
and the incidence of gray mold of fruit in cold storage. We visualize kiwifruit and
other kinds of fruit as recording devices that copy the environmental conditions as
latent infections. And in some cases, quantification of these latent infections can
predict postharvest disease (i.e. BOTMON (Botrytis monitoring in kiwifruit sepals
and/or fruit stems and in stems of grape berries) and ONFIT (overnight freezing
incubation technique in stone fruit, other fleshy fruit, and in nut crops)). The source
of inoculum that can drive an epidemic of a disease in the field can also affect
T.J. Michailides (), D.P. Morgan, and Y. Luo

Department of Plant Pathology, Kearney Agricultural Center, University of California-Davis,
9240 South Riverbend Ave, Parlier, CA 93648, USA
e-mail: themis@uckac.edu

in the 21st Century Vol. 2, DOI 10.1007/978-1-4020-8930-5_6,
70 T.J. Michailides et al.
the incidence of postharvest decay, by affecting the incidence of latent infection.

Reducing the source of inoculum (sanitation) can reduce the incidence of latent
infection of fruit, with the ultimate result in reducing postharvest disease (i.e., stone
and pome fruit). Environmental conditions during bloom in various crops can have
detrimental effects on the incidence of postharvest disease (i.e., grapes, pomegran-
ates, prunes, etc.) by affecting the levels of latent infection or affecting the plant
host directly. Altering cultural practices that may affect environmental conditions
in the field or the physiology and histology of fruit can also affect the incidence
of fruit diseases both in the field and postharvest. The development of efficient,
accurate, and rapid molecular techniques (including real-time PCR assays) can
facilitate the detection and quantification of disease inoculum and latent infec-
tion of fruit (i.e., stone fruit) and help predict incidence of postharvest disease.
In addition, development of allele-specific RT-PCR methods for rapidly detecting
fungicide resistant fungal pathogens will help growers to manage fungicide resis-
tance and make correct decisions to reduce postharvest disease. The goal of our
laboratory is to develop less expensive molecular techniques that determine latent
infections and assess populations of fungi resistant to fungicides and enable us to
process large numbers of samples at our laboratory and to provide the protocols
to private laboratories.
6.1 Definitions
Before proceeding with this review, we would like to define the various kinds of
infection. According to Verhoeff 1974) latent infection of plants by pathogenic
fungi is often considered of the highest levels of parasitism, since the host and para-
site coexist for a period of time with minimal or no damage to the host. In other
words, these infections are successfully established infections some of which will
perish while others will survive and resume development as the physiological char-
acteristics of the tissues where these infections reside change. A true latent infec-
tion involves a parasitic relationship that eventually induces macroscopic symptoms
(Verhoeff 1974). Quiescent infection, however, is microscopically visible although
mycelial development is arrested after infection and resumes only as the host plant
reaches maturity and/or senescence (Sinclair and Cerkauskas 1996). Latent con-
tamination, which we prefer the term inoculum load for it, involves fungal spores
(or other type of fungal propagules) on the host’s surface which fail to germinate
until the host reaches maturity or senescence, or wounded by insects and other
means.
Latent infection of plants by parasitic fungi is often considered one of the high-
est levels of parasitism, since the host and parasite coexist for a period of time with
minimal damage to the host. Latency involves an asymptomatic parasitic phase
that eventually gives rise to visible symptoms (Verhoeff 1974) if conditions are
favorable for disease development. For instance, if latent infections are at high
levels and most develop to active disease symptoms then a disease epidemic can
initiate.
6 Epidemiological Assessments and Postharvest Disease Incidence 71
6.2 Introduction
Postharvest diseases of fresh produce (fruit and vegetables) and mycotoxin

contamination of certain susceptible crops contribute to major economic losses to
growers, processors, marketers, and consumers. Therefore, management of postharvest
disease is needed to avoid losses and increase growers’ revenues.
Postharvest diseases are the result of latent infections that occur in the field dur-
ing the growing season and infections from wounding during harvest and handling
operations (Eckert and Summer 1967; Michailides and Manganaris 2009). Although
postharvest diseases are the continuum and or the result of diseases that initiate in
the field, some researchers have emphasized postharvest disease per se without
looking into the diseases caused by the same pathogens in the field. A good exam-
ple is the brown rot disease of stone fruit caused by Monilinia fructicola or M. laxa.
In California brown rot can have four distinct phases which in essence develop as
a continuum on stone fruit. At bloom, for instance, if weather is wet and relatively
warm, blossoms are infected and blighted: this is the blossom blight phase. Later
on, if conditions continue to be favorable for the pathogen, the fungus can invade
from the blossoms to the sustaining shoots causing cankers: this is the canker phase
(which is more common for M. laxa). If wet conditions prevail, green or maturing
fruit get infected and rot on the tree: this is the fruit rot phase. Finally, once fruit is
harvested and removed from the orchard still can decay and this is the postharvest
fruit rot phase which can occur either in storage, in the market (wholesale and
retailed stores), in the household refrigerator, or in customer’s fruit display basket.
Epidemiological knowledge of fungal diseases of fruit trees, nut crops, and
vines is essential to help predict disease risk during the growing season and/or
at harvest and during postharvest storage. Predicting plant disease is very chal-
lenging and requires major research efforts over several years to understand the
dynamics of the four main variables involved in disease development: the
presence/absence and quantity of the pathogen’s inoculum, stage and suscepti-
bility of the crop, environmental conditions, and growers who continuously
attempt to change the dynamics of the diseases in their agricultural systems.
These major factors are not steady but compoundedly change and make the
development of an accurate predictive model difficult, complex, and time con-
suming. Although increasingly accurate weather predictions are available
through the National Oceanic Atmospheric Agency (NOAA), the other aspects
of the disease triangle require a large database of information that includes the
pathogens’ inoculum, which can be quantified by either trapping spores or pre-
dicted based on disease incidence in previous seasons (historical disease data) or
by determining the incidence of latent infections. Susceptibility of the host and
the most susceptible stage of the crop can be determined experimentally with
periodic inoculations with the pathogen.
In many diseases of fruit trees, nut crops, and vines, latency is an important stage
in disease epidemiology and prediction. Although disease prediction depends on
the above mentioned factors, it is sometimes accurate, depending on the specific
disease and its nature, to predict the disease incidence and severity even without
any recording and consideration of the above mentioned parameters. This is

because the success for a latent infection to occur and develop to actual disease
depends on all these factors (inoculum level present, susceptibility of the host, and
environmental conditions conducive to infection) and these infections then can act
as recording devices that “record” and accumulate all these parameters that contrib-
ute to disease expression. In this contention, then an infective propagule that has the
capability to produce a latent infection and it could be considered as an alarm that
will go off as soon as the “right time” has reached based on the accumulated
“latency condition of the pathogen” has reached a minimum threshold. A “latency
condition” is defined as the status of the pathogen subjected to diminishing adver-
sity by the host as it changes physiologically and biochemically and changing
conditions of the environment that affect both the host and the pathogen. In this
case, the pathogen itself acts as a recording device that records all these changes (of
the environment and the host) and develops accordingly, with the final result the
expression of disease development. If the latency condition of the pathogen leads
to its death, then symptoms of disease will not develop.
Diseases we investigated that have a latency phase include Botrytis gray
mold in grapes and kiwifruit caused by Botrytis cinerea (Pers.:Fr.), brown rot
in stone fruit caused by Monilinia fructicola (G. Wint.) Honey and/or M. laxa
(Aderh.) Honey, panicle and shoot blight of pistachio caused by Botryosphaeria
dothidea (Moug.:Fr.) Ces. & De Not., and Alternaria late blight of pistachio
caused by three species of Alternaria. Latent infections of berries, stone fruit,
and nuts remain inactive until these fruit start maturing and environmental con-
ditions are favorable for disease development. However, if environmental con-
ditions are not favorable, the pathogen may not survive until the stage when the
crop becomes conducive to the development of latent infection to disease symp-
toms. The incidence of latent infections has been shown to correlate with dis-
ease incidence in the field at harvest of fruit and nut crops (Michailides et al.
2000) or with the incidence of decays that develop in storage (Michailides and
Morgan 1996a,b).
The degree of success in management of fruit tree diseases depends on the level
of disease itself, environmental conditions, effective cultural manipulations, and
proper timing and type of fungicides selected against a disease. Fungicide resistance
in fungal pathogens of tree fruit and vines also affects the outcome of disease
management as well as choices of postharvest treatments. If growers had access to
timely information on fungal resistance to fungicides, they could make correct
decisions on what fungicide to apply and what rotation schedule would be essential
to help avoid a failure of disease control in the field and/or prevent a build-up of
resistance in the pathogen’s population in the orchard or vineyard. To base this
discussion on personal experience, two examples will be emphasized as follows:
the Monilinia fructicola and M. laxa causing brown rot disease of stone fruit; and
the Botrytis cinerea causing gray mold of kiwifruit. These examples include good
methods for the detection of latent infection during the growing season that predict
disease at harvest and postharvest.
6.2.1 Brown Rot-Monilinia fructicola and M. laxa
Brown rot of stone fruit in California is caused by Monilinia fructicola and M. laxa.
The latter species was the predominant in the early nineteenth century while in late
century, M. fructicola became the dominant species, at least in prune orchards
(Michailides et al. 1987). The disease expresses itself in two major phases, infec-
tion of blossoms leading to blossom blight and infection of fruit leading to fruit rot
(both immature, green fruit and mature fruit infection). A third phase is the latent
infection that can occur from bloom to harvest. The occurrence of latent infection
by Monilinia spp. is known since the early 1960s various reports provided good
examples of latent infection on apricot (Wade 1956; Tate and Corbin 1978; and
Wade and Cruickshank 1992). On cherry (Curtis 1928; Förster and Adaskaveg
2000), and on peach (Tate and Corbin 1978; Michailides et al. 2000). The best
examples of latent infection by Monilinia spp. are on plum (Rosenberger 1983;
Northover and Cerkauskas 1994) and on dried plum (Luo and Michailides 2001,
2003; Luo et al. 2001). Specifically, on dried plum, infections by the brown rot
fungus can be divided in (1) latent infections in blossoms; (2) latent infections and
quiescent infections in green fruit, and (3) quiescent infections on leaves. Quiescent
infections on leaves are small brown spots that upon isolations on acidified PDA
the majority of them will produce colonies of Monilinia spp. Quiescent infections
on immature dried plum fruit (prunes) are raised pin-head black specks (Fig. 6.1).
The majority of the latent infections do not develop to disease; however, some can
overcome the inhibiting factors encountered in green fruit and form a brown decay
lesion in immature, green fruit. Latent infections will also affect directly the inci-
dence of postharvest decay (see below). Because of the direct relationships between
latent infection and brown rot disease, detecting latent infections could be a useful
assay to determine risk of brown rot at harvest and postharvest.
Fig. 6.1 Quiescent infections of Monilinia spp. on ‘Howard Sun’ plum

6.2.1.1 Conventional Methods Used to Detect Latent and Quiescent

Infections of Monilinia spp.
There are two types of methods for the detection of quiescent and latent infections.
These include conventional (direct isolation – incubation) and molecular (PCR and
real time PCR) techniques. These techniques have been used in our studies in the
last several years with great success.
Direct Agar Plating Technique (DAPT) This technique is commonly used to
isolate plant pathogens from plant tissues. The detection and quantification of the
Monilinia sp. pathogen in quiescent infections can be determined using this tech-
nique. The agar medium commonly used for the DAPT technique in our laboratory
is potato dextrose agar acidified with lactic acid (2.5 mL of a 25% vol./vol. lactic
acid per liter of medium), resulting in a pH of 3.5 that is inhibitory to the majority
of bacteria, but it allows the Monilinia spp. to grow when incubated at 20°–25°C
(68°F to 77°F) for several days. The traditional way is to cut the plant tissue in 3 ×
3 × 3 mm cubic pieces, surface disinfect them in a 5–10% chlorine solution
(prepared from household bleach, which is a 5.25% solution of NaOCl) for 1 to few
minutes, rinse them with sterile water once or twice, blot them on clean paper
towels, and place 5–10 pieces in a 55- or 90-mm in diameter Petri plate. The plates
are usually incubated at 25°C (77°F) for 5 to 7 days and recorded for the presence/
absence of Monilinia spp. from the isolations. The DAPT has been used for isolating
latent infections and quiescent infections from the skin of various stone fruits, fruit-
to-fruit contact areas, stylar and basal ends of the fruit, flower petals, and leaf
blades. For instance, when the weather is unusually wet, quiescent infections show
as small rusty spots on the leaf blade and 3 × 3 mm square pieces of the blade can
be surface sterilized and plated as described above.
Flower Incubation Technique (FIT) A second conventional technique for the
detection of latent infections of brown rot in flowers is by collecting 100–150 ran-
dom flowers per field, surface sterilized them in 1% chlorine solution (prepared
from household bleach containing 5.25% NaOHCl), and lay them either on wet
sterile paper towels or on sterile plastic screens in clean containers. Usually, the
containers used in our laboratory are made of hard plastic and measure 24.5 × 18.0
× 8.0 cm. The containers with the flowers are incubated at room temperature or at
25°C for 5 days when latent infections develop on the hypatheum showing charac-
teristic sporulation. The incidence of flowers with latent infection by M. fructicola
is then determined within 5–7 days incubation. This technique can also be per-
formed by using short (15–20 cm) twigs bearing flowers and placing flat four to
five twigs on top of the plastic screens in the plastic containers. All the other steps
for this procedure, incubation and determination of latent infections are the same as
those used above for individual flowers (Luo et al. 2001).
Overnight Freezing – Incubation Technique (ONFIT) (Table 6.1) This is
another conventional technique which developed and frequently used in our labo-
ratory and can detect and quantify latent infections of Monilinia fructicola and
M. laxa (the fungi that cause brown rot in stone fruit), Botrytis cinerea in grapes
Table 6.1 Protocol of overnight freezing incubation technique (ONFIT)a to reveal latent
infections by Monilinia fructicola or M. laxa in stone fruit
1. Collect 100 immature fruit from the orchard and bring to the laboratory in an ice chest.
2. Disinfect cleaned plastic screen racks and plastic containers (2 per site) in 1:20 bleach
(5.0% sodium hypochlorite: water) solution for 5 min.
3. Place 50 fruit in the plastic mesh-stretch bags, label as desired, and secured mesh bags with
plastic clips.
4. Prepare bleach solution in large (20 L) plastic containers. Solution is prepared 1:10 with 0.5
mL of Tween 20 per liter of tap water. Typical preparation is 5 L of solution with 0.125 mL
of Tween 20. Place the plastic container in the sink to minimize unwanted splashing of the
bleach solution.
5. Prepare 1 L of 70% ethanol in a 2 L beaker.
6. Place the samples in the ethanol solution for 10 s, shake off quickly and place in the bleach/
Tween-20 solution for 4 min. Swirl the bags in the solution for 5–10 s during every minute
the bags are in solution.
7. Remove the bags, shake off the excess liquid in the sink and quickly place the bags in the
appropriately labeled containers; replace the lid.
8. Arrange fruit in the containers under a hood, pour 200 mL of tap water, and cover the
containers.
9. Place all plastic containers at 3°–4°F (−16°C) freezer for about 15 h (17:00 h to 8:00 h)
overnight.
10. Remove containers from the freezer and place on a laboratory counter at about 77°F (25°C).
11. Record and count fruit showing brown rot symptoms and sporulation of the pathogen after
5–7 days.
ONFIT can be used for any stone fruit such as apricot, cherry, nectarine, peach, plum, and prune
a
causing bunch rot, and B. dothidea and Alternaria species causing blight diseases
in pistachio. The rationale of the technique is based on the fact that killing the fruit
tissues at a stage when the tissues do not favor disease development triggers the
development of latent infections to active disease symptoms or accelerates the
growth of hidden colonists. The herbicide paraquat (1,1´ diemthyl-4.4´ bipyridin-
ium dichloride) has been used in the past to kill plant tissues and trigger latent
fungal infections in soybean (Cerkauskas and Sinclair 1980) and modified later for
the detection of latent infections of plums by M. fructicola (Northover and
Cerkauskas 1994). We also used paraquat in our laboratory initially, but because it
is a toxic herbicide, we looked for an innocuous alternative. In contrast, the ONFIT
is a safe technique because it does not require the use of any noxious pesticides,
only surface disinfectants such as dilutions of household bleach and disinfectants,
such as ethyl alcohol, which are generally regarded as safe. But, it is still necessary
to thaw and then incubate the frozen fruit for a number of days under laboratory
conditions (75°–77°F) until latent infections develop into symptoms and signs of
the pathogen can easily be recorded (Fig. 6.2a) and results become available within
5–7 days. Latent infections of brown rot can also develop to disease symptoms, but
fruit needs to be incubated under high humidity for at least 2–3 weeks. Using the
ONFIT, a grower would need to wait only 5–7 days until he would be able to make
a decision on disease risk in the field (Luo and Michailides 2003). For instance, the
incidence of Monilinia spp. determined with the ONFIT performed with immature
Fig. 6.2 (a) ONFIT on French prunes to detect latent infections after 7 days incubation, (b) incu-
bation of surface sterilized French prunes without freezing for 4 weeks at room temperature
prune fruit collected in May/June correlated linearly with the incidence of the
brown rot developed in the field at harvest (Fig. 6.3). In other words, the incidence
of Monilinia determined with the ONFIT can predict the risk for fruit brown rot at
harvest. Waiting one week is still a long waiting time, therefore, more efficient and
quicker techniques than the conventional ones are urgently needed for the detection
of latent infections in tree fruits, nut crops, and vines in California. The timing for
performing ONFIT depends on various factors. One important factor is the inoculum
potential of Monilinia spp. in the orchard. For example, when the inoculum potential
is high in an orchard the timing of ONFIT can range from mid May to early July,
16
Percent branches with fruit rot (PBFR)

PBFR = −2.6 + 0.3355 ILI
14
r2 = 0.82, P = 0.002
12
10
0
0 10 20 30 40 50
Incidence of latent infection (%)
Fig. 6.3 Linear correlation between incidence of latent infections (ILI) and percentage of
branches with fruit rot (PBFR) caused by Monilinia fructicola on prune, detected by the ONFIT
technique. Each data point represents an average value of multiple locations and inoculations
when the inoculum potential is low, the best timing will be in mid June, and when
the inoculum potential is moderate, the best timing can be at any time in the month
of June. But in general, the critical period to determine latent infection for prunes
is in June (Luo and Michailides 2003).
Incubation of fruit after surface sterilization following the steps that are used for
the ONFIT procedure without freezing requires a long time until latent infections
in green fruit are triggered to develop. For instance, when immature prune fruit
were collected from two different sides of a large prune orchard, surface sterilized,
and incubated as above (without freezing the fruit), latent infections started devel-
oping after 8–10 days incubation, reached to 5–7% levels in 15 days and to a maxi-
mum after 30 days (Fig. 6.2b). Even this procedure if it is done in May provides
sufficient time for determining the risk for disease in an orchard and making deci-
sions for pre-harvest sprays. There is linear correlation of the incidence of latent
infection and postharvest decay for at least some of the stone fruit (i.e., prunes).
6.2.2 Botrytis Monitoring (BOTMON) - Botrytis cinerea

(Table 6.2)
The California kiwifruit industry and other kiwifruit industries suffer tremendous
losses due to postharvest gray mold caused by Botrytis cinerea. Although this disease
does not show any symptoms and or signs in the field in California, postharvest
gray mold is considered as the number one disease of kiwifruit in cold storage.
Since no disease symptoms develop in the field, it is most likely that infections
Table 6.2 Protocol of Botrytis monitoring (BOTMON) to reveal latent infections by Botrytis
cinerea by plating symptomless sepals and stem ends of kiwifruit
1. Harvest 60 kiwifruit from each 1-ha field (avoid any wounding) 4 months after fruit set (about
1 month before harvest).
2. Remove sepals (by hand) and stem end (with a cork borer) from each fruit.
3. Surface disinfect sepals and stem ends in 0.5% chlorine household bleach plus 2 drops of
Triton-X-100 surfactant (per liter water).
4. Rinse the above, surface-sterilized fruit sepals and stem ends in sterile water and dry them in
a positive-flow hood for 10–15 min.
5. Plate the above in Petri plates containing acidified potato-dextrose agar (pH = 3.2–3.5).
6. Incubate the petri Plates at 7°C for 6 days and record B. cinerea colonies growing from the
sepals and stem ends in each plate (first Botrytis recording).
7. Move and incubate the petri Plates at 23°C for 3 more days and record additional B. cinerea
colonies in each plate (second Botrytis recording).
8. Combine data from the two recordings (= total B. cinerea colonies) and determine incidence
(%) of colonization of sepals or stem ends.
9. Use prepared tables or regression lines for sepal or stem end colonization to predict Botrytis
gray mold after expected to develop after 3 or 5 months storage of fruit in controlled
atmosphere.
10. Make decisions: (a) yes or no preharvest fungicide spray(s); (b) which fruit to store longer; (c)
yes or no resorting and re-packing; etc.
by B. cinerea of kiwifruit are primarily latent. Michailides and Morgan (1996a) found
that these infections occur on the fruit sepals and receptacles during the growing
season starting 1 month after bloom and continuing until harvest. Furthermore, there
are major differences in the epidemiology of Botrytis gray mold of kiwifruit in New
Zealand and California. For instance, because of the dry climatic conditions in
California, latent infections is of primary importance for the postharvest gray mold
while in New Zealand, it is the infection of stem wound that affect the levels of the
postharvest decay and not the latent infections of sepals by B. cinerea. Therefore, in
California to develop more efficient methods for controlling gray mold in kiwifruit,
it was necessary to understand the relationship between B. cinerea latent infections
initiated in the field and the incidence of gray mold in cold storage. The BOTMON
technique was developed and used to detect B. cinerea, causing latent infections of
kiwifruit (Actinidia deliciosa) sepals in the field. The rationale of the technique is
based on the fact that the incidence of latent infection is a good predictor of gray mold
in cold storage (Michailides and Morgan 1996a,b; Michailides and Elmer 2000).
BOTMON involves the collection of fruit samples with stems attached from the
kiwifruit vineyard, removal of sepals or stem ends (receptacles; Fig. 6.4), and plating
the fruit samples in plates with APDA (Fig. 6.5). It was determined that 1 month
before harvest is the best time for sampling immature fruit to perform BOTMON
and gray mold prediction, since the correlation coefficients of B. cinerea colo-
nization of sepals and stem ends and gray mold in cold storage are the highest
(r = 0.92–0.98). Interpretation of the technique’s results was given in previous
Fig. 6.4 Sepals and stem-ends of kiwifruit cut from the fruit to be used in BOTMON to monitor
the incidence of colonization of Botrytis cinerea
Fig. 6.5 BOTMON technique: plates containing acidified potato-dextrose agar where sepals (left)
and stem ends (right) were plated to reveal colonization by Botrytis cinerea
publications (Michailides and Morgan 1996a,b). The results of the colonization of

sepals and/or stem ends by B. cinerea grown become available to the grower in 9
days. After obtaining the results, growers can use Table 6.3 to predict the levels of gray
mold in cold storage for a specific lot of fruit. In California more and more kiwifruit
growers have been using the BOTMON technique to make decisions on the need for
pre-harvest vinclozolin spray(s) since this technique uses fruit collected one month before
harvest, allowing for sufficient time to apply a fungicide. Additionally, packinghouse
Table 6.3 Relationship of levels of sepal and stem-end colonization by Botrytis cinerea in

kiwifruit samples collected 4 months after fruit set and number of vineyards with low (<1%),
moderate (1–3%), and high (>3%) levels of Botrytis gray mold decay after 3 months in controlled
atmosphere storage (31°F (−0.5°C) and eight parts per billion ethylene) in 2 years
Number of vineyards with fruit showing different levels of
postharvest gray molda
Sampled Colonization Low (£1%) Moderate (1–3%) High (³3%)
Plant part Level Colonization Yr 1 Yr 2 Yr 1 Yr 2 Yr 1 Yr 2
%
Sepals Low 0–15 6 3 0 1 0 0
Medium 16–50 0 0 2 3 1 0
High >50 0 0 0 0 0 1
Stem ends Low 0–15 4 2 0 0 0 0
Medium 16–50 2 1 2 3 0 0
High >50 0 0 0 1 1 1
a
In year 1 a total of nine vineyards were sampled and in year 2 a total of eight vineyards (fruit
from one vineyard in Butte County was not sampled because of prescheduled grower’s harvest).
Percentage of colonization was determined by plating 300 sepals and 60 stem ends per sampling
in each orchard.
operators and shippers can use the results to decide on the need for fruit sorting
and re-packing to minimize secondary spread of the disease in storage and plan on
the timing for marketing these fruit. When growers spray only when it is needed
and only those vineyards which have a high incidence of latent infections, they
reduce costs and contamination of the environment with pesticides. The only dis-
advantage of the technique is that it is still time consuming (6 days pre-incubation
of the plated sepals and stem ends on APDA at 45°F and 3 more days at 77° F (see
protocol in Table 6.2). Therefore, there is still a need for a quick technique that will
provide results within one day or even within a few hours. BOTMON has been used
also to detect B. cinerea in latent infections of apples, figs, grapes, pears, pistachios,
pomegranates, and various stone fruit (cherry, nectarine, peach, plum, and prune).
Prusky et al. (1981) developed a pre-harvest assessment of latent infections by
Alternaria alternata in mango fruit and found that there was a positive correlation
between the relative surface of fruit infected by latent Alternaria at harvest and the
incidence of black spots that developed on the fruit during postharvest storage.
6.2.2.1 Molecular Techniques
The polymerase chain reaction (PCR) and the development of thermocyclers have
revolutionized the molecular biology since they were first described in 1985. PCR-
based techniques have been used in various biological studies. Specifically in plant
pathology, PCR has been used in identifying pathogens and determining pathogen
population structures, taxonomy, and classification. Additionally, in the last several
years, techniques that quantify the DNA of pathogens have been developed, and
these can be very useful when the relationship of quantities of pathogens’ DNA,
latent infections, and disease levels are established. Such techniques could aid in
estimating spore inoculum that determines disease potential and levels of latent
infections that relate to disease risk at harvest or after postharvest storage. Following,
we present a few examples of molecular techniques we developed in the plant
pathology laboratory at the Kearney Agric. Center for the early detection of pathogens
and fungicide resistant pathogen genotypes and providing answers to critical
epidemiological questions that help predict disease risk in the field and pathogen
resistance to fungicides. We also discuss how these techniques could be used in
agribusiness as decision making tools.
PCR-based assays that detect M. fructicola and M. laxa in stone fruit Brown
rot, caused by M. fructicola and or M. laxa, is a destructive disease of stone fruit
(Prunus spp.) in California causing initially blossom blight and later fruit rot. When
the microclimatic conditions in the orchards are unfavorable for further disease
development, infected blossoms develop into young fruit with latent infections.
These fruits may drop naturally or be thinned, and when humidity is high, these
dropped fruit produce numerous conidia which can cause fruit infections in mid-
season. Later, when favorable conditions and maturation of the fruit occur, a num-
ber of the latent infections may develop into fruit rot. Inoculum potential in the
orchards is an important factor affecting both blossom blight and fruit infections
(Luo and Michailides 2001, 2003). Thus, determination of inoculum potential
(amount of pathogen’s spores in the orchard) in early- and mid-season is critical for
predicting and managing brown rot.
Inoculum potential is the most difficult parameter to determine in a stone fruit
orchard. Spore traps have historically been used to determine the spore density for
air-borne disease agents including M. fructicola. Because samples from traps
require microscopic examination, it is both very time consuming and requires spe-
cial training to recognize and count spores in the spore samples. Additionally, spore
counts may be an unreliable indicator of inoculum potential because of the abun-
dance of dust and other fungal species (i.e., Botrytis cinerea) having spores similar
to those of M. fructicola. Furthermore, culturing airborne spores collected on spore-
trap tapes or slides is also tedious and subject to frequent contamination problems.
Thus, such classical methods are impractical for recording large number of spore
trap samples required for a large-scale disease management.
PCR-based assays have the potential to monitor airborne inoculum levels of
plant pathogens because they are highly specific and sensitive. Recently, we devel-
oped a nested PCR method for the detection of M. fructicola on spore-trap tapes
(Ma et al. 2003b). Nested-PCR primer pairs (an external primer pair EMfF + EMfR
and the internal primer pair IMfF + IMfR) were designed based on the sequence of
a microsatellite region generated by a microsatellite primer M13 (5¢-GAG GGT
GGC GGT TCT-3¢). In specificity tests, we observed that the primer pairs EMfF +
EMfR and IMfF + IMfR amplified a 571- and a 468-bp DNA fragment, respec-
tively, from all tested M. fructicola isolates collected from different stone fruit hosts
at different locations in different years. No fragments were amplified from any

other fungus associated with stone fruit. Sensitivity tests showed that the nested
PCR assay could detect the specific fragment in as little as 1 fg of M. fructicola
DNA (Fig. 6.6a) or in DNA from only two spores of M. fructicola (Fig. 6.6b). This
nested PCR method can detect 200 spores in a spore-trap tape sample (equivalent
to two spores/PCR reaction) collected from a commercial prune orchard. Using
these species-specific primers, we can also detect latent infections in fruits caused
by M. fructicola within hours, while direct plating on agar media, or overnight
freezing-incubation technique would require at least one week (see details below:
Fig. 6.6 PCR using specific primers to detect (a) M. fructicola DNA and (b) DNA from spores
Table 6.4). Since the nested-PCR assay cannot quantitatively detect the number of
M. fructicola spores on a spore-trap tape, we are now working on a real-time PCR
technique that can quantify spores of M. fructicola. The efficient, accurate, and
feasible real-time PCR method could help growers in making timely decisions for
fungicide application and reduction of unnecessary sprays.
6.3 Use of Species-Specific PCR to Detect M. Fructicola

in Fruit and Flowers (Comparison of Conventional
with PCR Techniques)
In 2001, in a plum orchard cv. Howard Sun, the presence of numerous visible qui-
escent infections (Fig. 6.1) suggested the presence of even more latent (invisible)
infections. In order to compare the direct plating technique of visible quiescent
infections, with the ONFIT of invisible latent infections and a species-specific PCR
technique, in mid May fruit were observed in the field and their fruit-to-fruit con-
tact surface was marked with a permanent pen. All these fruit were collected and
brought to our laboratory at Kearney Agricultural Center, surface disinfected in
10% bleach solution for 3 min, rinsed with sterile water twice, and placed on clean
paper towels. The fruit samples were split in three subsamples; one subsample was
used for the DAPT, the second for the ONFIT, and the third subsample for the
species-specific PCR technique.
(a) For the direct plating technique, latent infections (small pieces of green tissue
of the fruit surface) and visible quiescent infections (small black specks on the fruit
skin) were excised with a sterile razor, and plated on APDA plates as described in
DAPT. (b) For the ONFIT, fruit without any visible infections were processed fol-
lowing the protocol in Table 6.1, and recorded for brown rot development on the fruit
after 7–9 days incubation. And (c) for the species-specific PCR method, invisible
latent and visible quiescent infections were excised as in (a) above, pre-incubated at
77°F for 1 day, and then DNA was extracted and diagnosed using M. fructicola
specific PCR following published protocols (Boehm et al. 2001). Results from the
PCR method after isolating fungal DNA can be completed in 6 h, i.e., in 30 h since
the initiation of the procedure (Table 6.4).
Table 6.4 Techniques to detect latent and quiescent infections by Monilinia fructicola in
‘Howard Sun’ plums
Time required for
Technique Latent infections (%) Quiescent infections (%) results (days)
PCR 7.9 60.5 1.25a
ONFIT 6.7 – 7–9
DAPT – 54.3 5–7
a
Time includes 1-day preincubation of sample.
Using the PCR technique, 7.9% of the samples with invisible latent infections
were positive for DNA of M. fructicola and 6.7% of the fruit processed with ONFIT
developed brown rot. Similarly, as expected when visible quiescent infections were
used, 60.5% were positive for M. fructicola with the PCR technique and 54.3% of
those plated on APDA developed colonies of M. fructicola. Interestingly, the tradi-
tional techniques required 5–9 days to completion while the PCR technique made
results known within only 1.25 days (Table 6.4).
In another experiment, plum flowers (cv. Royal Diamond) were collected in
March-April from a commercial orchard in Reedley, CA. Flowers were divided into
three groups based on visual symptoms: (+) flowers were heavily infected with
M. fructicola and showed obvious signs of fungal sporulation on the stem and calyx
surface; (+/−) flowers displayed brown patches on the petals but showed no external
signs of fungal sporulation; and (−) flowers were asymptomatic, without any
evidence of brown discoloration or fungal infection. DNA extractions from indi-
vidual plum flowers were obtained using the Fast-Prep System FP-120 biohomog-
enizer instrument, following the manufacturer’s instructions for plant DNA
extraction (Q-BIOgene, Inc.). In planta polymerase chain reaction (PCR) detection
from flowers used the species-specific primer pair 210F1 + 210R1 at the high
annealing temperature for retention of species specificity. The results indicated that
all (100%) of the (+) series of flowers had much stronger amplification signals
than either the (+/−) (80%) or the (−) flowers (only 10% of the flowers carrying
M. fructicola (Fig. 6.7). Thus in approximately 8 h, one can determine the percentage
of asymptomatic flowers carrying M. fructicola. Knowing the percentage of flowers
latently infected by M. fructicola should prove a useful estimate of the inoculum
potential in stone fruit orchards necessary for determining disease risk, assessment
and blossom blight incidence, and in developing pre-harvest and postharvest
chemical control strategies against brown rot. The PCR technique can replace the
flower incubation technique (FIT) that can also provide an estimate of inoculum
potential in stone fruit orchards.
6.4 Techniques to Monitor Resistance of Fungal Pathogens

to Fungicides
Fungicides are commonly used to manage plant diseases. However, the frequent
use of fungicides with single mode of action incurs a high risk of selecting resistant
genotypes of plant pathogens. To determine levels of resistance to fungicides in
fungal populations, the most common conventional technique used is the direct
plating of single-spore isolates in media amended with the fungicide of interest.
Single-spore isolates of the pathogen are grown on PDA or acidified PDA or other
specialized media under conditions that favor their sporulation. Media such as PDA
or water agar (WA) amended with increasing concentrations of the test fungicide
are used to determine either the EC50 of inhibition of hyphal growth (after placing
a 5-mm in diameter mycelial plug in the center of the Petri plate) and/or EC50 of
Fig. 6.7 PCR using specific primers to detect DNA of Monilinia fructicola in latent and quiescent
infections of plum flowers
spore germination (after spreading a 0.5 mL of a dense spore suspension of the

fungus on the surface of the plate) in comparison with the respective medium that
is not amended with the test fungicide. EC50 is the concentration of the fungicide
that provides 50% inhibition of mycelial growth or spore germination of the patho-
gen to be tested. This technique was used in our laboratory for detecting fungicide
resistance in plant pathogens such as M. fructicola and M. laxa, Botryosphaeria
dothidea, Fusarium moniliforme, B. cinerea, Alternaria alternata, A. tenuissima,
and A. arborescens. This technique is time consuming, and obtaining the results of
the tested isolates is usually very critical, especially since growers would rely on
such results to decide what fungicide programs to use, depending on the presence
and levels of resistance in a field.
Depending on the pathogen species, in order to obtain mycelial plugs or
adequate amounts of spores, the entire test can take 7–10 days, if not longer, if one
takes into account the time required to isolate the pathogens from infected plant
tissues. In 2004, a new technique was reported using a Spiral Plate® gradient dilu-
tion method to determine EC50 values that still requires 1–5 days for mycelial
growth assays and 14–20 h for the spore inhibition studies to reveal results plus 2–5
days for sporulation in culture (Förster et al. 2004). Although results with this tech-
nique are available sooner than the classical technique, both the high cost of the
Spiral Plate® equipment and the long time period to obtain results when one wants
to check multiple isolates are prohibitive. Therefore, more efficient and rapid tech-
niques are needed for checking resistance of pathogens to fungicides.
An example will be the resistance in Monilinia spp. Since benzimidazole resis-
tance in M. fructicola and M. laxa has been shown to be associated with point
mutations in the b-tubulin gene (Ma et al. 2003a), we developed an allele-specific
real-time PCR method for rapidly detecting benzimidazole-resistant M. fructicola
and M. laxa in stone fruit orchards. A similar procedure was used for the detection
of Alternaria resistance to azoxystrobin. This technique was used with blossoms

collected from peaches in spring (March) and successfully detected the level of
resistance to benzimidazoles in M. fructicola. Obviously, such rapid and quantita-
tive detections of fungicide resistance in the fungal pathogen populations will be
valuable for growers to manage fungicide resistance in fruit tree orchards and
vineyards.
6.5 Conclusions and Future Prospects
The presented examples represent a part of the methodology used at the Kearney
Agricultural Center to access latent infections to help predict diseases at harvest
and in postharvest storage and guide growers to disease management decisions and
packinghouse operators to proper marketing of fruit. Although the conventional
techniques can be accurate and may be less expensive because of specialized
reagents (enzymes and equipment are not needed), only minimal information con-
cerning a few isolates becomes available and only after 1–2 weeks. This “waiting-
for-results” time often can be a very critical time for growers who need to make a
decision on disease management, fungicide timing and frequency, type of fungi-
cide, and resistance management program selection in their fields. Because of these
serious drawbacks in the last 5 years we have focused our efforts on research to
develop molecular techniques, which although are more costly, they can replace the
conventional ones and have the potential to finally provide very accurate and timely
information for disease management decisions.
As shown from the examples above, molecular technology has proven very valu-
able in our plant pathology research program at the Kearney Agricultural Center.
One disadvantage of the molecular methodology is that it does require specialized
reagents, enzymes, and costly equipment (Polymerase Chain Reaction and Real
Time PCR machines) and may be more expensive than the conventional methodology.
However, when affordable, portable real-time PCR instruments and simple
protocols are developed, routine and efficient diagnosis of many crop diseases or
fungicide resistance in pathogen populations can be made on site and within one
day, thus reducing the total costs of such tests. In general, molecular technology
also helps us in understanding the biology and population structures of plant patho-
gens and provides quick and accurate answers to epidemiological questions on
plant diseases. Subsequently, these techniques help us in developing effective
strategies for disease control.
With many diseases, latent infections were correlated with disease levels in the
field and or postharvest. Although significant progress has been made in the dis-
covery of conventional methods that help detect latent infections, latent infection
detection is based mainly upon subjecting the infected tissues to surface sterilants,
and tissue damaging agents (paraquat) or conditions (freezing) followed by incuba-
tion. Additionally, there are many variations in the type, number, duration and
sequence of these processes. There is a need for faster and more efficient methods
of latent infection detection and the use of real time polymerase chain reaction
(RT-PCR) can provide the basis for efficient, accurate, and more rapid detection of
pathogens.
There are still gaps in our understanding on the trigger that activates the patho-
gen’s growth in latent infections. It is hoped though molecular techniques will
eventually replace conventional ones in other patho-systems, help elucidate gaps in
epidemiological research, and improve our understanding of plant disease. Future
goals of our research are to develop more efficient, accurate, and rapid molecular
procedures using RT-PCR and replace the conventional ones. Furthermore, our goal
is to reduce costs for processing samples by using such techniques in large numbers
of samples, a process that will provide accurate answers to epidemiological ques-
tions and allow the expansion of molecular epidemiology in plant disease.
Acknowledgments We thank M. Doster, D. Felts, L. Boeckler, H. Reyes, R. Puckett, and

J. Windh for technical assistance. We also thank Drs. Z. Ma, H. Avenot, and E. Boehm,
Postdoctoral Associates, and visiting Professor M. Yoshimura, for their research contributions.
Funding for this research was provided by the California Kiwifruit Commission, California
Pistachio Commission, California Dried Plum Board, California Tree Fruit Agreement, and the
California Fig Institute.
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receptacles to predict gray mold decay in storage. Plant Dis 80:248–254
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Chapter 7
Preharvest Strategies to Control Postharvest
Diseases in Fruits
N. Teixidó, J. Usall, C. Nunes, R. Torres, M. Abadias, and I. Viñas
Abstract Postharvest diseases on citrus and pome fruits are generally controlled
by chemical treatments applied in packinghouses before fruit storage. However,
there are some points that make pre-harvest strategies interesting practices to con-
trol or reduce postharvest rots. (a) Field practices and fruit manipulation in general
can play an important role in fruit susceptibility in front postharvest diseases. (b)
In some cases, infection of fruit occurs in the field prior to harvest and it could be
advantageous starting control at this point. (c) Pre-harvest strategies also decrease
fruit manipulation and subsequently potential damages and injuries, which are
necessary for some important fungi infection. (d) Additional contamination by
pathogenic fungi present in drenching solutions used in packinghouses would also
be avoided. Twelve years of research have allowed us to study different approaches
and some of them are summarized in this chapter.
The aim of this research was to enhance efficacy of preharvest biocontrol treat-
ments using different strategies: combination of biocontrol agents, combination
with low-risk substances such as ammonium molybdate, and finally increase envi-
ronmental stress tolerance of biocontrol agents. All these experiences have let to
conclude that pre-harvest practices can play an important role in postharvest dis-
ease control.
N. Teixidó (*), J. Usall, R. Torres, and M. Abadias

IRTA, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida,
Catalonia, Spain
e-mail: neus.teixido@irta.cat
C. Nunes
University of Algarve, Centro de Desenvolvimiento de Ciências e Técnicas de Produção Vegetal,
Faro, Portugal
I. Viñas
University of Lleida, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198,
Lleida, Catalonia, Spain

90 N. Teixidó et al.
7.1 Introduction
The development of resistance in fungal pathogens to fungicides (Dekker and

Georgopoulos 1982; Viñas et al. 1991, 1993) and the growing public concern over
the health and environmental hazards associated with high levels of pesticide use
into fruit orchards (Wisniewski and Wilson 1992) have resulted in a significant
interest in the development of alternative non-chemical methods of disease control.
Biological control using microbial antagonists has emerged as one of the most
promising alternatives, either alone or as part of an integrated control strategy to
reduce pesticide inputs.
Infection of fruit by postharvest pathogens often occurs in the field prior to har-
vest (Biggs 1995; Roberts 1994), thus it would be advantageous to apply antago-
nists before harvest which would reduce initial infection and then remain active and
suppress the pathogens in storage (Elad and Kirshner 1993; Leibinger et al.
1997).
When biocontrol agents are applied at postharvest, they often have difficulty
controlling previously established or incipient infections, which originated in the
field. Indeed, the effectiveness of the antagonist decrease in proportion as increase
the time since pathogen infection was originated and the antagonist was applied
(Ippolito and Nigro 2000). Biocontrol of fruit decay in storage with microbial
antagonists has so far been mainly studied under controlled environmental condi-
tions at postharvest: few studies have attempted to apply biocontrol agents to fruit
under field conditions with the purpose of controlling postharvest decay (Benbow
and Sugar 1999; Leibinger et al. 1997). The pathogens P. digitatum and P. italicum
infect fruit through wounds produced by mechanical injuries during the growing
season and harvest handling operations (Roberts 1994; Biggs 1995; Obagwu and
Korsten 2003). An antagonist applied in the field, prior to harvest, will have longer
to interact with the pathogen than one applied after harvest (Ippolito and Nigro
2000). It would therefore be advantageous to apply antagonists before harvest,
which would reduce initial infection, and then let them remain active and suppress
pathogens during storage (Elad and Kirshner 1992, 1993; Teixidó et al. 1998a).
However, an important consideration for the application of biocontrol agents at
preharvest is their ability to colonize the surface of fruit both in the field and during
storage and to persist, for as long as possible, in sufficient numbers on the fruit
surface to maintain an efficient decay control (Wisniewski and Wilson 1992).
Several works have demonstrated the ability of some microorganisms to survive
under field conditions and consequently provide effective control against posthar-
vest decay (Korsten et al. 1997; Tian et al. 2004). However, under field conditions,
rapid fluctuations in water availability and temperature are both characteristic of
this environment and constitute the main factors limiting the development of micro-
bial populations (Köhl and Fokkema 1998).
Some different strategies have been deeply studied by our group in order to
enhance biocontrol efficacy in preharvest applications of two biocontrol agents: the
yeast Candida sake CPA-1 and the bacterium Pantoea agglomerans CPA-2 and
7 Preharvest Strategies to Control Postharvest Diseases in Fruits 91
some examples have been described in this chapter: biocontrol agents combination,
combination with low risk substances and enhance environmental stress tolerance.
C. sake CPA-1 has demonstrated to be an effective biocontrol agent (BCA)
against major postharvest diseases on pome fruits (Viñas et al. 1998; Usall et al.
2000, 2001) and currently is commercialized in Spain as a liquid formulation
named Candifruit by SIPCAM-INAGRA S.A. P. agglomerans CPA-2 is an effec-
tive antagonist to the major postharvest fungal pathogens of pome and citrus fruits
(Nunes et al. 2001b, 2002b; Teixidó et al. 2001)and it is in commercialization pro-
cess in Spain as a solid formulation named Pantovital by BIODURCAL S.L.
7.2 Biocontrol Agents Combination
The yeast Candida sake CPA-1 (Viñas et al. 1998) and the bacterium Pseudomonas
syringae CPA-5 (Nunes et al. 2007a) were isolated from apple surface. These
microorganisms have been tested, for many years, for their control activity against
the major postharvest diseases of pome fruits.
The objective of this work was to determine the efficacy of pre-harvest applica-
tion of a combined treatment of C. sake CPA-1 and P. syringae CPA-5 to control
P. expansum decay of pear and apple fruits during cold storage and study the
population dynamics of each biocontrol agent.
A yeast and a bacterium have been used in order to have microorganisms with
different nutritional requirements. This fact could be an advantage in order to avoid
competence problems.
The results of pre-harvest treatments to control postharvest blue mold in pears
and apples are shown in Fig. 7.1. All treatments significantly reduced incidence
of P. expansum on pears and apples stored at 1°C for 4 months. On ‘Blanquilla’
pears treated only with C. sake CPA-1 or only with P. syringae CPA-5, P. expan-
sum incidence was reduced 53%. The combined treatment in pears was significantly
different from the application of each antagonist alone, and it reduced blue
mold incidence in 90%, and enhanced biocontrol activity of each antagonist in
78% (Fig. 7.1a).
On ‘Golden Delicious’ apples the biocontrol agent P. syringae CPA-5 reduced
blue mold incidence in 40%. Although no significant differences were observed
between the individual applications of C. sake CPA-1 or combined with P. syringae
CPA-5, blue mold was reduced 46% and 56% respectively. Regarding to the indi-
vidual application of P. syringae CPA-5 the combined treatment enhanced biocon-
trol activity in 26% (Fig. 7.1b).
The population dynamics of C. sake CPA-1 and P. syringae CPA-5 has been
recorded. At day 0 and 2 the population densities of both antagonists were higher
on pears than on apples. However, at the end of the experiment (day 120) popula-
tion densities reached similar levels. In pears at day 0 and 2, the population level of
P. syringae alone or in combination was higher than C. sake, while in apple the
opposite was observed at all sampling times.
a b
100 100
a 80
80
% Incidence
% Incidence
60 60 a
b b 40
40 b
c
c
20 20
c
0 0
Control CPA-1 CPA-5 CPA-1 Control CPA-1 CPA-5 CPA-1
+CPA-5 +CPA-5
Fig. 7.1 Incidence of blue mold on (a):‘Blanquilla’ pear and (b): ‘Golden Delicious’ apple pre-
harvest treated with C. sake CPA-1 (107 CFU mL−1), P. syringae CPA-5 (2 × 107 CFU mL−1), and
their combination in a proportion of 50:50. Fruits were wounded and treated in the field 2 days
before harvest. After harvest, fruits were sprayed with P. expansum (104 spores mL−1) and stored
at 1°C and 90% ± 5% RH for 120 days. Columns with the same letter are not significantly differ-
ent (P > 0.05) according to the least significant difference test (LSD). (Nunes et al. 2007b)
This study demonstrated that the pre-harvest treatment with a mixture of 50:50 of
C. sake CPA-1 and P. syringae CPA-5 enhanced biocontrol activity against P. expan-
sum on apples and pears in comparison with control by antagonists applied sepa-
rately. Similar results were obtained in postharvest treatments using a mixture of
C. sake CPA-1 and Pantoea agglomerans CPA-2 in apples and pears (Nunes et al.
2002c). In that work similar population level of C. sake was obtained. The ability of
both antagonists to colonize wounded fruits was not affected by the presence of the
other antagonist, since similar level was obtained either alone or in combination.
This result agrees with other authors (Janisiewicz and Bors 1995) that concluded that
the carrying capacity of the wounds is greater than the population of a single antago-
nist indicates. It seems that the enhancing effect of the mixture of both antagonists
appears to be due to the depletion of nutrients by their growth of both antagonists in
wounded fruits that does not allow the development of P. expansum.
In conclusion, our research showed that the pre-harvest application of a combi-
nation of C. sake CPA-1 and P. syringae CPA-5 results in an improvement of each
antagonist (Nunes et al. 2007b).
7.3 Biocontrol Combined with Low Risk Substances
The chemical environment can be manipulated to enhance biocontrol activity of the

antagonists, and in fact, several works report additives that enhance the effective-
ness of microbial antagonists (McLaughlin et al. 1990; Janisiewicz 1994; Wisniewski
et al. 1995; Janisiewicz et al. 1998).
Preliminary assays in vitro and in vivo (Nunes et al. 2001a) on the combination
of some nutrients with C. sake strain CPA-1 in order to enhance biological control
showed that the combination of the antagonist and ammonium molybdate reduced
blue mold decay more than other chemicals. However, the ability of ammonium
molybdate to control disease development has not been fully explored.
Ammonium molybdate affects metabolic processes in several organisms (Wang
et al. 1995; Bodart et al. 1999). The basis of its biological activity was reported to
be its ability to inhibit acid phosphatase which interferes with phosphorylation and
dephosphorylation (Glew et al. 1988), one of the most important processes of cell
regulating (Remaley et al. 1985; Hunter 1995).
The efficacy of preharvest applications of C. sake CPA-1 combined with ammo-
nium molybdate to control blue mold during cold storage on apples and pears was
evaluated. In apple assays, fruits were wounded in the field 2 days before harvest
and treated with the biocontrol agent, ammonium molybdate or the combination,
and in pear assays the treatments were applied 7 and 2 days before harvest on
unwounded fruits and wounds were made at postharvest. In both trials P. expansum
was artificially inoculated before cold storage.
In the case of apples the preharvest application of C. sake 107 CFU mL−1 com-
bined with ammonium molybdate (NH4-Mo) 1 mM did not improve postharvest
biocontrol of blue mold in comparison of each treatment applied alone and the
efficacy was lower than the obtained with postharvest treatments. However, about
54% rot reduction was achieved with these combined preharvest treatments (Nunes
et al. 2002d). The concentration of NH4-Mo used in this study was lower than that
used in pears and in postharvest trials because the application of 5 mM preharvest
caused spots on the fruit surface.
The preharvest application 5 mM did not affect blue mold decay development
in pears (Nunes et al. 2002a) and no differences in incidence of blue mold were
observed among preharvest application of NH4-Mo followed by postharvest
application of C. sake, postharvest application of NH4-Mo and postharvest appli-
cation of C. sake (90% rot reduction in all cases); therefore, the reduction
observed with treatments of preharvest application of NH4-Mo (at 7 and 2 days
before harvest) followed by postharvest application of C. sake seems to be due to
the effect of the antagonist. The preharvest application of NH4-Mo was made on
unwounded pears and the fact that this treatment did not reduce blue mold decay
is probably because a residue of NH4-Mo must remain on the wound to inhibit the
infection. Smilanick et al. (1999) found that the capacity of sodium carbonate or
bicarbonate to control green mold on citrus was significantly reduced when the
fruits were rinsed at high-pressure. They concluded that this reduction occurred
because the high-pressure removes the residues of the compounds necessary to
achieve control of green mold.
Toxicology date of NH4-Mo was determined by the “Centre d’Investigació i
Desenvolupament Aplicat” (Barcelona, Catalonia, Spain), calculating the rat oral
median lethal dose (LD50). This work showed that the LD50 of ammonium molyb-
date is 1714. 3 mg kg−1 of live weight of Sprague Dawley rat and, at this concentra-
tion, no mortality or alterations in tested animals were observed.
The C. sake enhancement achieved with the combination with ammonium

molybdate is better in postharvest treatments than in preharvest.
7.4 Enhancing Biocontrol Agents to Environmental Stress

Conditions
To improve stress response behaviour and stimulate mechanisms of environmental

survival of biocontrol agents, it is very important to optimise their efficacy and suit-
ability for practical conditions.
Survival of foodborne pathogenic microorganisms under different stress treat-
ments (e.g. heat, osmotic or water activity, low pH, etc.) have been extensively
studied and reported. Initial investigations carried out on bacteria such as
Escherichia coli (Poirier et al. 1998), Salmonella spp. (Mattick et al. 2000) and
Listeria spp. (Greenacre et al. 2003) have demonstrated that they possess an inher-
ent ability to adapt to unfavourable environments by the induction of various gen-
eral and specific stress responses. These stress responses are characterised by the
transient induction of general and specific proteins and by physiological changes
that generally enhance the particular organism’s ability to withstand more adverse
environmental conditions (Ang et al. 1991). In the case of osmotic stress, the sig-
nificant physiological changes reported in bacteria include the induction of stress
proteins as well as the accumulation of compatible solutes such as K+ ions, the
amino acids glutamate, glutamine, proline and alanine, the quaternary amines gly-
cine betaine, the tetrahydropyrimidine ectoine and sugars such as sucrose and tre-
halose (Csonka 1989; Kets et al. 1994; Ko et al. 1994). In yeasts and filamentous
fungi the accumulated compatible solutes are mainly low- (glycerol and erythritol)
and high- (arabitol and mannitol) molecular weight sugar alcohols (Beever and
Laracy 1986; Ellis et al. 1991; Van Eck et al. 1993). These compatible solutes allow
equilibration of the cytoplasmic water activity (aw) with the surrounding environ-
ment, thereby retaining water in the cell and thus, maintaining turgor pressure, and
helping to preserve protein function within cells (Yancey et al. 1982; Csonka 1989;
Van Eck et al. 1993).
Subjection to a mild stress can make cells resistant to a lethal challenge with the
same stress condition. Preadaptation to one particular stress condition can also
render cells resistant to other stress imposing conditions: this phenomenon is
known as cross protection (Sanders et al. 1999).
First studies conducted in this sense in biological control were conducted by
Hallsworth and Magan (1994a, 1995, 1996) on entomopathogenic biocontrol fungi,
demonstrating that it is possible to physiologically manipulate growth conditions,
carbon sources and carbon:nitrogen ratios to channel specific polyols into myce-
lium and fungal propagules, resulting in improved and more rapid germination and
(or) germ tube extension under water stress conditions. Conidia modified in this
way may be more pathogenic to target pests at low relative humidity (Hallsworth
and Magan 1994b). Physiologically manipulated inocula of the biocontrol agent
Epicoccum nigrum, containing high concentrations of glycerol and erythritol have

also been found to give better control of brown rot (Monilinia laxa) of peaches than
unmodified inocula (Pascual et al. 1996).
7.4.1 C. sake CPA-1 Enhancement
C. sake cells have been grown under mild or sublethal stress conditions in order
to adapt this biocontrol agent and render cells resistant to lethal or more stressful
conditions. Significant improvements in low aw tolerance were achieved by modi-
fying both the aw and nutrient concentration of growth media. The best results
were obtained with glucose and glycerol solutes, and the intracellular accumula-
tion of polyols and glucose and trehalose in these aw stress-improved cells was
significantly different than with unmodified control cells (Teixidó et al.
1998b,c).
In laboratory studies, the low aw-tolerant cells provided significantly better dis-
ease control as compared with the unmodified cells, and reduced the number of
infected wounds and lesion size by 75% and 90%, respectively, as compared with
nontreated controls (Teixidó et al. 1998a).
Unmodified and low water activity (aw) tolerant cells of C. sake CPA-1
applied before harvest were compared for ability to control blue mold of apples
(Golden Delicious) caused by P. expansum under commercial storage conditions
(Teixidó et al. 1998a). The population dynamics of strain CPA-1 on apples was
studied in the orchard and during storage following application of 3 × 106 CFU mL−1
of each treatment 2 days prior to harvest. In the field, population sizes of unmod-
ified treatment remained relatively unchanged, while the low aw-modified CPA-1
cells increased. During cold storage the populations in both treatments increased
from 10 3 CFUg−1 to 105 CFU g−1 after 30 days, and then declined to about 2.5
× 10 4 CFU g −1 apple. After 4 months in cold storage both unmodified and
low aw-tolerant cells of C. sake were equally effective against P. expansum on
apple (>50% reduction in size of infected wounds) Fig. 7.2. In these experi-
ments, it was also observed that unmodified yeast cells initially adhered better
to the apple surface than the two low aw-tolerant treatments. Little is known
about the characteristics of the polysaccharide matrix produced by yeasts such
as C. sake. However, previous studies with such polysaccharide matrices of
spores of other fungi suggest that they may have a number of important ecologi-
cal properties, including protection against temperature extremes, desiccation
and short wave radiation (Louis and Cooke 1983, 1985). It is possible that the
energy requirements for the production of high concentrations of endogenous
reserves during growth at low aw, such as polyols and trehalose (Teixidó et al.
1998b,c) could result in a modification of the amount or characteristics of the
matrix. Probably, if adherence of modified cells could be improved with specific
additives, population of C. sake on fruit surface could be increased and also
efficacy could be enhanced.
90 18
80 16
a % Infected
70 14
Lesion diameter (mm)

wounds
Infected wounds (%)

60 Lesion diam. 12
50 10
b
40 8
b b
30 6
20 4
10 2
0 0
CONTROL NYDB NYDB25+ NYDB50+
GLY GLU
Treatment
Fig. 7.2 Suppression of blue mold of Golden Delicious apples by Candida sake CPA-1 grown in
different media. Fruits were wounded in the field and suspensions of the antagonist (107 CFU
mL−1) were sprayed onto the apples 2 days prior to harvest. After harvest fruits were sprayed with
an aqueous suspension of P. expansum at 104 conidia mL−1 and kept in cold storage for 4 months.
Columns and lines with the same letter are not significantly different according to least significant
difference test (LSD) (P < 0.01). The letters apply to both, % infected wounds and lesion diameter.
Treatments: C. sake grown in unmodified NYDB (NYDB), in NYDB diluted by 75% with water
and amended with glycerol to modify aw to 0.96 (NYDB25 + GLY) and in NYDB diluted by 50%
with water and amended with glucose to modify aw to 0.96 (NYDB50 + GLU). (Teixidó et al.
1998a)
Other stress conditions, such as high temperature have been studied with this
biocontrol agent achieving thermotolerant cells that survived better under high
temperature conditions and spray-drying process (Cañamás et al. 2008b).
7.4.2 P. agglomerans CPA-2 Enhancement
The improvement of tolerance to low water activity (aw) and desiccation in P. agglo-
merans cells subjected to mild osmotic stress during growth was studied using
different solutes to change aw of growth media. It was shown that cells grown in
media at low aw using NaCl exhibited osmotic adaptation in solid media at low aw
obtaining high production level and maintaining biocontrol efficacy (Teixidó et al.
2006). Osmotic-adapted cells also demonstrated thermotolerance (Teixidó et al.
2005) and desiccation tolerance after spray drying (Teixidó et al. 2006).
The role of different compatible solutes in adaptation of the bacterium to
osmotic stress was determined and this study suggested that glycine-betaine and
ectoine play a critical role in environmental stress tolerance improvement (Teixidó
et al. 2005; Cañamás et al. 2007).
These osmotic adapted cells were used in preharvest treatments in order to

control the main postharvest diseases on citrus fruit.
In the first experiment (Cañamás et al. 2008a), it was observed that P. agglom-
erans cells (osmotic-adapted and non-adapted) were unable to maintain a stable
population on the fruit surface and consequently, preharvest applications resulted
ineffective against both naturally and artificially inoculated P. digitatum. This low
survival rates revealed the sensibility of P. agglomerans cells to environmental field
conditions. These results also led us to conclude that it is necessary a minimal
antagonist population level on fruit surfaces in order to guarantee competition with
pathogens for sites and nutrients, and to subsequently obtain an efficient control.
The establishment of bacterial populations on plant surfaces is a critical phase in
disease control (Ippolito and Nigro 2000). Microbes can be inactivated by several
environmental factors, including sunlight, temperature, humidity, leaf surface exu-
dates and competitors. They may also be physically lost from the target location
due to the action of wind, rain or leaching (Jones and Burges 1998).
The effect of main environmental factors on P. agglomerans cells, such as rela-
tive humidity and solar radiation was studied and different formulation strategies
were used in order to enhance survival on fruit surface under field conditions and
subsequently enhance biocontrol efficacy (Cañamás et al. 2008a).
Osmotic adapted P. agglomerans cells, specially when these cells grew at 0.98
aw, were more resistant than non-adapted cells when both were applied in oranges
and stored in chambers at a low Relative Humidity (43%). However, the minimum
values of relative humidity registered during the first field assay were between 15%
and 50%. These extreme values may restrict the survival and growth of P. agglom-
erans cells. The relative importance of each of these factors depends on why and
where a particular product is used. In applications in foliar environments (which
were our case), solar radiation, and especially the ultraviolet (UV) portion of the
spectrum, is probably the most important factor affecting the persistence of micro-
bial insecticides (Rhodes 1993; Filho et al. 2001). This detrimental effect of UV
radiation was also checked in a laboratory assay in which a major decrease in
P. agglomerans population was observed during exposure to sunlight.
In laboratory studies, different additives, such as summer oils, alginate, glycerol
and food additives at different concentrations were tested mixed with P. agglomerans
in order to study its compatibility. The non-toxic ones (Citroline, Summer oil,
Alginate, Sunspray, Glycerol, Siapton and Fungicover) were added to the biocon-
trol agent, sprayed on detached oranges and left outdoors. Population dynamics of
the antagonist on fruit surface was determined along the time (Fig. 7.3).
Fungicover was the most effective additive for improving the adherence and per-
sistence of P. agglomerans cells on oranges and it was also compatible with the
antagonist. Firstly, adherence was improved by Fungicover since the P. agglomerans
cell population just after application and drying (0 h), was greater than when cells
were only sprayed with water. It was also visually observed that spreading, wetting
and dispersion were also clearly improved. This could have been due to the fact that
the additive Fungicover contains fatty acid derivatives in an alcohol solution. These
components could have reduced the surface tension of the cell suspension and
5.0 a
4.5
r
b
4.0
bc bc
3.5
Log10(CFU cm−2)
cd
d d
3.0
d
2.5
2.0 s
s
1.5 st
st
1.0 st
t
0.5
t
0.0
P-SH alone P-SH P-SH P-SH P-SH P-SH P-SH P-SH
+Citroline +Summer Oil +Sunspray +Glycerol +Siapton +Alginate +Fungicover
Additives
Fig. 7.3 Effect of additives in the population level of non-adapted P. agglomerans cells on
oranges during exposure outside under springtime environmental conditions. Samples were recov-
ered 0 h (white bars) or 24 h (black bars) after spraying treatment. Treatments consisted in aqueous
suspension of P. agglomerans cells alone or in combination with a respective additive. Values
plotted at each time point are averages of three replications. Columns with different letters are
significantly different (P < 0.05) according to Duncan’s test. (Cañamás et al. 2008a)
thereby improved the spread and wetness of the spray over the plant surface
(Burges 1998). On the other hand, the persistence of P. agglomerans cells was also
improved outdoors under springtime environmental conditions in the presence of
Fungicover at 5%. It has not been possible to exactly elucidate the mechanism(s)
by which this additive was able to protect the antagonist population. The additive
Fungicover could also have protected P. agglomerans cells from solar radiation as
sunscreen, physically reflecting and scattering, or selectively absorbing radiation,
converting short wavelengths to harmless longer ones (Jones and Burges 1998).
Fungicover is an edible film-forming compound for fruits and vegetables to
reduce weight loss, delay senescence, improve natural brightness and reduce physi-
ological disorders. It also reduces droplet size and improves uniformity of distribu-
tion on the surface to be protected. This additive did not show any fungicidal effect
on P. digitatum (Cañamás et al. 2008a).
In this study it has also been demonstrated that inoculum formulation can influ-
ence the persistence of P. agglomerans cells. Bacterial treatments prepared with
lyophilised P. agglomerans cells become more resistant to environmental condi-
tions than fresh cells, as Stockwell et al. (1998) observed when the bacterial antago-
nists Pseudomonas fluorescens A506 and Erwinia herbicola C9-1R was applied
under field conditions.
Different strategies could be used to improve P. agglomerans cell survival on

oranges under non-controlled environmental conditions and all them were applied
in two representative field trials on ‘Lane late’ and ‘Valencia’ oranges, in order to
evaluate the effectiveness of different bacterial formulations of P. agglomerans
applied at preharvest for controlling postharvest decays caused by natural infection
and also by artificial infection (Penicillium digitatum) (Cañamás et al. 2008c).
Population dynamics of P. agglomerans during trials (under field conditions and
at postharvest) are shown in Fig. 7.4. In both experiments greater adherence and
persistence of populations was observed when the biocontrol agent cells were
sprayed using the additive Fungicover, being the population level similar to that
treatment applied at postharvest. The use of additives to improve the adherence and
persistence of biocontrol agents has been previously shown by Guijarro et al.
(2007), who added additives at different points of the production-formulation pro-
cess, and showed that they improved adhesion of Penicillium frequentans conidia
to fruit surfaces, providing more effective control against brown rot in peaches.
Results for the efficacy of preharvest treatments for artificial infection by P. digi-
tatum are shown in Fig. 7.5. In experiment 1 (Fig. 7.5a) the P25-LY + FC and
P25-SH + POST treatments were significantly more effective than the other prehar-
vest treatments and non differences were found between both after 15 days. In
experiment 2 (Fig. 7.5b) and after 15 days of storage, all the preharvest treatments
with the additive Fungicover showed effective control against P. digitatum with
decay values of below 12.5%. Treatment P25-LY also showed effective control
with 12.8% decay. However, only treatments P25-LY + FC, P98-LY + FC and
P97-LY + FC with decay values of between 7% and 5.3%, exhibited levels of con-
trol on P. digitatum no statistically different to the antagonist postharvest treatment
P25-SH + POST.
The protective effect of the additive Fungicover was again confirmed by results
and this effect varied according to whether or not the P. agglomerans cells had been
osmotic adapted and if the cells had been lyophilised or not. It is therefore likely
that adding Fungicover to formulations could protect cells against field conditions.
At the same time, it was possible to observe that populations of osmotic adapted
cells showed a higher level of survival than non-adapted cells when the additive FC
was used. Furthermore, the positive effect of applying lyophilised P. agglomerans
cells instead of fresh cells was also evident when treatments were combined with
Fungicover.
The ability of antagonists to survive at sufficient population levels on fruit sur-
faces after application is very important for achieving effective control (Benbow and
Sugar 1999). Results indicated that there was a close relationship between the popu-
lation level associated with a given treatment under field conditions and the level of
control achieved by this treatment during storage. In the first experiment with ‘Lane
late’ oranges, the P25-LY + FC treatment, which showed a high survival level, was
the most effective of the six bacterial preharvest treatments for controlling artificial
infection by P. digitatum showing a level of control not statistically different to the
postharvest treatment (P25-SH + POST). In the second experiment with ‘Valencia
late’ oranges, a relationship between population level and effectiveness was also found.
100 a
5 Harvest
Preharvest conditions 8C and 80% RH

Storage conditions at 20 ºC
4
Log10(CFU cm-2)
0
0 2 4 6 8 10 12 14 16 18 20 22
Time (days)
5 b
Harvest
Preharvest conditions Storage conditions at 20 8C and

20ºC and80%
80%RH
RH
4
Log10(CFU cm-2)
0
0 3 6 9 12 15 18 21
Time (days)
Fig. 7.4 Population dynamics of P. agglomerans treatments during field and storage conditions.
In Experiment 1(a) the following bacterial treatments were used: P25-SH (——), P-LY (——),
P25-LY (—°—), P98-LY (—∆—), P97-LY (—◊—), P25-LY + FC (---°---) and P25-SH +
POST(—´—). The bacterial treatments used in Experiment 2 (b) were P25-LY (—°—), P25-SH
+ FC (------), P-LY + FC (------), P25-LY + FC (---°---), P98-LY + FC (---∆---), P97-LY + FC
(---◊---) and P25-SH + POST (—´—). Bacterial treatments were prepared from lyophilized (LY) or
fresh (SH) and from non-adapted (P) or osmotic adapted P. agglomerans inocula in presence of
25 g L−1 of NaCl (P25) or at 0.98 or 0.97 aw in the medium (P98 and P97, respectively). The addi-
tive Fungicover (+FC) was used in some treatments at a concentration of 5% in order to check its
adherence and persistence effect on the populations of P. agglomerans cells. An adequate volume
of non-adapted or osmotic adapted P. agglomerans inocula for each bacterial treatment was mixed
into 30 L of water in a plastic recipient to obtain a final concentration of 2 ×108 CFU mL−1.
70
a
r
60
% Incidence of P. digitatum
rs
50
a rst
a st
st
40
t ab
ab
30
abc
u
c c
20
u
10 d
e v
0
CK P25-SH P-LY P25-LY P98-LY P97-LY P25-LY+FC P25-SH-POST IZ
70
b
60
% Incidence of P. digitatum
50
40
30
r
20
a s
s s
ab ab ab
10 t t
bc bc t t
c
c
d u
0
CK P25-LY P25-SH+FC P-LY+FC P25-LY+FC P98-LY+FC P97-LY+FC P25-SH+POST IZ
Treatments
Fig. 7.5 Effectiveness of preharvest treatments against artificial infection of the fungal pathogen
P. digitatum. Fruits were stored for 15 days at 20°C and 85% RH. The incidence of decayed fruits
was scored after 7 (white bars) and 15 days (grey bars) of storage and expressed as % decay pro-
duced by the P. digitatum pathogen on orange cultivars, ‘Lane late’ and ‘Valencia late’ in
Experiments 1(a) and 2(b), respectively. Different letters in the bars indicate significant differences
between means according to a Duncan’s Multiple Range Test (P < 0.05). (Cañamás et al. 2008c)
All preharvest treatments, which showed stable population levels on the surface of
orange fruits under field conditions, therefore also demonstrated greater effective-
ness than the control treatment. Moreover, only bacterial treatments, which were
Bacterial treatments were sprayed onto orange fruits cv ‘Lane late’ (EX-1) or ‘Valencia late’
(EX-2) 1 week before harvest. Treatment P25-SH + POST was applied at postharvest dipping
oranges in a solution at 1 ×108 CFU mL−1 before the storage period. Results are means of four
independent samples and vertical bars indicate standard deviations. (Cañamás et al. 2008c)
prepared from lyophilised and osmotic adapted cells, showed a level of control
comparable to postharvest treatments with the biological control P. agglomerans
when they were applied with additive Fungicover. These preharvest treatments
were also associated with population levels that were higher under field conditions.
These findings were in concordance with those of Tian et al. (2004) who found that
only Rhodotorula glutinis and Cryptococcus laurentii, whose populations remained
at high and stable levels, significantly reduced fruit decay during storage at 25°C.
Moreover, applications of Bacillus subtilis, aimed at establishing antagonistic bac-
teria prior to arrival of Pseudocercospora purpurea inoculum, resulted in sustained
control (Korsten et al. 1997).
We conclude that survival and stability of P. agglomerans populations
could be maintained under field condition by integrating certain formulation
strategies: adding additives, ecophysiological osmotic adaptation and lyophili-
sation. Thus, it has highlighted that it is very important to optimize both,
distribution of the biological control agent on the host surface and survival
under field conditions.
The additive Fungicover reduces droplet size and improves uniformity of distri-
bution on the surface to be protected. Furthermore, Fungicover seems to bring a
protective effect to the biocontrol agent against adverse environmental conditions.
Osmotic adaptation and lyophilisation also provided a better performance of cells
under field conditions. All these formulation strategies have consequently demon-
strated that the improved formulation of P. agglomerans provided good protection
for orange fruits against both natural and artificial infections, resulting preharvest
biocontrol treatments effective in the control of postharvest diseases. However,
although preharvest treatments were applied with the objective to control the infec-
tions produced in the field and consequently obtain a better control respect to apply
biocontrol agents in postharvest treatments, the results showed that no significant
differences in the level of control were found between the applications of biocontrol
agent at preharvest or postharvest.
The results of this work suggest that it is possible to broaden the spectrum of use
of the biocontrol agent P. agglomerans and to thereby develop practical uses for this
biocontrol agent under preharvest conditions.
The results presented here are an example that the induction of stress adaptation
responses is a useful and practical tool, that could broaden new possibilities for
improving performance of biocontrol agents to other hosts and diseases and it could
improve their antagonistic activity in a wide range of conditions.
In this chapter it has been tried to compile some examples to show the interest
of preharvest strategies in controlling postharvest diseases and it has been demon-
strated that it is worthwhile to go ahead on this kind of research.
Acknowledgements The authors are grateful to Spanish government (Ministerio de Ciencia y

Tecnología) for grants AGL-2002-01137 and AGL-2005-02510 and to FEDER (Fondo Europeo
de Desarrollo Regional) and COST Action 864 for their financial support. Authors are also grate-
ful to company BIODURCAL S.L. for providing the Fungicover product.
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Chapter 8
New Developments in Postharvest Fungicide
Registrations for Edible Horticultural Crops
and Use Strategies in the United States
J.E. Adaskaveg and H. Förster
Abstract New developments in postharvest fungicide registrations of fresh fruit

and vegetable crops and use strategies in the United States are discussed for
preventing decay and crop losses while minimizing the potential of selection of
resistant pathogen populations. Postharvest fungicides used on agricultural com-
modities are among the most rigorously tested and regulated chemicals in the world
and their risk assessment analysis and residue limits are extensively reviewed by
multiple regulatory agencies. Novel products and pre-mixtures increase the
spectrum of fungal decays managed and the number of crops labeled allowing
global marketing of crops. These product registrations are part of a continuum of
integrated approaches of handling agricultural commodities designed for steward-
ship of products and their safe usage in the worldwide distribution of fresh produce.
Optimized postharvest usage strategies of fungicides include integration with other
fungicides (i.e., pre-mixtures) and sanitation treatments to optimize performance
while allowing identification of methods that reduce the selection of resistant sub-
populations of pathogens.
The competitive global marketing of fresh fruit crops that demands decay-free fruit
and often involves long-distance shipping makes postharvest decay management a
challenging task. In an integrated approach of decay management, cultural, prehar-
vest, harvest, and postharvest practices are essential components that influence the
complex interaction between host, pathogen, and environment. Orchard practices
mainly affect crop health and pathogen inoculum levels, however, preharvest fun-
gicide applications can also directly reduce the development of fruit decay. Among
postharvest practices, postharvest fruit treatments with fungicides are the most
J.E. Adaskaveg (*)
Department of Plant Pathology and Microbiology, University of California, Riverside,
CA 92521, USA
e-mail: address: jim.adaskaveg@ucr.edu
H. Förster
Department of Plant Pathology, University of California, Davis, CA
108 J.E. Adaskaveg and H. Förster
effective means to reduce decay (Eckert and Ogawa 1988; Adaskaveg et al. 2002).
Ideally, these fungicides protect the fruit from infections that occur before
treatment, including quiescent infections, as well from infections that are initiated
after treatment during postharvest handling, shipment, and marketing.
Problems that had been arising with the use of the ‘older’ postharvest fungicides
and that led to the cancellation of their registration or reduced usage initiated our
extensive research on the identification and development of alternative treatments
in the 1990s (Adaskaveg et al. 2005). These problems included high non-target
toxicity as for ortho-phenylphenol and biphenyl or lack of high efficacy and need
for high usage rates as for captan or sulfur. For other fungicides such as iprodione
and triforine, re-registration was not pursued because of potentially exceeding
the exposure limit that is set for each pesticide. Furthermore, widespread resistance
in pathogen populations has arisen against other materials such as benomyl,
thiabendazole, and imazalil. In the development of new fungicides, emphasis was
placed on minimizing human health risks and environmental toxicity. With mam-
malian toxicity concentrations of generally more than 2,000 mg/kg, these new
treatments are the safest fungicides ever developed for postharvest use. Fungicides
that meet these standards are classified as ‘reduced risk fungicides’ by the United
States Environmental Protection Agency (EPA; EPA 2003). To reduce the potential
of resistance development against the new treatments, application strategies have
been developed based on experiences in developing postharvest fungicide treat-
ments of agricultural commodities made in the past.
8.1 Risk Assessment, Maximum Residue Limits,

and Postharvest Fungicide Registration
Fresh fruits and vegetables are considered essential for a high-quality human diet.
The use of postharvest fungicides dramatically reduces crop losses and allows
worldwide distribution of fresh market fruit and vegetable crops. Registration of
synthetic postharvest fungicides, like any other agricultural pesticide, includes a
risk assessment analysis by governmental regulatory agencies to ensure that the
product is safe to users, consumers, and the environment, as well as to the crop
itself. Pesticides used in agriculture are among the most rigorously tested and
regulated chemicals in the world. Risk assessment analysis involves evaluating
the potential hazard characteristics of the product including the active ingredient
and breakdown products, as well as carrier or inert ingredients in the formulation.
Toxicological, ecotoxicological, and physical properties of the active ingredient
and formulated product are evaluated to safeguard the use of the product, and
analytical procedures for measuring the active ingredient are established during
product development. In regulatory assessments of single active ingredient prod-
ucts in the United States, multiple exposure tests assess acute and chronic dietary,
as well as short-, intermediate-, and long-term occupational, residential, and rec-
reational exposure. In many countries, comprehensive reviews of data packages
8 New Developments in Postharvest Fungicide Registrations for Edible Horticultural 109
are done at national and regional levels for each country where the product will
be sold. Furthermore, pesticides in general are subject to a re-review after some
period of time. The prospect of world-wide pesticide registration is a daunting
task for any manufacturer and only recently has regulatory harmonization across
regions and among regulatory groups been considered.
For any pesticide, concentration limits on a given commodity are set to ensure
safe usage and minimal risk for consumers. In the United States, the EPA along
with the Food and Drug Administration (FDA) evaluates human risk associated
with pesticide exposure and sets its own standards for pesticide tolerances using
chemical hazard and exposure assessment procedures in the risk assessment pro-
cess as described previously. The Codex Alimentarius Commission was created in
1963 by the Food and Agriculture Organization (FAO) and the World Health
Organization (WHO) to develop food standards and guidelines in order to protect
consumer health, ensure fair trade practices, and to promote coordination of all
food standards. The Codex Committee on Pesticide Residues (CCPR) develops and
maintains acceptable maximum residue limits (MRLs) of pesticides for food com-
modities. Residue limits are established by CCPR and the Joint Meeting on
Pesticide Residues by FAO and WHO based on toxicological data and estimates for
average daily intake (ADI) and acute reference dose (ARfD) for humans, as well as
data on a pesticide’s metabolism, environmental fate, and use patterns according to
Good Agricultural Practices (GAPs). Postharvest fungicide residues are generally
several times lower than the MRL (Table 8.1). Furthermore, MRLs and typical use
residues for postharvest fungicides are similar to residues found when the fungi-
cides are used preharvest. Any crop that exceeds the MRL must be destroyed fol-
lowing strict procedures. Residue monitoring is routinely done on agricultural
commodities and ensures the distribution of safe, high-quality, disease-free fresh
produce around the world. Unfortunately, many countries including the United
States and the European Union have not accepted Codex MRLs as an international
standard and instead have relied on their own national or import MRLs. These
countries still have to follow Codex MRLs when exporting agricultural commodities
to destination markets that have adopted these guidelines.
Table 8.1 Maximum residue limits (MRLs) and typical use residues of common postharvest
fungicides of fruit crops
Typical use residues
Postharvest fungicide Crop MRL (mg/kg) (mg/kg)
Fludioxonil Nectarine, plum 5 0.25–0.5
Peach 5 1–1.25
Pome fruit 5 0.5–1
Pyrimethanil Pome fruit 3 (15) a 1 (3–5)
Tebuconazole Sweet cherry 4 0.5–1
MRL for pyrimethanil recently changed to 15 mg/kg to accommodate different application
a
methods.
8.2 Registration of New Postharvest Fungicides

in the United States
After the cancellation of iprodione in 1996, the sterol biosynthesis inhibitor (SBI)
tebuconazole was first in an unprecedented succession of new postharvest registra-
tions that occurred during the years since that date and this process is still ongoing
(Table 8.2). Tebuconazole (Elite®) was approved for use on sweet cherry in 1997.
Also in 1997, the ‘reduced risk’ phenylpyrrole fludioxonil (Scholar® or Graduate®)
was approved for use on stone fruit, and later on pome fruit, pomegranate, kiwifruit,
and citrus. This was followed by registrations of the ‘reduced risk’ hydroxyanilide
fenhexamid (Judge®), the anilinopyrimidine pyrimethanil (Penbotec®), and the QoI
fungicide azoxystrobin (Diploma®), as well as the SBI propiconazole (Mentor®) on
a range of fruit crops as indicated in Table 8.2.
Each of the postharvest fungicides registered or in development has a distinc-
tive spectrum of activity as indicated in Table 8.2 and for each crop, treatments are
being made available to manage all major decays. Thus, brown rot (Monilinia
spp.), gray mold (Botrytis cinerea), and Rhizopus rot (Rhizopus stolonifer) of
stone fruit, gray mold (B. cinerea) and Penicillium decays (Penicillium expansum
and other species of Penicillium) of pome fruit, and Penicillium decays (P. digi-
tatum, P. italicum) and sour rot (Geotrichum citri-aurantii) of citrus fruit all can
be effectively managed using minimal rates that result in very low fungicide resi-
dues on the crop.
For several crops, more than one fungicide, each belonging to a different chemi-
cal class, is available. This is being done to expand the spectrum of activity of
postharvest treatments and to accommodate export markets with specific residue
tolerance requirements. In addition, the availability of multiple active ingredients
for management of the same pathogen is critical for crops where a high risk for
resistance development in pathogen populations is present (e.g., citrus and pome
fruit) and where the use of prudent resistance management strategies is essential
(see below). As also indicated in Table 8.2, the range of crops where new posthar-
vest fungicides are being registered is expanding. Thus, for example, planned reg-
istrations include fludioxonil on tuber crops, tomato, as well as tropical fruits such
as pineapple, papaya, and mango, azoxystrobin on potato, and propiconazole on
tomato where currently resistant populations limit the use of registered compounds
or where no highly effective compounds for decay management are available. For
example, TBZ-resistant populations of Fusarium and Helminthosporium species
occur on potato tubers causing dry rot and silver scurf, respectively, whereas on
tomato no other fungicide is registered after the cancellation of ortho-phenylphenate.
Furthermore, additional fungicides are needed for a number of crops where numerous
decay organisms are not successfully managed. For instance, difenoconazole is
proposed for use on pome fruit and tuber crops for managing Bull’s eye rot and
decays caused by Fusarium spp., respectively.
Table 8.2 Current and future postharvest fungicides for selected agricultural crops in the United States
Fungicide Class Crops registered Crops planned Spectrum of activity
Azoxystrobin* QoI Citrus Potato Penicillium decays
Difenoconazole SBI-triazole – Pome fruit, tuber Penicillium decays, bull’s eye rot, Rhizopus rot,
crops Fusarium decay
Fenhexamid* Hydroxyanilide Stone fruit, pome fruit, – Brown rot, gray mold
pomegranate, kiwifruit
Fludioxonil* Phenylpyrrole Stone fruit, pome fruit, Tuber crops, Brown rot, gray mold, Rhizopus rot, Penicillium
pomegranate, kiwifruit, tomato, decays
citrus tropical fruit
Imazalil SBI-imidazole Citrus – Penicillium decays
Propiconazole SBI-triazole Stone fruit Citrus, tomato Penicillium decays, brown rot, gray mold, sour rot
Pyrimethanil* Anilinopyrimidine Citrus, pome fruit Stone fruit Penicillium decays, brown rot, gray mold, bull’s
eye rot
Tebuconazole SBI-triazole Sweet cherry – Brown rot, Rhizopus and Mucor decays
Thiabendazole Benzimidazole Citrus, pome fruit, potato – Penicillium decays, gray mold, Fusarium rot
* - Reduced risk classification by the United States Environmental Protection Agency (US-EPA). See http://www.epa.gov/opppmsd1/PR_Notices/pr97-3.html.
8 New Developments in Postharvest Fungicide Registrations for Edible Horticultural
111
8.3 Risk of Fungicide Resistance Development

in the Postharvest Environment
Fundamental to understanding the resistance potential of a fungal species against a

fungicide is the fact that resistant populations develop primarily by selection from
a very small number of naturally occurring, less sensitive individuals within the
population (Brent and Hollomon 1998). These variants arise by continuous random
changes or mutations that occur within a species. Some of these changes may be
lethal or render the individual less fit while others have no apparent effect. The
naturally occurring variations are a mechanism for survival of a species in an ever
changing environment that applies selection pressures (for example, the use of
fungicides) on sub-populations of the species. Once the size of a less sensitive,
competitive sub-population reaches a threshold, treatments with the fungicide will
no longer be effective. The relative frequency of these less sensitive isolates within
the population is the resistance frequency, whereas the resistance factor describes
the increase in fungicide sensitivity as compared to the baseline sensitivity
(Beresford 1994). The extent of potential fungicide resistance development within
a population is influenced by intrinsic properties of the fungicide and the pathogen
in a given environment. In addition, a range of packinghouse practices that affect
fungicide efficacy also affects the risk of resistance development.
Because all of the new postharvest fungicides are single-site mode of action
compounds that affect a single metabolic process, the selection of less sensitive
or resistant individuals is more likely than for multi-site mode of action com-
pounds. In our studies with P. digitatum on citrus we directly demonstrated that
single-site mode of action fungicides may differ in their resistance frequency and,
based on laboratory and packinghouse selection studies, were determined to
range from 1 × 10−4 to 7 × 10−6 and from 1 × 10−6 to 1 × 10−9 for fludioxonil and
pyrimethanil, respectively. Because resistance frequencies are generally very low,
resistant individuals will more likely be selected for if the pathogen population is
large. Among postharvest decay pathogens, this is certainly true for species of
Penicillium that have a high reproductive potential due to abundant spore produc-
tion as well as a short generation time and that developed resistance to the ‘older’
postharvest fungicides registered on citrus (i.e., imazalil and thiabendazole) and
pome fruit (i.e., thiabendazole). Still, most of the other major decay fungi also
have large population sizes.
8.4 Fungicide Usage Strategies for Preventing Fungicide

Resistance in the Postharvest Environment
The goal in using postharvest fungicides is to minimize the incidence of decay (sur-
vivorship) and to limit the reproduction of any surviving pathogen individuals that
cause fruit decay (anti-sporulation properties). Commodities that are being stored
for extended periods of time before marketing and that commonly develop decay in
storage, benefit from the use of fungicides that suppress sporulation on decaying
fruit. For example, on citrus and pome fruit, fludioxonil is more effective in inhibit-
ing sporulation of Penicillium species than is pyrimethanil (Adaskaveg et al. 2004,
Kanetis et al. 2008a). Fludioxonil, however, has a reduced post-infection or reach-
back activity (inhibition of infections that occur at and after harvest) as compared to
pyrimethanil and azoxystrobin due to its contact properties that limit penetration into
the fruit tissue. Because of often large harvest volumes and long transportation time
from orchard to packinghouse, citrus fruit may not be processed and treated with a
fungicide in a packinghouse until 24–36 h after harvest. Thus, postharvest fungicides
with post-infection activity are needed for fruit that arrive from the field. With excel-
lent reach-back but reduced anti-sporulation activity, pyrimethanil applications
should be combined with other fungicides that provide sporulation control. A flu-
dioxonil-azoxystrobin pre-mixture (Graduate A+®) for use on citrus fruit combines
the anti-sporulation activity of fludioxonil with the post-infection activity of azox-
ystrobin and thus, will provide maximum decay control while reducing spore inocu-
lum in citrus storage facilities, similar to imazalil before widespread resistance
developed against this latter compound. If fludioxonil is used alone, applications
during packing of lemon fruit allow for immediate protection of fruit handling inju-
ries as fruit are removed from storage and processed for marketing. Post-infection
activity is not required at this stage because of the short time interval between
removal of fruit from storage and fungicide treatment.
Packinghouse practices that increase the likelihood of resistance development
include all methods that lead to sub-optimal fungicide coverage (i.e., uneven distri-
bution over the fruit surface) and residue concentrations at infection sites (e.g.,
mostly fruit injuries). These practices include improper application methods due to
non-calibrated equipment or due to cost-saving or using improper fungicides-fruit
coating mixtures. The Fungicide Resistance Action Committee (FRAC) considers
that reducing the treatment rate can enhance the development of resistance and that
recommended rates must be maintained (Brent 1995). Based on the use of single-
or multiple-site mode of action fungicides and on the ratio between resistant and
surviving wild-type sensitive isolates in the pathogen population, contrary beliefs,
however, exist about whether reduced-dose treatments will increase or decrease the
selection of less-sensitive sub-populations (Brent 1995). The thought is that when
one single-site mode of action compound is used and disease is managed, but not
to zero levels, that the surviving sensitive wild-type population will eventually
replace the resistant sub-population. Experimental data are very difficult to gener-
ate to prove this hypothesis. Predictive models are usually based on the assumption
that the same fungicide is being used repeatedly. Because cross-resistance between
different fungicide classes is rare and several effective fungicide classes are now
available for many fruit crops, fungicides can be rotated or mixed effectively to
obtain a high degree of decay control while minimizing the risk for resistance
development.
The registration of multiple new fungicides allows stipulation of the philosophy
that any fruit lot should only be treated once with a fungicide of the same class or
Imidazole Anilinopyrimidine Philabuster®

Imazalil
+ pyrimethanil
= citrus - registered
Phenylpyrrole QoI Graduate A+®

Fludioxonil
+ Azoxystrobin = citrus - registered
SBI
Azoxy- Citrus –
Fludioxonil + strobin
Propicon- = in development
azole
Phenylpyrrole SBI Pome fruit -

Fludioxonil
+ Difenoconazole = in early development
Fig. 8.1 Registered and planned pre-mixtures of postharvest fungicides in the United States as a
strategy for increasing spectrum of activity and reducing the potential of resistance development
in target pathogen populations
mode of action. Ideally, rotations of mixtures should be used for fruit crops that are
being treated more than once, such as some citrus and pome fruits. New and
planned postharvest fungicide pre-mixture registrations accommodate this strategy.
Currently two pre-mixtures, i.e., imazalil-pyrimethanil (Philabuster®) and Graduate
A+® are fully registered on citrus in the United States, however, several additional
ones are in development (Fig. 8.1). Thus, for citrus a triple pre-mixture between
azoxystrobin, fludioxonil, and propiconazole is in preparation. In this triple pre-
mixture, all three components are effective against Penicillium decays, whereas
propiconazole is also active against sour rot caused by G. citri-aurantii. For pome
fruit, a pre-mixture of fludioxonil and difenoconazole is in development, with both
components being very active against Penicillium decays, fludioxonil also effective
against gray mold, and difenoconazole also effective against bull’s eye rot caused
by several species of Neofabrea. In mixture applications, the resistance potential is
much reduced as compared to applications with single active ingredients because
of a lower resistance frequency. For example, assuming resistance frequencies for
fungicides A and B of 10−6 and 10−9, respectively, the resistance frequency of the
mixture will be 10−15. Although these numbers are extremely low, the risk for resis-
tance development will never be zero, and the full spectrum of integrated strategies
should be employed.
Maintaining a high efficacy of postharvest treatments is essential to minimize
the incidence of decay and the number of surviving pathogen propagules.
Application equipment has to be routinely monitored for optimal performance.
Low- and ultra-low-volume in-line fungicide spray applications to wet, washed
fruit have been the standard treatment method of fruit industries for many
years because run-off is limited and, consequently, few disposal problems arise.
New application technologies are being developed for some crops where
Table 8.3 Comparison of postharvest application methods in an experimental packingline study

on inoculated Bartlett pear fruit
Incidence of
decay gray Incidence of decay blue
Treatment and rate Application method molda molda
Control – 100 a 100 a
Fludioxonil 225 mg/L Drench 4.2 d 5.6 c
Dip 23.6 bc 11.1 c
Low-volume spray 38.9 b 36.1 b
Pyrimethanil 500 mg/L Drench 5.6 d 7c
Dip 15.3 cd 5.6 c
Low-volume spray 13.9 cd 34.7 b
Fruit were wound-inoculated with conidia of Botrytis cinerea (gray mold) or Penicillium
expansum (blue mold), incubated for 13–16 h at 20°C, and treated using an in-line drencher or a
low-volume spray applicator at 20 gal/200,000 lb (or 75 L/91,000 Kg), or by a 30-s dip. All treat-
ments were applied as aqueous solutions. Fruit were then incubated at 20°C for 6 days.
a
For each decay, values followed by the same letter are not significantly different based on an
analysis of variance and least significant mean separation procedures.
efficacious fungicide residues are not easily obtained, such as for nectarines,
plums, or some pome fruit that have a smooth, waxy epicarp. We evaluated the
use of postharvest in-line re-circulating drench applications on several crops
(Förster et al. 2007; Kanetis et al. 2008b). As shown for a trial with Bartlett
pear in Table 8.3, treatment efficacies obtained in drench applications were
generally significantly higher as compared to low-volume spray applications
or dip treatments. This treatment strategy has been established for the use on citrus
fruit in California but is also starting to be more widely used on other crops.
8.5 Sanitation and Fungicide Resistance Management
Appropriate sanitation practices are another key aspect of resistance management.

The goal is to keep postharvest pathogen populations that are exposed to fungicide
selection pressure at a minimum. Sanitation includes the sorting of fruit to remove
any decay and the proper disposal of treated decayed fruit during packinghouse
handling. Thus, treated fruit should never be disposed of in the orchard to prevent
the introduction of fungicide-exposed pathogen populations into the field.
Sanitation also involves removal of fungal inoculum from air or water by filtration,
as well as inactivation of inoculum on fruit, equipment, and in water handling sys-
tems using heat or chemical sanitizing agents. Chemical sanitation treatments
inactivate microbial propagules brought into solution from fruit and equipment
surfaces and thus, reduce the amount of pathogen inoculum that subsequently is
being exposed to the fungicide. Thus, based on the concept of resistance frequency
Table 8.4 Compatibility of citrus postharvest fungicides with sanitizers

Sodium
Fungicide hypochlorite Peroxyacetic acid Sodium bicarbonate
Azoxystrobin + + −
Fludioxonil + + +/−
Imazalil − + +
Pyrimethanil − + +
Thiabendazole + + +
Ratings: − = incompatible or negative effects on fruit (phytotoxicity) or fungicide efficacy
+ = compatible or not phytotoxicity on fruit and/or decrease in fungicide efficacy. Rates of sodium
hypochlorite, peroxyacetic acid, and sodium bicarbonate were 100 mg/L (HOCl), 2700 mg/L
(H2O2), and 30,000 mg/L or 3%, respectively.
discussed above, the probability for selection of resistance is reduced when only a
small population is exposed to a selection pressure (i.e., use of a fungicide).
In addition to sanitation of fruit and equipment, re-circulating fungicide solu-
tions for fruit treatment also have to be sanitized (Kanetis et al. 2008b). Although
only washed and sanitized fruit are being treated, spore inoculum will eventually
build up in solutions during prolonged usage. Depending on the agricultural com-
modity, associated usage patterns, and costs, fungicide solutions can be filtered to
remove inoculum, heated to kill temperature-sensitive inoculum, or chemically
treated, most commonly with oxidizing agents. As indicated in Table 8.4, sanitizing
agents including sodium hypochlorite (chlorine), peroxyacetic acid, or sodium
bicarbonate are not all compatible with all postharvest fungicides. Thus, sanitizing
treatments have to be selected depending on the fungicide used.
8.6 Epilogue
The development of highly effective and regulated postharvest fungicides that can
be quantified and tested and that can be re-evaluated over time has been a sound
approach in the prevention of postharvest crop losses by fungal decay. The registra-
tion of active compounds with different modes of action in the pathogen has fol-
lowed a trend to extremely low use rates. The increased arsenal of fungicides
provides an increased spectrum of activity, allows for registration on additional
commodities with less dependency on any single active compound, and facilitates
global marketing. Usage patterns have to be optimized for each crop to obtain
maximum decay control. The near simultaneous registration of several compounds
for a single crop allows for new postharvest usage strategies to minimize selection
of resistant pathogen sub-populations. This scenario is distinctly different from
historical events where postharvest fungicides for agricultural commodities were
introduced sequentially after resistance to the previously registered fungicide had
already developed. The registration of fungicide pre-mixtures especially for crops
where resistance to the ‘older’ registered compounds has developed is a built-in

resistance management strategy. Integration with other practices such as sanitation,
and the usage philosophy that any fruit lot should only be treated once with a fun-
gicide of the same class should minimize any risk losing the new treatments due to
the development of resistance. In addition to these pre-registration and usage strate-
gies of postharvest fungicides, among other pivotal components in resistance man-
agement is the routine monitoring for fungicide sensitivity in the pathogen
population (Adaskaveg et al. 2004). The goal is to detect any shifts in sensitivity as
compared to baseline values at an early stage before field (practical) resistance and
lack of fungicide efficacy occurs and when corrective measures can still alleviate
the spread of resistance. Fungicide manufacturers and packinghouse managers real-
ize the need for judicial usage patterns because the identification and registration of
new compounds that qualify for the same the high standards in safety and perfor-
mance is difficult due to the paucity of appropriate materials and the regulatory
safeguards. Thus, every effort should be made to extend the usage time of any reg-
istered postharvest fungicide.
References
Adaskaveg JE, Förster H, Sommer NF (2002) Principles of postharvest pathology and manage-
ment of decays of edible horticultural crops. In: Kader A (ed) Postharvest technology of hor-
ticultural crops, 4th edn. UC DANR Publ. 3311. Oakland, CA, pp 163–195
Adaskaveg JE, Kanetis L, Soto-Estrada A, Förster H (2004) A new era of postharvest decay con-
trol in citrus with the simultaneous introduction of three new “reduced-risk” fungicides. Proc
Int Soc Citriculture Vol. III: 999–1004
Adaskaveg JE, Förster H, Gubler WD, Teviotdale BL, Thompson DF (2005) Reduced-risk fungi-
cides help manage brown rot and other fungal disease of stone fruit. Calif Agric 59:109–114
Beresford R (1994) Understanding fungicide resistance. Orchardist 67:24
Brent KJ (1995) Fungicide resistance in crop pathogens: how can it be managed? FRAC
Monograph No. 1. GIFAP, Brussels
Brent KJ, Hollomon DW (1998) Fungicide resistance: the assessment of risk. FRAC Monograph
No. 2. GIFAP, Brussels
Eckert JW, Ogawa JM (1988) The chemical control of postharvest diseases: deciduous fruits, ber-
ries, vegetables and root/tuber crops. Ann Rev Phytopathol 26:433–469
EPA (2003) Reducing pesticide risk. www.epa.gov/pesticides/controlling/reducedrisk
Förster H, Driever GF, Thompson DC, Adaskaveg JE (2007) Postharvest decay management for
stone fruit crops in California using the “reduced-risk” fungicides fludioxonil and fenhexamid.
Plant Dis 91:209–215
Kanetis L, Förster H, Adaskaveg JE (2008a) Comparative efficacy of the new postharvest fungi-
cides azoxystrobin, fludioxonil, and pyrimethanil for managing citrus green mold. Plant Dis
91:1502–1511
Kanetis L, Förster H, Adaskaveg JE (2008b) Optimizing efficacy of new postharvest fungicides
and evaluation of sanitizing agents for managing citrus green mold. Plant Dis 92:261–269
Chapter 9
New Approaches for Postharvest Disease
Control in Europe
M. Mari, F. Neri, and P. Bertolini
Abstract Alternative methods to fungicide treatments have been studied in order

to prevent fruit losses in the postharvest phase. Within these methods the appli-
cations of: (a) biological control agents (BCAs), (b) plant bioactive compounds,
and (c) physico-chemical methods showed interesting results but still far from a
practical application in Europe. Actually, despite the substantial progress obtained
with BCAs, any biofungicide has been registered in Europe to control postharvest
pathogens, moreover because of their insufficient and inconsistent performance.
The use of plant bioactive compounds has shown that the treatment conditions
(concentration, form of application, formulation, exposure time, time of treatment,
etc.) can deeply influence their efficacy. The different responses found in many
studies indicate a cultivar specificity in the product-pathogen-volatile interaction.
A barrier to use the plant bioactive compounds may not be efficacy, but rather
the off-odours caused in fruits and vegetables and/or the phytotoxicity. Physico-
chemical methods include heat, ionising and ultraviolet C irradiation, food addi-
tives inducers of resistance. Heat treatments by hot water dips, hot dry air, vapour
heat or very short water rinse and brushing appear promising. To overcome the
drawbacks that have arisen with the these methods, the integration of the antagonist
with other treatments such as low toxic substances (GRAS), heat, etc. has been
proposed; this strategy could produce an additive or synergistic effect on disease
control and obtain satisfactory levels of disease reduction.
Keywords Botrytis cinerea • fruit • Monilinia spp. • Penicillium spp. • Phlyctema

vagabunda
M. Mari (*), F. Neri, and P. Bertolini

CRIOF – DIPROVAL, University of Bologna, Via Gandolfi, 19, 40057, Cadriano, Bologna, Italy
e-mail: marta.mari@unibo.it
120 M. Mari et al.
9.1 Introduction
Fruit production for human consumption requires time and money and is not only
a biological process but also an important part of the market economy. Any waste
due to spoilage and pest infestation, in the field and during the postharvest phase,
represents economic losses which are greater the closer they are to the sale of the
fruit. Fruit perishes quickly, and if care is not taken in its harvesting, handling and
transport, it will soon decay and become unfit for human consumption (FAO 1989).
The extent of postharvest losses varies in relation to commodities and country;
although few up-to-date data are available (Amorim et al. 2008), they can be esti-
mated as ranging between 4–8% in countries where refrigeration facilities are well
developed to 50% where these facilities are minimal (Eckert and Ogawa 1985).
Microbial decay is one of the main factors that determine losses, also compromis-
ing the quality of the fresh produce. In the past the use of new fungicides and
modern storage technologies such as controlled atmosphere, ultra low oxygen
atmosphere, dynamic atmosphere, etc. have extended the shelf-life of fresh fruit,
reducing postharvest losses. However, in the last 20 years, the concern about pub-
lic health and the environment has considerably limited the use of fungicides after
harvest and consumer demand for fungicide-free fruit has also led the multiple
retailers to pursue a policy of eliminating residues, following a new ‘zero residue’
integrated pest and disease management programme implemented by their pro-
duces (Cross and Berrie 2008). The interest in finding alternative approaches to
control postharvest disease fitting well with the concept of safe food for human
health has thus greatly increased. There are three main research fields (biological
control with microbial antagonists, natural bioactive compounds, physico-chemical
methods) characterized by a great number of studies and widely reviewed
(Janisiewicz and Korsten 2002; Spadaro and Gullino 2004; Mari et al. 2007a).
9.2 Biological Control with Microbial Antagonists
The first studies on biocontrol of postharvest diseases appeared over 20 years ago and
since then important progress has been achieved. These investigations have produced
commercial products: Aspire™ (Ecogen Inc., Langhore, PA) based on the yeast
Candida oleophila; BioSave™ 100 and 110 (JET Harvest Solution, Longwood, FL)
based on a strain of Pseudomonas syringae; YeldPlus™ (Anchor Yeast, Cape Town)
based on Cryptococcus albidus; Shemer™ (AgroGreen, Asgdod) based on
Metschnikovia fructicola. These biofungicides have been registered for postharvest
use in the United States (Aspire™, BioSave™), in South Africa (YeldPlus™), in Israel
(Shemer™) but not in Europe. CANDIFRUIT™ (SIPCAM INAGRA, S.A. Valencia)
based on Candida sake is commercially available only in Spain from the 2008 season
for pome fruits against postharvest pathogens. Despite these efforts biological control
agents (BCAs) are still not routinely applied because of their insufficient and incon-
sistent performance, the difficulty in obtaining an adequate formulation and the dif-
9 New Approaches for Postharvest Disease Control in Europe 121
ficulty in controlling previously established infections. The registration process for a

biofungicide in Europe is more difficult than elsewhere. In fact to register a BCA, the
EPA (Environmental Protection Agency), which is in charge of biocontrol agent regu-
lation in the USA, needs an average of 2 years while in Europe the registration of the
same products takes almost 7 years. To accelerate the registration process, the European
Community has supported a policy action, called REBECA (www.rebeca-net.de),
that reviews the possible risks of biocontrol agents, compares regulations in the EU
and the USA and proposes alternative less bureaucratic and more efficient regulation
procedures maintaining the same level of safety for human health and the environ-
ment but accelerating market access and lowering registration costs.
9.3 Natural Bioactive Compounds
Plants possess a diverse array of secondary metabolites which function as fungal

inhibitors. The aroma component of horticultural products or the essential oils of
spices and herbs commonly used in human diet appear interesting to control decay,
because of their safety at low concentrations. The high volatility and low solubility
in water of these compounds make them particularly suitable for application in their
vapour phase by a new process defined as ‘biofumigation’. Recently, packing systems
that deliver volatile antimicrobials to the packaged product, known as ‘antimicrobial
active packaging’, have been evaluated (Ayala-Zavala et al. 2008). Absorption of volatile
compounds in solid substances (starch, alumina, etc.) or their micro-encapsulation
on the internal cavity of b-cyclodestrine could be interesting for the stabilization and
controlled release of the antifungal compounds in active packaging. However,
selected film types need to be used to prevent both loss of volatile compounds from
permeation and accumulation of excessive concentration of carbon dioxide and fer-
mentative metabolites causing off-flavours in products. The latter aspect is particu-
larly important for fruits and vegetables with a high respiration rate especially, when
kept at ambient temperature. Several bioactive compounds inhibited the growth of
fruit and vegetable postharvest pathogens (Plotto et al. 2003; Tsao and Zhou 2000;
Neri et al. 2006a,b,c, 2007, 2009; Mari et al. 2002a, 2008). The most consistent
fungicidal activity was found with vapour of some isothyocyanates (ITCs) such as
allyl-ITC and benzyl-ITC, followed by trans-2-hexenal, carvacrol, citral and trans-
cinnamaldehyde. Nevertheless, the fungal inhibition by these compounds was not
always confirmed in vivo, showing that the treatment conditions (concentrations,
temperature, form of application, exposure time, time of application in relation to the
establishment of conidia in wounds, etc.) can considerably affect fruit and vegetable
response to treatment (Fan et al. 2006; Spotts et al. 2007; Sholberg and Randall
2007). Plant volatile compounds generally have an intense and specific odour, they
can be absorbed and metabolized by the commodities, often altering their sensory
properties; moreover, especially when used at high concentrations, they can also
be toxic for plant tissue (Mari et al. 2008; Neri et al. 2008). In spite of their
consistent efficacy in culture, a barrier to the use of plant bioactive compounds
122 M. Mari et al.
as non-conventional methods for disease control may be the development of

off-odours and/or phytotoxicity in treated products (specie and cultivar). There are still
very few studies on the effect of treatment on sensory quality, and more investiga-
tion is required to avoid detrimental effects of plant bioactive compounds on texture
and flavour of fruits and vegetables. In addition, allyl-ITC and other ITCs are avail-
able as synthetic compounds but can be produced from Brassica defatted meal with
similar results in their efficacy against pathogens (Mari et al. 2008); this is an impor-
tant aspect showing that biofumigation could be used for industrial applications. The
use of bio-based chemicals obtained from renewable natural resources fits well with
the goals of the European Technology Platform “Plants for the Future’’, and the
beneficial effect of the use of Brassica plants in pharmacology has also been docu-
mented (Mennicke et al. 1998; Stoner et al. 1999).
9.4 Physico-Chemical Methods
The use of heat, ionising and ultraviolet C (UV-C) irradiation has recently been pro-
posed to control postharvest diseases. Physical stress can have a dual effect: disinfec-
tion of fruit skin and induction of resistance against future infections. The concept of
induced resistance in plants to pathogens is not new (Chester 1933) but has been
ignored for a long time. However, the induced/acquired resistance has recently been
considered a preferred strategy for achieving integrated pest management (Kuc
2000). A pre- or postharvest treatment with chemical, physical or biological elicitors
may reduce or suppress postharvest diseases. Salicylic acid (SA), methyl jasmonate,
acibenzolar, chitosan, and phosphonate are some of the chemical elicitors tested and
reviewed by Terry and Joyce (2004). Their activity has sometimes proved inconsis-
tent (Yu et al. 2007), with only a fungistatic effect (El-Ghaouth et al. 1992) and
related to treatment timing and the plant development stage (Huang et al. 2000). In
addition, SA can be incompatible with integrated pest management since phytotoxic
effects on leaf margin (Reglinski et al. 1997) or on fruit skin (Mari data not published)
were observed. Heat treatments by hot water dips, hot dry air, vapour heat or very
short water rinse and brushing have been used with success against numerous post-
harvest pathogens and reviewed by Lurie (1998) and Fallik (2004); the authors
pointed out the beneficial effects of a pre-storage heat treatment: (a) easy to use; (b)
kills the pathogens on the surface of fruits: (c) economical (d) environmentally safe.
However, the physiological responses can be different with respect to cultivar, season
and growing location, and a thorough evaluation of temperature and dipping time is
therefore necessary. Heat may also inhibit pathogens infecting fruit prior to harvest
and reduce rot development on organic produce, maintaining fruit quality.
Common food additives generally recognised as safe (GRAS) include salts such
as sodium carbonate, sodium bicarbonate, potassium sorbate, sodium propionate,
etc., allowed with no restrictions for many applications in European and North
America regulations. They have been tested in numerous trials and appear to be
interesting tools to manage postharvest decay because in addition to their consistent
antimicrobial activity, they are inexpensive, readily available, and suitable for the
postharvest handling practices that use water to float fruit out of field bins and to
remove field heat from fruit by hydrocooling. Although these salts appear fungi-
static, not very persistent and with minimal risk of injury to the fruit overall when
used as a heated solution, the combination of GRAS and BCA is better, being more
effective than either treatment alone as shown in numerous trials (Teixido et al.
2001; Conway et al. 2004; Karabulut et al. 2005).
9.5 Main Postharvest Diseases in Europe
The main fungal pathogens that cause important postharvest losses on fruits in
European growing areas are: Penicillium expansum and Phlyctema vagabunda on
pome fruits, Monilinia sp. on stone fruits, P. digitatum and P. italicum on citrus
fruits, Botrytis cinerea on table grape, strawberries and kiwifruits.
9.5.1 Blue Mould
Blue Mould (P. expansum Link) is one of the main postharvest diseases in pome
fruit (Jones and Aldwinckle 1990). In Europe, the pathogen causes extensive decay
(Amiri et al. 2008), particularly in pears penetrating through wounds and micro-
wounds, that frequently occur during harvesting and handling (Spotts et al. 1998).
Control of blue mould is based on the use of thiabendazole and imazalil, applied as
a drencher or on line-sprayer treatment prior to cold storage. However, in the last
decade the development of resistance to these fungicides has reduced the effective-
ness of such treatments, making them useless (Vinas et al. 1993; Baraldi et al. 2003).
In this situation, the need for new and alternative means to control blue mould is
more and more urgent. The results obtained with BCAs in packinghouse tests on
pome fruits showed a control level of natural infections of P. expansum not com-
mercially acceptable, even less than 50%. In addition, a different level of efficacy
was observed when the same BCA was applied to fruits derived from different
orchards (Torres et al. 2006). This probably depends on fruit quality, inoculum den-
sity, level of fruit susceptibility to infection and the time elapsing between inocula-
tion and treatment (Droby et al. 2003). The formulation of BCA can also considerably
influence the efficacy of the antagonist but also make its application easier and less
expensive (Fravel et al. 1998). Torres et al. (2006) tested seven liquid and one wet-
table powder formulations of C. sake (strain CPA-1) on pome fruit against blue
mould and only one, a liquid formulation, was selected for commercial trials,
because it was cheap to produce and easily suspended in water; however all C. sake
formulations showed the same efficacy as fresh cells. To enhance the antifungal
activity of BCA, the application of yeast mixtures or GRAS (sodium bicarbonate)
and antagonist mixture was tested with a decay reduction of 84–97.4% compared to
124 M. Mari et al.
the P. expansum drench alone (Janisiewicz et al. 2008). The efficacy of a biocontrol
yeast, Cryptococcus laurentii, was increased by the combination with SA (Ting et al.
2007). P. expansum is a necrotrophic pathogen and can start the active pathogenic
process immediately (within the first 12–24 h of incubation), after spores land on the
wounded tissue (Prusky and Lichter 2007); this may explain the inefficacy of SA
treatment in some host-pathogen interactions. But when SA was used in combina-
tion with C. laurentii, the antagonist acted as the primary defence line against the
pathogen, rapidly colonizing the wounds, utilizing available nutrients and inhibiting
the initial attack of fungus, while SA was able to reinforce the decay control by
activation of fruit natural resistance after 48 h of incubation.
Within natural bioactive compounds, AITC and trans-2-exenal showed consis-
tent fungicidal activity against P. expansum (Mari et al. 2002a; Neri et al. 2006a).
In ‘Golden Delicious’ apples, the blue mould control by trans-2-exenal was par-
ticularly interesting, treatment applied 24 after inoculation significantly reduced
decay and patulin content in fruits, without causing negative effects on quality traits
(Neri et al. 2006a). In contrast, treatment with trans-2-exenal concentrations, effec-
tive against blue mould, caused phytotoxic symptoms on ‘Abate Fetel’ pears and
affected fruit flavour in ‘Conference’ and ‘Barlett’ pears and ‘Royal Gala’ apple
(Neri et al. 2006b).
Another important aspect is the accumulation of a toxin: patulin in pome fruit
infected by P. expansum. Morales et al. (2008) found that the use of two BCAs
(C. sake and Pantoea agglomerans) seemed to have a positive effect on decay con-
trol and patulin accumulation after apple cold storage. On the other hand, a yeast,
Rodotorula glutinis (strain LS11), appears to metabolize patulin in vitro and in a
model emulating decaying apple tissue. Further, the BCA is able to decrease accu-
mulation of this toxin in P. expansum-infected apples. If confirmed, these results
could pave the way for the development of new technologies for the prevention and/
or detoxification of patulin contamination in apple-based juices (Castoria et al.
2005). The same effect on patulin accumulation was also recently observed in
‘Golden Delicious’ and ‘Granny Smith’ apples treated with natural biocides, quer-
citin and umbelliferone phenolic compounds (Sanzani et al. 2008).
9.5.2 Lenticel Rot
Lenticel Rot [Neofabrea alba (EJ Gutrie) Verkley, anamorph P. vagabunda Desm.,
syn. Gloeosporium album Ostew] is one of the most frequent and damaging dis-
eases occurring in stored apples (more rarely in pears) in Italy, France and other
European apple growing countries (Pratella 2000; Amiri et al. 2008). Fruit infection
occurs in the orchard, but disease symptoms appear only several months after har-
vest. ‘Pinova’, ‘Topaz’ and several late maturing cvs of apples such as ‘Gold Rush’
and ‘Pink Lady’ are particularly susceptible to the disease, with an incidence that
can exceed 15–30% after 120 days of cold storage, particularly in organically
grown fruit (Bompeix and Cholodowski-Faivre 1998; Mari et al. 2002b; Maxin
et al. 2005; Weibel et al. 2005). Current measures to control N. alba infection
include pre- and postharvest treatments with synthetic fungicides. In Italy, the use
of thiabendazole fungicide is allowed in postharvest treatments only for apples and
pears stored longer than 31 December. Among non-chemical means to control
lenticel rot in apples, hot water treatment has shown good efficacy, and seems par-
ticularly interesting for organic production (Maxin et al. 2005; Neri et al. 2008b).
Dips in water at 45°C for 10 min led to a consistent reduction of infection both on
artificially inoculated cv ‘Golden Delicious’ (80% of efficacy after 90 days of storage)
and naturally infected cv ‘Pink Lady’ (90% of efficacy after 135 days of stor-
age), without causing any damage to the fruit (Neri et al. 2008b). The efficacy of
the treatment is likely due to effects on the pathogen; however, effects on fruit
(induced resistance) could also be involved. Shorter treatments (10–30 s) would be
optimal to accelerate fruit handling in packing houses and would be better for com-
mercial application than 45°C for 10 min. However, a temperature of at least 50°C
is needed to significantly reduce N. alba mycelial growth for short exposure treat-
ment (1 min) (Neri et al. 2008b), and apples dipped at 50–55°C for 30–180 s
showed skin damage, with sensitivity varying among cultivars (Maxin et al. 2005;
Burchill 1964; Spalding et al. 1969).
Fumigation with plant volatile compounds (carvacrol, trans-2-hexenal, trans-
cinnamaldehyde and citral) showed consistent efficacy only in vitro (Neri et al.
2008b). The failure of these volatiles in lenticel rot control on apples may be due
to their insufficient vapour pressure, making the volatiles unable to penetrate
through fruit lenticels and reach the pathogen site. Other studies on lenticel rot
control reported the inefficacy of several natural compounds applied in dipping
treatment at concentrations much higher than those used in this study and a signifi-
cant disease control (64–84%) was only found with 2,000 ppm L-carvone or
eugenol treatment in hot water (Bompeix and Cholodowski-Faivre 1998). However,
similar or better control of the decay was found with a single hot water treatment
(Bompeix and Coureau 2008).
9.5.3 Brown Rot
Brown rot (Monilinia sp.) is one of the main diseases in stone fruit occurring in the
field during both pre- and postharvest. In Europe there are substantially two causal
agents of brown rot, Monilinia laxa (Aderhold and Ruhland) and M. fructigena
(Aderhold and Ruhland); a third M. fructicola (G. Winter) has recently been
detected in Spain and France, but it is not yet widespread in Europe. Losses depend
on weather conditions and are especially severe if high humidity, warm tempera-
tures and abundant rainfall prevail prior to harvest (Bonaterra et al. 2003). In some
cases, infections occurring in the field can remain quiescent until fruit ripens, pro-
voking important losses in the postharvest phase, estimated between 5% and 10%
or more (Margosan et al. 1997). In European countries, this plant pathogen is con-
trolled by fungicide spray programs only in the field, since postharvest treatment
126 M. Mari et al.
is not allowed. In the last two decades numerous studies have indicated the efficacy
of BCAs against M. laxa on peach and nectarine; most of these studies refer to
yeasts (Karabulut and Baykal 2003), bacteria (Bonaterra et al. 2003) and fungi
(Mari et al. 2007b) alone or integrated with food additives (Qin et al. 2006) or
physical methods (Karabulut and Baykal 2002). Pre-harvest treatments can be
fundamental as a consequence of disease epidemiology and the importance of
controlling latent infections (Larena et al. 2005). For this reason, a scheduled
programme of brown rot management could consider a turnover of active ingredients,
including BCA in an integrated control strategy. However, applied in the posthar-
vest, BCA does not appear able to control previously established infections (latent
or quiescent) that often develop within 24–48 h after harvest and their eradication
could be possible with postharvest physico-chemical treatments such as hot water
(Margosan et al. 1997) or potassium sorbate (Gregori et al. 2008).
Fumigants are good candidates for postharvest brown rot control since their use
entails minimal handling of the food product and absence of the fruit wetting
involved in vapour treatments. Several studies on antifungal activity of plant aroma
compounds against M. laxa and M. fructicola have been carried out in vitro and
in vivo trials. Trans-2-hexenal (Tsao and Zhou 2000; Neri et al. 2007), hexanal
(Song et al. 2007), acetic acid (Liu et al. 2002), and isothiocyante (Mari et al. 2008)
treatments obtained satisfactory control of the pathogen. However, to achieve opti-
mal effects, it is important to establish an effective volatile concentration and expo-
sure duration combination. Compared with previous studies, stone fruit were found
to be more sensitive to trans-2-hexenal injury than were pome fruit; a differential
sensitivity was also observed among stone fruit species, increasing from plum to
nectarine and peach, to apricot (Neri et al. 2007). Others bioactive molecules
derived from the development of an endophytic fungus Muscodor albus applied on
peaches in bugged cartons had an interesting effect; the biofumigation treatment
reduced brown rot by 72% with respect to untreated fruits (Schnabel and Mercier
2006). The fungus, growing on previously colonized desiccated rye grain, produces
at least 28 volatile compounds and this mixture can be more effective than other
fumigant agents, which are used as single compounds.
9.5.4 Green and Blue Moulds
Green and blue moulds (P. digitatum Pers.: Fr. Sacc. and P. italicum Wehmer respec-
tively) are the most important disease of citrus fruit in growing areas characterised
by low summer rains (Eckert and Eaks 1989). Both pathogens grow at the optimal
temperature of 24°C and although green mould is predominant at room temperature,
blue mould is more important on cold-stored citrus fruit, since P. italicum grows faster
than P. digitatum below 10°C (Brown and Eckert 2000). The fungi are wound para-
sites and can infect fruit in the grove, the packinghouse and during distribution and
marketing. The fungal inoculum is practically always present on the surface of fruit
during the season and after harvest can build up high levels unless appropriate pack-
inghouse sanitisation measures are adopted (Kanetis et al. 2007). Application of
synthetic fungicides is generally the main method to control green and blue moulds
where the frequency of fungicide-resistant isolates is low; however, the intense use
of fungicides such as sodium o-phenylphenate, thiabendazole, and imazalil (the only
fungicides registered for use on citrus fruits in the European community) has led to
the proliferation of fungicide-resistant isolates (D’Aquino et al. 2006). The alterna-
tives to conventional fungicides for the control of green and blue moulds in citrus
fruit have been widely reviewed in a recent article by Palou et al. (2008). According
to their nature, these alternatives can be physical, chemical or biological. As far as
physical control methods are concerned, heat (Plaza et al. 2003), ultraviolet (UV-C)
(D’hallewin et al. 1999) and ionising (Sommer et al. 1964) radiation and ozoned
atmosphere storage (Palou et al. 2001) controlled P. digitatum and italicum; never-
theless, each of them showed drawbacks and disadvantages. Typical heat treatment
by curing considers the exposure of fruit for 2–3 days to an air atmosphere heated to
temperatures higher than 30°C and high relative humidity. Although this procedure
showed satisfactory control of green mould, blue mould was less controlled when
fruit were cold-stored for long periods after treatment (Plaza et al. 2003). In addition,
fruit weight loss and heat phytotoxicity can be potential risks for heat-treated fruit
with respect to cultivars, season, growing location and initial conditions while fruit
quality traits may not be affected by heat treatment (Porat et al. 2000). The exposure
to low doses (0.5–8 kJ/m2) of UV-C or ionising radiation has significantly reduced
the incidence of green and blue moulds (D’hallewin et al. 1999) and on-line UV-C
equipment for fruit treatment was developed (Wilson et al. 1997). Despite this,
numerous issues have to be addressed before a practical use of this system: the entire
area of the fruit has to be rapidly treated and the system should be sufficiently flex-
ible in relation to fruit attributes and retail destination (Palou et al. 2008). Ionising
radiation is associated with some beneficial effects on fruit, such as the stimulation
of synthesis of antifungal compounds that extend shelf-life by delaying ripening and
senescence, although the doses required exceeding 1,000 Gy (100 krad) induce
apparent rind injury (Barkai-Golan 1992). Ozone (O3) is a potent biocide and has
recently been approved for many food contact applications, but at non-phytotoxic
concentrations (0.3–1 mL/L) it is not able to control P. digitatum and P. italicum,
obtaining only a fungistatic effect; however, inhibiting aerial mycelium growth and
sporulation can reduce the proliferation of fungicides-resistant strains of these
pathogens (Ariza et al. 2002).
Biological control using microbial antagonists is a promising alternative to fun-
gicides to control postharvest disease of citrus fruit. During the last 20 years, strains
of yeast, bacteria and filamentous fungi have been isolated, tested, selected, identi-
fied and characterized as BCA effective against citrus green and blue moulds (Palou
et al. 2008). This considerable research work has led to the production and registra-
tion of three biofungicides for use against postharvest citrus pathogens. Aspire™
(C. oleophila, limited to the USA and Israel), BioSave™ (P. syringae, limited to the
USA) and Shemer™ (Metschnicowia fructicola, limited to Israel) are now avail-
able. Other products, such as Biocure, Bio-Coat (based on a strain of C. saitoana),
will soon be available on the marketplace. However, the commercial use of these
128 M. Mari et al.
products is and remains limited and accounts for only a very small fraction of the
potential market. As discussed in several reviews (Droby and Wisniewski 2003;
El-Ghaouth et al. 2004; Janisiewicz and Korsten 2002), the combination of BCA with
other alternative control methods can be a promising approach to overcome some
drawbacks in BCA activity, enhancing their efficacy. The combination of BCAs with
heat (Porat et al. 2002), GRAS (Usall et al. 2001), and UV-C (Stevens et al. 1997)
produced a synergic effect and was superior to all the treatments alone in controlling
green and blue moulds. A sequence of treatments proposed by Usall et al. (2008)
comprises initial treatment with a heated solution of sodium bicarbonate or sodium
carbonate followed by the application of BCA. Such combined treatments can be
easily implemented on a commercial scale in many citrus packinghouses because
they are compatible with existing facilities and postharvest handling practices.
9.5.5 Grey Mould
Grey mould (B. cinerea Pers. Fr.) is an important postharvest disease because envi-
ronmental conditions prevailing during storage facilities are favourable to its devel-
opment. B. cinerea is a widespread pathogen on many crops but is the main
problem in cold storage and subsequent shipment for table grape, strawberry and
kiwifruit. On table grape, B. cinerea is responsible for severe losses in the field and
after harvest and is the main threat to its long-distance transport and storage
(Romanazzi et al. 2007). Control of the disease is especially important in storage
because it develops at low temperatures (−0.5°C) and spreads quickly among ber-
ries, forming ‘nests’ that can spread over the entire bunch. Canopy management,
pre-harvest fungicide applications and postharvest sulphur dioxide fumigation are
the current practices to control grey mould (Droby and Lichter 2004); however,
alternatives to sulphur dioxide are urgently needed because sulfites can be harmful
to allergic people and also because sulphur dioxide has been removed from the
GRAS compound list by the FDA (Anonymous 1986) and is not allowed in
European countries. In an integrated pest management strategy, some physico-
chemical methods have been tested with interesting results. Dipping bunches after
harvest in ethanol has prevented B. cinerea infection partially due to the high sen-
sitivity of conidia to ethanol (Lichter et al. 2003), although ethanol is unsuitable for
latent infection. Others treatments, such as vapour heat (Lydakis and Aked 2003),
hexanal fumigation (Archbold et al. 1997), UV-C (Nigro et al. 1998), resveratrol
(Gonzalez Ureña et al. 2003), and chitosan (Romanazzi et al. 2006) alone or in
combination with ethanol (Romanazzi et al. 2007) were found to significantly
inhibit the development of B. cinerea. Several reports demonstrated the efficacy or
BCAs against prevention of grey mould after harvest (Lima et al. 1997; Karabulut
and Baykal 2003), although no antagonist seems to be efficient enough against
natural infections (Schena et al. 2003).
Strawberry is a highly perishable non-climacteric fruit and has to be harvested
at full maturity to achieve maximum quality (Hernández-Muñoz et al. 2008).
Botrytis rot is one of the most important diseases of strawberry in Europe; infections
initiated in the field during flowering remain quiescent until fruit ripens, causing
heavy economic losses during shipment or storage. The use of synthetic fungicides
in the field is the main method for reducing postharvest diseases and involves two
applications of botrycides during peak flowering periods; however these measures,
when weather conditions are favourable to the pathogen, have limited effects on
postharvest disease control. Biocontrol of grey mould has been attempted for 10
years now and preliminary findings have been obtained (Lima et al. 1997; Karabulut
et al. 2004; Hernández-Muñoz et al. 2008). BCAs are more efficient against infec-
tions that are established during the postharvest stage; since Botrytis rot originates
mainly from latent infections of floral parts, the application of the antagonists at the
flower stage seems the best strategy for effective control of grey mould (Lima et al.
1997). Antagonist treatment under field conditions may reduce pathogen sporulation
on crop debris and consequently flower and fruit infection.
Stem-end rot is the main symptom produced by B. cinerea on kiwifruit; the fun-
gus colonizes fruit sepals and receptacles (Michailides and Elmer 2000) and pene-
trates by picking wound at, or soon after, harvest. The disease appears after 3–4
months of storage at 0°C or sooner if the fruit is kept at higher temperatures. Pre-
harvest fungicide treatments are not always effective and easy to apply; alternative
control methods suggest good orchard hygiene to remove the inoculum of B.
cinerea, summer pruning to increase sunlight and air exposure ( Brigati et al. 2003a)
and careful postharvest handling to avoid fruit injury. A delay between harvest and
cool storage results in a significant decrease of B. cinerea infections during storage.
This method, also called ‘curing’, consists of keeping fruit at ambient temperature
for at least 2 days before packing or cold storage and it has been implemented over
the years. In Italy, ‘curing’ is now carried out directly in cold storage rooms, decreas-
ing the temperature from 10 to 1°C in 8 days and delaying establishment of the
controlled atmosphere regimes to 30–40 days after harvesting. This method reduced
the incidence of stem end rot and at same time any negative effects were observed
on the fruit quality (Brigati et al. 2003b). Few data are reported on the biocontrol of
B. cinerea in kiwifruit; the attempts to use some antagonists such as Bacilus subtilis
and P. syringae (Michailides and Elmer 2000), Aureobasidium pullulans and
C. oleophila (Lima et al. 1997) revealed a variable activity. A study by Kulakiotu
et al. (2004) showed the potential for successful biological control of grey mould by
volatiles of ‘Isabella’ grape. However, the antibiotic action of the ’Isabella’ volatiles
against B. cinerea was tested only at two temperatures (10°C and 21°C) and was
stronger at 21°C than at 10°C.
9.6 Conclusions
The increasing interest in alternative methods to fungicides to control postharvest

diseases has produced numerous studies over the last few decades. The results
obtained show some significant progress in the reduction of pesticide use; however,
130 M. Mari et al.
some critical points have still to be considered. It’s unrealistic to assume that bio-
logical control agents have the same fungicidal activity as pesticides; studies to
improve the formulation, compatibility with current handling and storage practices
and the integration with GRAS, hot or resistance elicitors treatments are in progress
and new registrations of biofungicides are expected. In the future, all of these
methods will be fundamental for organically grown crops that, untreated with
chemical fungicides, suffer due to a relatively high rate of decay in the postharvest
phase. Research should provide appropriate tools (BCAs, natural substances,
GRAS, curing) to tailor a complete integrated disease management strategy spe-
cific for each situation (species, climatic and seasonal conditions, destination
market, etc.).
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116:339–345
Chapter 10
Quo Vadis of Biological Control of Postharvest
Diseases
Wojciech J. Janisiewicz
Abstract Research on Biological Control of Postharvest Diseases (BCPD) has

been conducted for over two decades and successes, present and future direc-
tion are being discussed. The BCPB has been accepted by the fruit and vegetable
industry as the stand alone or in combination with other commercial treatments,
depending on fruit and vegetable. BioSave has been on the market since 1996, and
its use is expanding to control more postharvest diseases of fruits and vegetables.
World-wide efforts in developing BCPD resulted in the registration of more prod-
ucts recently. The number of scientific publications is also increasing steadily. As
postharvest biocontrol products are coming to the market, their anticipated limita-
tions are become apparent, and much of the current research is focused on address-
ing these limitations. Combining antagonists with various substances Generally
Regarded as Safe (GRASS), such as sodium bicarbonate, calcium chloride, diluted
ethanol, or with physical treatments such as heat, or UV irradiation are typical
examples of approaches being used. A mixture of two compatible biocontrol agents
often showed an additive or synergistic effect in controlling fruit decays. Biocontrol
agents developed for the control of fruit decays have also been shown to inhibit
growth of foodborne human pathogens. This aspect gains in importance as new
outbreaks are reported with increasing frequency, and fresh cut fruit and vegetables
are particularly vulnerable to colonization by foodborne pathogens. Biocontrol
agents can also control decays originating from wounds made during mechanical
harvesting of fruits. Currently, available biocontrol products were developed for
the control of decays originating from the infection of fruit wounds, but the next
greatest challenge for BCPD research is the development of the next generation of
biocontrol products that control latent infections. Many important diseases of tem-
perate, subtropical and tropical fruit, including those caused by Monillinia spp. and
Colletotrichum spp.., originate from these infections in the orchard and cause decay
on fruits in storage. This research requires broadening the pool of microorganisms
W.J. Janisiewicz
Agricultural Research Service, U.S. Department of Agriculture, Appalachian Fruit Research
Station, 2217, Wiltshire Road, Kearneysville, WV, 25430, USA
e-mail: wojciech.janisiewicz@ars.usda.gov
138 W.J. Janisiewicz
screened for biological control activity to include, in addition to those occurring

naturally on fruit, microorganisms from different plants and plant parts, as well as
microorganisms from different habitats. Some programs are already focusing in this
direction, and there is great hope and optimism that at the next ISPP Congress in
2013 in Beijing, we will have reports on the significant progress in this area.
Keywords Biocontrol, Biocontrol products, Emerging biocontrol, Postharvest decay
10.1 Historical Perspective
This is the fourth consecutive evening session on Biological Control of Postharvest

Diseases (BCPD) at the ICPP and it is axiomatic that, after more than two decades
of research on BCPD, we should reflect on the direction of the current research and
what we can expect in the foreseeable future. A SCOPUS survey of the last 10 years
of research on BCPD indicates an increasing number of peer-review publications
from 19 in 1998 to 54 in 2008 (a projected number based on 38 publications by the
end of August, 2008). Most of the research has been conducted in Europe (mainly
Belgium, Italy and Spain, and Sweden), United States, Israel, South Africa, and
more recently in China. Many smaller programs developing in other countries also
contribute significantly to progress in this field, and further indicate the growing
interest in BCPD. The expansion of this research began with the report by Pusey
and Wilson (1984) on the successful control of brown rot of peach and other stone
fruits caused by Monilinia fructicola after harvest using soil isolated Bacillus sub-
tilis (strain B3). Postharvest application attempts were made after the field applica-
tion of this bacterium to peach trees from bloom to harvest failed to control this
disease. The authors concluded that controlled environmental conditions in the
postharvest system provide a more stable environment for the antagonist resulting
in a high level of disease control (Wilson and Pusey 1985). Results from pilot tests
on the control of brown rot of peach, conducted in commercial packinghouses,
were encouraging, but this antagonist was never commercialized (Pusey et al.
1988). The discovery of bacterial and yeast antagonists effective against various
postharvest diseases of pome fruits among resident microflora of apple and pear
provided a new source of antagonists. It also made us realize that potential antago-
nists on the surfaces of these fruits have long been consumed by humans without
any apparent adverse effects (Janisiewicz 1987). The next milestone was the
registration by the United States Environmental Protection agency (EPA) and com-
mercialization of the first two biocontrol agents: a yeast Candida oleophila, used in
Aspire™ (Droby et al. 1998), and a saprophytic strain of Pseudomonas syringae,
used in BioSave™ (Janisiewicz and Jeffers 1997) in 1995. In this biotechnology
era, each year seems to be a millennium, and the success of new commercial bio-
control products is often measured by their life span on the market. Three years of
commercial use seems to be the breaking point for many biocontrol products. In
10 Quo Vadis of Biological Control of Postharvest Diseases 139
this context, the last milestone was achieved in 2006, the 10-year anniversary of the
large scale commercial use of BioSave. There have been many important advances
in BCPD during the past decade, especially in the area of mechanisms of biocon-
trol, manipulation of antagonist physiology for the benefit of biocontrol, develop-
ment of formulations, and integration with other control methods. Only time will
indicate the full impact of these advances on the increased use of BCPB.
10.2 Biocontrol Products
Aspire™ and BioSave™ lead the way in commercial application of biocontrol agents
to fruit. Other products such as YieldPlus™ based on Cryptococcus albidus,
Avogreen™ based on Bacillus subtilis, and Shemer™ based on Metschnikowia fruc-
ticola are also on the market in various countries. Aspire™ has been registered in
the United States for postharvest application to citrus and pome fruits. This product
was taken off the market 3 years after its large scale commercial introduction.
BioSave™ (two strains of P. syringae) was originally registered for postharvest
application to pome and citrus fruits, and this was later extended to cherries, pota-
toes, and more recently to sweet potatoes. YieldPlus™ was developed in South
Africa for postharvest application to pome fruits but the success of this product is
largely unknown and there is no published literature or information available to
determine extent of its use. Avogreen™ has been used for control of postharvest
disease of avocado. Its use has been limited (L. Korsten, personal communication),
possibly due to inconsistent results. More recently, Shemer™ was registered in
Israel for both pre- and postharvest application on various fruits and vegetables
including apricot, citrus, grapes, peach, pepper, strawberry and sweet potato. There
are three more products coming to the market: Candifruit™ based on Candida sake,
developed in Spain; Boni-Protect®, based on Aureobasidium pullulans, developed
in Germany and NEXY, based on Candida oleophila, developed in Belgium. All
of these products have been registered for control of postharvest diseases of pome
fruits. These new products are further testimony that increasing interest in BCPD is
not just a matter of scientific curiosity but have resulted in diligent efforts to imple-
ment this approach. Gradual removal of the major regulatory barriers to registration
of antagonists for BCPD in different countries is also very encouraging.
10.3 Postharvest System
BCPD now encompasses the use of antagonists to control postharvest diseases of

fruits, vegetables and grains. Postharvest biocontrol of flower diseases is another
area awaiting exploration. Early attempts to use a metabolite (pyrrolnitrin) of a
bacterial antagonist to control cut rose flower infections were very encouraging
(Hammer et al. 1993) but little else has been done in this field. Biocontrol of grain
spoilage during storage in silos has made significant advances and appears to be on
the way to commercialization (Peterson and Schnurer 1995; Peterson et al. 1999;
Druvefors 2004). Examples from commercially used biocontrol products indicate
that biocontrol agents developed for BCPD on one commodity could also be effec-
tive against the same or different pathogens on other commodities. The activity of
biocontrol agents is not as universal as fungicides but is less specialized than origi-
nally anticipated. Broadening the application of biocontrol agents is a good strategy
for commercial success. It not only makes the product more profitable and allows
for a quicker return on the investment made in the commercialization of the prod-
uct, but it also allows a buffer in the fluctuation in the market due to registration of
new fungicides or other alternative products. As postharvest biocontrol products are
coming to the market, their anticipated limitations are elucidated and much of the
current research is focused on addressing these limitations. The most commonly
used approach is combining antagonists with various substances Generally
Regarded as Safe (GRASS), sodium bicarbonate (SBC), calcium chloride, or etha-
nol (Bertolini 2008; Karabulut et al. 2003). These combinations both reduced the
fluctuation and increased the level of decay control. Combining antagonists with
other alternatives to fungicide treatments also showed good results, but its imple-
mentation often requires modifying currently used postharvest practices or adds
substantially to the cost of the treatment. For example, a heat treatment of apples
with hot air at 38°C for 4 days or oranges at 30°C for 1 day after harvest in combi-
nation with antagonists gave superior control (including eradicative activity) of
blue mold and gray mold, respectively, compared to the individual treatments
(Huang et al. 1995; Leverentz et al. 2000, Leverentz et al. 2003c). However, this
approach will require adding heating equipment and changing the temperature in of
storage rooms. Some packinghouses use high temperatures to sanitize empty bins
and storage rooms, but even this practice is still very limited. It is also well estab-
lished that the proper selection of the combination of two antagonists can provide
superior decay control to either antagonists applied individually (Calvo et al. 2003;
Janisiewicz and Bors 1995). Although using this approach is very attractive, it
doubles the cost of registration because each antagonist must be registered sepa-
rately. This problem could be eliminated if biocontrol products currently on the
market could be combined resulting in additive or synergistic effects.
There is increasing consensus that, in the future, non-fungicidal control will rely
on the combination of various treatments with biocontrol. This combination of treat-
ments is similarly to the hurdle concept developed by Leistner in 1978 for the reduc-
tion of food contamination with foodborne pathogens, where each additional treatment
further reduces the possibility of food contamination (Leistner 1978, 2000).
10.4 Challenges of Latent Infection
All biocontrol agents currently registered for postharvest application control fruit
decays originating from wound infections made during or after harvest (see review
by Janisiewicz and Korsten 2002). Although for some fruits, such as pome or citrus
(depending on the region) fruits, wounds are the main court of entry for postharvest
decay causing fungi, many postharvest decays of stone fruits and subtropical fruits
develop in storage from latent infections occurring in the orchard. These infections
are difficult to control because the intimate relationship of the pathogen with the
host has been already established, and melanized appressoria often formed by these
fungi on fruit surface are very resistant to environmental factors and penetration by
fungicides. Control of latent infection with microbial antagonists is the next big
challenge to BCPD. Earlier attempts to reduce decay originating from latent infec-
tions on mango fruit indicate that microbial antagonists could be effective against
this type of infection (Koomen and Jeffries 1993), however this work did not con-
tinue beyond initial reports, suggesting difficulties in making further progress. New
screening and fruit testing approaches must be developed to address latent infec-
tion. One of the approaches used in our laboratory employs in vitro screening for
biocontrol activity against M. fructicola and Colletotrichum accutatum on crystal-
line cellulose membranes impregnated with waxes isolated from apple, plum or
nectarine fruit. Both fungi produce melanized appressoria on these membranes
when inoculated as an aqueous suspension of the conidia, and can infect fruit from
these appressoria if the membrane is placed on the fruit. Bacteria and yeast can be
applied to the membrane surface with the appressoria, and those showing growth
around appressoria and/or reducing fruit infection from these membranes are
selected for further testing directly on fruit. Our current challenge is to make this
procedure more quantitative. Unlike with wound infecting fungi, where screening
for antagonists was independent of the mechanism of biocontrol, screening against
latent infections could benefit from focusing on organisms exhibiting mechanisms
of biocontrol which could most likely succeed against the melanized structures of
the pathogen. The primary candidates are microorganisms producing volatile
substances and enzymes capable of degrading the melanized structure of the fungi.
Predacious yeasts may also be useful. Antifungal volatiles produced by the antagonist
Muscodor albus, discovered as an endophyte of Cinnamomun zeylanicium in
Honduras, have been shown recently to control major postharvest pathogens on a
variety of fruits (Strobel 2006; Strobel et al. 2001; Mercier and Jiménez 2004;
Mercier and Smilanick 2005). Focusing the search on this mechanism could greatly
enhance opportunities of finding antagonists with volatiles capable of penetrating
melanized structures of the fungi. This example also indicates that microflora of
exotic plants, especially endophytes, is an untappped resource and should be
explored for their biocontrol potential. With the exception of grapes, pome, citrus
and several other fruits, information about resident microflora of fruit and fruit tree
plants is very limited and their potential for BCPD is largely unexplored.
10.5 Biological Control Mechanism
Studies of the ecology and physiology of antagonists were the main driving forces
during initial development of BCPD that lead to commercial products, but research
on the mechanisms of biological control (MBC), that have been lagging behind, has
made significant progress especially during the past decade. This subject is covered
in Chapter 12 by R. Castoria and Sandra A.I. Wright. Here, I would like to point
out a few factors that have contributed to this progress. More focus and resources
have been put into studying mechanisms of BCPD after the first commercial prod-
ucts began to be marketed on a large scale in late 1990s. The limitations to BCPD
have become a reality in commercial settings and the knowledge of the MBC has
been look upon as one of the ways that may address some of these limitations.
Advances in molecular biology have allowed us to address, in a more direct man-
ner, questions about the role of some putative MBC e.g. lytic enzymes or reactive
oxygen species (ROS) (Castoria et al. 2005; Chan et al. 2007; Friel et al. 2007;
Macarisin et al. 2007; Massart and Jijakli 2006; Xu and Tian 2008). In most cases
more than one mechanism of biocontrol has been implicated and the significance
of different mechanisms of biocontrol still needs to be established. Development of
a bacterial and yeast model systems, where genes for different MBC traits can be
expressed, and where the transformants can be tested on fruit for biocontrol poten-
tial, could be very helpful in many cases. A good examples of what could be
accomplished in the fruit system is work with, Saccharomyces cerevisiae and Picha
pastoris, non-antagonistic yeasts which were transformed with genes coding for
antifungal peptides cercopin (defensin from insects) and Psd1 (defensin from pea
seeds), respectively. These transformants greatly reduced postharvest decays on
tomato and pome fruits (Jones and Prusky 2002; Janisiewicz et al. 2008). There are
genes currently available that could be tested in such a system, e.g. genes coding
for antifungal pyrolnitrin, which were isolated from Pseudomonas fluorescent, and
also produced by Pseudomonas cepacia (now Burkholderia cepacia), an excellent
biocontrol agent against various postharvest decays on pome, stone and citrus fruits
(Janisiewicz and Roitman 1988; Hammer et al. 1997). Other good candidates are
genes responsible for the production of various lytic enzymes, ROS or sidero-
phores. Eventually, this approach may lead to improvement of existing antagonists
or the development of new ones (as with cercopin and Psd1 defensins), however for
this to have a practical outcome, the hurdles of safety and public acceptance will
have to be overcome (Janisiewicz 1998).
The discovery of a very effective antagonist producing antifungal volatiles indicates
that a single MBC can be sufficient to provide adequate control of a fruit decay.
This also justifies screening for a single MBC, if an effective mechanism can be
identified, and makes in vitro screening more meaningful and effective because it
allows the screening of vast numbers of organisms in a short period of time before
resorting to more expensive and time consuming tests on fruit.
10.6 Emerging New Areas in Postharvest Biocontrol
During the past decade, more emphasis has been put on the safety of fruits and
vegetables as an increasing number of outbreaks with foodborne human diseases
are reported each year following consumption of various fruit and vegetables. The
situation is worsened by growing consumption of fresh-cut fruits and vegetables,
which provide a conducive environment for the growth of various bacterial human
pathogens. This also creates an opportunity for the use of microbial antagonists to
combat these foodborne pathogens. For example, Pseudomonas syringae used in
Bio-Save can prevent the growth of E. coli in apple wounds (Janisiewicz et al.
1999), and several other biocontrol agents, e.g. Gluconobacter asaii, Discofaerina
fagi, or Metschnikowia pulcherrima, developed for control of postharvest decays,
not only prevented growth but even reduced populations of the foodborne patho-
gens Listeria monocytogenes and Salmonella enterica sv. Poona on fresh cut apples
(Leverentz et al. 2006). The concept and the potential for using various microorgan-
isms against human foodborne pathogens on minimally processed fruits and vege-
tables has been discussed (Leverentz et al. 2003b). Lactic acid bacteria (LAB) such
as Lactobacillus plantarum, Lactococcus lactis, Leuconostoc sp., or Weissella sp.,
occur frequently on fruits and vegetables and many strains of these LAB prevented
growth or even reduced populations of the major foodborne pathogens on apples
and lettuce (Trias et al. 2008). Lytic bacteriophages can be very effective in reducing
populations of foodborne pathogens (Leverentz et al. 2001). They can be combined
with other bacterial or yeast antagonist or the bacteriocin, niacin, to achieve the
targeted level of a 6 log unit reduction in populations of the foodborne pathogens
(Leverentz et al. 2003a, Fig. 10.1). In 2005, the United States Environmental
Protection Agency approved, for the first time, the use of bacteriophages as a food
additive to reduce contamination of foods with foodborne pathogens.
It is not incidental that the control of foodborne pathogens has recently been the
topic of various sessions, including the Plenary Session, at the APS Annual
Meetings, as the growth of these pathogens can be intertwined with the growth of
plant pathogens. Recent studies indicate that a diseased tissue can be more prone to
colonization by foodborne human pathogens than the healthy tissue. For example,
apple tissue infected with Glomerella cingulata, causing bitter rot, becomes less
acidic allowing colonization by L. monocytogenes (Conway et al. 2000).
Labor shortages, especially in the United States and other developed countries,
have increased the demand for mechanical harvesting of fruits and vegetables.
Significant progress has been made during the past two decades and some fruits are
currently harvested mechanically, not only for processing, but also for the fresh market.
Despite these major advances, fruit wounding is still a problem and is the major
limitation for developing this harvesting technology. Since the majority of posthar-
vest pathogens of temperate fruit crops infect fruits through wounds which results in
decay in storage, this presents an ideal opportunity for the use of biological control.
Considering that the most successful BCPD to date is against wound invading patho-
gens, this area is worth of exploring (Janisiewicz and Korsten 2002). The first
attempt to biologically control decay originating from wounds made during mechan-
ical harvesting suggests that this approach is feasible (Janisiewicz and Peterson
2004). Stem loss (stempulls) on mechanically harvested apples range from 20–57%,
depending on cultivar. If a portion of apple skin is removed together with the stem,
flesh tissue is exposed, creating a potential entry site for the pathogen. The P. syrin-
gae (ESC-11) strain that is used in BioSave reduced blue mold decay on mechani-
cally harvested ‘Empire’ apples with stempulls from 41% to 3.3% and this
antagonist completely eliminated decay on other, less susceptible, cultivars
8
Lm
Lm+Phage 7
Lm+G.asaii
Lm+Phage+G.asaii 6
logCFU/plug
5
1
0 2 5 7
Storage Time (d)
Treatment 0 2 5 7
L 2.64a 1z2 4.45ay 7.06ax 7.474ax

LP 2.74az 3.01bz 5.54y 6.38bx
LG 2.49ay 4.01ax 3.95cx 3.65cx
LP G 2.79ax 3.07bx 2.50dxy 1.61dy
1 Treatment means within Da y with different a, b, c, d letters are different at the 0.05
significance level.
2 Da y means within Treatment with different x, y, z letters are different at th e 0.05
significance level.
Fig. 10.1 Recovery of Listeria monocytogens from honeydew wedges treated with the pathogen
(104 CFU/mL) alone (L) or in combination with phage (LP), Gluconobacter asaii (LG). or a
combination of the two (LPG) and stored at 10°C over 7 days (Hong Y, Leverentz B, Coway WS,
Janisiewicz WJ, Abadias M, Camp MJ, unpublished)
(Table. 10.1). A similar situation may occur with harvested sweet cherries where the
first mechanical prototypes are already being used commercially and all of the fruit
is harvested without stems (Peterson and Wolford 2001). Commercially mechanically
harvested blueberry may also provide an opportunity for biocontrol, and the devel-
opment of blackberry mechanical harvesters has made significant progress recently
(Peterson et al. 1997; Peterson and Takeda 2003; Takeda et al. 2008).
10.7 Conclusions
We are entering into a new era in BCPD with expanding scientific interest and new
products coming to the market. Commercial use of the pioneering products indi-
cates that the fruit and vegetable industry will accept biological control, as long as
Table 10.1 Development of blue mold in the stem cavity area of mechanically harvested apples
with and without stems (stempulls) after inoculation with 25 µl of Penicillium expansum suspension
(5 × 105 conidia/mL) alone or in combination with the antagonist Pseudomonas syringae (ESC-
11). Fruit were stored at 1°C for 2 months (Janisiewicz WJ, Peterson DL, unpublished)
it provides adequate control. The persistent efforts in many programs focusing on

development of new biocontrol agents and/or studying mechanisms of biocontrol
are beginning to pay off. Regulatory agencies in an increasing number of countries
are accepting biocontrol products as safe alternative to fungicide treatments.
Currently, most of the biocontrol products are registered in individual countries, but
expanding registration to other countries would facilitate greater use of the products
and possibly promote the use of mixtures of some products if more than one product
is registered in a country, as combinations of compatible antagonists often provided
improved decay control compared to individual antagonists used alone. Examples
presented earlier indicates that greater use of BCPD can be achieved by expanding
its use to new commodities, new applications, and by integrating biocontrol with
other treatments. Current successes with biocontrol of decays originating from fruit
wounds gives us optimism that the next great challenge, the control of latent infec-
tions, also can be achieved. This, most likely, will require broadening the pool of
microorganisms screened for biocontrol activity to include epiphytes and endo-
phytes from different plants and plant parts, as well as microorganisms from differ-
ent habitats. With research in this direction already under way, there is great hope
and optimism that at the next ISPP Congress in 2013 in Beijing we will have reports
that the next generation of biocontrol agents are capable of controlling latent infec-
tions of fruit.
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Chapter 11
Improving Formulation of Biocontrol Agents
Manipulating Production Process
J. Usall, N. Teixidó, M. Abadias, R. Torres, T. Cañamas, and I. Viñas
Abstract There are several reasons of the limited number of commercial available
biocontrol agents, such as the difficulties in developing a shelf-stable formulated
product that retains biocontrol activity. This chapter shows that it is possible to
improve the formulations during the production process and describes several
examples of improving liquid and dry formulations using different strategies such
as grow microorganisms in aw modified media, under sublethal thermal stress con-
ditions or preservation in isotonic solutions.
Liquid formulation of C. sake was improved growing the cells in molasses
medium with aw modified to 0.98 with the addition of sorbitol and preserved with
an isotonic trehalose solution. After 180 days of storage at 4°C, the viability of this
formulate was 100% and the efficacy against Penicillium expansum on apples was
more than 95% rot reduction.
Spray drying formulations were improved by modifying growth media or tem-
peratures during growing period. The biocontrol agent Pantoea agglomerans grown
during 48 h in NaCl 0.97 aw modified medium could increase their viability after
spray drying formulation from 6% in unmodified medium to near 30% without
affecting their biocontrol potential.
In contrast Candida sake cells grown in unmodified molasses medium exposed to
mild heat treatments at 30°C or 33°C during mid or late-exponential or early or mid-
stationary growth phases showed an increase of survival when are exposed to lethal
shock at 40°C, but only a very reduced improvement after spray drying formulation.
Finally the combination of thermal and osmotic stress was studied in order to
improve fluidized bed drying formulations of P.agglomerans. The results showed
than using NaCl to adjust aw to 0.988 in the growth medium and increasing the
temperature to 35°C during 1 h in the early stationary phase could get a good
formulate with only 0.5 log reductions during fluidized bed drying process.
J. Usall (*), N. Teixidó, M. Abadias, R. Torres, and T. Cañamas

IRTA, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198, Lleida, Catalonia
e-mail: josep.usall@irta.cat
I. Viñas
University of Lleida, UdL-IRTA Centre, XaRTA-Postharvest, 191, Rovira Roure Av, 25198,
Lleida, Catalonia
150 J. Usall et al.
Keywords Liquid formulation • spray drying • thermal stress • fluidized bed drying
• osmotic stress • isotonic solutions • candida sake • pantoea agglomerans
11.1 Introduction
Biological control of postharvest diseases of fruits has advanced greatly during

the past decade. Concerns regarding human health and environmental risks asso-
ciated with chemical residues in foods have been the main driving force of the
search for new and safer control methods (Droby et al. 1998). Among the pro-
posed alternatives, the use of naturally occurring antagonistic microorganisms
has been the most extensively studied. Several microbial antagonists, based on
either yeast or bacteria were developed and commercially tested. However the
success of these products remains limited and just a few microorganisms are
commercial available to control postharvest decay of citrus and pome fruits such
as Bio-Save 10 (Pseudomonas syringae ESC-10, Jet harvest solutions, USA),
Shemer (Metschnikowia fructicola, Agrogreen, Israel), Boniprotect (Aureobasidium
pullulans, Bio-protect, Germany) and Candifruit (Candida sake CPA-1, Sipcam
Inagra, Spain).
There are several reasons of this limited success, such as the inconsistency,
variability of the efficacy under commercial conditions, the limited tolerance of
fluctuating environmental conditions and the difficulties in developing a shelf-
stable formulated product that retains biocontrol activity similar to that of the
fresh cells (Janisiewicz and Jeffers 1997). Dehydration of the product and main-
tenance in a dry environment is one of the best ways to formulate microbial
agents so that they can be handled using the normal distribution and storage chan-
nels (Rhodes 1993). Unfortunately, not all microorganisms are amenable to drying
and many tend to lose viability during both the drying process and storage. The
classical dehydration processes are: freeze-drying, spray-drying and fluidised bed
drying. These processes change the physical state of water by varying the tem-
perature or pressure or by the combined action of both parameters. Cells dehy-
drated in this way are therefore subjected to simultaneous temperature and water
activity stress.
Survival of foodborne pathogenic microorganisms under different stress treat-
ments (e.g. heat, osmotic or water activity, low pH, etc.) have been extensively
studied and reported. Initial investigations carried out on bacteria such as
Escherichia coli (Arnold and Kaspar 1995; Poirier et al. 1998), Salmonella spp.
(O’Driscoll et al. 1996; Mattick et al. 2000) and Listeria spp. (Jorgensen et al.
1995; Greenacre et al. 2003) have demonstrated that they possess an inherent
ability to adapt to unfavourable environments by the induction of various general
and specific stress responses. These stress responses are characterised by the
transient induction of general and specific proteins and by physiological changes
that generally enhance the particular organism’s ability to withstand more adverse
11 Improving Formulation of Biocontrol Agents Manipulating Production Process 151
environmental conditions (Ang et al. 1991). In the case of osmotic stress, the
significant physiological changes reported in bacteria include the induction of
stress proteins as well as the accumulation of compatible solutes such as K+ ions,
the amino acids glutamine, proline, glycine betaine, carnitine and sugars such as
sucrose and trehalose (Csonka 1989; Kets et al. 1994; Ko et al. 1994). In yeasts
and filamentous fungi the accumulated compatible solutes are mainly low-
(glycerol and erythritol) and high- (arabitol and mannitol) molecular weight
sugar alcohols (Beever and Laracy 1986; Ellis et al. 1991; Van Eck et al. 1993;
Hallsworth and Magan 1994, 1996). These compatible solutes allow equilibration
of the cytoplasmic water activity (aw) with the surrounding environment, thereby
retaining water in the cell and thus, maintaining turgor pressure, and helping to
preserve protein function within cells (Yancey et al. 1982; Csonka 1989; Van
Eck et al. 1993).
Subjection to a mild stress makes cells resistant to a lethal challenge with the
same stress condition. Preadaptation to one particular stress condition can also
render cells resistant to other stress imposing conditions: this phenomenon is
known as cross protection (Sanders et al. 1999).
There is significant interest in the mechanisms used by such microorganisms to
tolerate some stress. It has been shown that exposure of cells to a low level of envi-
ronmental stress may induce endogenous adaptation strategies, which can facilitate
resistance to exposure to elevated levels of the same stress, e.g. temperature. As a
result, it has been demonstrated that thermotolerance in yeasts to extreme heat
treatment can be achieved following a brief shift in the incubation temperature
(Tiligada et al. 1999). Control of the resistance of microorganisms to temperature
stress may have potential practical benefits in the process of formulation where
enhanced thermotolerance is required (Ross et al. 2005). The heat stress response
is also usually characterized by the transient induction of general and specific pro-
teins and by the physiological changes that generally enhance an organism’s ability
to withstand more adverse environmental conditions (Ang et al. 1991).
Some of these stresses could be improved during production process. There is
little published research concerning the impact of growth culture on the viability of
a formulated biocontrol agent. Some studies have demonstrated that growth of
C. sake in a commercial molasses-based medium or in the same medium with low-
ered aw (0.98) showed high viable counts, and increased the intracellular concentration
of arabitol and glycerol and the water stress resistance of the cells (Abadias et al.
2001). Moreover, the intracellular water potential of cells decreased with lowered
aw of the culture medium (Abadias et al. 2000).
Candida sake CPA-1 has been demonstrated to be an effective biocontrol agent
(BCA) against major postharvest diseases on pome fruits (Viñas et al. 1998; Usall
et al. 2000; Usall et al. 2001). Detailed studies have shown that the strain CPA-2 of
Pantoea agglomerans – previously classified as Erwinia herbicola – is an effective
antagonist to the major postharvest fungal pathogens of pome and citrus fruits
(Teixidó et al. 2001; Nunes et al. 2001, 2002) and it is in commercialization process
in Spain as a solid formulation named Pantovital by BIODURCAL S.L. Commercial
152 J. Usall et al.
and technical formulations of these two biocontrol agents are been developed in the
Postharvest Unit of UdL-IRTA Center, Lleida, Catalonia.
This chapter describe several examples of improving liquid and dry formulations
of these two biocontrol agents using several strategies during the production pro-
cess such as grow microorganisms in aw modified media, in sublethal thermal stress
or preservation in isotonic solutions.
11.2 Liquid Formulation
In this work we will describe how to improve a liquid formulation of C. sake by

growing the cells in several media and preserved in a liquid with the same water
potential of the cells (isotonic solutions) using different substances to adjust aw and
the efficacy of these liquid formulations against P. expansum in Golden Delicious
apples (Abadias et al. 2003).
11.2.1 C. sake CPA-1 Formulation Using Isotonic Solutions
Viability of the postharvest biocontrol agent Candida sake CPA-1 stored as liquid
formulation was evaluated by studying the effect of growth, preservation medium,
and temperature. C. sake was grown in molasses medium with unmodified water
activity (aw) and in the same medium with aw modified to 0.98 with the addition of
several solutes (glycerol, glucose, NaCl, proline or sorbitol). Low water potential
solutions used as preservation media were prepared with arabitol, erythritol, glyc-
erol, glycine, mannitol, proline, sorbitol and trehalose. These solutes were added to
de-ionized water at the concentration to obtain the same (isotonic) water potential as
the cells.
The best growth media were the unmodified one and those modified to 0.98 aw
with the addition of glycerol or sorbitol. For all growth media, the best preservation
medium was the isotonic solution prepared with trehalose.
Generally, for each growth medium, viability of C. sake cells was significantly
higher when cells were preserved in the trehalose isotonic solution than when cells
were maintained in the other trehalose solutions (Table 11.1).
Viability of cells preserved in water decreased greatly (<3%) after 30 days of
storage at 4°C. Generally, at concentrations below the isotonic one, increased con-
centration of trehalose in the preservation medium implied an increase of the viabil-
ity of C. sake cells. The best viability result was obtained when cells were grown in
the sorbitol modified medium and preserved with the isotonic trehalose solution,
with 110% and 77% of cells remaining viable after 180 and 210 days of storage at
4°C, respectively.
Table 11.1 Percentage of viable cells preserved at 4°C in relation to growth medium and
trehalose concentration in the preservation solution
Storage time at 4°C (days)
Trehalose
concentration, M
Growth medium (mol L−1) 30 60 90 120 150 180 210
% Viability
Unmodified 0 51a 17c 7d 3d NDx ND ND
0.03 55a 39a 32c 24c 18c 18b 13c
0.14y 54a 41a 54a 43a 42a 32a 28a
0.81 32b 40a 38bc 39ab 37b 35a 29a
0.96 38b 33b 43b 36b 34b 34a 21b
Glycerol 0.98 aw 0 42c 5b 1d 1b ND ND ND
0.03 68a 26a 35c 29a 22b 14c 6b
0.14 79a 36a 48b 35a 38a 32b 20a
0.81y 65ab 37a 55ab 33a 47a 45a 20a
0.96 51bc 36a 58a 31a 40a 32b 20a
Sorbitol 0.98 aw 0 23b 1e 2e 2d ND ND ND
0.03 90a 45d 36d 16d 10c 27b 2d
0.14 76a 65c 60c 37c 38b 40b 9c
0.81 75a 109b 105b 92b 118a 111a 63b
0.96y 96a 125a 121a 118a 108a 110a 77a
Viability was not determined due to its low value
x
y
Values in bold represent the trehalose solution that is isotonic with C. sake cells.
For each growth medium and storage time, LSD test for separation of means has been made
(P < 0.05). Different letters (a, b, c, d, e) indicate statistical differences among means.
ND-Not determinated
11.2.2 Viability of C. sake CPA-1 After Storage at 25°C and 4°C
The viability of cells preserved in Phosphate Buffer or in the isotonic solutions of

glycerol or proline was similar to that obtained with water. Therefore, only results
of water and trehalose are shown (Fig. 11.1). More cells remained viable at 4°C
than at 25°C. The number of viable cells decreased rapidly during the first 10 days
of storage at 25°C, and cells grown in sorbitol-modified media and preserved with
the isotonic trehalose solution obtained again the highest viability after 10 days of
storage (25%). The viability of all treatments after 30 days of storage was <7%.
After 4 months of storage at 4°C and regardless of the growth media used,
viability of cells preserved with the isotonic trehalose solution was >21% while
cells preserved with water shown viabilities <2%. Consequently, only viability
of cells preserved with isotonic trehalose solution was evaluated until 7 months
of storage.
Viability of C. sake cells grown in the unmodified molasses medium
(Fig. 11.1a) and preserved with the isotonic trehalose solution at 4°C decreased
progressively and was 72%, 59%, 30%, 21% and 9% after 30, 60, 90, 120 and
154 J. Usall et al.
210 days of storage, respectively. Similarly, the number of viable cells grown in the
glycerol modified molasses medium and preserved with the isotonic trehalose solu-
tion was 70%, 70%, 56%, 46% and 10% after 30, 60, 90, 120 and 210 days of
storage at 4°C (Fig. 11.1b).
In contrast, the number of C. sake cells grown in the sorbitol modified medium
and stored at 4°C with the isotonic trehalose solution (Fig. 11.1c) remained stable at
about 100% during the 210 days of storage period. Generally, cells grown in the
sorbitol modified medium showed higher viability than cells grown in the unmodified
a 9
log10 cfu ml−1
6
0 30 60 90 120 150 180 210
b 9
log10 cfu ml−1
6
0 30 60 90 120 150 180 210
c 9
log10 cfu ml−1
6
0 30 60 90 120 150 180 210
Time (days)
Fig. 11.1 Viable counts of C. sake cells preserved in water (■) or in the isotonic trehalose solu-
tion (▲) at 4°C ( — ) or at 25°C ( --- ). Cells grown in (a) the unmodified molasses medium,
(b) in the medium modified to 0.98 aw with the addition of glycerol, and (c) in the molasses
medium modified to 0.98 aw with the addition of sorbitol. Vertical bars indicate standard error of
means. When they are not visible, they are smaller than symbol size
medium or in that modified with glycerol. The growth medium can influence the
pattern of intracellular solutes accumulated in yeast (Teixidó et al. 1998a,b) or
conidia (Hallsworth and Magan 1994). In some cases, such modifications
resulted in improved tolerance to water stress with retained biocontrol activity
(Teixidó et al. 1998a,b). However, no significant changes in the pattern of some
polyols were obtained when C. sake grew in the sorbitol-modified medium
(Abadias et al. 2000).
Generally for each storage time and growth medium, the increase of trehalose
concentration in the preservation medium involved an increase of C. sake viability
at trehalose concentrations below the isotonic ones. This could suggest that tre-
halose concentration is not the only factor involved in maintaining C. sake viabil-
ity, but probably it interacts with other factors in the growth medium. It is known
that intracellular trehalose exerts a protective effect on yeasts under extreme envi-
ronmental conditions such as dessication, freezing, osmotic stress and heat shock
(Rapoport et al. 1998) and it also provides thermal stability to the cells (Attfield
et al. 1992). In recent years evidence has been accumulated that trehalose may
protect cell viability during drying by replacing the water around polar residues
in enzymes and other essential proteins, thus maintaining their integrity in the
absence of water (Leslie et al. 1994). However, there are no studies on the role
played by the external addition of this sugar in protecting yeast cells in liquid
formulations. Teixidó et al. 1998b found that when C. sake was grown in a aw
modified medium with the addition of trehalose, the intracellular level of treha-
lose increased. It is possible that C. sake could have a trehalose-specific carrier
in the cells.
11.2.3 Efficacy of Trehalose Isotonic Formulations Stored

at 4°C for 7 months
Efficacy of the best liquid formulations stored for different periods was tested
against infection by Penicillium expansum on apples. Regardless of the growth
medium, isotonic trehalose formulations of C. sake stored at 4°C for 7 months
showed good biocontrol effect when they were tested against P. expansum infection
of apples with incidence of decay lower than 18%. In contrast, decay incidence for
untreated fruits was 98%. Isotonic trehalose formulation of cells that grew in the
sorbitol-modified medium performed as well as fresh cells in controlling blue mold
and only 2% of inoculated apples rotted.
In this study (Abadias et al. 2003) an improved liquid formulation of the biocon-
trol agent C. sake has been found (grown in sorbitol-modified media and preserved
with the isotonic trehalose solution) with retained viability and efficacy for 7
months, which will cover all the pome fruit season, and it is composed of safe sub-
stances. However, it should be stored and distributed under refrigerated conditions.
The storage and transport of cells at 4ºC appears not to be an obstacle as other
similar products are handled in this way.
156 J. Usall et al.
11.3 Spray Drying
In this work we will describe how to improve spray drying formulations of P.

agglomerans or C. sake modifying growth media or the temperatures during
growth period.
11.3.1 Improving Spray Drying Formulation of Pantoea

agglomerans CPA-2 by Osmotic Treatments
This research demonstrated that cells of P. aggolerans grown in media at low aw

using NaCl exhibited osmotic adaptation in solid media at low aw obtaining high
production level and maintaining biocontrol efficacy (Teixidó et al. 2006). Osmotic-
adapted cells also demonstrated thermotolerance (Teixidó et al. 2005) and desicca-
tion tolerance after spray drying (Teixidó et al. 2006).
The microorganism was cultured in an unmodified liquid (control) or in aw-
modified media with the non-ionic solutes glycerol, glucose and polyethylene
glycol (PEG 600) to 0.98, 0.97, 0.96 and 0.95 aw, and viability of these cells was
evaluated on unstressed (0.995) and aw-stressed (NaCl 0 96 aw) solid media, in
order to check total viability and aw-stress tolerance, respectively.
Significant improvements in viability on unmodified medium were observed
with cells grown for 24 h in NaCl 0.98 aw, glycerol 0.98 aw and 0.97 aw and for 48
h in NaCl 0.98 aw and 0.97 aw modified media. Both yield improvements and water
stress tolerance were achieved with low aw-media. Cells grown for 24 h in NaCl
0.98 aw or for 48 h in NaCl 0.98 aw, 0.97 aw and 0.96 aw, glucose 0.97 aw and glyc-
erol 0.97 aw showed improved aw-stress tolerance in comparison with control cells
(data not shown). The best results were obtained with NaCl treatments (0.98 aw and
0.97 aw), for that reason a detailed time course study was conducted from 16 to 32
h and the viability of these cells was evaluated on unstressed (0.995) and aw-
stressed (NaCl 0 96 aw) solid media, in order to check total viability and aw-stress
tolerance, respectively.
Control cells (grown in unmodified medium) achieved maximum viable counts
after 18 h incubation with about 4 × 109 CFU mL−1 in unstressed solid media and
9x108 CFU mL−1 in aw-stressed solid media. Their population then progressively
decreased under 5 × 108 CFU mL−1 after 32 h (Fig. 11.2). However, the highest
viability from the experiment was achieved with cells grown in NaCl 0.98 medium,
with 7 × 109 CFU mL−1 after 22 h incubation and 3 × 109 CFU mL−1 after 26 h in
unstressed and aw-stressed solid media respectively. In this case, the stationary
phase was more stable than in the control cells and – throughout the process –
viabilities remained greater than 1 × 109 CFU mL−1. The maximum viability of
NaCl 0.97 cells under aw-stress conditions was obtained after 32 h rising more than
1 × 109 CFU mL−1.
10 a a
a a a a a a
a
b r a b
b b b
rb r b r
r r r
r b s
9 b s r
r r
Viable count, log (CFU ml-1)
s r s s c c b c
s
s t t
8 s
t s
s
7 c
s c
t
6 c
c
5
14 16 18 20 22 24 26 28 30 32 34
Incubation time (hours)
Fig. 11.2 Viability of P. agglomerans cells grown in different media on unstressed (0.995 aw,
closed symbols) and water-stressed (0.96 aw, open symbols) solid media throughout incubation.
Studied growth media were unmodified (❍, ●), NaCl 0.98 (∆, ▲) and NaCl 0.97 (❑, ■). Points
represent the means for four replications. Statistical analysis was performed within (stressed or
unstressed) solid growth media and points labelled with the same letter are not significantly
different (p < 0.05) according to LSD Test
Sodium chloride treatments appeared to play a significant role in improving low

aw tolerance and obtaining high production levels. This result contrasted with those
obtained by Abadias et al. (2000) and Teixidó et al. (1998b), who reported that
C. sake suffered greater inhibition by NaCl than by glycerol, glucose or proline-
amended media.
Cells grown in NaCl modified media which showed the best low aw adaptation
were tested in spray-drying trials to check desiccation tolerance. P. agglomerans
was grown on unmodified, and NaCl 0.98, 0.97 and 0 96 aw modified basic media
for 24 and 48 h at 30°C and 150 rpm agitation on a rotary shaker. Harvested cells
were resuspended in MgSO4 10% suspension to obtain an initial concentration of
1 × 1010 CFU mL−1. These suspensions were then incubated for 30 min at room
temperature, constantly shaken to allow cell adaptation and then spray-dried at an
inlet temperature of 140°C and a delivery rate of 500 mL h−1. The powder obtained
was rehydrated with reconstituted nonfat skimmed milk (10%), which acted as a
rehydration medium. Used methodology was optimized by Costa et al. (2002).
Significant differences between growth treatments were found with respect to
the survival of spray-dried cells (Fig. 11.3). The best survival was achieved with
158 J. Usall et al.
35
a
30
25 b
Viability %
20
15
c
10
c
d
5 de
e
0
Unmodified NaCl 0·98 NaCl 0·97 NaCl 0·96
growth media
Fig. 11.3 Effect of growth media and incubation time (24 h ■ and 48 h ❑) on the survival (%
Viability-relation among the viable bacterial counts before and after drying) of P. agglomerans
cells after spray-drying. The separation of means was conducted according to the LSD procedure.
Columns with the same letter are not significantly different (p < 0.05). Vertical bars indicate
standard deviations
cells grown for 48 h in NaCl 0.97 medium (29%), followed by cells grown for
48 h in NaCl 0.98 (23%). The survival rate of cells growth in unmodified medium
was always less than 7%. In the case of NaCl, the survival of pre-stressed cultures
improved when cells were incubated for 48 h instead of 24 h. The opposite
tendency was observed with control cells. Our research demonstrated that the treat-
ments that showed the best adaptation to low aw, also presented better survival
during the spray-drying process than control cells. These results confirm others obtained
by Prasad et al. (2003), who found that when pre-stressed with either heat (50°C) or salt
(0.6 M NaCl), Lactobacillus rhamnosus HN001 showed a significant improvement in
viability compared with a non-stressed control culture after storage at 30°C in its dried
form. The drying technique applied in this case was fluidised bed drying.
Although desiccation improvement of modified NaCl cells is clear, it is not suf-
ficiently good in practical terms to consider spray-drying as an appropriate strategy
for dehydrating this biocontrol agent.
Regardless of the growth medium, all P. agglomerans treatments showed an
excellent biocontrol efficacy of P. expansum and P. digitatum on both apples and
oranges. The efficacy of unmodified and of the two sodium chloride modified (0.98
and 0.97 aw) treatments was not statistically different and reductions of rot inci-
dence higher than 77% and 73% were respectively observed on artificially wounded
and inoculated apples and oranges.
This investigation revealed that it is possible to improve the low aw tolerance of
P. agglomerans by manipulating growth conditions. Improved cells also showed
better survival during the spray-drying process. These results also suggest that it is
possible to improve the stress tolerance of the microorganism and thus its behaviour
under non-controlled environmental conditions and/or during its formulation process
without affecting its biocontrol potential.
11.3.2 Impact of Mild Heat Treatments on Induction of

Thermotolerance in the Biocontrol Yeast Candida sake
CPA-1 and Viability After Spray-Drying
Other way to improve the tolerance of biocontrol agents of desiccation at high

temperature during spray drying is to expose the microorganisms to mild tempera-
tures during growth (Cañamás et al. 2008).
Studies were conducted on the biocontrol agent C. sake cells grown in molasses
medium at 25°C and exposed to mild temperatures of 30°C and 33°C during mid
(16 h), late-exponential(24 h), early- (30 h) and mid-stationary (36 h) growth phases.
The effect on viability was determined after exposed to lethal shock at 40°C for up
to 120 min and after spray-drying.
The log (N N0−1) values obtained from cells first exposed to mild heat treatments
at 30 or 33°C and then exposed to lethal shock at 40°C for up to 120 min is shown
in Fig. 11.4. Cells heat-adapted at 30 or 33°C survived exposure to 40°C from all
studied phases of growth significantly (P £ 0.05) better than the control cells. This
pattern was similar for all mild heat treatments although heat-adapted cells at 33°C
survive lethal shock better at 40°C than those adapted at 30°C. Mild heat treatments
at 33°C on cells in mid- or late-exponential, early- and mid-stationary phases of
growth survived 0.91-log, 1.17-log, 1.26-log, 1.21-log, respectively, more than
control cells when they were subjected to 40°C for 120 min.
In order to determinate the effect on survival of cells after spray-drying C. sake
was grown as previously described and then resuspended into 50 ml in 10% w/v
reconstituted nonfat-skimmed milk. Suspended yeast cells were incubated at 150
rev min−1 for 30 min at room temperature. Subsequently, each sample was spray-
dried at an inlet temperature of 150°C and a delivery rate of 500 mL h−1 as described
Abadias et al. ( 2005). The spray-dried powder was rehydrated with 5 mL of 10%
nonfat-skimmed milk. The dried cells were shaken vigorously for 1 min and then
allowed to rehydrate for 9 min.
Survival after spray-drying of heat-adapted and non-adapted control cells of
C. sake is shown in Fig. 11.5. The results showed that only mild heat treatments at
33°C from the stationary phase of growth (early or mid) were able to increase sur-
vival significantly after spray-drying in comparison to control cells. At 30°C, there
was no effect on induced resistance after spray-drying. Prasad et al. (2003) also
observed that during fluidised bed drying the viability of Lactobacillus rhamnosus
HN001 cells adapted from stationary phase were superior to cells that were heat-
adapted from the exponential phase. In contrast to this, Teixeira et al. (1997)
showed that heat adaptation increased the survival of Lactobacillus bulgaricus cells
during spray-drying only when heat stress was applied to cultures in the exponen-
tial phase of growth.
The possible role of heat-shock proteins (HSPs) biosynthesis in induced thermo-
tolerance and the role of sugars and sugar alcohols were also determined and cyclo-
heximide and chloramphenicol were used to examine the role of HSPs and HPLC
was used to analyse accumulation of sugar and sugar alcohols.
160 J. Usall et al.
a 0
Log10 (N No -1)
−1
−2
0 30 60 90 120
time (min)
b 0
Log10 (N No -1)
−1
−2
0 30 60 90 120
time (min)
Fig. 11.4 Thermotolerance of nonheat-adapted (♦) or heat-adapted cells of C. sake CPA-1

incubated at 30°C (a) or 33°C (b) from mid-exponential (16 h;❍), late-exponential (24 h; ❑),
early-stationary (30 h; ∆) or mid-stationary (36 h; ×) phase of growth until 40 h of incubation.
Heat lethal shock was performed at 40°C. Levels of thermotolerance were expressed as loga-
rithmic value of relative survivor fraction log (N N0-1). Initial cell count was c. 1 × 107 CFU
mL-1. Results are the mean of at least three-independent measures and vertical bars indicate
standard deviations
It was found that mild heat-adapted cells at sublethal temperature of 33°C from
early- or mid-stationary growth phase still showed higher viability, independently
of the presence of chloramphenicol or cycloheximide. This suggests that protein
synthesis is not responsible for the acquision of thermotolerance in the yeast
C. sake CPA-1. Other research studies mainly with bacteria have shown evidence
20 Control Mild heat treatments at 30 8C Mild heat treatments at 33 8C

b
15 b
% Viability
10 a
a a
a
5 a
a
a
0
CS mid-exp late-exp early-sta mid-sta mid-exp late-exp early-sta mid-sta
Treatments
Fig. 11.5 Survival (%) after spray-drying of control (CS) and heat-adapted cells of Candida sake
CPA-1 at 30°C or 33°C (mild heat treatments) from mid-exponential (mid-exp), late-exponential
(late-exp), early-stationary (early-sta) or mid-stationary (mid-sta) growth phase until 40 h of incu-
bation. Results are the mean of at least three-independent spray-drying trials. Columns with
different letter are statistically different according to Duncan’s multiple range test at P £ 0.05
of the relationship between protein synthesis and acquired thermotolerance. Thus,

Periago et al. (2002) showed that for heat-adaptive response in Bacillus cereus
ATCC 14579 de novo protein synthesis is required. Ananta and Knorr (2004) with
Lactobacillus rhamnosus GG, De Angelis et al. (2004) with Lactobacillus plan-
tarum and Prasad et al. (2003) with Lactobacillus rhamnosus HN001 (DR20) all
found a role of protein biosynthesis in the induction of heat tolerance. However, in
yeast this link may still be tenuous. This is because some studies have reported data
that confirmed that incubation of cells at up to optimum growth temperature
induced thermotolerance and synthesis of stress proteins (McAlister and Finkelstein
1980; Tiligada et al. 1999), while others e. g. Hall (1983), showed that when micro-
organisms were incubated with inducer substances of HSPs, they did not show
thermotolerance. These results suggested that the stress proteins induced by these
analogous substances are not sufficient condition for the acquisition of thermotoler-
ance. It was in concordance with other reported data which is concluded that HSPs
were not the direct responsible compounds of thermotolerance acquired by cells
(Watson et al. 1984; Swan and Watson 1999).
In this study, the correlation between acquired thermotolerance and accumula-
tion of intracellular components, such as arabitol and glucose, was only observed
when heat stress was applied to cells in stationary phase at 36 h or in log-phase at
16 or 24 h. Hottiger et al. (1989), Piper (1993), and Swan and Watson (1999) have
all showed that in yeast neither HSPs nor trehalose are closely correlated with heat-
stress tolerance. The present study suggests that for C. sake, there is no consistent
role for intracellular components in thermotolerance of cells.
162 J. Usall et al.
11.4 Fluidized Bed Drying
In this study, adaptation stress responses (osmotic, thermal and the combination)
are studied to improve survival of cells after fluidized bed drying process.
11.4.1 Screening of Thermal-Stress Treatments
In the first part of the present research, the heat stress response of biocontrol agent
P. agglomerans CPA-2 was analyzed subjecting cells to different thermal stress
treatments (35°C and 40°C) at mid exponential (mid-ex), late exponential (late-ex),
early stationary (early-st) or mid stationary (mid-st) phases of growth until the end
of incubation time (24 h).
Results indicated that thermal stress treatments induced thermotolerance in P.
agglomerans cells which meant that cells were more resistant to a lethal temperature
of 45°C. Heat-inducible thermotolerance allows bacteria, after a non-lethal heat
shock, to tolerate a second heat stress higher in intensity (Boutibonnes et al. 1992).
It was demonstrated that 40°C was more effective than 35°C to induce thermo-
tolerance on P. agglomerans cells under in vitro conditions (Fig. 11.6). Results also
indicated that the highest thermotolerance was obtained when thermal stress treat-
ments were applied in late exponential or early stationary phase of growth.
Consequently, it could be concluded that the application of a mild heat treatment
protects P. agglomerans cells from death during subsequent more extreme heat
exposure. This fact could be interesting for our objectives because extreme heat
conditions could happen during formulation process and also at field conditions.
The induction of thermotolerance by thermal stress treatments in bacteria has
been studied by other authors such as Teixeira et al. (1994) and Gouesbet et al.
(2002) who reported that heat adaptation increased the thermotolerance of lactoba-
cilli. Similar approach has also been used to improve tolerance of the biocontrol
agent Candida sake (Cañamás et al. 2008) as it has been shown above in
Section 11.3.2 of this chapter.
11.4.2 Improving Fluidized Bed Drying Formulation

of P. agglomerans CPA-2 by Osmotic-Stress Treatments
In the second part of the present study we analyzed the effect of osmotic treatments
on the viability of cells after fluidized bed drying process. Assayed treatments were
the same described above in Section 11.3.1 using NaCl to adjust aw (0 g/l Na Cl
(0.99 aw)- P, 35 g/L NaCl (0.98 aw)-P980 or 53 g/L NaCl (0.970 aw)-P970) plus
another treatment with 25 g/NaCl (0.988 aw )- P988. For an optimal level of
growth in P, P988, P980 and P970 treatments, incubation times were 20, 20, 22 and
Thermal stress treatments Thermal stress treatments

P T35-mid-ex T35-late-ex T35-early-st T35-mid-st P T40-mid-ex T40-late-ex T40-early-st T40C-mid-st
a 0.0 b 0.0
−0.5 −0.5
−1.0 −1.0 c
c
Log10 (N N0 )
Log10 (N N0-1)
-1
−1.5 −1.5
−2.0 c −2.0
−2.5 −2.5 b
b
b b
−3.0 −3.0
−3.5 −3.5 a
a a
−4.0 −4.0
Fig. 11.6 Thermotolerance of non-adapted (P) or thermal adapted P. agglomerans cells. During

growth process cultures were subjected to thermal stress treatment at a sublethal temperature of
35°C (a) or 40°C (b) in mid exponential (mid-ex), late exponential (late-ex), early stationary
(early-st) or mid stationary (mid-st) phase until cultures achieved 24 h of incubation. Subsequently,
a heat lethal shock was performed at 45°C for 60 min. Initial cell count was approximately 1 ×
108 CFU/mL and viabilities were measured after 60 min of exposition to heat lethal shock. Levels
of thermotolerance were expressed as logarithmic value of relative survival fraction log10 (N/N0).
Results are the means of three independent measures. Columns with different letter are statisti-
cally different according to Duncan’s multiple range test at P < 0.05
30 h, respectively. For P980 and P970 cultures were also obtained after 48 h of
incubation, corresponding this time to an optimal level of osmoresistance of P.
agglomerans cells (P980 + 48 h and P970 + 48 h treatments) as it had been demon-
strated by Teixidó et al. (2006).
Results pointed out those treatments which were effective to improve survival of
P. agglomerans cells during drying process. The best survival values of P. agglomerans
cells were achieved with osmotic treatments when cells grown at aw of 0.98 and
0.97 for 48 h using NaCl to adjust aw (Fig. 11.7). These treatments also shown the
highest viabilities following spray-drying process in studies carried out by Teixidó
et al. (2006). However, P988 osmotic treatment was chosen for further experi-
ments because it was a cheaper and optimized production treatment. Although
P980 + 48h and P970 + 48h treatments gave higher viabilities than P988 osmotic
treatment, they showed lower levels of biomass production. Other authors such as
Prasad et al. (2003) obtained also higher viabilities in osmotic shocked cells of
Lactobacillus rhamnosus HN001 (DR20) than non-adapted after drying in fluid-
ized bed dryer.
11.4.3 Improving Fluidized Bed Drying Formulation

of P. agglomerans CPA-2 by Combination of Thermal
and Osmotic-Stress Treatments
To our knowledge there has been no previous study in which thermal and osmotic
stresses were combined to improve survival of cells after drying process. In the present
study the combination of osmotic and thermal stress treatments on P. agglomerans
164 J. Usall et al.
Fig. 11.7 Survival of non-osmotically (P treatment) and osmotically adapted cells of P. agglomerans

after dehydration in a fluidized bed dryer for 20 min at 40°C. Levels of viability after drying
process were expressed as logarithmic value of survival fraction log10 (N/N0). Results are the
means of at least two independent fluidized bed drying trials with two independent replications
per trial. Columns with different letter are statistically different according to Duncan’s multiple
range test at P < 0.05
cells resulted in improved viabilities especially when short mild heat treatments
were applied in early stationary phase of growth.
Log reduction values showed that non-significant differences (P > 0.05) were
found between survival fractions of osmotically adapted cells from P988 treat-
ment compared with survival fractions of osmotically and thermal adapted cells
from P988 treatment combined with thermal treatments T35-early-st or T40-
early-st (Data not shown). The combination of osmotic treatment P988 with
thermal stress treatment (T40-late-ex) showed survival values statistically lower
than osmotic treatment P988 but this survival value was significantly higher than
that obtained with P treatment (non-osmotic cells). The combination of osmotic
and thermal treatments also resulted in concentration of viable cells higher than
non-adapted cells, ranging these values between 1.2 × 1010 and 5.6 × 1010 CFU/g
of fluidized product.
On the other hand, osmotic P988 treatment combined with short thermal treat-
ments, T35-early-st-1h or T35-early-st-3h, showed survival values higher than non-
adapted cells after fluidized bed drying process (Fig. 11.8). In this case, log N/N0
values obtained from P988 + T35-early-st-1h combined treatment were signifi-
cantly higher than when osmotic P988 treatment was applied alone with log reduc-
tion of –0.51. T35-early-st-1h or T35-early-st-3h had concentration of viable cells
between 5.5 × 1010 and 1.1 × 1011 CFU/g of fluidized product, respectively. The
moisture content in fluidized-dried product from osmotic and thermal treatments
Fig. 11.8 Viability of osmotically (P988) or thermal-osmotically-adapted cells of P. agglomerans

after dehydration in fluidized bed drying. Thermal-osmotic-adapted cells were obtained applying
osmotic P988 treatment and subjecting P. agglomerans cells in early stationary phase of growth
to sublethal temperature of 35°C for 1 or 3 h (P988 + T35-early-st-1 or P988 + T35C-early-st-3
treatment, respectively). Levels of viability after drying process were expressed as logarithmic
value of relative survival fraction log10 (N/N0). Results are the means of at least two independent
fluidized bed drying trials with two independent replications per trial. Columns with different
letter are statistically different according to Duncan’s multiple range test at P < 0.05
ranged between 9.2% and 11.8%. Interestingly, when osmotic treatment was
combined with thermal stress treatment applied in early stationary phase of growth
at sublethal temperature of 35°C for 1 h higher viabilities were obtained than when
osmotic treatment P988 was applied alone.
These results demonstrated that the combination of different stress treatments
had an accumulative effect on the resistance of cells, resulting osmotic and thermal
adapted P. agglomerans cells more resistant than cells only adapted with a simple
stress treatment.
Protective additives are used in drying processes to protect cells against environ-
mental damages (Corcoran et al. 2005). These additives are often native substances
that the microorganisms themselves produce, such as disaccharides, amino acids
and amino acid derivates. Production of these compounds is often induced upon
stress or pre-conditioning (Ross et al. 2005). The accumulation of glycine-betaine
and carnitine has been shown to enhance survival of L. plantarum after drying (Kets
et al. 1994). Results reported by Cañamás et al. (2007) confirmed that P. agglomerans
cells accumulate different compatible solutes under osmotic and thermal stress.
Osmotic adapted P. agglomerans cells using NaCl as solute mainly accumulated
glycine-betaine. However, there was no increase in quantities of glycine-betaine
and ectoine under heat stress (Cañamás et al. 2007).
166 J. Usall et al.
Overall, the present study it has been demonstrated that osmotic and thermal
stress treatments are a practical tool to improve survival of P. agglomerans cells
under stress conditions. Moreover, the final fluidized product obtained showed
adequate viabilities to formulate P. agglomerans cells by fluidized bed drying pro-
cess (between 5.5 × 1010 and 1.1 × 1011 CFU/g of fluidized product).
11.5 Conclusions
The importance to develop a shelf-stable formulated product that retains biocon-

trol activity is clear in order to obtain satisfactory commercial biocontrol agents.
The results shown in this chapter indicate that it is possible to improve liquid and
solid formulations using several strategies such as growing microorganisms in aw
modified media, in sublethal thermal stress or using preservation in isotonic
solutions.
This study also demonstrates that technologically sensitive cultures as P. agglomerans
or C. sake can potentially be manipulated to become more robust for survival under harsh
conditions, such spray drying or fluidized bed drying processes.
The use of these new techniques could help the companies to put in the market
more stable and effective biocontrol products.
Acknowledgements The authors are grateful to Spanish government (Ministerio de Ciencia y

Tecnología) for grants AGL-2002–01137 and AGL-2005–02510 and to FEDER (Fondo Europeo
de Desarrollo Regional) for their financial support.
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Chapter 12
Host Responses to Biological Control Agents
Raffaello Castoria and Sandra A.I. Wright
Abstract Host responses in stored fruits induced by biocontrol agents (BCAs)

i.e. by non-pathogenic yeasts and bacteria, share many features with the defence
mechanisms that are induced in actively growing plant tissues. The perception
of a microorganism is accompanied by the production and activation of reactive
oxygen species (ROS), antioxidant enzymes, phytoalexins, phenylalanine ammo-
nia lyase and enzymes that degrade fungal cell walls. The responses of harvested
fruit to BCAs do not fit with the existing division of induced resistance pathways
into Systemic Acquired Resistance (SAR) and rhizobacteria-mediated Induced
Systemic Resistance (ISR), nor are the roles of salicylic or jasmonic acid clear.
These responses seem to carry elements of both pathways. Moreover, successful
BCAs need to be able to resist environments rich in toxic ROS; hydrogen perox-
ide being the dominant species, generated both during the induction of resistance
(as in the defence of citrus fruit against Penicillium digitatum) and during the
attack of some necrotrophic pathogens (as in the case of Penicillium expansum
invading apples). Application of BCAs to fruits can result in increased production
of antioxidant enzymes (by either organism), which protect living cells from the
potential damage of ROS. Induction of resistance has usually not been considered
an important mechanism in the activity of postharvest biocontrol agents. A deeper
understanding of fruit responses that BCAs provoke of the infection process by
necrotrophic pathogens during postharvest and of the accompanying host responses
is needed. In the following chapter, we present examples from diverse plant-patho-
gen-BCA systems and suggest approaches for future research.
R. Castoria (*)
Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via Francesco
De Sanctis, 86100, Campobasso, Italy
e-mail: castoria@unimol.it
S.A.I. Wright
Dipartimento di Scienze Animali, Vegetali e dell’Ambiente, Università del Molise, Via Francesc
De Sanctis, 86100, Campobasso, Italy, and
Department of Plant and Environmental Sciences, University of Gothenburg, 461SE, 405 30,
Göteborg, Sweden
172 R. Castoria and S.A.I. Wright
Keywords Biological control • induced resistance • pathogenicity strategies of

postharvest pathogenic fungi.
12.1 Introduction
Biological control of plant diseases is a three-way interaction in which pathogen,

plant tissue(s) and biocontrol agent (BCA) are involved. The plant tissue cannot
merely be considered as the battlefield where pathogen and biocontrol agent
confront each other, since it appears to perceive the presence of both the pathogen
and the BCA. Perception of the pathogen or of the BCA is followed by deployment
of an active response that results in induced resistance that can be localized or sys-
temic. The localized response is based on rapid cell death, reinforcement of cell
walls and accumulation of phytoalexins. Systemic resistance also involves the syn-
thesis and accumulation of antifungal depolymerases that attack the fungal wall.
Even if induced resistance is normally considered to be a response of non-senesc-
ing, growing plant tissues and intact plants, the phenomenon has also been reported
in BCA-treated and stored fruits and some of the responses have commonalities
with those observed during the induced resistance reactions of intact, growing
plants (see below).
A handful of biocontrol products for postharvest use are at present available
commercially, and they represent only a small fraction of the potential market for
control of postharvest diseases (Fravel 2005). Some examples of these products
are Bio-Save®10 LP and Bio-Save®11 LP, which are based on strains ESC 10 and
ESC 11 of the bacterium Pseudomonas syringae, respectively, and are registered
in the USA by JET Harvest Solutions (Longwood, FL, www.jetharvest.com). Bio-
Save®10 LP is active on postharvest rots caused by Penicillium digitatum,
Penicillium italicum and Geotrichum candidum of citrus fruit, rots caused by
Penicillium expansum, Botrytis cinerea and Mucor piriformis of pears and apples,
and rots in cherries caused by P. expansum and B. cinerea. Another biocontrol
product for use in postharvest systems is Shemer® (www.agrogreen.co.il/shemer.
asp), based on a strain of the yeast Metschnikowia fructicola, and is registered in
Israel by AgroGreen Ltd (Ashdod, Israel).) Registrations in the USA in and
Europe are currently pending. Shemer® is effective against postharvest rots of
apricot, grapes, strawberry, citrus, peach, pepper and sweet potato caused by spe-
cies of Aspergillus, Botrytis, Penicillium and Rhizopus. BoniProtect® and
BoniProtect® Forte are based on mixtures of two strains of the yeast Aureobasidium
pullulans, developed and marketed in Germany by Bio-Protect (Konstanz, www.
bio-protect.de). BoniProtect® is effective against postharvest rots in apple caused
by B. cinerea, P. expansum and Pezicula malicortici. BoniProtect® Forte is applied
at bloom to strawberries and protects them during postharvest storage against the
development of grey mould caused by B. cinerea. Candifruit® is based on a strain
of the yeast Candida sake, developed by Centre UdL-IRTA (Lleida, Catalonia)
and marketed in Spain by SIPCAM INAGRA SA (Valencia, www.sipcam.es).
12 Host Responses to Biological Control Agents 173
Candifruit® is effective against postharvest rots in apple and pears caused by P.

expansum, B. cinerea and Rhizopus stolonifer.
In stored fruits, the defence capability becomes weaker with time. As the physiol-
ogy of the fruits changes during maturation and senescence, inhibitors of fungal
growth decrease and the fruit becomes more susceptible to postharvest pathogens,
especially to necrotrophic ones. A clear example is the antrachnose disease of avo-
cado caused by Colletotrichum gloeosporioides, in which fungal growth undergoes
a transition from the quiescent to the necrotrophic stage, a condition that appears to
be associated with fruit ripening. During this transition, the level of a preformed
antifungal diene decreases in the fruit as a consequence of the decrease of the level
of the protective antioxidant epicathechin (Karni et al. 1989). Physiological changes
such as fruit tissue softening and the shift of plant cells to a more oxidized state
negatively affect fruit resistance to necrotrophic pathogens and the antagonistic
activity of biocontrol yeasts (Castoria et al. 2003). Most if not all of the studies on
fruit responses to treatment with BCAs have been carried out with biocontrol yeasts
or yeast-like organisms. As a consequence, the present chapter will focus on the
interaction with this kind of microorganisms. The chapter is not to be taken as a
comprehensive review of all the literature in this area, but an attempt to give a suc-
cinct summary of the information at present available on fruit responses to BCAs,
and to present new perspectives for research in postharvest biocontrol.
12.2 Modes of Action of Postharvest Biocontrol Agents
As a general introduction to mechanisms of action, some definitions will be

presented in order to envisage the dynamic context in which fruit responses to
BCAs operate. The modes of action of beneficial microorganisms can be divided
into direct antagonism and indirect antagonism. These mechanisms do not exclude
each other since they are frequently described as co-occurring within the activity of
the same BCA. Direct antagonism comprises those mechanisms that are a direct
result of the action of an individual BCA: competition for space and nutrients,
secretion of lytic enzymes (depolymerases) such as b-1,3 glucanases and chitinases
that degrade the polymers of the pathogen cell wall and mycoparasitism, the latter
often documented to require direct physical interaction between the cells of the
BCA and the hyphae of the pathogenic fungi (Castoria et al. 1997; Janisiewicz and
Korsten 2002; Wisniewski et al. 1991). Indirect antagonism implies mechanisms
which are not a direct result of the activity of the BCA, but are the consequence of
the response of the fruit tissue to the presence of these beneficial microorganisms,
ultimately resulting in induction of resistance (see Section 12.2.1). Competition for
space and nutrients by the BCA mainly takes place when the BCA colonizes
wounds of fruit, caused by bruising during handling and transportation. These
wounds represent the main ports of penetration of pathogenic fungi. In order to
successfully out-compete the pathogens, postharvest BCAs need to possess a
sound wound colonization competence, i.e. the ability to rapidly colonize and
thrive in fruit wounds (Droby and Chalutz 1994). Since wounding in apple fruits
causes the production of hydrogen peroxide (H2O2) and superoxide anion (•O2−), it
has been suggested that resistance to the oxidative stress caused by these reactive
oxygen species (ROS) plays a role in wound competence of biocontrol yeasts and,
as a consequence, in their competition with the pathogen for space (and nutrients).
As with other climacteric fruits, the physiology of stored apples changes during
maturation and senescence. One of these changes is represented by the increased
production of ROS when the apple tissue becomes wounded during storage. The
increased production of ROS in apple wounds negatively affects both wound
colonization by BCAs and their antagonistic activity (Castoria et al. 2003).
Mycoparasitism is mediated by the physical attachment of the biocontrol agent to
the hyphae of the pathogen, and is associated with the secretion of cell wall depoly-
merases in causing damage to the hyphae of the pathogenic fungi, as in the case of
the interaction of Trichoderma spp. with Rhizoctonia solani (Harman et al. 2004).
This mechanism has not been reported to be common for postharvest BCAs but,
when present, as in the case of the attachment of cells of Pichia guilliermondii to
hyphae of B. cinerea, it appears to be associated with the secretion of cell wall
depolymerases that cause damage to the hyphae of the pathogenic fungi (Wisniewski
et al. 1991). In addition, depolymerases have been suggested to contribute to the
antagonistic activity of BCAs, even in the absence of clear mycoparasitism
(Castoria et al. 1997, 2001).
12.2.1 Induced Resistance Signalling Pathways
Induced resistance in plants is termed localized when this response and the mecha-
nisms it relies on occur at the same site of pathogen inoculation or treatment with
any triggering factor such as elicitors (i.e. compounds that elicit a hypersensitive
response) or BCAs. Conversely, resistance is systemic when it occurs at a site that
is distal from the one where perturbation of the plant homeostasis initially takes
place. Systemic resistance can be the consequence of local pathogen inoculation, in
which case it is referred to as systemic acquired resistance (SAR). Alternatively,
another form of systemic resistance, called rhizobacteria-mediated induced sys-
temic resistance (ISR), is not a result of a localized resistance event induced by
pathogens, but is triggered by beneficial microbes such as rhizobacteria, chewing
insects, wounding or by biotic elicitors. SAR and ISR are generally thought to oper-
ate through different signal transduction pathways: SAR is activated through sali-
cylic acid (SA)-dependent pathway, ISR through the jasmonic acid (JA) and
ethylene – dependent pathway, which is also involved in wound-induced resistance
(WIR) (Kessler and Baldwin 2002) caused by tissue damage as a result of insect
feeding. Another general idea is that resistance induced by JA (and ethylene) or by
SA has its own respective pattern in defence mechanisms elicited: SA-mediated
responses are characterized by the synthesis of pathogenesis-related (PR) proteins,
whereas JA (and ethylene)-mediated responses are not (Vallad and Goodman
2004). Studies on defined mutants of Arabidopsis thaliana have suggested that

specific plant defence responses may be active on specific types of pathogens:
SA-dependent defences are effective against biotrophs, JA (and ethylene)-depen-
dent responses are effective against necrotrophs (Glazebrook 2005). The latter
comprises a category that includes most postharvest wound pathogens. However,
the actual situation seems to be more complex, since there is some cross-talk
between these two major signalling pathways (De Vos et al. 2005; Glazebrook
2005), through NPR1 (Non-expressor of PR-genes 1). They can thus be turned on
simultaneously, producing an additive effect, and sometimes SAR seems to have a
negative effect on the JA/ethylene pathway (reviewed in Durrant and Dong 2004).
In the following paragraphs of this chapter, findings on localized and systemic
resistance induced in fruits by BCAs will be reviewed, and the roles of plant resis-
tance responses and pathogenicity determinants of major postharvest pathogens
will be discussed.
12.2.1.1 Localized and Systemic Resistance Induced in Fruits by BCAs
An early report on the induction of local resistance mechanisms in stored fruits

(Arras 1996) showed that the phytoalexin scoparone was synthesized in oranges
pre-treated with the yeast Candida famata strain FS35. Ippolito et al. (2000)
reported a transient induction of glucanase, chitinase and peroxidase (POD) in
apple wounds treated with A. pullulans strain L47, which, although not demonstrated,
they attributed to the apple tissue. This yeast-like fungus was used to successfully
protect stored apple fruits from infection by P. expansum and B. cinerea (Ippolito
et al. 2000). The results obtained by Castoria et al. (2001) when using a different
strain of A. pullulans, strain LS30, further substantiated the notion that BCAs can
induce apple tissues to produce glucanases. Some of the glucanase activity recorded
in this study most probably derived from the apple tissue, since the activity was
detected also in wounds that were only treated with a preparation of cell walls of
P. expansum (i.e. in the absence of any living microorganism). Tian et al. (2007)
cloned two b-1,3 glucanase genes (Glu-1 and Glu-2) from Jujube fruit (Ziziphus
jujuba Mill), and showed that the expression of Glu-1 was induced by the yeast
Cryptococcus laurentii, starting 12 h after application of this BCA. The expression
study was complemented with a biochemical analysis by assessing b-1,3 glucanase
activity (although it was not ascertained if the source of enzyme activity was the
yeast, the fruit or both). b-1,3 glucanase activity was the highest following treatment
with the yeast at all time points tested and correlated positively with the
protection against P. expansum and Alternaria alternata achieved by the yeast. It
is not clear if this is an example of localized or systemic resistance, since it seems
as if the authors extracted fruit tissue as a whole, not only the wounded area.
The first convincing evidence of BCA-induced systemic resistance in fruit
was presented by Droby et al. (2002) and El Ghaouth et al. (2003), who studied
C. oleophila applied to grapefruit and C. saitoana applied to apples, respectively.
In both cases, the assessment of systemic resistance was based on the application
of a Candida strain in freshly generated wounds, inoculation of the pathogen in

wounds that were spatially separated from the wounds in which the BCAs had been
applied and on the evaluation of disease incidence and/or severity. Droby et al.
(2002) showed that the biocontrol yeast C. oleophila induced systemic resis-
tance to P. digitatum in grapefruit. Protection took place 24 h after the application
of the yeast, it was BCA concentration-dependent and did not occur in wounds
made more than 4 cm from the wounds in which the BCA was applied. Living yeast
cells were needed to ensure protection, which correlated to the induction in the fruit
tissue of defence responses such as phenylalanine ammonia lyase (PAL) activity,
accumulation of b-1,3 glucanase and chitinase, biosynthesis of ethylene and the accu-
mulation of the phytoalexins umbelliferone, scoparone and scopoletin. The authors
hypothesized that the dependence of systemic protection on the BCA cells being
alive when applied might be due to the involvement of recognition processes
between the yeast and the fruit cells and the secretion of elicitors by the BCA. The
presence of BCA-derived elicitors was also tested in another yeast-plant system:
plants of A. thaliana treated with autoclaved baker’s yeast were proposed to recognize
compounds deriving from the yeast that could act as PAMPs or MAMPs (pathogen-
associated or microbe-associated molecular patterns). These compounds induced
resistance in A. thaliana to the necrotrophic pathogen B. cinerea (Raacke et al. 2006).
El Ghaouth et al. (2003) demonstrated the induction of systemic protection of
apples by the yeast C. saitoana, which partially protected them from infection by
B. cinerea. The protection, expressed as the reduction of diameters of lesions
caused by B. cinerea, occurred when the pathogen was inoculated not earlier than
48–72 h after the application of the BCA and correlated with the timing of induction
of b-1,3-glucanase and chitinase activities in the systemically (distally) protected
fruit tissue, i.e. where the yeast had not been applied. The capability of inducing
systemic resistance depended on the age of fruits, monitored as the efficacy of
protection against infection by B. cinerea, which correlated with the induction
of b-1,3-glucanase and chitinase activities appeared to occur only in fresh (young)
but not in stored (old) apples. The authors hypothesized that a systemic signal was
produced at the site of BCA treatment.
As of now, no unequivocal evidence has been reported on the involvement of SA
or JA in yeast-induced resistance in fruits. In this regard, Raacke et al. (2006)
showed that A. thaliana mutants impaired in the SA-dependent or JA-dependent
pathways were not negatively affected in resistance to the necrotrophic fungus
B. cinerea induced by autoclaved baker’s yeast, suggesting that this resistance is
independent of the SA and JA pathways. Moreover, SA and JA have been tested as
possible inducers of resistance in postharvest horticultural crops. A comprehensive
review of these studies is presented in Terry and Joyce (2004).
SA or a derivative of JA, methyl jasmonate (Me-JA), have also been directly
applied on fruits and, in some reports, their effects have been compared with those
induced by BCAs. However, it is not clearly described if the examined biochemical
markers of induced resistance were measured at the sites of treatment and/or in
other fruit parts. In sweet cherry fruit, moderate protection from infection by
Monilinia fructicola was achieved by pre-harvest treatment with 2 mM SA or 0.2
mM Me-JA, and was correlated with the induction of glucanase, PAL and POD
activities, thus suggesting that enzymes commonly known as PR proteins are
induced by both SA and Me-JA (Yao and Tian 2005). However, the methods used
for measuring and/or detecting the induced enzyme activities do not allow the
distinction between localized and systemic resistance. In the same fruit, pre-treatment
with 0.5 mM SA or the BCA Pichia membranefaciens yielded some protection
against P. expansum. Both treatments appeared to influence antioxidant enzyme
activities, which either increase (superoxide dismutase, SOD, through dismutation
of superoxide anion, O2− to H2O2) or decrease (catalase, CAT, which converts
hydrogen peroxide to molecular oxygen and water; POD, which breaks down H2O2
when using it as a cosubstrate in the oxidation of phenolic substrates) the concen-
tration of H2O2. Following inoculation with the pathogen, yeast treatment increased
SOD activity but decreased CAT activity, suggesting a role for the steady-state level
of ROS in the protection achieved. The authors suggested that resistance induced
by the BCA might share some commonalities with that induced by SA, but that the
situation was complex and needed to be clarified (Chan and Tian 2006). It is known
that ROS, in particular H2O2, and the oxidative stress these chemical species cause
is involved in the signalling cascade in the induction of resistance in plants. The
oxidative burst contributes to SAR expression in Arabidopsis (Alvarez et al. 1998)
and in pathogenesis of necrotrophic pathogens (van Kan 2006; Hadas et al. 2007).
Therefore, the complex pattern of antioxidant enzyme activities reported in sweet
cherry suggests that direct measurements of H2O2 and O2− are needed to shed more
light on the processes involved in protection by SA and the BCA in this system.
A role for ROS in the BCA-induced defence mechanisms and in the attack on
fruit by postharvest necrotrophic pathogens is also indirectly suggested in the
results obtained by Chan et al. (2007) who showed the induction of antioxidant
enzymes following treatment of peach fruit with 0.5 mM SA or P. membranefa-
ciens. Differences in proteomes after the fruits had been treated with SA and the
yeast correlated with the difference in protection against P. expansum provided by
treatments with SA and the yeast. Although it is not clear whether or not extracted
tissue contained the wound site and the yeast cells, the expression of seven proteins
(28% of those identified), comprising antioxidant and PR proteins were regulated
by both the BCA and SA. Protection with these two treatments correlated with a
higher production of PR-9, PR-10, CAT (as confirmed also at a transcriptional and
enzyme activity level) and glutathione peroxidase (PR-9, also confirmed at an
enzyme activity level). Conversely, a more rapid increase of SOD[Mn] production
was induced by the BCA as compared with that induced by to SA, although this
finding was not in agreement with the level of enzyme activity detected.
Castoria et al. (2003) hypothesized that biocontrol yeasts could also act as exog-
enous suppliers of antioxidant activity to the apple tissue, thus partially accounting
for their protective ability, by providing it with additional defence tools counteract-
ing the production or induction of ROS by B. cinerea. Although it is not yet estab-
lished that induction of oxidative damage to plant tissues could be a general
phenomenon of fungal necrotrophic attack, results by Xu et al. (2008) appear to
support this notion. Treatment of peach fruits with different biocontrol yeasts
(C. guillermondii, C. laurentii, Rhodotorula glutinis and P. membranefaciens)

reduced M. fructicola attack. As in the case of SA treatment that offered protection
to sweet cherries from attack by P. expansum, as mentioned above (Chan and Tian
2006), protection achieved by the BCAs seemed to be accompanied by induction
of CAT, POD (i.e. enzymes that lower concentration of H2O2), and b-1,3-glucanase
activities as well as the expression of the respective genes. Although control treat-
ments of peaches not inoculated with Monilinia were not performed – and thus it
is not possible to know if the antioxidant enzymes, chitinase and glucanase mea-
sured were of plant or yeast origin – there might have been a decrease in protein
damage caused by oxidative stress when infected peaches were treated with the
BCAs, as compared to infected fruits not treated with the different yeasts. The
authors concluded that the proteins were protected by an antioxidant response, and
that this could constitute mechanisms of protection of peach fruits by the microbial
biocontrol agents (Xu et al. 2008). However, more studies are needed to confirm
these results and to clarify if the BCA-dependent decrease of oxidative damage to
peach proteins is an (indirect) indication of M. fructicola-induced oxidation linked
to the infection process, i.e. an interference with the pathogenicity strategy of the
fungus, or just a reduction of symptom development, i.e. a mere epiphenomenon.
Furthermore, as mentioned above, , there was no assessment of the possible oxidative
stress caused by the BCAs, i.e. the oxidative damage in healthy peaches treated and
untreated with the BCAs (e.g. as a consequence of ROS-mediated signalling) was
not reported. In this regard, unpublished results by Droby et al. have recently shown
that application of the biocontrol yeast M. fructicola in wounded tissue of apple and
citrus induces generation of H2O2. In addition, the BCA itself produces O2 in
apples, peaches and citrus.
12.3 Concluding Remarks
The study of fruit responses to postharvest biocontrol agents is in its infancy. Even
if application of SA and JA appears to improve fruit protection, a role for these
compounds in BCA-induced systemic (or even localized) resistance is yet to be
established. In stored fruits, the defence responses observed do not neatly fit with
the classical division into SA-dependent and JA-dependent signal transduction
pathways. In contrast to the responses noted in intact plants, stored fruits produce
antifungal hydrolases (and other PR proteins) following either SA or (Me)JA treatment.
Furthermore, both SA and JA seem to induce defence responses that provide
resistance to necrotrophs, although the precise pathways are not known.
BCAs could play a role in the oxidative phenomena that occur during the induction
of resistance, but more studies are needed to confirm this hypothesis. Even so, some
evidence appears to support the possibility that the induction of an antioxidant
response may play a role in the BCA-mediated resistance of fruit to necrotrophic
fungi. Further studies are needed also in this case. Sclerotinia sclerotiorum
produces oxalic acid, a pathogenicity determinant that induces programmed cell
death at pHs in the range of 5 to 6, and which is necessary for a successful infection
of the plant tissue, via the induction of ROS. As oxalic acid accumulates in the
tissues, the pH is lowered and at the lower pH the host oxidative burst is
down-regulated. During this phase of the infection process, after the ROS-mediated
attack, Sclerotinia is thus able to avoid an excessively H2O2-rich environment
induced by the plant (Kim et al. 2008). H2O2, a toxic reactive oxygen species, can
in some cases be employed by plants as a defence strategy and in other instances
by the necrotrophic pathogen as a weapon mediating its invasion. In the P. expansum-
apple fruit system, acidification through production of gluconic acid by P. expansum
positively affects transcription of fungal genes encoding lytic enzymes that attack
the walls of fruit cells. During this phase, there is also an accumulation of H2O2
(since it is a by-product that is formed during gluconic acid synthesis) at the limit
of the decaying area (Hadas et al. 2007), thus suggesting a direct role of H2O2 in
the infection process, as in the case of necrotrophic attack by B. cinerea (van Kan
2006, Williamson et al. 2007). In contrast, Torres et al. (2003) suggested the pos-
sible involvement of fruit-derived H2O2 as a mechanism of resistance of young
apples to P. expansum. Similarly, the resistance of citrus fruits to the incompatible
pathogen P. expansum is associated with a large local accumulation of H2O2, sug-
gested to be of citrus origin, which ultimately leads to lignification and HR
(Macarisin et al. 2007). The compatible pathogen, P. digitatum is instead able to
suppress the citrus fruit defence reactions and actively lowers the accumulation of
hydrogen peroxide in wounds as compared to non-inoculated but wounded con-
trol fruit. As additional weight to the above theory, exogenously added CAT
enhanced the pathogenicity of both P. expansum (it became a pathogen) and P. digitatum
on citrus fruit.
The elucidation of the role of hydrogen peroxide in the pathogenesis of different
Penicillium species pathogenic to apples and citrus fruits will be facilitated when
defined mutants of P. expansum and P. digitatum become available. Taken together,
healthy and infected wounds of harvested fruits present an environment that is rich
in ROS, an environment in which BCAs need to operate. It is therefore an advantage
when BCAs are able to resist locally produced ROS, whatever the role(s) of ROS is
– as inducers of fruit resistance through ROS production and signalling or as activa-
tors of an antioxidant response in the fruit that counteracts the oxidative attack by
necrotrophic pathogens. Induction of resistance is not usually considered to be a
major mechanism of postharvest biocontrol agents, most probably because it has
been difficult to monitor and because the BCA and the pathogen are usually applied
at the same site. As has been demonstrated, this area of research is emerging. As more
information on the mechanisms of pathogenesis of postharvest (necrotrophic) patho-
gens becomes available, the exact dynamics of the plant-pathogen signalling events
and the environment that BCAs encounter will become clearer. In addition, more
information on the nature of fruit responses to the BCAs is needed, preferably by
using defined plant mutants, so the responses can be organized into pathways, much
like has been done for intact plants. BCAs consst of diverse microorganisms. It com-
prises both yeasts and bacteria. For the most part, they are considered to be non-
pathogenic, and in this case, according to the established induced resistance path-
ways of intact and actively growing plants, SAR should not be involved. However,
as was shown by Giobbe et al (2007), a yeast strain (Pichia fermentans) that is a
BCA of Monilinia brown rot of apple is a pathogen when applied to peach fruit. It
is plausible that other BCAs could be pathogens in as of yet unidentified pathosys-
tems, and if so, they would in fact be regarded as incompatible pathogens in the plant
test system used to monitor biocontrol activity. This category of BCAs would be
expected to cause an oxidative burst and the onset of HR-related defence mecha-
nisms in the fruits as a pathway for induction of resistance. Future research efforts
need to deepen our knowledge in all three areas mentioned - on the nature of BCAs,
the responses of fruits by using defined mutants and on the mechanisms of patho-
genesis of the postharvest pathogens – in order for resistance pathways to be estab-
lished and characterized also in harvested fruits.
Acknowledgements The authors are sincerely grateful to David B. Wright for critical reading of
the manuscript and assistance with language editing. This work was funded by the Italian Ministry
of Education, University and Scientific Research (MIUR) PRIN 2006 “Pilot study on innovative
systems for the reduction of patulin contamination in pome fruits”, project number 2006072204,
and through the MIUR-funded: “Incentivazione alla mobilità di studiosi stranieri e italiani
residenti all’estero” (DM 1.2.2005, n.18).
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Chapter 13
Non-fungicidal Control of Botrytis Storage Rot
in New Zealand Kiwifruit Through
Pre- and Postharvest Crop Management
M.A. Manning, H.A. Pak, and R.M. Beresford
Abstract Storage rot of ‘Hayward’ kiwifruit, caused by Botrytis cinerea, became

a serious problem in New Zealand during the 1980s, costing the then NZ$200
million industry about NZ$10 million per year. Fruit are healthy at harvest and
become infected through the picking wound during postharvest handling. Disease
symptoms develop after 4–8 weeks of storage at 0°C. Ethylene produced by a
single rotting fruit in a tray can cause the whole tray to soften prematurely. Control
attempts with pre-harvest fungicides led to resistance in B. cinerea to dicarboxim-
ide and benzimidazole fungicides. In the orchard, B. cinerea is visible mostly on
flower petals early in the season, although it occurs on all plant surfaces, in under-
story weeds and in necrotic kiwifruit leaves. Research into storage rot risk factors
revealed a relationship between rot incidence and the incidence of B. cinerea on
necrotic leaves in the orchard around harvest time. Orchard populations of B. cinerea
were quantified by assessing the incidence of necrotic leaf discs that were colonised
by B. cinerea after incubation. From this, a predictive system has been developed
that can identify high risk orchards. The botrytis problem has largely been solved
by vine management that avoids dense leaf canopies. This prevents the build-up of
the necrotic leaf tissue on which B. cinerea multiplies. It was also found that stor-
age rot incidence can be greatly reduced by “curing” the fruit after harvest. This
involves storing fruit for 48 h at ambient temperature before cooling. Storage rot
incidence is also reduced by harvesting fruit when they are more mature (7–8°Bx),
as riper fruit are much less susceptible to botrytis than immature fruit. The botrytis
storage rot problem has thus been avoided by a combination of pre-harvest orchard
management and postharvest handling practices, without the need for intervention
with fungicides.
M.A. Manning and R.M. Beresford (*)

The New Zealand Institute for Plant & Food Research Limited
(Plant & Food Research, formely HotResearch),
Mt Albert Research Centre, Private Bag Auckland, 1142, New Zealand
e-mail: robert.beresford@plantandfood.co.nz
H. Pak
Avocado Industry Council, 13 267, Tauranga, 3141, New Zealand
184 M.A. Manning et al.
13.1 Botrytis Storage Rot – Historical Perspective
13.1.1 Economic Impact
Exports of New Zealand kiwifruit (Actinidia spp.) currently have an annual value
of NZ$700–800 million from a production base of about 12,000 ha (HortResearch
2007). Although kiwifruit production suffers from relatively few plant diseases,
there was a time from the late 1970s to the early 1990s when a storage rot disease
(stem end rot), caused by Botrytis cinerea, led to important economic losses
(Beever et al. 1984; Pennycook 1985). At that time New Zealand kiwifruit produc-
tion consisted almost entirely of the A. deliciosa cultivar ‘Hayward’ (marketed as
ZESPRI™ GREEN), with an export value of about NZ$200 million. Botrytis stor-
age rot had a significant economic impact, particularly from the cost of inspecting
and repacking affected lines of fruit, e.g. in 1991, Cheah and colleagues (Cheah
et al. 1993) reported a cost to the industry of NZ$10 million per annum. Even a low
incidence of botrytis rot affects the marketability of fruit because ethylene pro-
duction from a single infected fruit in a tray can cause the whole tray to soften
prematurely (Manning and Pak 1993). The tolerance for storage rot incidence,
above which lines of fruit must be repacked, is one diseased fruit per three trays,
or about 1%.
From the mid-1990s until the present, the A. chinensis cultivar ‘Hort16A’
(marketed as ZESPRI™ GOLD), which is not prone to botrytis storage rot, has been
increasing in importance, with 20 million tray equivalents produced in 2006/07.
However, ZESPRI™ GREEN, which is susceptible to botrytis storage rot, is still the
main kiwifruit product exported from New Zealand, with 60 million tray equiva-
lents in 2006/07.
13.1.2 Etiology
When botrytis storage rot was first recorded as a problem in 1978 (Beever et al.
1984) the etiology and epidemiology of the disease were not understood. Fruit that
developed storage rot were symptomless at harvest and the first sign of disease
appeared after about 4 weeks of storage at 0°C (Pennycook 1985; Manning et al.
1995). It was initially supposed, by analogy with the etiology of botrytis fruit rot in
strawberry, that infection of the fruit occurred at or just after flowering, from flower
parts colonised by B. cinerea that adhered to the fruit. Infection that supposedly
occurred around flowering was believed to remain latent during the fruit develop-
ment period, with symptoms appearing only during cool storage (Pennycook 1985).
However, field observations and early epidemiological studies could not show
consistent relationships between flower botrytis and storage rot incidence. It was
subsequently determined that infection occurs through the picking wound during
harvest, as a result of B. cinerea spores (conidia) deposited at the picking wound
13 Non-fungicidal Control of Botrytis Storage Rot in New Zealand 185
during orchard handling, packing and packhouse grading operations (Pennycook

1985). The conidia germinate in the uppermost ruptured cell layers of the picking
wound and the germ tubes grow rapidly into the fruit’s vascular tissues (Hallett and
Sharrock 1993).
13.1.3 Attempts at Control
Early attempts to control botrytis storage rot used fungicides applied in the orchard
before harvest. Although reductions in rot incidence could be demonstrated experi-
mentally (Pennycook 1988), fungicides were only partly effective (Beever et al.
1984; Pyke et al. 1994). When the etiology of the disease was understood, it was
realised that field-applied fungicides only indirectly affect botrytis storage rot by
reducing inoculum in the orchard. They cannot directly prevent infection of the
picking wound. The use of dicarboximide and benzimidazole fungicides in the
orchard led to the development of populations of B. cinerea in kiwifruit orchards
that were resistant to both these groups of fungicides (Manning and Pak 1995).
Although postharvest treatment of fruit with fungicides showed some ability to
reduce storage rot incidence (Pennycook 1985; Pyke et al. 1994), this was never
adopted as a commercial practice because the New Zealand kiwifruit industry did
not wish to market fruit treated with postharvest fungicides. Postharvest hot air
treatment and hot water treatment were also investigated experimentally (Cheah
et al. 1993). Hot air treatment at temperatures ranging from 38–60°C for times
ranging from 2–40 min gave no reduction in botrytis storage rot. Although hot
water treatment at 46–48°C for 8–15 min gave a significant reduction in storage
rot without apparent detrimental effects to the fruit, this approach was not adopted
by the kiwifruit industry. The postharvest systems used by the kiwifruit industry
were designed for handling dry fruit and hot water treatment was not compatible
with this.
13.2 Epidemiology of Botrytis cinerea in Kiwifruit Orchards
13.2.1 Introduction
In order to resolve the botrytis storage rot problem, new control strategies were
required that did not depend on pre- or postharvest fungicides, nor on other control
methods that were incompatible with the fruit handling and marketing requirements
of the kiwifruit industry. Epidemiological information about B. cinerea in orchards
was required, to determine whether orchard management practices could be manip-
ulated in any way to lessen the risk of storage rot.
Although it was understood as early as 1985 that the picking wound was the
infection site by which B. cinerea entered the fruit (Pennycook 1985), it was not
known where the sources of inoculum for infection were. B. cinerea had been found
on kiwifruit flower parts, necrotic leaves, wind damaged canes, cane prunings on
the ground, fallen fruit and in understory weeds. However, there was no knowl-
edge about how B. cinerea populations from these sources change during the
season, particularly in relation to harvest date and the likelihood of infection of
the picking wound.
A series of studies to resolve the epidemiology of botrytis storage rot was com-
missioned in the early 1990s by the then New Zealand Kiwifruit Marketing Board,
which subsequently became the kiwifruit marketing company, ZESPRI™ Group.
Considerable government-funded research into botrytis storage rot also occurred
during the 1980s and early 1990s, which at its peak, involved up to 11 plant pathol-
ogists. In 1992 the two government departments carrying out botrytis storage rot
research, Plant Diseases Division of the Department of Scientific and Industrial
Research and MAF-Technology of the Ministry of Agriculture and Fisheries, were
dis-established and were replaced by the Crown Research Institute, HortResearch.
From 1990 onwards government funding came from a single contestable pool
administered by the Foundation for Research Science and Technology. These
changes resulted in a substantial shift in research funding on botrytis storage rot
from mostly government to mostly industry and they were accompanied by an
increased clarity of purpose to solve the storage rot problem. The botrytis epide-
miology research from the 1990s was detailed in kiwifruit industry reports and an
overview of the methodologies and key outcomes from these studies is sum-
marised below.
13.2.2 Methods
Populations of B. cinerea in the leaf canopy of ‘Hayward’ kiwifruit orchards, vine

canopy characteristics and factors affecting infection of fruit through picking
wounds at harvest were studied in commercial orchards in South Auckland and the
Bay of Plenty in 1992 and 1993. These studies were made during the late fruit
development period (April–May, just prior to commercial harvest) in orchards with
high or low risk of botrytis storage rot, as determined by the history of storage rot
incidence over the previous 4 years. In each region the high and low risk orchard
blocks were approximately 1 ha in area, were within 10 km of each other and all
used pergola training systems.
13.2.2.1 Manipulating Vine Canopy Density in Orchard Plots
To determine how vine canopy management influenced B. cinerea populations, two

canopy density treatments were imposed, each in ten replicated single vine plots in
each orchard. Canopy density was manipulated with different pruning strategies,
whereby vines in the low density treatment were pruned to 2.4 canes/m in winter
and received a standard commercial summer pruning regime, whereas vines in the
high density treatment were pruned to 3.3 canes/m in winter and received no
summer pruning.
Canopy density was measured weekly in each plot using 0.5 × 0.5 m wire quad-
rats at four locations per plot. Total number of leaves, total leaf area, mean leaf area
index (LAI) and percentage of leaf tissue that was necrotic were determined. Total
and necrotic area was measured with a LICOR leaf area meter.
13.2.2.2 Measuring B. cinerea on Leaves in the Orchard
The amount of B. cinerea colonisation of necrotic leaf tissue was determined by

weekly sampling of 40 discs (12 mm diameter) from necrotic leaf lesions on other-
wise healthy leaves in each plot. After collection, discs were incubated at room
temperature under moist conditions for 16–24 h and the degree of B. cinerea colo-
nisation was determined as the proportion of discs with B. cinerea conidiophores.
13.2.2.3 Effect of Fruit Handling Method at Harvest on Infection

by B. cinerea
To test the hypothesis that infection of the fruit resulted from the transfer of
B. cinerea conidia from the fruit surface to the picking wound, and to determine
whether postharvest fruit handling could influence the amount of botrytis storage
rot, three different methods of handling fruit after picking were investigated in 1992
and 1993: (1) direct picking into trays, (2) picking into bags followed by transfer to
trays, (3) picking into bags, then jostling the fruit in the bags followed by transfer
to trays. Thirty trays of fruit (33 fruit/tray) were subjected to each treatment. Fruit
were assessed for incidence of botrytis stem end rot after 10 weeks of storage at
0°C in 1992 and after 20 weeks in 1993. These fruit came from an orchard with a
history of high botrytis storage rot incidence.
13.2.3 Results
13.2.3.1 Necrotic Leaf Tissue in Relation to Vine Canopy Density
Quadrat sampling showed that the mean number of leaves with necrosis and the
percentage of leaf area that was necrotic increased markedly during the month lead-
ing up to commercial harvest (early May) in both years of the study. Data for
necrotic leaf area from the high risk block in South Auckland in 1992 are shown in
Fig. 13.1. Necrotic leaf area continued to increase between harvest and leaf fall
(data not shown).
Mean necrotic leaf area (%) 12
10
Y = 1.5428 * Ln(X) + 5.9096

6 R2 = 0.98
4
6/04 / 1992
1/05 / 1992
6/05 / 1992
11/04 / 1992
16/04 / 1992
21/04 / 1992
26/04 / 1992
11/05 / 1992
Fig. 13.1 Increase in the mean percentage of necrotic leaf area per quadrat with time in a high
risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992. Values are the means of 10 single
vine plots (4 quadrats per plot). The X-variate for the fitted line is days from 7 April 1992
The development of necrosis appeared to be associated with leaf senescence

arising from shading and ageing of leaves within the canopy. Although a substantial
proportion of the necrotic leaf tissue eventually became colonised by B. cinerea, it
is believed that B. cinerea was a secondary invader and not the primary cause of the
leaf necrosis.
The amount of leaf necrosis was greater in plots with greater canopy density, as
measured by LAI. For measurements made on 13 May 1992, there was an exponen-
tial increase in both number of dead leaves and necrotic leaf area with increasing
LAI (Fig. 13.2).
13.2.3.2 B. cinerea Populations on Necrotic Leaves
B. cinerea populations on necrotic leaf tissue in the orchard increased with time
during the weeks leading up to harvest, as shown by the increasing incidence of
necrotic leaf discs that were colonised by B. cinerea (Fig. 13.3).
However, the amount of necrotic leaf tissue alone was not a good predictor of
the B. cinerea population, because necrotic tissue may or may not be colonised. The
total size of the B. cinerea population (percentage of leaf canopy area colonised per
plot) was estimated as the product of the proportion of leaf discs colonised by
B. cinerea and the percentage of leaf canopy area per quadrat that was necrotic. The
total B. cinerea population increased exponentially in the period leading up to har-
vest in both high and low risk orchards, although it occurred at a greater rate in high
6
No. necrotic leaves
Y = .56 + exp(5.54 * X - 15.90)

4 R2 = 0.94
0
2.2 2.4 2.6 2.8 3.0 3.2 3.4
Leaf area index
b
1300
Necrotic leaf area (cm2)
1100
Y = 476.4 + exp(3.17 * X - 3.64)
900 R2 = 0.73
700
500
300
2.2 2.4 2.6 2.8 3.0 3.2 3.4
Leaf area index
Fig. 13.2 Mean number of necrotic leaves (a) and mean necrotic leaf area (b) per quadrat in rela-
tion to leaf area index, for the high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992
risk orchards (Fig. 13.4). This showed that the botrytis storage rot history from the
high and low risk orchards was strongly correlated with the pre-harvest B. cinerea
populations.
A strong relationship was found between the B. cinerea population in the
orchard, measured using necrotic leaf discs, and storage rot incidence (Fig. 13.5).
This relationship was robust for leaf disc samples taken at various times, including
the average B. cinerea incidence for six weekly samples prior to harvest (Fig. 13.5),
a single sample at harvest (data not shown) or the average of four samples during
the week after harvest (data not shown). Any of these explained >70% of the varia-
tion in storage rot incidence.
Incidence of leaf discs with B. cinerea (%)

0.9 Y = 0.06 + exp(0.04 * X - 1.92)
R2 = 0.79
0.7
0.5
0.3
0.1
6/04 / 1992
1/05 / 1992
6/05 / 1992
11/04 / 1992
16/04 / 1992
21/04 / 1992
26/04 / 1992
11/05 / 1992
16/05 / 1992
Fig. 13.3 Increase over time in the incidence of leaf discs colonised by Botrytis cinerea in the
high risk ‘Hayward’ kiwifruit orchard at South Auckland in 1992. The X-variate for the fitted line
is days from 7 April 1992
3.0
High risk orchard
Canopy area colonised (%)
2.5 Low risk orchard
2.0 Y = 0.15 + exp(0.055 * X - 2.8)

R2 = 0.85
1.5
1.0
0.5 Y = 0.02 + exp(0.046 * X - 3.0)

R2 = 0.88
0.0
14/03 / 1993
24/03 / 1993
3/04 / 1993
13/04 / 1993
23/04 / 1993
3/05 / 1993
13/05 / 1993
23/05 / 1993
2/06 / 1993
Fig. 13.4 Increase in percentage of leaf canopy area colonised by Botrytis cinerea in high and
low risk ‘Hayward’ kiwifruit orchards at South Auckland in 1993. The X-variate for the fitted
lines is days from 15 March 1993
20
Incidence of botrytis storage rot (%) 15
10
Y=9.577 + 5.944 log(X+0.1)

5 R2= 0.79
0
0 2 4 6 8
Incidence of leaf discs with B. cinerea (%)
Fig. 13.5 Incidence of botrytis storage rot in relation to incidence of Botrytis cinerea on leaf discs
in the orchard, as the average of six samples taken weekly leading up to harvest in the high risk
‘Hayward’ kiwifruit orchard at South Auckland in 1992
13.2.3.3 Effect of Fruit Handling Method at Harvest on Infection

by B. cinerea
Direct picking of fruit into trays resulted in lower storage rot incidence in both 1992
and 1993 than treatments that involved further handling in picking bags (Table 13.1).
Jostling of fruit resulted in the greatest disease incidence, showing that further
handling increases the opportunity for transfer of inoculum to the picking wound.
Although commercial grading of fruit was not included as a treatment, it is reason-
able to assume that the additional handling would further increase the opportunity
for transfer of inoculum to the picking wound.
Table 13.1 Incidence of botrytis rot in cool stored ‘Hayward’ kiwifruit in 1992 and 1993 in rela-
tion to three fruit handling treatments. After picking, fruit were either placed directly into trays,
or into picking bags with and without jostling and then into trays. In 1992 the fruit were assessed
after 10 weeks at 0°C and in 1993, after 20 weeks at 0°C
Botrytis storage rot Botrytis storage rot
Fruit handling treatment incidence (%) 1992 trial incidence (%) 1993 trial
Placed into trays directly 7.0 a a 1.6 a
Placed into picking bags 15.5 b 4.4 ab
Placed into picking bags then jostled 22.1 c 7.2 b
a
Means within a column followed by the same letter are not significantly different (LSD0.05).
13.2.4 Discussion
These studies showed that the ‘Hayward’ kiwifruit that are most at risk from botrytis
storage rot are those within dense kiwifruit vine canopies, where necrotic leaf tissue
is colonised to a high degree by B. cinerea. Fruit that are least at risk are those
within open canopies that are exposed to low B. cinerea inoculum levels. The most
likely source of inoculum for botrytis storage rot is from populations of B. cinerea
that increase on dead leaf tissue during the period leading up to harvest. Leaf necro-
sis within the vine canopy increases during this period as a result of ageing and
shading of leaves. The denser the canopy, the greater the rate at which necrosis
increases.
Although the amount of necrotic leaf tissue determines the potential B. cinerea
population, this tissue may or may not become colonised by B. cinerea. Colonisation
presumably depends on suitable environmental conditions for growth and spread of
the fungus, with more dense leaf canopies likely to provide a more favourable envi-
ronment. It appears that B. cinerea is a secondary invader of necrotic tissue and not
the primary cause of the necrosis. The leaf disc method was developed to quantify
the degree of colonisation of necrotic leaf tissue and it was subsequently incorpo-
rated into a prediction system for botrytis storage rot (see below).
13.3 Prediction and Management of Botrytis Storage Rot
13.3.1 Prediction Systems
The understanding of botrytis storage rot resulting from epidemiological studies

underpinned the development of prediction systems designed to identify which
lines of fruit were at greatest risk. Several postharvest prediction methods were
investigated, including protocols for withdrawing sample trays from lines of fruit
and accelerating the development of rots in the sample at warm temperatures.
Another method used agar plugs dabbed on to the picking wounds to assess
B. cinerea presence at the infection site. For various practical reasons, these meth-
ods were not taken up by the kiwifruit industry.
The method that was adopted, and is still used, by the kiwifruit industry is the
assessment of leaf discs, as described above, to assess pre-harvest B. cinerea inocu-
lum in the orchard (Manning and Pak 1993). Relationships of the sort shown in
Fig. 13.5 allow prediction of storage rot risk in individual lines of fruit. This infor-
mation can be used to make management decisions about the fruit inventory from
a particular harvest season. For example, it is possible to identify which lines of
fruit should be inspected for rots before shipping or which lines are less suitable for
long term controlled atmosphere storage.
13.3.2 Vine Canopy Management to Lessen Disease Risk
The demonstration of relationships between leaf canopy density, leaf necrosis and
botrytis storage rot risk led to the use of canopy density manipulation as the pri-
mary means to control storage rot. Canopy density is manipulated through winter
and summer pruning strategies. However, reduction in LAI for storage rot control
must be balanced against the need to retain adequate LAI to sustain economic kiwi-
fruit yield, as discussed by Buwalda and colleagues (Buwalda et al. 1992). The
mechanism by which canopy density influences B. cinerea colonisation is presum-
ably through restricted air movement, reduced radiation and greater humidity
favouring growth and sporulation of B. cinerea.
13.3.3 Management of Fruit Susceptibility to Disease
13.3.3.1 Effect of Harvest Date on Fruit Susceptibility
During the 1980s there were anecdotal indications that harvest and postharvest
management practices could influence the amount of botrytis storage rot. Harvest
date, was one factor that came into focus, particularly the maturity at which kiwi-
fruit were harvested. Until the early 1990s it was preferred to harvest fruit as early
as possible, to exploit market opportunities and to avoid the risk of damage from
early autumn frosts. Harvesting at a fruit soluble solids concentration of 6.2°Bx was
recommended although there was a trend towards earlier harvesting, at about
6.0°Bx. An analysis of field data collected at eight orchards over 4 years showed
that storage rot incidence tended to be greater in fruit harvested at lower soluble
solids concentrations (Fig. 13.6). It was clear that fruit harvested later, at 7–8°Bx,
60
1993
50
Botrytis storage rot
1994
incidence (%)
1995
40
1996
30
20
10
0
5 6 7 8 9 10
Soluble solids conc. at harvest (8Brix)
Fig. 13.6 Incidence of botrytis storage rot in kiwifruit after about 13 weeks of storage at 0°C in
relation to the mean concentration of soluble solids in fruit at harvest. Data were collected from
eight commercial ‘Hayward’ kiwifruit orchards (two orchards per year) in South Auckland and
the Bay of Plenty in New Zealand over 4 years
were much less susceptible to botrytis than fruit harvested earlier. Thus by harvesting
fruit at a higher soluble solids concentration, it has been possible to lessen the risk
of botrytis storage rot greatly.
13.3.3.2 Curing of the Picking Wound
Botrytis storage rot incidence is greatly reduced when the picking wound is “cured”
by storing fruit for 48 h at ambient temperature before cooling. This phenomenon
was first reported anecdotally during the late 1980s and was defined experimentally
by Pennycook and Manning (1992). They inoculated picking wounds of fruit with
B. cinerea conidia and held fruit at c. 14°C for different times before placing in cool
storage at 0°C. They demonstrated a decrease in the susceptibility of fruit to botrytis
storage rot when fruit were held at 14°C over a 7-day period (Fig. 13.7). Lallu et al.
(1997) subsequently showed that the majority of curing occurs within the first 48 h
after picking. They also showed that excessive curing times can lead to increased
incidence of fruit softening, although a further delay of up to 48 h between packing
and pre-cooling was not detrimental. The efficacy of curing against botrytis storage
rot appeared to be enhanced by a slower rate of cooling when the fruit were brought
down to storage temperature.
The mechanism by which curing works is not well understood, although it
appears to be related to loss of water from fruit. Curing does not occur if there is
no weight loss during the curing period, and it is more effective at low humidity and
where there is air movement around fruit. It does not appear to be caused merely
by suberisation of the picking wound.
30
Botrytis storage rot incidence (%)
25
20
15
10
0
0 2 4 6 8
Delay before start of cool storage (days)
Fig. 13.7 Incidence of botrytis storage rot in ‘Hayward’ kiwifruit after 13 weeks of storage at 0°C
in relation to delay before cooling. Fruit were kept at ambient temperature (14–18°C) for various
times after picking, inoculated with Botrytis cinerea conidia, then placed into storage at 0°C
13.4 Conclusions
Botrytis storage rot was a serious problem affecting production and marketing of
‘Hayward’ kiwifruit in New Zealand from the late 1970s to the early 1990s.
Pre-harvest fungicide sprays used in early attempts at control failed because they
did not protect the picking wound against infection and because fungicide resistance
developed in B. cinerea field populations. The kiwifruit industry rejected use of
postharvest fungicides to prevent infection because it did not wish to market
fungicide-treated fruit. A concerted research effort, investigating interactions
between the biology of B. cinerea and the physiology of kiwifruit vines and fruit, led
to an understanding of pre- and postharvest factors that cause botrytis storage rot.
B. cinerea is ubiquitous in kiwifruit orchards, growing mainly on dead leaf
tissue in the vine canopy. Spores accumulate on the fruit during the growing season
and are transferred to the picking wound at harvest, where they infect. More
B. cinerea inoculum occurs in dense vine canopies because restricted light penetra-
tion leads to increased leaf necrosis. There is also less air movement in dense cano-
pies and this provides humid conditions favourable for production of B. cinerea
inoculum. High-inoculum orchards can be identified using the leaf disc method and
this has been incorporated into a prediction system to determine lines of fruit that
are at risk of storage rot. However, good vine canopy management means the pre-
diction system is not often required.
The successful control of this disease without fungicides has been achieved by
vine canopy management that avoids dense leaf canopies. Harvesting fruit at a
higher sugar content minimises fruit susceptibility and delayed postharvest cooling
to “cure” the fruit also reduces their susceptibility. These research findings have
been incorporated into the botrytis management programme in the industry’s
manual on integrated pest management. This research has ultimately led to the
current situation where botrytis storage rot is a relatively minor problem for
the New Zealand kiwifruit industry.
Acknowledgments Thanks are due to Zespri™ Group for permission to summarise and present
the data contained in kiwifruit industry reports and to the Foundation for Research Science and
Technology for funding research presented in this chapter.
References
Beever DJ, McGrath HJW, Clarke DL, Todd M (1984) Field application and residues of fungicides
for control of botrytis storage rot of kiwifruit. NZ J Exp Agric 12:339–346
Buwalda JG, Meekings JS, Curtis JP (1992) Where in the canopy is photosynthesis highest?
NZ Kiwifruit 95 (November):14–15
Cheah LH, Irving DE, Hunt AW (1993) Postharvest heat treatments for botrytis control.
NZ Kiwifruit 96 (February):26
Hallett I, Sharrock K (1993) Penetrating the picking scar’s defences. NZ Kiwifruit 96
(February):7–9
HortResearch 2007 Fresh facts New Zealand Horticulture (2007) HortResearch – the Horticulture
and Food Research Institute of New Zealand Ltd, Private Bag 92169 Auckland (www.
hortresearch.co.nz), pp 33
Lallu N, Manning M, Pak H (1997) Best curing practices for orchard and packhouse. NZ Kiwifruit
J 120 (March/April):35–36
Manning MA, Pak HA (1993) Production of botrytis rots. NZ Kiwifruit 96 (February):22–23
Manning MA, Pak HA (1995) Botrytis storage rot of kiwifruit: efficacy of pre-harvest sprays
in orchards with dicarboximide-resistant botrytis populations. Proceedings of the 48th
New Zealand Plant Protection Conference, pp 22–26
Manning MA, Pak HA, Pennycook SR (1995) Timing condition checking to catch Botrytis rots.
NZ Kiwifruit 107 (February/March):24–25
Pennycook SR (1985) Fungal fruit rots of Actinidia deliciosa (kiwifruit). NZ J Exp Agric
13:289–299
Pennycook SR (1988) Some straight talking on botrytis control. NZ Kiwifruit 43 (February):15
Pennycook SR, Manning MA (1992) Picking wound curing to reduce botrytis storage rot of
kiwifruit. NZ J Crop Hortic Sci 20:357–360
Pyke NB, Manktelow DG, Elmer PAG, Tate KG (1994) Postharvest dipping of kiwifruit in iprodi-
one to control stem-end rot caused by Botrytis cinerea. NZ J Crop Hortic Sci 22:81–86
Chapter 14
The Peach Story
Paloma Melgarejo, Antonieta De Cal, Inmaculada Larena, Iray Gell,

and Belen Guijarro
Abstract One of the most important postharvest disease of peaches is brown rot caused
by different species of the fungus Monilinia. Anamorphs dominate as inoculum sources
especially in the Mediterranean areas of Europe, where brown rot in peaches is caused by
M. laxa and M. fructigena. A third species, M. fructicola causes brown rot in other parts
of the world and is included in the A2 list of quarantine organisms for Europe (organ-
isms present in the EPPO region, but contained, under official control) because its broad
dissemination in Europe would be devastating especially for peach and nectarine. These
fungi overwinter and produce mycelium in fruit mummies and infected wood. This pro-
duces conidia under favorable conditions or from stromata that produce ascospores in the
case of M. fructicola. Fruit infection by conidia of Monilinia spp. can occur secondarily
from any infected tissue in which the moisture content is sufficient for sporulation. When
the microclimate is unfavorable, infections may remain latent until conditions favor
disease expression, which finally leads to fruit rot. Correlation between the incidence of
rotting and latent infection caused by Monilinia spp. has been reported. Management of
orchards focused to decrease postharvest brown rot will be treated here. Detection and
identification methods, inoculum sources, and epidemiological factors affecting latent
fruit infections and postharvest brown rot will be described, together with the models
available to predict disease risk. Different integrated control strategies are presented.
14.1 Introduction
Peaches are commodities produced in template areas of the world. Production in

Europe is mainly restricted to Mediterranean countries such as Italy, Spain, Greece
and France. In Spain crop surface is increasing in the last years being approx.
12,000 ha in 2007, with a production of approx. 110,000.000 kg.
P. Melgarejo (*), A. De Cal, I. Larena, I. Gell, and B. Guijarro

Department of Plant Protection, Instituto Nacional de Investigación y Tecnología Agrariay
Alimentaria, Crtra. De La Coruña km. 7, 28040, Madrid, Spain
e-mail: melgar@inia.es
198 P. Melgarejo et al.
Several pests and diseases may cause severe losses in peach orchards. Brown rot
caused by Monilinia spp. is a serious disease in Mediterranean areas. Direct yield
losses result from infection of flowers (flower and twig blight) and from fruit rot at
preharvest, harvest and postharvest.
Three Monilinia species may cause brown rot of peaches. M. fructigena and
M. laxa have been extensively reported in Europe. Monilinia fructicola occurs in
North and South America, Japan and Australia (EPPO 2007) and has been recently
introduced in France (Lichou et al. 2002), Austria (NPPO of Austria 2002) and
Spain (Petroczy and Palkovics 2006). This species, however is listed by EPPO as a
quarantine pest within the European Union (EPPO 2007) because a broad dissemi-
nation of this species in Europe would be devastating especially for peach, nectar-
ine and apricot. In addition this is the first time in which the three species coexist
in the same area.
14.2 Management of Brown Rot
The first step to manage brown rot is the detection and identification of the spe-
cies of Monilinia causing the disease. This issue is not only important for quar-
antine purposes, such in the case of M. fructicola to avoid new introductions in
areas free of this fungus, but it is also essential for managing brown rot caused
by all three Monilinia spp. Currently, identification of Monilinia spp. is based on
cultural and morphological characteristics and fungal isolation from the plant
material (De Cal and Melgarejo 1999), and molecular DNA-based methods. We
designed a detection protocol that combined a set of universal primers [(IMon3.1
(GCTCGCCAGAGAATAAYY) and IMon3.2 (AGACTCAATACCAAGCTGT)]
and the inclusion of the plasmid pGMON as an Internal Control for diagnosis
of all Monilinia spp, and three sets of primers to discriminate the three spe-
cies of Monilinia [ILaxaS (TGAGCACGAGTGAATGTATAG) and ILaxaAS
(TGAGCACGAGGGCATATC) for M. laxa, IGenaS (TGCTCTGCCCGTACCCAG)
and IGenaAS (GGATTTATTGTGATGTAGTTTCG) for M. fructigena, and IColaS
(GAGACGCACACAGAGTCAG) and IColaAS (GAGACGCACATAGCATTGG) for
M. fructicola] (Gell et al. 2007a).
In Spain control of brown rot is managed at pre- and postharvest. Pre-harvest
strategies include sanitary measures (removing of mummies and diseases twigs and
branches) and fungicidal treatments (at flowering and pre-harvest), and postharvest
con trol is base on a careful management of fruit, a quick coolness after harvest and
store at 0°C, and, in some cases, a washing with chloride solutions. Biological
control is also a new tool to be applied.
We have developed two biological control agents, Epicoccum nigrum (strain
282, EPI282) and Penicillium frequentans (strain 919, PF919) from the resident
mycoflora of peach twigs (Melgarejo et al. 1985). Application of conidia and
14 The Peach Story 199
mycelium of these fungal antagonists to peach tress reduced twig blight

caused by M. laxa (De Cal et al. 1990; Madrigal et al. 1994). We have also
developed an efficient mass production method of conidia of these fungi by solid
fermentation (De Cal et al. 2002; Larena et al. 2004). Different conidia formula-
tions of each biocontrol agents with enhanced adherence to fruit, dispersal in
water or/and stability were produced and tested for controlling brown rot (Larena
et al. 2005; Guijarro et al. 2007). Some of them had also and improved efficacy
in controlling the disease. In the following several examples are shown of our
biological control research.
14.3 Biological Control
Seven field experiments were carried out in peach orchards located in Spain, Italy
and France in 2001 and 2002 to develop an effective and practical method of con-
trolling brown rot disease caused by Monilinia spp. by pre-harvest applications of
EPI282 treatments in the framework of BIOPOSTHARVEST EU-Project (Larena
et al. 2005). Fresh or formulated (106–7 conidia mL−1) of EPI282 needed to be
applied twice both at bloom and pre-harvest to reduce postharvest brown rot
(Table 14.1). Chemical fungicides reduced disease in French and Italian trials but
not in a Spanish trial (Table 14.1). Integrated control (biological and chemical) was
efficient in controlling the pathogens (Table 14.1).
Postharvest treatments with EPI282 were also tested in Italy on natural and
artificial infections to nectarine over 3 years. EPI282, as fresh or formulated cells,
at a concentration of 108 conidia mL−1, were effective, significantly reducing the
incidence of brown rot compared to control, both under artificial and natural infec-
tion, from 43 to 100% (Mari et al. 2007) (Fig. 14.1).
Four wettable powder formulations of PF919 conidia with measurable viability
of 1 year and an improved adherence to peach surfaces were applied to fruit either
as postharvest treatments or before harvest in field experiments to peach trees
(Guijarro et al. 2007). In the case of postharvest treatments to fruit, reductions of
brown rot were obtained with all PF919 formulations. Treatments applied before
harvest were tested in six field experiments in peach orchards in Spain. PF919
formulations significantly reduced the inoculum density of the pathogen in five tri-
als out of the six tested, better than a chemical fungicide that only showed a reduc-
tion of the pathogen conidia in two of the six trials (Table 14.2).
All these trials showed that both, EPI212 and PF919, are interesting potential
biocontrol agents against brown rot of peaches, but that it is necessary to improve
their efficacy. Different approaches can be used such as to getting better formula-
tions, using integrated strategies (i.e. pre-harvest treatments and physicochemical
postharvest treatments). But, a better knowledge of the disease epidemiology is
undoubly important. We have begun a project to study the epidemiological basis to
200
Table 14.1 Percentage of decayed fruit by Monilinia spp. in trials carried out in different countries during 2001 and 2002 after different treatments
Spain Italy France
Treatmenta SP1 2001 IT1 2001 IT2 2001 IT3 2002 FR1 2001 FR2 2001 FR3 2002
T1 (CB106 × 4) 46 ± 10 39 ± 3 37 ± 4 74 ± 8 64 ± 2 68 ± 3 61 ± 8
T2 (CB107 × 4) – – – 72 ± 6 – – –
T3 (CBF106 × 4) – – – 70 ± 3 – – –
T4 (CB106 × 2) 77 ± 8 59 ± 3 54 ± 15 – – – –
T5 (CB106 × 2) 72 ± 11 61 ± 4 57 ± 5 – – – –
T6 (CQ) 59 ± 10 29 ± 2 28 ± 6 69 ± 2 34 ± 2 44 ± 6 36 ± 5
T7 (CI1) – 42 ± 1 40 ± 7 66 ± 3 – – 70 ± 12
T8 (CI2) – 28 ± 1 29 ± 2 57 ± 7 – – 32 ± 7
T9 (NT) 80 ± 6 64 ± 2 58 ± 11 84 ± 4 67 ± 4 62 ± 5 74 ± 8
Note: Data are the mean of four replicates ± standard error of the mean.
a
CB = biological treatments; CB(106 × 4) = biological treatments applied four times at 106 conidia mL−1; CB(106 × 2) = biological treatments applied twice
at 106 conidia mL−1; NT = untreated; CBF = formulated cells of EPI282; CB(107 × 4) = biological treatments applied four times at 107 conidia mL−1;
CQ = chemical treatments; CI = Integrated treatments
P. Melgarejo et al.
100
a b
80 a
a a
a
Infected fruits (%)
60
b b
a* a
b
40
b
20 b
a a a
nd b nd b
0
2001 2002 2003 2001 2002 2003
Control Fresh cells Dry cells FOR4
Fig. 14.1 Effects of postharvest treatments with Epicoccum nigrum fresh cells (FC), dry cells
(DC) or formulation FOR1 at different concentrations on artificially (a) and naturally (b)
Monilinia laxa infected nectarine. FC (at a concentration of 106 conidia mL−1) were applied in
2001; FC (107 conidia mL−1) or DC (106 conidia mL−1) in 2002, and FC or FOR1 (108 conidia
mL−1) in 2003. nd: not determined. *Different letters, within the same year, show a significant
difference (P < 0.05) according LSD Test
control postharvest brown rot of peaches caused by Monilinia spp. Different

aspects have been already studied.
14.4 Epidemiology of Brown Rot in Spanish Conditions
The sources of primary inoculum were studied in nine commercial orchards of the
Ebro Valley region of Spain during 6 years (2003–2005). Mummified fruit, twigs
and pits have been identified as plant organs carry the pathogen from year to year
in peach grown Spanish orchards. However, no relationships between any of these
sources and the numbers of conidia on the fruit surface, or incidence of latent infec-
tion, or brown rot were found (Gell et al. 2008).
To evaluate the effect of conidial density of Monilinia spp on fruit surface on the
incidence of latent infection and brown rot in peaches eleven field surveys were
performed in commercial orchards located in Cataluña, Spain, over four growing
seasons from 2002 to 2005 (Gell et al. 2009). There was a significantly positive
relationship (r = 0.69) between the numbers of conidia of Monilinia spp. on the
fruit surface and the incidence of latent infections caused by Monilinia spp. in stone
fruit. The effect of environmental temperature (T), intensity of solar radiation (SR),
202
Table 14.2 Effect of PF909 formulations on population of Monilinia spp. (number of conidia per fruit) in different orchards where chemical and biological
treatments were applied along crop during 2003 to 2005
2003 2004 2005
Treatmenta ALF03 SU03 ALF04 SU04 ALF05 SU05
FOR3 42,969 ± 5,750 0.0 ± 0.0 2,930 ± 1,870 4,883 ± 1,224 – –
FOR4 59,570 ± 6,046 6,835 ± 3,335 – – – –
FOR7 – – 4,883 ± 976 – – –
FOR8 – – 0.0 ± 0.0 1,953 ± 738 4,400 ± 1,871 488 ± 488
Chemical 39,062 ± 5,289 2,930 ± 1,870 4,639 ± 2,008 2,930 ± 1,224 4,394 ± 2,497 5,859 ± 1,476
Untreated 48,828 ± 6,478 6,836 ± 4,331 24,170 ± 7,191 7,812 ± 1,808 23,926 ± 6,196 12,207 ± 3,417
Note: Data are the mean of four replicates with five fruit per replicate ± standard error of the mean.
P. Melgarejo et al.
rainfall (R) and wind speed (WS) on the area under the number of conidia of
Monilinia spp. curve (AUncC) on peach surfaces was analysed using a multiple
regression model. The results of regression analysis revealed that T, SR, R, and WS
could account for 99% of area of the AUncC on peach surfaces, following the
equation:
AUncC (T , WS , SR, R ) = -1.9x10 7 - 4.4x10 5 xSRxRxWS +
5.6x10 4 xTxSRxR + 2.5x10 5 xWS 2 xSR - 9.8x10 4 WS 2 xT (1)
(2.0 × 106) (4.9 × 104) (5.6 × 104) (3.9 × 104) (3.3 × 104)
(R = 0.986)
2
The main finding from this study is that in order to reduce the incidence of latent
infection and brown rot it is essential not only to remove the sources of primary
inoculum but to reduce also the number of Monilinia spp. conidia on the fruit surface.
Furthermore, the sources of airborne conidia of Monilinia spp. that are deposited
on fruit surfaces should be taken into consideration in disease management
programmes in Spain.
The correlation between latent infections and postharvest brown rot was demon-
strated in cherries infected with M. laxa and M. fructigena (Xu et al. 2007), and
plums and nectarines infected with M. fructicola (Emery et al. 2000; Luo and
Michailides 2001). Five field experiments were performed in commercial orchards
located in Cataluña (Spain) over three growing seasons, 2000–2002, in order to
estimate the relationship between the incidence of latent infection caused by
Monilinia spp. in peaches and the incidence of postharvest brown rot in Spanish
conditions (Gell et al. 2008). No latent infection was recorded at popcorn and the
maximum incidence occurred pre-harvest; in some orchards a second peak was
detected during the pit hardening period. M. laxa is the most prevalent species iso-
lated from peaches with brown rot. There was a positive correlation between the
incidence of latent infection and that of postharvest brown rot:
[Arcsine(z) = - 0.39 + 3.48 Arcsine(y)] (2)
(3.77) (0.45)
The average incidence of latent infection during the crop season explained 55% of
the total variation in the incidence of postharvest brown rot. The effect of tempera-
ture (T) and duration of wetness (W) on the incidence of latent infection (y) in peach
and nectarine orchards was analysed using multiple regression. The regression
analysis indicated that T and W jointly explained 83% of the total variation in the
incidence of latent infection (y) by the equation:
Arcsine (y) = 12.53 – 0.35WT + 0.002T2W2 + 0.00019T3W (3)
(4.138) (0.081) (0.0004) (0.00006)

24
latent 18
100
50
0 12
25
W
20
15 6
T 10
5
0
0
Fig. 14.2 Response surfaces predicting the incidence of latent infection (%) in peach and nectar-
ine fruit at a range of durations of wetness (W) and temperatures (T). The surface was generated
using Eq. (3)
The model predicts no latent infections when T < 8°C, and >22 h wetness required
when T = 8°C but only 5 h at 25°C necessary for latent infection to occur (Fig. 14.2).
The incidence of brown rot and latent infection of peaches caused by M. laxa under
controlled experimental conditions was also affected by T and W, as well as by fruit
maturity and inoculum concentration. Latent infections were produced in fruit
when T was not suitable for the development of brown rot symptoms. In these
experiments more than 4–5 h of daily wetness were required after embryo growth
in fruit sprayed to run-off with an inoculum concentration higher than 104 conidia
of M. laxa per ml for brown rot and latent infections to develop. The fitted model
obtained from the field data was able to predict the observed data obtained under
controlled environmental conditions. Present results demonstrate that latent infec-
tion should be taken into consideration in disease management programmes in
Spain. Although brown rot may not be severe at harvest, it could develop later
because of the high incidence of latent infection. A control strategy should be based
on estimation of the risk of latent infection, assessed on the basis of the effects of
temperature and duration of wetness. The quantitative relationships of latent infec-
tion with temperature and duration of wetness could be used to develop a risk
assessment and disease prediction system for brown rot.
Finally, a study of the genetic diversity of M. laxa populations in peach orchards
in Spain was carried out using 144 RAPD markers (59 polymorphic and 85 mono-
morphic) on 21 isolates collected from several orchards (subpopulations), in various
years and in various hosts (Gell et al. 2007b). The analysis of population structure
Fig. 14.3 The UPGMA dendrogram showing the genetic similarity among isolates of Monilinia
laxa sampled in different orchards in Spain, and analyzed by RAPD. The percentages below the
branches are the frequencies with which a given branch appeared in 1,000 bootstrap replications.
Bootstrap values below 50% are not displayed
revealed that genetic diversity within orchards (HS) accounted for 97% of the total
genetic diversity (HT), while genetic diversity among orchards represented only 3%.
The relative magnitude of gene differentiation between subpopulations (GST) and
the estimate of the number of migrants per generation (Nm) averaged 0.032 and
15.1, respectively. The results obtained in dendrograms were in accordance with the
gene diversity analysis (Fig. 14.3). Grouping of isolates in the dendrogram was
independent of whether they came from the same or different orchards. There was no
relationship between clustering among isolates from distinct years and hosts.
Our analysis of M. laxa populations using RAPD markers revealed that most of the
genetic diversity is present within subpopulations (orchards). The hypothesis of
geographic subdivision, with orchards as subpopulation units, is based on the use
of distinct management practices in different orchards. Continuous gene flow must

occur between orchards, thereby preventing differentiation between subpopula-
tions. Our results confirm that the spread of disease from one orchard to another
occurs frequently. We estimated the number of migrants between orchards to be
Nm = 15.1, indicating the importance of migration in preventing genetic differentia-
tion. This flow of migrants has severe implications for the management of brown
rot, in particular for the management of sanitation practices. Orchards having good
sanitation practices which maintain them free or with low densities of inoculum can
be infected by inoculum coming from other orchards. Disease control strategies
should be designed at the local or regional scale because practices have implica-
tions on other orchards.
Another important factor to be taken into account is the potential high risk of
rapid spread of M. fructicola from one orchard to another. This species has recently
been detected in Spain (Petroczy and Palkovics 2006). If M. laxa inoculum easily
spread from orchard to orchard, it is assumed that the same will occur with
M. fructicola. Once entry of a quarantine organism has been achieve in a new area,
the pathogen will encounter favourable conditions for spread and establishment.
The economic impact of establishment of M. fructicola in Spain, and hence in
Europe was estimated as considerable (van Leeuwen et al. 2000). Direct fruit losses
will increase and export markets will be affected. Cost of control will increase, and
control measures might become less efficient because of the development of fungi-
cide resistance. In addition, and as M. fructicola shares common hosts with M. laxa
and M. fructigena, interactions between the different species may occur, even at the
fruit or the flower scale. At the moment M. laxa is the prevalent species in orchards
in Spain, but, after introduction and establishment of M. fructicola, this situation
may change. M. fructicola grows faster and sporulates more abundantly than
M. fructigena and M. laxa, having a better ability for dispersal (van Leeuwen and
van Kesteren 1998; De Cal and Melgarejo 1999).
Screening of fungicide resistance to benzimidazole and dicarboximide fungi-
cides was made among populations of M. laxa in Spain. No isolate out of 114 tested
was found resistant to either group of fungicides (Gell 2008).
Acknowledgements We thank Dr. Usall, Dr. Torres, N. Lamarca, and M. Mari for collaboration
in experiments. This work was supported by projects AGL2002-4396-CO2, RTA2005-00077-CO2
from the Ministry of Science and Innovation (Spain), and QoL-PL-1999-01065 from the European
Commission. We thank X. O. de Eribe and growers for their support and collaboration.
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Index
A Chitosan, 34, 122, 128

Acibenzolar, 32–33, 122 Cladosporium, 5, 32
Active defense, 18–19 Colletotrichum
Alternaria, 3–5, 32–35, 44, 46, 47, 72, 75, 80, acutatum, 141
85, 86, 175 gloeosporioides, 4–10, 45–48,
Alternaria alternata, 3, 8, 32–35, 46, 47, 80, 52, 173
85, 175 Control postharvest diseases, 31–37, 72, 86,
Alternative methods, 58, 90, 108, 120, 121, 89–102, 137–145
123, 127–129
Antagonists, 22, 90, 120, 138, 150, 173, 199
Anthracnose, 4, 5–9 D
Antifungal substances Decaying fruits, 32, 43–53, 90, 102, 107, 112,
avocado preformed, 2, 9–10 140, 142
induced, 2, 10
mango preformed, 2–9
Aspergillus, 49, 51, 172 E
Elicitors, 14, 17, 31–37, 52, 58, 122, 130,
174, 176
B Epidemiological assessment,
b-Aminobutyric acid (BABA), 34 69–87
Bioactive compounds, 120–122, 124 Epidemiology, 69–87, 125, 184–192,
Biocontrol agents 199, 201–206
formulations, 99, 102, 120, 123, 129, 139, Ethylene (ET), 10, 16, 19–23, 25, 52, 58–65,
149–166, 199, 201, 202 80, 156, 174–176, 184
Biocontrol treatments, 90–94, 102, 140, 145,
158–162, 173, 178, 199, 200
Biological control, 32, 33, 35–37, 58, 90, 93, F
94, 102, 120–121, 127–129, 137–145, Fruit resistance, 1–10, 31–37, 58, 86, 124,
150, 171–180, 198–201 125, 173, 175–180
Biotrophic pathogens, 15, 18–19, 44–45, Fruits
52–53, 64 avocado, 2, 9–10, 45–47, 139
Botryodiplodia, 5, 9 citrus, 49, 57–65, 91, 110, 123, 139,
Botryosphaeria dothidea, 72, 75, 85 150, 172
Botrytis cinerea, 13–25, 47, 64, 72, 110, 123, mango, 2–9, 32, 34, 80, 110, 141
172, 184 melons, 31–37, 46
peach, 32, 73, 75, 80, 86, 95, 99, 109, 125,
126, 138, 139, 172, 177, 178, 180,
C 197–206
Candida sake, 90–96, 120, 123, 124, 139, pome, 70, 80, 91, 109–115, 120, 123, 124,
150–157, 159–162, 166, 172 126, 138–142, 150, 151, 155
209
210 Index
Fruits (cont.) P
stone, 70–75, 77, 80–85, 110, 111, 123, Pantoea agglomerans, 90–92, 96–102, 124,
125, 126, 138, 141, 142, 201 151, 156–158, 162–166
tomato, 13, 16–19, 21–25, 34, 46, 51, 52, Pathogenicity, 20, 45, 47, 49, 51, 52, 175,
110, 111, 142 178–179
Fruit susceptibility, 123, 193–195 Penicillium
Fungicide residues, 14, 32, 109, 115 Penicillium digitatum, 47, 49, 57–65, 90,
Fungicide resistance, 14, 32, 72, 85, 86, 97–99, 101, 112, 123, 126, 127, 158,
112–116, 195, 206 172, 176, 179
Fusarium moniliforme, 85 Penicillium expansum, 47, 49–51, 91–93,
Fusarium rot, 32–35, 85, 110–111 95, 96, 110, 115, 123–124, 145, 152,
155, 158, 172, 173, 175, 177–179
Penicillium italicum, 47, 49, 58, 90, 110,
G 123, 126, 127, 172
Geotrichum, 32, 110, 172 pH modulation
Global regulation of genes, 57–65 acidifying fungi, 45, 47–51
alkalizing fungi, 45–47
Phoma, 9
I Phomopsis, 9
Induced resistance, 13–25, 31–37, 122, 125, Phyotalexins, 2, 15, 24, 172, 176
159, 172, 174–179 Plant hormones, 19–20, 25
Plant resistance, 14–16, 18, 20, 21, 25, 175
Polygalacturonase-inhibiting proteins (PGIP),
L 15–17
Latent infection, 32, 70, 126, 140–141, 201 Polygalacturonases (PGs), 17, 23
Low risk substances, 91–94 Postharvest application, 33, 36, 93, 115,
138–140
Postharvest fungicide, 107–117, 185, 195
M Postharvest pathogens, 1–10, 33, 43–53,
Mango 57–65, 90, 91, 112, 115, 120–122, 141,
latex, 2–8 151, 173, 175, 180
Mechanisms of resistance, 13–25, 32, 36, 37, Postharvest treatment, 8–9, 32, 33, 35, 72,
175, 179 92–94, 99, 102, 110, 114, 122, 124,
Microbial antagonists, 90, 92, 120–121, 127, 125, 185, 199, 201
141, 143, 150 Potato, 32, 74, 78, 79, 110, 111, 139, 172
Monilinia fructicola, 71–77, 81–86, Prediction systems, 192, 195, 204
125, 126, 138, 141, 177, 178, 198, Preharvest application, 90–93, 97, 199
203, 206
Monilinia laxa, 71–77, 81–83, 85, 95,
125–126, 198–199, 201, 203–206 Q
Monitoring resistance, 84–86 Quiescent infection, 2, 4, 9, 44, 45, 52, 70,
Mucor rot, 32, 35 73–77, 83–85, 108, 125, 128
Quiescent stage, 44–45
N
Necrotrophic infection, 17, 20, 51–52 R
Necrotrophic pathogens, 14–15, 17–23, 25, Registration of new postharvest fungicides,
123, 173, 176, 177, 179 107–117, 120–121, 127
Non-fungicidal control, 140, 183–195 Residue limits, 108–109
Resistance, 1–10, 13–25, 31–37, 52, 58, 72,
90, 108, 122, 151, 172, 195, 206
O Resistance management, 86, 110, 115–117
Osmotic treatments, 97, 150, 156–158, Rhizopus rot, 32, 35, 110, 111, 172–173
162–166 Risk assessment, 84, 108–109, 204
Index 211
S T
Sanitation, 70, 115–117, 126, 140, 198, 206 Thermal-stress treatments, 162–166
Sclerotinia sclerotiorum, 47, 49–51, 178 Trichothecium, 32
Silicon, 33–34 Tuber crops, 110, 111
Storage rot, 34, 35, 183–195
Systemic acquired resistance (SAR), 18–19,
32–35, 174, 175, 177, 179

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Postharvest Pathology

Plant Pathology in the 21st Century:

For other titles published in this series, go to

ISBN 978-1-4020-8929-9 e-ISBN 978-1-4020-8930-5

Library of Congress Control Number: 2009932670

© Springer Science+Business Media B.V. 2010

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

1 The Role of Pre-formed Antifungal Substances

2 Mechanisms of Induced Resistance Against B. cinerea.......................... 13

3 Induced Resistance in Melons by Elicitors for the Control

4 Mechanisms Modulating Postharvest Pathogen

5 Global Regulation of Genes in Citrus Fruit in Response

6 Epidemiological Assessments and Postharvest

7 Preharvest Strategies to Control Postharvest

8 New Developments in Postharvest Fungicide Registrations

9 New Approaches for Postharvest Disease Control in Europe.............. 119

10 Quo Vadis of Biological Control of Postharvest Diseases..................... 137

11 Improving Formulation of Biocontrol Agents Manipulating

12 Host Responses to Biological Control Agents........................................ 171

13 Non-fungicidal Control of Botrytis Storage Rot in New Zealand

14 The Peach Story....................................................................................... 197

This collection of papers includes some of the presentations given at the

Nimal Adikaram, Chathurika Karunanayake, and Charmalie Abayasekara

Abstract Plants contain secondary metabolites with antifungal properties. In fruits

N. Adikaram (*), C. Karunanayake, and C. Abayasekara

D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology 1

1.1 Pre-formed Antifungal Substances

Plants produce a range of chemically diverse secondary metabolites with antifungal

1.2 Preformed Antifungal Compounds in Mango

forms quiescent infections. The concentration of resorcinols in the unripe mango

1 The Role of Pre-formed Antifungal Substances in the Resistance of Fruits 7

1.2.4 Pre- and Postharvest Treatments Enhance Fruit

Exposure of harvested mangoes to CO2 at a flow rate of 100 mL of CO2/min for 24 h

(Duncan’s Multiple Range Test)

1.3 Avocado (Persea americana) Fruit

Anthracnose disease caused by Colletotrichum gloeosporioides (Penz.) Penz. &

Hassan KM (2006) Constitutive alk(en)ylresorcinols and resistance to postharvest diseases in

Tesfaye Mengiste, Kristin Laluk, and Synan AbuQamar

Abstract Botrytis cinerea is a widespread pre-and postharvest pathogen of diverse

Keywords Botrytis cinerea • genetic resistance • necrotrpohic pathogens • induced

B. cinerea is a ubiquitious fungal pathogen with relative host unspecificity primarily

T. Mengiste (*), K. Laluk, and S. AbuQamar

D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology 13

2.2 Plant Resistance to B. cinerea

Plant resistance to diseases is controlled by a multitude of environmental as well as

2.3 The First Line of Defense Against B. cinerea

Recent studies illuminate differences that underpin host responses to pathogens

2.4 Active Defense Against Botrytis cinerea Contrasts

2.5 The Plant Hormones Jasmonate and Ethylene Play

2.6 The Emerging Role of Absciscic Acid as a Regulator Plant

and Mauch 2005). Exogenous application of ABA caused susceptibility to B.

2.7 Resistance to B. cinerea and Cross-Talk

with a particularly strong reduction in bos3. Production of the phytoalexin camalexin

2.8 Mechanisms of B. cinerea Resistance Are Conserved

The predominant mechanism of B. cinerea resistance appears to be conserved between

cytoplasmic kinase that is localized to the plasma membrane. Pathogen infection,

2.9 Phyotalexins in B. cinerea Resistance

The role of induced chemical compounds in relation to B. cinerea resistance has

2.10 Changes in Genome Wide Gene Expression

of an average of 621 genes representing roughly 0.48% of the Arabidopsis

2.11 Conclusion and Perspective

Genetic approaches in Arabidopsis and tomato defined some components of the

1.1 Pre-formed Antifungal Substances

1.2 Preformed Antifungal Compounds in Mango

1.2.4 Pre- and Postharvest Treatments Enhance Fruit

1.3 Avocado (Persea americana) Fruit

2.2 Plant Resistance to B. cinerea

2.3 The First Line of Defense Against B. cinerea

2.4 Active Defense Against Botrytis cinerea Contrasts

2.5 The Plant Hormones Jasmonate and Ethylene Play

2.6 The Emerging Role of Absciscic Acid as a Regulator Plant

2.7 Resistance to B. cinerea and Cross-Talk

2.8 Mechanisms of B. cinerea Resistance Are Conserved

2.9 Phyotalexins in B. cinerea Resistance

2.10 Changes in Genome Wide Gene Expression

2.11 Conclusion and Perspective

3.2 Chemically Induced Resistance

3.3 Physically Induced Resistance

3.4 Biologically Induced Resistance

4.2 The Quiescent Stage

4.3 pH Modulation of the Environment by Postharvest Fungal

4.4 Effectors That Activate the Transition from Quiescent

the incidence of postharvest decay, by affecting the incidence of latent infection.

6.2.1 Brown Rot-Monilinia fructicola and M. laxa

6.2.1.1 Conventional Methods Used to Detect Latent and Quiescent

6.2.2 Botrytis Monitoring (BOTMON) - Botrytis cinerea

6.3 Use of Species-Specific PCR to Detect M. Fructicola

6.4 Techniques to Monitor Resistance of Fungal Pathogens

6.5 Conclusions and Future Prospects

7.2 Biocontrol Agents Combination

7.3 Biocontrol Combined with Low Risk Substances