Infection of grapevine tissue by Botryosphaeria species

Nicholas Amponsah, Eirian Jones, Hayley Ridgway and Marlene Jaspers

Bio-Protection and Ecology Division, Lincoln University, PO Box 84 Lincoln University,
Canterbury, New Zealand

Abstract
The Botryosphaeria species isolated from symptomatic grapevines, B. lutea (Neofusicocum
luteum), B. australis (Neofusicocum australe) B. parva (Neofusicocum parvum) B. stevensii
(Diplodia mutila) and Botryosphaeria sp. were inoculated onto to wounded green shoots of
grapevines on which they were shown to be pathogenic. The brown lesions that developed
were able to exude numerous conidia for all Botryosphaeria species tested except B. parva.
Non-grapevine isolates of B. australis, B. stevensii and a Botryosphaeria sp. were also found
to be equally pathogenic on grapevines. The shoots of the grapevine cultivars, Chardonnay,
Pinot noir, Riesling, Cabernet sauvignon and Sauvignon blanc were also equally diseased by
mycelium of B. lutea, B. parva and B. stevensii. When mature trunks of 18 month Pinot noir
plants were inoculated with B. lutea conidia and mycelium the plants showed no visible
symptoms, however the pathogen was found to have progressed through the plant to the
pruned tip, from where it then caused dieback. This research has shown that the
Botryosphaeria species from infected grapevines and other woody hosts are able to produce
symptomatic infections on green grapevine shoots of the commonly-grown varieties.
However on woody shoots, the infections are initially endophytic and symptomless,
progressing quickly through the shoots to pruning wounds from which they cause dieback.

1 Introduction
Pathogenic Botryosphaeria species cause economic losses world-wide on a wide range of
woody plant hosts of agricultural, horticultural and forestry significance. On grapevines,
Botryosphaeria spp. are often found associated with dieback of trunks, pruned or trimmed
shoots and canes, as well as incomplete graft unions, bud mortality and a general loss of
vigour termed ‘grapevine decline’. Worldwide, at least 11 Botryosphaeria species have been
identified as important grapevine pathogens, but species distribution and their pathogenicity
appears to vary between countries, possibly due to climatic factors.

Very little research has been conducted on the infection processes of the Botryosphaeria
spp. in grapevines and the factors that enhance infection risk, knowledge that is essential to
the development of control strategies. However, the reported research on Botryosphaeria
diseases of other crop species, and observations in vineyards has provided some relevant
information. In Botryosphaeria diseases of other crops, spores were shown to infect through
pruning wounds, and to cause similar symptoms to those in grapevines. However, the
reported grapevine infection studies have mostly used mycelial plugs applied to the wounds.
Since it is more likely that spores such as conidia are the main inoculum source in vineyards,
they should also be tested. This research reports on the preliminary infection studies that
tested the infection potential of the Botryosphaeria species found within New Zealand against
the main grape varieties grown here. To ensure that the research was robust and relevant,
both conidium and mycelial inoculation methods were used.

2 Materials and Methods

2.1. Pathogenicity of common Botryosphaeria species from New Zealand vineyards
Three isolates each of B. lutea (Neofusicocum luteum), B. australis (Neofusicocum australe)
B. parva (Neofusicocum parvum), B. stevensii (Diplodia mutila), two isolates of B. obtusa
(Diplodia seriata) and one isolate of a Botryosphaeria sp. were cultured on potato dextrose
agar (PDA) and incubated at 24oC in continuous darkness for 3 days. Mycelium agar discs
(3 mm) cut from the pathogen cultures were inoculated onto wounded sections of 20-25 cm
long green Pinot noir shoots cut from actively growing field vines. Controls were inoculated
with sterile agar plugs. The base of each shoot was inserted into a Universal bottle
containing sterile distilled water in an enclosed transparent chamber at room temperature
(15-23°C), with frequent misting for the first 3 days. The assessments of lesion length were
made on all plants at 10 days, with isolations made from three plants per treatment. The
lesions on the three remaining plants from each isolate were surface sterilised, air dried and
put in a moist container, to induce pycnidium formation and oozing of conidia (spores). Data
of lesion lengths were analysed by one way ANOVA using GENSTAT 9.0.
A B

Figure 1A. In vitro inoculation of a grapevine shoot with a mycelium colonised agar plug (a)
1B. Lesion length at 7 days on an inoculated plant (b), and no lesion (c) on a control
plant.

2.2. Grapevine cultivar susceptibility to three Botryosphaeria species

The shoot susceptibility of five grapevine cultivars: Chardonnay, Pinot noir, Riesling,
Cabernet sauvignon and Sauvignon blanc was studied using mycelium colonised agar plugs
of B. lutea, B. parva and B. stevensii isolates, one each, which were initially isolated from
New Zealand grapevines. Prior to the inoculations, the green shoots were either wounded
by cutting a 3 mm wide and 2 mm deep hole or non-wounded. The discs cut from the
Botryosphaeria cultures were inserted mycelium side first into the wounds in the centres of
the grapevine shoots (Fig. 1A), six shoots per isolate, which were incubated as described
previously. For non-wounded treatments, the agar plugs were placed onto the shoots and
held in place with cling film. Control plants were inoculated with sterile agar.
2.3. Pathogenicity of non-grapevine Botryosphaeria isolates on grapevine shoots
The 10 isolates of Botryosphaeria species found in symptomatic non-grapevine hosts (Table
3) were inoculated onto green shoots of Pinot noir, as described above. There were six
replicates per isolate with plants arranged in a completely randomised design. Assessment
of lesion length was made on all six plants and three were used for pathogen isolation. The
lesion areas from the remaining three plants per isolate were dried at room temperature,
moistened and incubated to induce pycnidium and conidium production.
2.4. Susceptibility of mature canes to infection by B. lutea
Potted Pinot noir grapevine plants (18 months old) were wounded by drilling 3.5 mm
diameter holes into the wood and inoculated with Botryosphaeria lutea (the most pathogenic
species) using mycelium agar discs (3 mm) or conidia (104/mL). The 10 replicate plants per
treatment were arranged in a randomised complete block design within a shade house.
Disease assessment was carried out monthly for 4 months. Due to difficulty seeing the
lesions, pathogen position was determined by isolating from wood segments taken at 10 mm
intervals from the inoculation point.

3. Results and Discussion

3.1. Pathogenicity of common Botryosphaeria species from New Zealand vineyards

There were significant differences (P≤0.05) in the pathogenicity of the Botryosphaeria
isolates and species, as shown by the mean lesion lengths (Table 1 and Fig 2). At 10 days
after inoculation, the B. lutea isolates [M (13) 2, G-14 and N (12)2] had the longest lesions
followed by isolate I-1 (Botryosphaeria sp.) and then B. australis isolates Mel-2, J-3 and Kat-
1. The next most pathogenic isolate Q-2 (B. parva) was followed by Iso-2 (B. stevensii), and
both were significantly different (P≤0.05) from the other B. parva isolates [I (15)2 and I (15) 3]
and B. stevensii isolates [F (12)2 and Q]. Similar differences in pathogenicity among isolates
of the same species have been reported by many researchers. In contrast to many overseas
reports of the B. obtusa, the isolates L-I and L (16)2 were not pathogenic to the grapevine
shoots since the lesions extended little beyond the wounded inoculated points, being similar
(P≥0.05) to the controls.

Table 1. Mean lesion lengths caused by Botryosphaeria isolates on wounded green shoots
of Pinot noir.
Species name Isolate Mean lesion
length (mm)
B. lutea N(12)2 76.13 g1
B. lutea M(13)2 71.12 fg
B. lutea G-14 68.98 efg
B. australis Kat-1 65.93 defg
B. australis J-3 65.85 defg
B. australis Mel-2 62.66 cdefg
B. parva Q-2 58.70 cdef
B. parva I(15)2 53.04 cd
B. parva I(15)3 48.53 c
B. stevensii Iso-2 55.42 cde
B. stevensii F(12)2 26.33 b
B. stevensii Q 24.23 b
B. obtusa L(16)2 10.07 a
B. obtusa L-1 8.38 a
Botryosphaeria sp. I-1 67.28 defg
Sterile agar Control 0.00 a
LSD (P≤0.05) 13.36
1
Means followed by different letters are different (P≤0.05) by Fisher’s protected LSD test.
Figure 2. Shoot lesions at 10 days after inoculation with three isolates each of (A) B. lutea
(B) B. australis (C) B. parva (D) B. stevensii and (E) B. obtusa.
3.2. Grapevine cultivar susceptibility to three Botryosphaeria species
The non-wounded shoots of all cultivars had no significant lesions for all the Botryosphaeria
isolates tested, being similar to that of the controls (data not included). However with the
wounded inoculated shoots, symptoms of brown to dark brown shoot lesions (as in Fig 1B)
were observed on all five cultivars. After 10 days, lesion lengths differed significantly
(P≤0.05) among the pathogens (B. lutea, B. parva and B. stevensii) used to test for the
cultivar susceptibility (Fig 3), and all caused shoot death by 2-3 weeks. However, there was
no significant difference in lesion length of the five grapevine cultivars tested (P>0.05; Fig.4),
indicating that they were equally susceptible to the Botryosphaeria species tested. The
inoculated isolates were all re-isolated from the shoots together with a few other fungi.

30
Mean lesion lengths (mm)

25 C
20

15 B
10 A

5 D
0
B. stevensii B. parva B. lutea Control
Botryosphaeria species

Figure 3. Mean lesion lengths, indicating pathogenicity of B. lutea, B. parva and B.

stevensii on five grapevine cultivars (Bars with the same letters do not differ
significantly (P≤0.05) by Fisher’s protected LSD test).

Figure 4. Mean lesion lengths at 10 days after inoculation with mycelium plugs of B. lutea,
B. parva and B. stevensii, indicating susceptibility of grapevine cultivars (bars
with the same letters do not differ significantly (P≤0.05) by Fisher’s protected LSD
test)
3.3. Pathogenicity of non-grapevine Botryosphaeria isolates
All the Botryosphaeria isolates from the non-grapevine hosts were able to infect grapevine
shoots, producing similarly long lesions to the grapevine isolates and differed significantly
(P≤0.05) from the controls (Table 2). This indicates the potential of other hosts to provide
inoculum sources for infection of vineyards. Pathogen re-isolation yielded 100% recovery of
the inoculated isolates together with a few other fungi. All of the isolates were able to
produce pycnidia with copious conidium ooze exuding from them after incubation of the
infected shoots under moist conditions (Fig. 5).

Table 2. Pathogenicity of Botryosphaeria isolates from non-grapevine.

Species name Isolates Hosts Lesion
length
(mm)
B. australis J-3 Broom 68.4 f1
B. stevensii Iso-2 Native ngaio 57.9 e
B. stevensii J-4 Oak 51.0 de
B. stevensii A-1 Plum 39.6 bc
B. stevensii F-1 Willow 40.7 bcd
B. stevensii A-2 Apple 34.0 b
Botryosphaeria sp. O-3-1 Cherries 46.1 cd
Botryosphaeria sp. O-1 Pine 44.3 bcd
Botryosphaeria sp. C-4 Olive 43.8 bcd
Botryosphaeria sp. I-1 Lemon wood 69.8 f
Control Sterile agar 0.0 a
LSD (P≤0.05) 10.03
1
Means followed by different letters are different (P≤0.05) by Fisher’s protected LSD test.

Figure 5. Pycnidia oozing conidia of Botryosphaeria sp. after incubating lesion shoots
under moist condition.
3.4. Susceptibility of mature canes to infection by B. lutea
Pathogen position within the wood showed that after conidium inoculations, the pathogen
progression was slower than with the mycelium inoculations. With both types of inoculation,
the pathogen progressed through the tissue, moving faster in the upward than the downward
direction (Fig. 6). By the 4th month the pathogen had entered the first side shoots just past
the inoculation point, still progressing without any visual symptoms. Once the pathogen
progressed to the tip of the shoot, symptoms of dieback began to appear at the pruned tip.

100 Conidia ▲
Mycelium ▲
Distance moved by pathogen

80 Conidia ▼
Mycelium ▼

60

40
(mm)

20

-20

-40
Dec-07 Jan-08 Feb-08 Mar-08 Apr-08
Dates on which pathogen was assessed

Figure 6. Botryosphaeria lutea progression upwards and downwards from points of

inoculation with conidia and mycelium discs. in 18 months old Pinot noir
grapevine trunks.

4. Conclusion
This research has shown that the Botryosphaeria species present in and around New
Zealand vineyards were pathogenic to the commonly grown grapevine cultivars. They were
able to produce numerous spores that could infect through wounds in green shoots, on which
they caused severe lesions, and in the mature canes, where visible symptoms did not
developed immediately at the infection points. In the mature wood, the pathogen progressed
rapidly through the plant to the pruned tip from which it caused then dieback. This has clear
implications for development of pruning strategies and the need to protect pruning wounds
from potential infection.

5. Acknowledgements
The authors gratefully acknowledge the many New Zealand grape growers who provided
practical help for this project, and New Zealand Winegrowers and Lincoln University who
provided funding.