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STUDIES ON LECTINS FOR SUPPRESSION OF

SOIL-BORNE PATHOGENS

JAYALAKSHMI K.

DEPARTMENT OF PLANT PATHOLOGY


COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES
DHARWAD-580 005

MAY, 2014
ii

STUDIES ON LECTINS FOR SUPPRESSION OF


SOIL-BORNE PATHOGENS

Thesis submitted to the


University of Agricultural Sciences, Dharwad
in partial fulfillment of the requirements for the
Degree of

Doctor of Philosophy

in

Plant Pathology

By

JAYALAKSHMI K.

DEPARTMENT OF PLANT PATHOLOGY


COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES
DHARWAD-580 005

MAY, 2014
iii

DEPARTMENT OF PLANT PATHOLOGY


COLLEGE OF AGRICULTURE, DHARWAD
UNIVERSITY OF AGRICULTURAL SCIENCES,
DHARWAD
CERTIFICATE
This is to certify that the thesis entitled “STUDIES ON LECTINS FOR SUPPRESSION OF
SOIL-BORNE PATHOGENS” submitted by Ms. JAYALAKSHMI K., for the degree of DOCTOR
OF PHILOSOPHY in PLANT PATHOLOGY to the University of Agricultural Sciences,
Dharwad, is a record of research work carried out by her during the period of her study in this
University, under my guidance and supervision, and the thesis has not previously formed the
basis for the award of any degree, diploma, associateship, fellowship or other similar titles.

DHARWAD (S. LINGARAJU)


MAY, 2014 CHAIRMAN

Approved by :

Chairman : ____________________________
(S. LINGARAJU)
Members :
1. __________________________
(S. T. NAIK)

2. __________________________

(M. M. JAMADAR)

3. __________________________

(K. BASAVANA GOUD)

4. __________________________

(RAMESH BHAT)
iv

Acknowledgement

.......Gratitude is the memory of heart!


At last the movement has come to look into the deeper layers of heart, which is filled
with the feelings of togetherness, loveliness, consolation and satisfaction, a sign of relief and
a sense of fulfilment. One would not achieve whatever he is now. No scientific endeavour is a
result of individuals’ efforts. Therefore it is a matter of pleasure to recall all the faces and
spirits in the form of teacher, friends, near and dear ones. But it is often difficult to put ones
feelings into words and is the most difficult to accomplish and to express all my feelings and
sense of gratitude in words.

I am highly grateful to the Rajeev Gandhi National Fellowship for SC/ST students,
university grants commission (UGC), New Delhi for providing me financial assistance in the
form of Senior Research Fellowship (SRF) and University of Agricultural Sciences, Dharwad
for providing me an opportunity to pursue my Doctoral programme.

Diction is not enough to express my deep and profound sense of gratitude and
indebtedness, reverence and heartfelt thanks to my major advisor and Chairman of Advisory
Committee Dr. S. LINGARAJU, Professor of Plant Pathology and Head, Institute of Organic
Farming, University of Agricultural Sciences, Dharwad. I accomplish his constructive criticism,
inspiration, insightful suggestion with privilege and heartfelt gratitude, without which I could
not have completed this work. For his meticulous guidance, transcendent suggestions and
inspiring guidance at every step, constant supervision, this not only moulded my research
work in a right form but also my overall personality. I confess that, it has been a rare privilege
for me to be associated with him during my Doctoral programme. I feel proud to have him as
my chairman of advisory committee, who treated me as a daughter rather than as student. I
seek his blessings forever.

It is rather difficult to express in words my sincere and heartfelt gratitude to


Dr. RAMESH BHAT, Associate Professor, Department of Biotechnology, College of
Agriculture, University of Agricultural Sciences, Dharwad, for his encouragement, scholarly
advice and kind help at every stage of the investigation to completetion of my Doctoral
programme. Infact, I was very fortunate to have him as member of my advisory committee
and his contribution in the completion of the research and thesis was indispensable. I avail
this opportunity to express my deep sense of reverence and gratitude to my member of my
advisory committee Dr. S. T. NAIK, Professor and ADE, University of Agricultural Sciences,
Dharwad for his moral support during course of my research work. I express my sense of
respect and sincere thanks to Dr. M. M. JAMADAR, Associate Professor of Plant Pathology,
A. C. Bijapur, UAS, Dharwad for his kind help and encouragement in pursuit of the study. I
take this opportunity to express my deep sense of gratitude to Dr. K. BASAVANA GOUD,
Professor, Department of Agricultural Entomology, UAS, Dharwad. I am highly grateful to
them for critically going through the manuscript and for their valuable suggestions for the
improvement of the thesis.

It is with immense pleasure that I imprint my profound sense of gratitude and


indebtedness to DR. A. S. BYADAGI, Professor and Head, Department of Plant Pathology,
UAS, Dharwad, and Dr. V. B. Nargund, Dr. Yashoda R. Hegde, for their keen interest,
persistent encouragement and valuable suggestions throughout my stay in the department,
during study and research.
v

I express my deep reverence to my teachers, Dr. V. I. Benagi, Dr. M. S. Patil, Dr. M.


S. L. Rao, Dr. Virupaksha Prabhu, Dr. S. Harlapur for their kind cooperation extended to me
during the course of my study and research. Dr. A. S. Patil

On my personal note with deep respect from bottom of my heart to express my


sincere gratitude to my loving mother Puttamma, friendly father Krishnappa, brother Yogesh
and sister Sudha for their love, sacrifices blessings, care and affection showed on me during
the study period, may I be worthy to live up to their desires and expectations. I also would like
to thank all the members of family and relatives who always supported me throughout my life.

I owe heartfelt sense of gratitude to my best friend Raju, brother Raghu, and my
lovely sisters Reena and Roopa who have given sound and fruitful advice and helped me in
my research work and without them I would have not finished research work.

On a personal note, I am very glad to mention sincere mental support and words of
encouragement of my beloved friends Rajani, Nethra, Savitha, Nagashree, Veena, Gunjan,
Poornima, Smitha, Sowmya, Pavithra, Kumuda, Harish, Ranganath, Prashanth, Ganesh,
Yogesh, Pradeep P. E, Vinod , sisters Uma, Ashwini, Divya, Basamma, Shwetha, Renuka,
Kumari and brothers Shreeshail, Sachin, Ravichandran and Shivakumar.

I will be failing in my duty if I do not mention the help, their valuable suggetions and
co-operation during research of my class mate Yogesh Bhagat and my seniors Sagar D,
Madhu Giri.
I have been highly fortunate in having many friends in whose company; I never felt
the burden of my studies. Their helping hand was evident at every stage of tension, anxiety
and achievement. To mention the names of only a few petals this together as a flower
scented my life with an elegant fragrance. I must begin with my classmates Arun Kumar,
Hemachandra, Gurupad and Suresh, juniors Sumangala, Vinamratha, Pushpa, Veena,
Rajalaxmi, Anusha, Kavya, Sangeetha, Ramya, Sukrutha, Nazia, Rohini Kolekar,
Santhoshreddy, Ranganath Swamy, Nagaraj, Huligappa, Anil, Pradeep Manyam, Madhu,
Druva, Anand, Chidananda, Swamy and my seniors Tippesh, Shadakshari, Yogesh Gowda
who have helped me in one way or the other for which I am ever greatful to them.

I would like to thank the non-teaching staff Shri Nadaf, Shri S. N. Patil, Shri
Gangadhar, Shri Barigidad, Shri. Arvind, Shri Hazarat, Smt. Shobha Nargund, Shri Manju
(Biotech) for their help during my research.

As a matter of external compliance, at the outset, I bow to thank Lord Ganesha, one
of the incarnations of Almighty who enabled me through his liberal blessings to accomplish
this venture.

I convey my whole hearted thanks to Mr. Arjun and Mr. Kalmesh (M/s. Arjun
Computers), for their meticulous typing of the manuscript neatly, timely and more vitally his
cooperation and affection towards me and Mr. Kumbar, for the neat binding of the manuscript.

Finally I share my sincere thanks and inspirations to all those seen and unseen hand
and minds.

……….any omission in this short manuscript doesn’t mean lack of gratitude.

DHARWAD
MAY, 2014 (JAYALAKSHMI K.)
vi

Affectionately Dedicated
to
My beloved parents
Shri. Krishnappa, Smt. Puttamma,
Brother Yogesh and Sister Sudha
vii

CONTENTS

Sl.
Chapter Particulars
No.
CERTIFICATE
ACKNOWLEDGEMENT
LIST OF TABLES
LIST OF FIGURE
LIST OF PLATES
1. INTRODUCTION
2. REVIEW OF LITERATURE
2.1 Sources of lectins
2.2 Plant lectins
2.3 Fungal lectins
2.4 Lectins in plant defence
2.5 Evaluation of plant and fungal lectins for in vitro suppression of
some common soil-borne pathogens
2.6 Influence of lectins on biocontrol agents in the suppression of
soil-borne pathogens in vitro
2.7 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens
2.8 Mechanism of action of lectins in the suppression of soil-borne
pathogens
3. MATERIALS AND METHODS
3.1 Collection and isolation soil-borne pathogens
3.2 Expression of RVL1 and SRL1 in E. coli and its isolation
3.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)
3.4 Evaluation of plant and fungal lectins for the in vitro suppression
of some common soil-borne pathogens
3.5 Influence of lectins on biocontrol agents in the suppression of
soil borne pathogens in vitro
3.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens
3.7 Evaluation srl1 and rvl1 transgenics and non-transgenics lines
for root infection by Meloidogyne incognita and its development
3.8 Mechanism of action of lectins in the suppression of soil-borne
pathogens
4. EXPERIMENTAL RESULTS
4.1 Collection and isolation of soil-borne pathogens
4.2 Expression of RVL1 and SRL1 in E. coli
4.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)
4.4 Evaluation of plant and fungal lectins for the in vitro suppression
of some common soil-borne pathogens
4.5 Influence of lectins on biocontrol agents in the suppression of
soil borne pathogens in vitro
viii

4.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens
4.7 Evaluation of rvl1 and srl1 transgenics and non-transgenics for
root infection by M. incognita and its development and
reproduction
4.8 Mechanism of action of lectins in the suppression of soil-borne
pathogens
5. DISCUSSION
5.1 Collection and isolation of soil-borne pathogens affecting
tomato
5.2 Expression of RVL1 and SRL1 in E. coli
5.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)
5.4 Evaluation of plant and fungal lectins for the in vitro suppression
of some common soil-borne pathogens
5.5 Influence of lectins on biocontrol agents in the suppression of
soil borne pathogens in vitro
5.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens
5.7 Evaluation of rvl1 and srl1 transgenics and non-transgenics for
root infection by M. incognita and its development and
reproduction
5.8 Mechanism of action of lectins in the suppression of soil-borne
pathogens
6. SUMMARY AND CONCLUSION
REFERENCES
ix

LIST OF TABLES

Table
No. Title

1 Growth phase studies of Ralstonia solanacearum

Morphological and biochemical characterization of Ralstonia


2
solanacearum

3 Important characters of perineal pattern of tomato root-knot nematode

4 Heamagglutination assay for E. coli-expressed SRL1 and RVL1

5 Determination of titer of the antiserum of SRL1 by micro-precipitation test

6 Anti-SRL1 titre determination by DAC-ELISA

Evaluation of plant and fungal lectins against F. oxysporum and


7
S. rolfsii through poison food technique
Evaluation of plant and fungal lectin against F. oxysporum,
8
S. rolfsii and R. solani through spread plate technique

Evaluation of plant and fungal lectin against F. oxysporum and


9
S. rolfsii through inhibition zone assay

10 Inhibition of F. oxysporum spores germination

Germination of mature and immature sclerotia of S. rolfsii on Byrde’s


11
medium

12 Germination of sclerotia of R. solani on Byrde’s medium

In vitro evaluation of plant and fungal lectins against


13
R. solanacearum

14 Effect of SRL1 and RVL1 on hatching eggs of M. incognita

15 Effect of RVL1 and SRL1 on juvenile mortality of M. incognita

In vitro evaluation of antagonists against S. rolfsii, R. solani and


16
F. oxysporum (without lectins)

In vitro evaluation of SRL1 and RVL1 lectin on T. viride and


17
P. lilacinus

Effect of SRL1 and RVL1 on inhibition spore germination of


18
P. lilacinus and T. viride

19 Growth phase studies of Pseudomonas fluorescens and Bacillus subtilis

In vitro evaluation of bacterial and fungal bioagents against Ralstonia


20
solanacearum
Antimicrobial activity, MIC, MBC and MAC of SRL1 and RVL1 against
21
bacterial biocontrol agents
x

22 Effect of SRL1 and RVL1 lectin on P. fluorescens and B. subtilis strains

23 Activity of SRL1 and RVL1 lectin on P. fluorescens and B. subtilis

Influence of bacterial and fungal antagonists’ in combination with lectins


24
on R. solanacearum

Effect of bacterial antagonists’ culture filtrates with lectins on eggs


25
hatching of M. incognita
Effect of bacterial antagonists’ culture filtrates with lectins on juvenile
26
mortality of M. incognita

Effect of lectins and fungal antagonists on hatching of eggs of


27
M. incognita

Effect of lectins and fungal antagonists on juvenile mortality of


28
M. incognita

29 Heamagglutination activity of srl1 and rvl1 expressed transgenic events

Biocontrol potentiality of srl1 and rvl1 expressed transgenic events


30
against different pathogens (alone and in combinations)
Infection and development of M. incognita in srl1 and rvl1 expressed
31
transgenic events

Binding of histochemical dyes to exudates of Meloidogyne incognita


32
juveniles

33 Binding of FITC conjugated lectins


xi

LIST OF FIGURE

Figure
No. Title

1 Plan showing dilutions of virus and antiserum in microprecipitin test

2 Growth phase studies of Ralstonia solanacearum

3 Activity of different concentrations of SRL1 and RVL1 on Ralstonia


solanacearum

4 Effect of SRL1 ad RVL1 on hatching eggs of Meloidogyne incognita

5 Effect of RVL1 and SRL1 on juvenile mortality of Meloidogyne


incognita

6 Evaluation of antagonists' against S. rolfsii, R. solani and


F. oxysporum (without lectins)

7 Effect of SRL1 and RVL1 on inhibition spore germination of


P. lilacinus and T. viride

8 Growth phase studies of P. fluorescens and B. subtilis

9 Effect of SRl and RVL1 lectin on P. fluorescens and B. subtilis


strains

10 Influence of bacterial and fungal antagonists’ in combination with


lectins on R. solanacearum

11 Effect of lectins and fungal antagonists’ on hatching of eggs of M.


incognita

12 Effect of lectins and fungal antagonists' on juvenile mortality of M.


incognita
xii

LIST OF PLATES

Plate
Title
No.
1 Tomato root diseases from which the pathogens were isolated
2a Root pathogens and the growth in vitro
2b Their hyphae and spores
3 Ralstonia solanacearum: growth in vitro, morphology, biochemical
charecterisation and hypersensitivity
4 Meloidogyne incognita: Eggmass and perineal pattern
5 Mass multiplication (Giant culture)
6 Heamagglutination assay of E. coli-expressed RVL1 and SRL1
7 SDS-PAGE analysis of E. coli-expressed RVL1 and SRL1
8 Titre of polyclonal antibody against SRL1antigen determined by
DAC-ELISA
9 In vitro evaluation of SRL1 and RVL1 against F. oxysporum,
S. rolfsii and R. solani using poison food technique
10 In vitro evaluation of SRL1 and RVL1 against F. oxysporum,
S. rolfsii and R. solani using spread plate method
11 Evaluation of SRL1 and RVL1 against F. oxysporum, S. rolfsii and
R. solani employing inhibition zone assay
12 Germination of mature and immature sclerotia on Byrde’s medium
13 Germination of micro-sclerotia on Byrde’s medium
14 In vitro evaluation of plant and fungal lectins against
R. solanacearum through well diffusion assay
15 Determination of Minimum inhibitory concentration of SRL1 and
RVL1 against R. Solanacearum
16 Evaluation of SRL1 and RVL1 against R. solanacearum
17 Activity of SRL1 and RVL1 on R. solanacearum
18 Paecilomyces lilacinus: growth in vitro and spores and sporophores
19 In vitro evaluation of antagonists against S. rolfsii, R. solani and F.
oxysporum (without lectins)
20 In vitro evaluation of SRL1 and RVL1 on T. viride and
P. lilacinus
21 In vitro evaluation of bacterial and fungal bioagents against Ralstonia
solanacearum (without lectins)
22 Determination of Minimum inhibitory concentration for bacterial
biocontrol agents
23 Effect of SRL1 and RVL1 on P. fluorescens and B. subtilis strains
24 Influence of bacterial and fungal antagonists’ in combination with
SRL1 And RVL1 on R. solanacearum
xiii

25 Swollen invaded egg bearing a conidiophore and chains of conidia


(400 X)
26 PCR confirmation of srl1 and rvl1 expressed transgenic events
27 Haemagglutination assay with rvl1 and srl1 expressed in transgenic
tomato lines
28 SDS-PAGE of crude protein from srl1 transgenic events
28a SDS-PAGE of crude protein from rvl1 transgenic events
29 Performance of srl1 and rvl1 transgenic events against combined
inoculation of tomato root pathogens
30 Meloidogyne incognita infection and development in srl1 and rvl1
transgenic events
31 Histopathology of Meloidogyne infected transgenic and non-
transgenic root system
32 FITC–conjugated SRL1, anti SRL1 and RVL1 labelling of Sclerotium
rolfsii
33 FITC–conjugated SRL1, anti-SRL1 and RVL1 labelling of
Rhizoctonia solani
34 FITC-conjugated SRL1, anti SRL1 and RVL1 labelling of Fusarium
oxysporum
35 FITC -conjugated SRL1, anti SRL1 and RVL1 labeling of
Trichoderma viride
36 FITC -conjugated SRL1, anti SRL1 and RVL1 labelling of
Paecilomyces lilacinus
37 Immunolocalization of anti SRL1 in sclerotia and microsclerotia of
Sclerotium rolfsii and Rhizoctonia solani respectively
38 Staining of amphidial and excretory secretions of M. incognita (400x)
39 Binding of FITC conjugated SRL1, anti SRL1 and RVL1 to the
Meloidogyne incognita juveniles
xiv

LIST OF APPENDIX

Appendix
Title
No.
I Composition of different growth media/reagents/indicators used
1. INTRODUCTION

Protecting the crop plants from pests and pathogens is one of the most important
aspects in reaping higher crop productivity. Soil-borne pathogens are important in the
context of Indian agriculture as they cause high crop yield losses. Various soil-borne
pathogens are known to cause reduction in crop yields. For instance, Fusarium
oxysporum, an ubiquitous and polyphagous soil-bone fungus brings down the yield in
tomato in the range of 5 to 94 per cent. Similarly ubiquitous soil-borne bacterium
Ralstonia solanacearum and nematode Meloidogyne incognita cause respectively, yield
reduction of 10 to 100 and 5 to 95 per cent in the same crop. Despite the fact that a
number of management options are employed by crop growers, crop yield losses are
mounting. Alternate management strategies will have to be environmentally non-
hazardous. In this direction, researches involving lectins for suppression of soil-borne
pathogens damage will be worthwhile.

Stillmark in 1888 while investigating the toxic effects of castor bean extract
(Ricinus communis) on blood noticed that the red blood cells (RBCs) agglutinated: the
protein from castor beans called “ricin” capable of agglutinating the RBCs of human and
animals were identified. Initially, the ‘agglutinins’ were found only in plants. Later, when
they were found in other organisms and agglutinating cells other than erythrocytes, the
term “lectin” was proposed by William Boyd (Boyd and Slapeigh, 1954). Present definition
of lectins states that they are "proteins /glycoproteins possesing at least one non-catalytic
domain which bind reversibly to a specific mono or oligosaccharide" (Goldstein et al.,
1980). They are ubiquitous and have been isolated from plants, viruses, bacteria, animals
(Weis and Drickamer, 1996; Fang et al., 2010) and fungi (Gold and Balding, 1975). Their
detection and quantification are based on the agglutinating property and purification uses
specific immobilized sugar affinity columns (Gabius et al., 1993; Goldstein and Poretz,
1986; Lis and Sharon, 1986).

Many biological processes in nature are triggered and nurtured by protein


carbohydrate recognition and protein-protein interactions. Lectins mediate cell-cell and
host-pathogen interactions through the specific recognition of carbohydrates present on
the cell surface. Lectins are thought to act as defense proteins in plants against pests and
2

pathogens. The amino acid sequences of several lectins are reported. Since lectins have
been implicated in several disease conditions, they have applications in diagnosis and
biomedical research. Lectins with specific carbohydrate specificity have been purified
from various plant tissues and other organisms. They can be classified on the basis of
their carbohydrate specificity. They can also be categorized according to the overall
structures into merolectins, hololectins, chimerolectins and superlectins, or be grouped
into different families (legume lectins, type II ribosome-inactivating proteins, monocot
mannose-binding lectins and other lectins). Some of them include GNA (Galanthus nivalis
agglutinin), PSA (Pisum sativum agglutinin), ConA (Canavalia ensiformis agglutinin), PHA
(Phaseolus vulgaris agglutinin), WGA (Triticum vulgaris agglutinin). Also, many lectins
have been identified and isolated from fruiting body-forming fungi of the phyla
Basidiomycota and Ascomycota (Guillot and Konska 1997; Wang et al., 1998; Dam and
Brewer 2007). Some of these lectins which have been characterized biochemically and
structurally include Agaricus campestris lectin, Agaricus bisporus lectin (Goldstein and
Poretz, 1986), Botrytis cinerea lectin (Kellens et al., 1992); Pleurotus ostreatus lectin
(Chattopadhyay et al., 1999); and Rhizoctonia solani lectin (Candy et al., 2001).
Characteristic features of this type of lectins are their water solubility, synthesis in the
cytoplasm and composition of one or more subunits of low molecular weight.

Lectins consumed in the diet may be innocuous, since most of them are usually
denatured by cooking and proteolytically digested upon consumption. Further, the lectins
from vegetable(s) are expected to contain “safe lectins”, which can have immense
application in transgenic research. During the last few years, lectins from
Monocotyledonaceae have attracted greater attention because of their anti-insect, anti-
nematode and anti-pathogen properties. Mannose-binding lectins and the gene encoding
them have been extensively studied from many plant families like Amaryllidaceae,
Araceae, Alliaceae and Orchidaceae.

Despite their extensive biochemical and structural characterization, the


physiological function of these fungal lectins is not well understood. Some fruiting body
lectins were shown to have mitogenic, antiproliferative, antitumour, antiviral and
immune-stimulating activity towards mammalian cells (Singh et al., 2010). With regard
to the fungi themselves, proposed functions of fruiting body lectins are storage or roles
3

in morphogenesis and development (Guillot and Konska, 1997; Rosen et al., 1997). The
latter functions are based on the temporal and spatial regulation of gene expression
during fungal development. The synthesis of these lectins is often restricted to
reproductive structures such as fruiting bodies or sclerotia, in which these proteins
represent a high percentage of the total soluble protein content (Boulianne et al., 2000;
Candy et al., 2001; Nowrousian and Cebula, 2005; Walti et al., 2008).

Whereas, lectins from plants have been extensively studied; many plant lectins
have been demonstrated to interact with carbohydrates of other organisms either in
symbiosis or in defence processes (Peumans and Van Damme, 1995; Van Damme et
al., 2004; De Hoff et al., 2009). One of the most important functions of plant lectins is
their role as effectors in the defence against parasites and herbivores (Murdock and
Shade, 2002; Vasconcelos and Oliveira, 2004). It has been shown that plant lectins
possess fungicidal, insecticidal and nematicidal properties and are also toxic to higher
animals (Peumans and Van Damme, 1995; Oka et al., 1997; Macedo et al., 2002). The
expression of these lectins is regulated both temporally and spatially. It can be tissue-
specific or systemic, constitutive or induced upon stress, herbivory or pathogen infection
(Qin et al., 2003; Chen, 2009; Subramanyam et al., 2008; Vandenborre et al., 2009).

Genes encoding plant lectins have even been used in transgenic crops to
confer resistance against insect pests, plantpathogenic nematodes and fungi (Burrows
et al., 1998; Powell et al., 1998 and Ripoll et al., 2003). A well-studied example is the
snowdrop lectin (GNA) that binds to terminal mannose residues on glycoproteins in the
gut of aphid larvae and other insects (Trebicki et al., 2009). Because fungi are similar to
plants with regard to their non-motile nature, it is likely that they have evolved similar
mechanisms to defend themselves against predators and parasites (Spiteller, 2008;
Rohlfs and Churchill, 2011). Fungivory plays a significant role in shaping the structure
and function of natural fungal communities and represent a strong selective force for the
evolution of chemical defence systems in fungi (McGonigle, 2007). Accordingly, a wide
variety of chemical compounds, either constitutively produced or wound-activated, have
been identified in fungi (Wicklow, 1988; Spiteller, 2008), and many of these secondary
metabolites are believed to have evolved to protect saprophytic fungi
4

from being used as a food source by amoebozoa, nematodes and other invertebrates
(Rohlfs et al., 2007; Fox and Howlett, 2008). On the other hand, it has been shown that
proteins are responsible for most of the insecticidal activity of mushrooms (Wang et al.,
2002). Accordingly, entomotoxic effects have been demonstrated for Xerocomus
chrysenteron lectin (XCL) and Rhizoctonia solani agglutinin (when added to the artificial
diet of Drosophila melanogaster and Acrythosiphon pisum) and the cotton leafworm
Spodoptera littoralis, respectively (Trigueros et al., 2003; Hamshou et al., 2009), as well
as for the Sclerotinia sclerotiorum agglutinin (SSA) fed to Acrythosiphon pisum
(Hamshou et al., 2010).

In general, mannose-binding monocot lectins having tetrameric structure have


shown more potent insecticidal property and antifungal activity (Barre et al., 1996). By
virtue of multivalency, such lectins are probably capable of interacting more strongly
with either simple or complex mannose-containing glycoconjugates. In this context, it is
interesting to note that the tetrameric snowdrop lectin is a more potent insecticidal lectin
than the trimeric daffodil lectin and the dimeric garlic lectin (Powell et al., 1995; Barre et
al., 1996). Available data indicate that the lectins from Araceae family are tetrameric
with high structural and functional similarity. For example, two members of Araceae,
Arisaema jacquemontii Blume and Colocasia esculenta contain tetrameric lectins which
exhibit potential insecticidal and fungicidal activity (Van Damme et al., 1995; Kaur
et al., 2006). Remusatia vivipara belonging to Monocotyledoneae (Fam: Araceae), is
commonly found in the western ghat region. The tubers of R. vivipara are cooked and
consumed as a vegetable in many parts of the world.

Recently Remusatia vivipara (Roxb.) Schott, lectin (RVL1) has been purified
from its tubers (Bhat et al., 2010a). It is a mannose-binding lectin which agglutinates only
rabbit erythrocytes and possesses specificity for mucin and asialofetuin. The possibility
that it is cooked and therefore is non-toxic to human beings can be ruled out because of
the fact that the tuber lectin of R. viviapara (RVL1) is heat stable even at 80˚C for 30 min
under wide range of pH (2.0–9.3). Therefore, the gene coding for tuber lectin of R.
vivipara could be considered for transgenic development. rvl1 gene coding for RVL1 was
5

cloned for the first time at the Department of Biotechnology, University of Agricultural
Sciences, Dharwad (Neekhra, 2009).

Sclerotium rolfsii, a soil borne plant pathogenic fungus produces lectins that act as
signaling molecule in interaction with antagonists (Barak et al., 1985; Barak and Chet,
1990; Inbar and Chet, 1994) and germination of sclerotial bodies (Swamy et al., 2004).
Till date, 45 kDa (Inbar and Chet, 1994), 55 kDa (Barak and Chet, 1990), 60 kDa (Barak
and Chet, 1990) and 17 kDa lectins have been purified from S. rolfsii (Swamy et al.,
2001). Considering the use of such fungal lectins in pharmaceutical and crop
improvement applications, a lectin coding gene was cloned from S. rolfsii at the
Department of Biotechnology, University of Agricultural Sciences, Dharwad
(Chandrashekar, 2007; Bhat et al., 2010b). The gene was named as srl1 and the
deduced protein as SRL1. For functional validation, srl1 was cloned and expressed in E.
coli. SRL1 expressed in E. coli showed nematicidal effect on second-stage juvenile (J2) of
Meloidogyne incognita (Bhat et al., 2010b).

Of the several means of managing the pests, genetic engineering of crop plants is
considered promising for several reasons. Six transgenic events that were developed in
Department of Biotechnology, University of Agricultural Sciences, Dharwad using
Agrobacterium-mediated tomato transformation with pNR68 binary vector carrying srl1
and rvl1 gene in pHS100 (Bhagat, 2010; Patil, 2011) were used in the present study.
Expression of SRL1 and RVL1 was confirmed by agglutination assay with rabbit
erythrocytes. Agglutination activity of SRL1 and RVL1 were inhibited by only mucin
among various sugars and their derivatives tested. Transgenic tomato were analysed for
the toxicity of srl1and rvl1 to common root-knot nematode (Meloidogyne incognita).
Bioassay with J2 of M. incognita showed that the transgenics were moderately resistant
as against highly susceptible control plants.

With this background, the present investigation was carried out to know the effect
of E. coli-expressed Remusatia vivipara lectin (a plant lectin) and Sclerotium rolfsii lectin
(a fungal lectin) on the development and inhibition of some soil-borne pathogens like
Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum, Ralstonia solanacearum,
Meloidogyne incognita with the following objectives.
6

Objectives

1. Evaluation of plant and fungal lectins for the in vitro suppression of some common
soil-borne pathogens.

2. Influence of lectins on biocontrol agents in the suppression of soil-borne


pathogens in vitro.

3. Evaluation srl1 and rvl1 transgenics and non-transgenics for the suppression of
tomato soil-borne pathogens.

4. Mechanism of action of lectins in the suppression of soil-borne pathogens.


2. REVIEW OF LITERATURE

Lectins have become the focus of intense interest for biologists


considering the research and their applications in agriculture and medicine
(Movafagh et al., 2013). These proteins with unique characteristics have found
use in diverse fields of biology and as more lectins are being isolated and their
role in nature elucidated, they continue to occupy an important place in
agricultural and therapeutic areas of research. Lectins have been implicated in a
variety of roles dealing with the defense mechanism of plants. They are shown to
protect plants against bacterial, fungal, and nematode pathogens. Presently, very
little literature is available in India on lectins of Remusatia vivipara and
Sclerotium rolfsii. Hence the available literature on these lectins is being
reviewed hereunder.

Lectins were first described by Stillmark (1888) working with castor bean
protein extracts, which were referred to as haemagglutinins or phytoagglutinins
(ricin) because of their ability to agglutinate erythrocytes. Later, a number of
haemagglutinins from various plant seeds were described. Screening of such
proteins for blood group-specific agglutinating activity led to the general concept
that each plant haemagglutinin binds specifically to erythrocytes of a particular
human blood group within the ABO system. Later, such haemagglutinins were
termed “lectin”, subsequently lectins were also shown to agglutinate blood cells
of rabbit, sheep, goat etc. because of their specific sugar binding activity. As
mentioned already, they are shown to protect plants from different pathogens.
This could be due to; a) the presence of lectins at the potential invasion sites by
infectious agents, b) the binding of lectins to various fungi inhibiting their growth
and germination; and c) lectins binding to the chemoreceptors and glycoproteins
present in the digestive tract of nematode have either antifeedant effect or result
in mortality or retarded growth.

2.1 Sources of lectins

Lectins are widely found and extensively distributed in nature. Several


hundreds of these molecules have been isolated from plants, fungi, viruses,
bacteria, invertebrates and vertebrates (Peumans and Van Damme, 1995).

2.1.1 Classification of lectins


8

While all lectins have a carbohydrate recognition domain (CRD) that


determines their specificity, their classification depends on the source, i.e. plant,
animal, microbial, etc. The animal lectins are classified based on their aminoacid
sequence homology and evolutionary relatedness, while the plant lectins are
grouped according to the plant family. Lectins found in microorganisms are
classified according to the function, e.g. adhesins or haemagglutinins and toxins.

Based on the overall structure and carbohydrate-binding sites, lectins are


distinguished as merolectins, hololectins, chimerolectins and superlectins.
Merolectins consists of a single carbohydrate-binding domain (monovalent),
hence cannot precipitate glycoconjugates or agglutinate the cells. Hololectins
also are built exclusively of carbohydratebinding domains but contain two or
more such domains that are either identical or homologous and bind either the
same or structurally similar sugar(s). Majority of all known plant lectins are
hololectins and behave as haemagglutinins. Chimerolectins are fusion proteins
possessing a carbohydrate-binding domain tandemly arrayed with an unrelated
domain which has catalytic activity that can bind to carbohydrates. Depending on
the number of carbohydrate-binding sites, chimerolectin could be like merolectin
or hololectin. Superlectins like hololectins, consist excluisively of two
carbohydrate-binding domains. However, unlike the hololectins, the
carbohydrate-binding domains of the superlectins recognize structurally
unrelated sugars. Therefore, superlectins can also be considered as a special
type of chimerolectin composed of two tandemly arrayed structurally and
functionally different carbohydrate-binding domains

2.2 Plant lectins

Plant lectins are usually considered to be a large and


heterogeneous group of proteins with specific carbohydrate-binding activity. They
differ in physico-chemical properties, molecular structure and carbohydrate-
binding specificity. In plants, lectins are distributed in different types of tissues
(Peumans and Van Damme, 1995). High amount of lectin is found in storage
organs like seeds; however low quantities are also found in leaves, stem, roots
and flowers (Rudiger, 1998). The major intracellular
9

location of plant lectins is inside protein bodies, but lectins also occur in the
cytoplasm and in the intercellular space. Plant lectins have been subdivided on
the basis of their carbohydrate-binding specificity into mannose-,
mannose/glucose-, mannose/maltose-, Gal/GalNAc-, GlcNAcc/ (GlcNAc)n-,
fucose-, and sialic acid-binding lectins. The rapid progress in the structural
analysis of lectins and the molecular cloning of lectin genes have provided
detailed sequence information of over 100 lectins. Analysis of available
sequences distinguishes seven families of evolutionarily related proteins. Four of
these families, namely, the legume lectins, the type 2 ribosome-inactivating
proteins (RIP), the chitin-binding lectins containing hevein domains and the
monocot mannose-binding lectins are considered to be 'large' families. The
amaranthins, the cucurbitaceae phloem lectins and the jacalin related lectins
comprise only a small number of individual lectins and accordingly are
considered 'small' families. Several lectin genes have been cloned on the basis
of structural similarity. Most of the monocot mannose-binding lectins have been
cloned and characterized from families of angiosperms such as Amaryllidaceae,
Araceae, Alliaceae, Orchidaceae, Liliaceae, Iridaceae, Bromeliaceae and
Taxaceae families of gymnosperms. Recently tarin1 lectin isolated from
Colocasia esculenta L. Schott is well characterized mannose binding type plant
lectin, mainly expressed in corm storage parenchyma cells.

Monocot mannose-binding lectins have exclusive recognition of mannose


and mannooligosaccharides. The first member of this family was the lectin
isolated from snowdrop (Galanthus nivalis) bulbs, a member of the
Amaryllidaceae family (Van Damme et al., 1987). By means of biochemical
analysis and molecular cloning, identification and characterization of related
lectins from other member families, namely, Alliaceae, Araceae, Bromeliaceae,
Orchidaceae, Liliaceae and Zridnceae have been identified (Barre et al., 1996;
Van Damme et al., 2000). Other than unique specificity for mannose, these
lectins have resemblance with respect to their amino acid composition, sequence
and molecular structure.

Recently, some of the monocot mannose-binding lectins are reported to


have antiproliferative and apoptosis inducing effects on different tumor cells (Liu
et al., 2009). Monocot mannose-binding lectins are being explored extensively for
10

crop protection in plant biotechnology as many of them are reported to exhibit


potent toxic effects on sucking pests and nematodes. Their strict specificity for
mannose sets them apart from the Glc/Man/Gal specific family of dicotyledonous
legume lectins and the C-type mannose binding animal lectins. Very few lectins
are characterized from Araceae species as compared to several lectins reported
from other monocot families (Shangary et al., 1995; Mo et al., 1999).

2.2.1 Remusatia vivipara lectin

It is a mannose-binding lectin present in the tubers of Remusatia vivipara,


a monocot vegetable plant. Remusatia vivipara is an epiphyte found in Western
Ghats of South India and all its parts viz., tubers, leaves and the stalks are
edible. Tubers are traditionally used as folk medicine for treating inflammation
and arthritis. Recently, Remusatia vivipara lectin (RVL1) has been purified from
its tubers (Bhat et al., 2010) which agglutinates only rabbit erythrocytes, and
possesses specificity for mucin and asialofetuin. RVL1 activity was stable up to
80°C and under wide range of pH (2.0–9.3). RVL1 was purified by a single-step
affinity chromatography on asialofetuin-Sepharose 4B. rvl1 gene coding for RVL1
was cloned for the first time at the Department of Biotechnology, University of
Agricultural Sciences, Dharwad (Neekhra, 2009). Sequence analysis indicated
an opening read frame of 771 bp with a deduced polypeptide of 256 amino acids
corresponding to 28.2 kDa. Post-translational cleavage of RVL1 precursor into
two mature polypeptides of 116 and 117 amino-acid residues was evident by N-
terminal sequencing, which gave two different sequences. However, RVL1
seems to be a tetramer (49.5 kDa) with two dimers. Each dimer consists of two
distinct subunits of 12 kDa and 12.7 kDa.

2.2.2 Functions of plant lectins

Plant lectins have a wide range of biological functions. They function as a


nitrogen source or recognition factor. Legume lectins are involved in rhizobial
attachment and root nodulation. Some plant lectins may be involved in plant
signaling pathway. Recent studies show that lectins are involved in the defense
mechanisms of the plant against viruses, bacteria, fungi, nematodes and insect
pests (Etzler, 1985; Chrispeels and Raikhel, 1991; Van Damme et al., 1994a and
Van Damme et al., 1994b).
11

2.3 Fungal lectins

Occurrence of lectin in fungi is known for long time (Gold and Balding,
1975) that had been isolated and characterized from different fungi. Many of
them play a biological role in antitumour, immunomodulatory and insecticidal
activities. The first lectin to be purified from fungus (including mushroom and
yeast) was from the fruiting bodies of the meadow mushroom, Agaricus
campestris and the common (commercial) mushroom, Agaricus bisporus. Lectins
have also been found in phytopathogenic fungi such as Botrytis cinerea,
Pleurotus ostreatus, Rhizoctonia solani, in different members of Sclerotiniaceae,
in the nematode trapping fungus Arthrobotrys oligospora, and in few species of
yeasts like Saccharomyces cerevisiae and Kluyveromyces bulgaricus.
Traditionally, some fungi have been used to control insects. For example,
Lycoperdon spores have been used to anaesthetize bees, Amanita muscaria will
kill houseflies when added to sugar solution, and the powder of Trametes
odorata keeps insects away from clothing. These observations suggest that
some fungi could contain repellent, antifeedant or even toxic compounds,
possibly active against a wide range of insects. Mier et al. (1996) screened 175
fungal species, and showed that nearly half of them were toxic. Wang et al.
(2002) further analyzed 14 most toxic species and found that mushroom fruit
body proteins were responsible for most of the insecticidal activity. A 15 kDa
lectin was purified from Xerocomos chrysenteron, an edible mushroom which
was found to be most toxic to the insects (Trigueros et al., 2003). In the recent
past several fungal lectins were purified and characterized (Kellens et al., 1989;
Pemberton, 1994; Kawagishi, 1995; Guillot and Konska, 1997; Wang et al.,
1998). Majority of the fungal lectins were isolated from the fungal fruiting bodies
and rarely from the vegetative mycelia (Kellens et al., 1992; Giollant et al., 1993;
Wang et al., 1998; Oda et al., 2003).

2.3.1 Sclerotium rolfsii lectin

Sclerotium rolfsii is a plant-pathogenic fungus which causes severe soil-


borne diseases in various crops. Sclerotial bodies of S. rolfsii secrete a cell wall-
associated, developmental-stage specific lectin (Swamy et al., 2004). This 17
kDa S. rolfsii lectin (SRL) was purified (Swamy et al., 2001). SRL is expressed on
12

the mycelia at the time of sclerotial body formation and facilitates the aggregation
of the mycelium to form sclerotial bodies by interacting with its endogenous
glycosyl ceramide receptor(s), which have specific carbohydrate moieties. SRL is
a monomer under acidic conditions (pH 4.3) and all experimental evidences so
far suggest that it forms a dimer at neutral or basic pH (Swamy et al., 2001). SRL
displays a clear specificity for Galβ1-3GalNAc-α- (TF, Thomsen-Friedenreich
antigen) and GalNAc-α- (Tn antigen) glycan structures (Wu et al., 2001).
Immunolocalization and expression studies on SRL have lead to the identification
of putative endogenous glycosphingolipid receptor, which recognizes and
interacts with SRL during the functional role of development and morphogenesis
of S. rolfsii (Swamy et al., 2004). Both the crystal structure and the amino acid
sequence have been reported to explain structural basis of carbohydrate
recognition (Leonidas et al., 2007). The crystal structure of SRL in its free form
and in complex with N-acetyl-D-galactosamine (GalNAc) and N-acetyl-D-
glucosamine (GlcNAc) was determined at 1.1 Å, 2.0 Å, and 1.7 Å resolution,
respectively. The data for the free SRL were the highest resolution data for any
protein of this family. The protein structure was composed of two β-sheets, which
consisted of four and six β-strands, connected by two α-helices. The crystal
structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc,
which differ only in the configuration of a single epimeric hydroxyl group,
provided the structural basis for its carbohydrate specificity. SRL has two distinct
carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the
primary site, whereas GlcNAc binds only at the secondary site.

Thus, SRL has the ability to recognize and probably bind at the same time
two different carbohydrate structures. Further, sequence ambiguities were
resolved using mass spectrometry (Sathisha et al., 2008). Both the crystal
structure and the amino acid sequence of SRL revealed that SRL is similar to
fungal lectin XCL (Birck et al., 2004). Cloning a lectin gene from S. rolfsii
revealed an open reading frame (ORF) of 429 bp corresponding to a polypeptide
of 142 residues with molecular weight of 16 kDa. Deduced polypeptide had
76.1% sequence similarity with SRL. Despite 34 amino acid mismatches, both
primary and secondary carbohydrate-binding sites remained unchanged
(Chandrashekar, 2007; Bhat et al., 2010b). It acts as signaling molecules in
13

interaction with antagonists (Barak et al., 1985; Barak and Chet, 1990; Inbar and
Chet, 1994). Immunolocalization and expression studies on SRL have lead to the
identification of putative endogenous glycosphingolipid receptor, which
recognizes and interacts with SRL during the functional role of development and
morphogenesis of S. rolfsii (Swamy et al., 2004).

2.3.2 Functions of fungal Lectins

Many fungal lectins play a biological role in antitumour,


immunomodulatory and insecticidal activities. Fungal lectins are involved in
morphogenesis and development of fungi and mediate host-parasite interactions
(mycoparasitism) (Hostetter, 1994; Fukazawa and Kagaya, 1997; Rudiger,
1998). Fungal lectins also function as storage proteins (Kellens and Peumans,
1990), and involved in morphogenesis and development of the fungi. Despite
reports on their diverse biological functions and knowledge of molecular
characterization, limited information is available on plant pathogen interaction in
defense of fungal lectin compared to plant and animal lectins. Their effects on
mammalian physiology as antitumor and anticancer have been well
characterized.

2.4 Lectins in plant defence

Recent studies show that lectins are involved in the defense mechanisms
of the plant against viruses, bacteria, fungi, nematodes and insect pests (Van
Damme et al., 1994a). This is primarily based on the fact that lectins bind to
glycoconjugates of other organisms. Although many plant lectins are able to bind
simple sugars such as glucose, mannose or galactose, they have a much higher
affinity for oligosaccharides, which are not common or totally absent in plants.
For instance, chitin-binding plant lectins recognize a carbohydrate that is typical
constituent of the cell wall of fungi and the exoskeleton of invertebrates. A
circumstantial argument in favour of a defense role of plant lectins is their marked
stability under a wide range of pH, heat and exposure to proteases. However, the
direct evidence that lectins play a role in plant defense was obtained using
purified protein in artificial diet or by using transgenic plants.
14

2.5 Evaluation of plant and fungal lectins for in vitro suppression of


some common soil-borne pathogens

2.5.1 Fungi

Soybean lectin, which recognizes D-galactose and N-acetyl-D-


galactosamine and the peanut lectin, specific for D-galactose, bound to young
spores and mature regions of hyphae of Penicillium and Asergillus spp. All three
lectins were found to interfere with growth and delayed spore germination of
Aspergillus ochraceus. It was proposed that the lectins may protect plants
against fungal pathogens during seed imbibitions, germination and early growth
of the seedlings (Mirelman et al., 1975). A potato lectin, with a specificity similar
to that of WGA (Wheat germ agglutinin) inhibited the hyphal extension and spore
germination in Botrytis cinerea (Callow, 1977). WGA itself binds to the hyphal tips
and septa of young spores of a number of species of chitin containing fungi in the
Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes (Golan et al.,
1978).

Under in vitro the zoospores adhere to the root region behind the cap
cells, germinate and penetrate the epidermis and cortex. When the root surface
carbohydrate was modified by oxidation, it completely destroyed the root’s
capacity for zoopore adhesion (Green and Northcote, 1978).

Lis and Sharon (1981) showed inhibition of fungal growth can occur
through lectin binding to hyphae resulting in poor absorption of nutrients as well
as by interference on spore germination process.

Antifungal property of potato tuber lectin was studied against early


developmental stages of Fusarium oxysporum, a fungal pathogen of potato.
Lectin did not inhibit mycelial growth but irreversibly inhibited conidia germination
and altered the germ tubes (Gozia et al., 1993).

Fungal lectins act as storage proteins in fungal-fungal interactions


(mycoparasitism), in host parasite interactions and also involved in
morphogenesis and development of the fungi (Cooper et al., 1997; Rudiger,
1998). Antifungal activity of lectins may vary depending on the species, AMML
lectin with binding affinity for
15

Plant lectins with fungicidal properties

Lectin Lectin specificity Fungus / fungi suppressed Reference

Amaranthus viridis (AML) Asialofetuin, fetuin, T- Botrytis cinerea, Fusarium oxysporum (Kaur et al., 2006)
antigen, N-acetyl-D-
lactosamine, N-acetyl-D-
galactosamine

Annona muricata (AML) Glucose/mannose Fusarium solani, F. oxysporum, Colletotrichum musae (Damico et al., 2003)

Artocarpus incisa (AIL) GlcNAc Fusarium moniliformae, Saccharomyces cerevisiae (Santi-Gadelha et al.,
2006)

Artocarpus integrifolia (AIL) GlcNAc Fusarium moniliformae, S. cerevisiae (Trindade et al., 2006)

Astragalus mongholicus Lactose/D-Gal Botrytis cincerea, Fusarium oxysporum, Colletorichum sp., (Yan et al., 2005)
(AMML) Drechslera turcicum

Bauhinia monandra (BmoRoL) Carbohydrate Fusarium solani, F. oxysporum (Souza et al., 2011)

Complex

Capsicum frutescens (CFL) D-mannose, glucose Aspergillus flavus, Fusarium Moniliformae (Ngai and Ng, 2007)

Curcuma amarissima (jackin N-acetylglucosamine Fusarium oxysporum, Exserohilum turcicum, Colectrotrichum (Kheere et al., 2010)
and frutackin) cassiicola

Contd…
16

Gastrodia data (GNA) α-Man/ GlcNAc Botrytis cinerea, Ganoderma lucidum, Gibberella zeae, (Xu et al., 1998)
Rhizoctonia solani, Valsa ambiens

Hevea brasiliensis (Hevien) Chitotriose Botrytis cinerea, Fusarium culmorum, F. oxysporum f. sp. pisi, (Van Parjis et al., 1991)
Phycomyces blakesIeeanus, Pyrenophora triticirepentis,
Pyricularia oryzae, Septoria nodorum, Trichoderma hamatum

Indigofera heterantha Seeds Carbohydrate Aspergillus niger, A. oryzae and Fusarium oxysporum (Sakeena et al., 2013)
(IHL)
Complex

Myracrodruon urundeuva (MUL) GlcNAc Fusarium solani, F. oxysporum, F. moniliformae, F. (Sa et al., 2009a)
decemcellulare, F. lateritium, F. fusarioides, F. verticiloide

Ophiopogon japonicus Man Gibberella saubinetii, Rhizoctonia solani (Tian et al., 2008)

Opuntia ficus indica (OFL) Glc/Man Colletrotrichum gloesporioides, Candida albicans, Fusarium (Santana et al., 2009)
oxysporum, F. solani

Phaseolus coccineus (PCL) Sialic acid Helminthosporium maydis, Gibberell sanbinetti, Rhizoctonia (Chen et al., 2009)
solani, Sclerotinia sclerotiorum

Pisum sativum (PSA) Man Aspergillus flavus, Fusarium oxysporum, Trichoderma viride (Sitohy et al., 2007)

Pouteria torta (PTL) Carbohydrate Fusarium oxysporum (Boleti et al., 2007)

Complex

Contd…
17

Sebastiania jacobinensis (SBL) Glycan complex Fusarium moniliforme, F. oxysporum (Vaz et al., 2010)
carbohydrate

Sophora alopecuroides (SAL) Carbohydrate Penicillium digitatum, Alternaria alternata (Li et al., 2012)

Complex

Talisia esculenta (TEL) Man Colletotrichum lindemuthianum, Fusarium oxysporum, (Freire et al., 2002)
Sacchromyces cerevisiae

Talisia esculenta (TEL) N-acetylglucosamine Sacchromyces cerevisiae, Fusarium moniliforme and (Damico et al., 2003)
Colletotrichum lindemuthianum

Talisia esculenta (TEL) N-acetylglucosamine Fusarium oxysporum, Colletotrichum lindemuthianum and (Maria et al., 2002)
Sacharomyces cerevisiae

Triticum vulgaris (WGA) GlcNAc Fusarium graminearum, F. oxysporum (Ciopraga et al.,


1999)

Ulex europaeus (UEA-1) L-fucose Phytophthora cinnamomi (Callow, 1987)

Urtica dioica (UDA) GlcNAc Botrytis cinerea, Colletotrichum lindemuthianum, Phoma (Broekaert et al.,
betae, Phycomyces blakesleeanus, Septoria nodorum, 1989)
Trichoderma hamatum, T. viride

Urtica dioica L. isolectin I (UDA- GlcNAc Botrytis cinerea, Trichoderma viride, and Colletotrichum (Does et al., 1999)
1) lindemuthianum
18

galactose and its derivatives in relation to Rhizoctonia solani and Mycospharella


arachidicola in concentration up to 200 µg/ml were not found antifungal activity
(Yan et al., 2005).

Chumkhunthod et al. (2006) reported SCL (Schizophyllum commune) lectin


binding affinity to N-acetylgalactosamine and showed antifungal activity against
Saccharomyces cerevisiae, Candida albicans and Penicillium digitatum at
concentration of 1 mg/ml. Lectin from manioc leaf powder showed fungicidal
properties against Fusarium oxysporum at 100 µg /ml (Silva et al., 2010).

Amano et al. (2012) reported that AAL is an L-fucose-specific lectin


produced in the mycelia and fruiting bodies of ascomycete fungus Aleuria aurantia
showed interaction with Mucor racemosus. AAL specifically bound to the hyphae of
M. racemosus and inhibited the growth at 1 µm and disrupted the growth at 7.5 µm.

2.5.2 Antibacterial activity

Lectins have a role in the plant defense against bacteria, it may be through
an indirect mechanism that is based on interaction with cell wall carbohydrate or
extracellular glycans. For instance, the potato lectin (which is considered as a cell
wall protein) immobilized virulent bacterial strains of Pseudomonas solanacearum
by agglutinating the cells (Sequeira and Graham, 1977).

Bourne et al. (1994) showed antibacterial activity on Gram-positive and


Gram-negative bacteria occurs through the interaction of lectin with components of
the bacterial cell wall including teichoic and teicuronic acids, peptidoglycons and
lipopolysaccharides: the study revealed that the isolectin I from Lathyrus ochrus
seeds bound to muramic acid and muramyl dipeptide through hydrogen bonds
between ring hydroxyl oxygen atoms of sugar and carbohydrate binding site of
lectin and hydrophobic interactions with the side chains of residues Tyr100 and
Trp128 of isolectin I.

Morus alba lectin exhibited antibacterial activity against Pseudomonas


syringae pv. mori and the bacterial agglutination was also inhibited by the
glycoproteins, fetuin and tyroglobulin (Ratanapo et al., 2001).
19

Leal-Bertioli et al. (2003) reported that transgenic tobacco plants expressing


tarin1 inhibited the growth of Pseudomonas syringe pv. tomato.

Iris et al. (2005) reported that Zuccagnia punctata leaf extract was active
against all assayed bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus
mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii,
Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas
maltophilia) with minimal inhibitory concentration (MIC) values ranging from 25 to
200 µg/ml. Minimal bactericidal concentration (MBC) values were identical or two-
fold higher than the corresponding MIC values.

Talas-Ogras et al. (2005) reported that cationic peptide isolated from


Robinia pseudoacacia seed inhibitied the 50% growth of Corynebacterium
michiganense, Staphylococcus aureus, Bacillus subtilis, Erwinia carotovora subsp.
carotovora, Pseudomonas syringae pv. syringae, Xanthomonas campestris pv.
campestris, and Escherichia coli at 20 and 120 µg/ml concentrations.

Santi-Gadelha et al. (2006) reported antibacterial activity of N-acetyl-D-


glucosamine-binding lectin isolated from Araucaria angustifolia seeds on
phytopathogenic bacteria revealed by reduction in the colony forming units (CFU).
The lectin was more effective against the Gram-positive Clavibacter michiganensis
(80% of reduction) than on Gram-negative Xanthomonas auxonoopodis (60%
reduction).

Oliveira et al. (2008) reported that purified EuniSL (Eugenia uniflora seed
lectin) strongly inhibited the growth of Staphylococcus aureus, Pseudomonas
aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) 1.5
µg/ml and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp.
and Escherichia coli with a MIC of 16.5 µg/ml.

Silva et al. (2009) reported antibacterial activity of leaf, root and fruit extracts
of Bixa orellana against Mycobacterium tuberculosis, Salmonella typhimurium,
Proteus mirabilis, Bacilus cereus, Staphyloccocus aureus and Pseudomonas
aeruginosa. MIC was assessed by diffusion and colorimetric analysis.

Selvi et al. (2011) reported that seed extract of Bixa orellana was
comparatively less efficacious against most of the tested pathogens except
20

Brucella sp. that was inhibited (15±0.1 mm) appreciably. Minimum inhibitory
concentration (MIC) of leaf extract was determined as 15.62 µg/ml against
Staphylococcus aureus and 31.25 µg/ml for Klebsiella pneumoniae, Pseudomonas
aeruginosa, Enterococcus fecalis and Staphylococcus typhi, on average. Among
the dermatophytes 78.2 per cent inhibition was seen in Trychophyton
mentagrophytes and Trychophyton rubrum.

2.5.3 Antinematode activity

Plant parasitic nematodes such as root-knot nematode infect nearly every


food crop, and are among the world's most damaging agricultural pests. For
example, root-knot nematodes parasitize more than 2,500 species from diverse
plant families and pose a tremendous threat to crop production world-wide. A
number of approaches hold promise, the best described being the expression of
lectins to disrupt nematode digestion (Victoria et al., 2008).

Rich et al. (1989) reported that ricin from castor bean inhibited more than 90
per cent of the mobility of M. incognita. Wheat germ agglutinin (WGA), pokeweed
(Phytolacca americana L.) and Bauhinia purpurea lectins were lethal at low
concentrations to juveniles of corn borer and root worms (Czapla and Lang, 1990).
This interaction of lectins with the receptors could result in anti-feedant effect, or
retarded growth or mortality (Peumans and Van Damme, 1995). Similar effects
were seen with antibodies, which on binding to the surface coat antigens of M.
javanica J2 affected both penetration and movement of nematode in Arabidopsis
thaliana roots (Sharon et al., 2002).

In vitro studies carried out at the Department of Biotechnology, University of


Agricultuyral Sciences, Dharwad showed the nematicidal effect for both native and
E. coli expressed forms of RVL1 and SRL1 on juveniles of Meloidogyne incognita
and also Sclerotium rolfsii lectin sharing a considerable homology, common
structural topology, glycan specificity, and carbohydrate-binding sites with XCL
when tested in PBS solution showed nematicidal activity against M. incognita (Bhat
et al., 2010a and Bhat et al., 2010b).

Bhat et al., (2010) showed efficacy (1:25, 1:50, 1:75 and 1:100 dilutions) of
tarin 1 (Colocasia esculenta L. Schott) on the activity of M. incognita juveniles.
21

Recently both native and E. coli expressed forms of Remusatia vivipara


lectin was found to be nematicidal and showed more than 85 per cent M. incognita
mortality at 1: 25 dilution (Neekhra et al., 2011).

2.6 Influence of lectins on biocontrol agents in the suppression of soil-


borne pathogens in vitro

Mirelman et al. (1975) showed wheat germ aggglutinin (WGA) inhibited the
growth of Trichoderma viride mycelium and spore germination by interacting with
fungal wall. WGA has a secificity for chitin oligomers of β-(1-4)-N-acetyl-D-
glucosamine and it interfered with chitin synthesis that lead to inhibition of fungus
growth. Successful biological control of Sclerotium rolfsii was achieved by applying
antagonistic species of Trichoderma. The interaction between S. rolfsii and
Trichoderma spp. was found to be mediated by the adsorption of S. rolfsii lectin (55
and 60 kDa) to conidia of Trichoderma (Elad et al., 1982).

Elad et al. (1983) showed the lectin activity in a host-mycoparasite


relationship in respect of Rhizoctonia solani and Trichoderma harzianum.
Attachment of ‘O’ but not ‘A’ and ‘B’ erythrocytes to hyphae occurred on R. solani
but not on its mycoparasite. This phenomenon, which was Ca2+ and Mn2+
dependent was prevented by galactose present in T. harzianum cell walls and by
fucose. This inhibition was also thought to be due to contaminating chitinases in
the lectin preparation (Schlumbaum et al., 1986).

Lectins of R. solani and S. rolfsii were found to be essential for the recognition
and adhesion of their mycoparasite, Trichoderma (Elad et al., 1983; Barak et al.,
1985; Barak and Chet, 1990).

Cytochemical localization of N-acetylglucosamine residues with WGA/Ricin


gold complex in cell walls of Fusarium oxysporum f.sp. lycopersici and Rhizoctonia
solani was hydrolyzed by an extracellular chitinase produced by Trichoderma that
leads to disorganization of the cellwall structure, promoted the internal osmotic
imbalanes that inturn, triggered intercellular disorders, such as retraction of the
plasma membrane and cytoplasm aggregtion (Cherif and Benhamou, 1990;
Benhamou and Chet, 1993). However implicating of S. rolfsii lectin (GalNac) with
Trichoderma harzianum leads to mycoparasitism (Neethling and Nevalainnen,
1996).
22

Parasitism of Trichoderma atroviride on Meloidogyne javanica nematode egg


masses, eggs and juveniles was enhanced when antibodies were incorporated into
in vitro bioassays. This shows that carbohydrate residues such as fucose, on the
surface of the nematode and fungal conidia are involved in the antibody and lectin
mediated improved parasitism (Edna et al., 2009).

2.7 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens

Virulent strains were not recognized by the lectin, escaped attachment to the
cell wall and therefore were able to multiply and spread over the plant. Another
indirect defense mechanism is the blocking of the movements of normally motile
bacteria at the air-water interface by the thorn apple (Datura stramonium) seed
lectin (Broekaert and Peumans, 1986).

Marban-Mendoza et al. (1987) provided the first indication that lectins may
have anti-nematode activity when application of concanavalin A lectin (Con A) to
the roots of tomato plants reduced the amount of galling inflicted by M. incognita by
75% compared with control plants. Similarly, Mannose-/glucose-specific lectin
Concanavalin A (Con A) reduced ability Globodera rostochiensis to locate host
diffusates on agar plates and (McCulloch et al., 1989).

Pusztai et al. (1990) showed the effect of the expressed lectins in Phaseolus
vulgaris on non-target organisms that feed on or live in the surroundings of the
plants i.e toxic to human and animal consumers of raw beans. Canola plants
carrying the 35S-chitinase-gene exhibit increased resistance to infection by the root
and stem rot pathogen Rhizoctonia solani. Seedlings constitutively expressing the
bean chitinase gene showed delayed development and progression of disease
symptom (Broglie et al., 1991).

ConA and WGA lectins expressed plants enhanced the penetration of


Radopholus citrophilus into citrus root segments than the control roots (Kaplan and
Davis, 1991). Lectin prevents the invasion of seedling roots by potentially harmful
bacteria by counteracting the chemotactic movement of soil bacteria toward the
germinating seed. Since studies of the binding of plant lectins to bacterial cell wall
23

peptidoglycans indicated that several legume seed lectins strongly interact with
muramic acid, N-acetylmuramic acid and muramyl dipeptide, the involvement of
lectins in the plant's defense against microbes may have been underestimated
(Ayouba et al., 1994).

Constitutive expression of tomato chitinase gene under CaMV 35S promoter


provides field tolerance to canola plants against Cylindrosporium concentricum,
Phoma lingam and Sclerotinia sclerotiarum (Grison et al., 1996). In tomato hairy
roots both Oc-I and Oc-I ∆D86-expressing roots leading to a reduction in size of
Globodera pallida females to a level at which fecundity is profoundly affected
(Urwin et al., 1995). Similarly Oryzacystatin protreinase inhibitor showed high level
of protection against M. incognita (Urwin et al., 1997).

Several efforts have been made to check toxic effect of lectins when
expressed in transgenic plants. Potato or oilseed rape expressing Galanthus nivalis
lectin (GNA) were shown to be partially resistant to Pratylenchus neglectus and
cyst-nematodes Globodera pallida and Heterodera schachtii (Burrows et al., 1998).

Griffiths et al. (2000) have done tests with transgenic potatoes expressing
GNA and ConA. Both in pot and field experiments no changes in the
decomposition of organic matter or negative consequences for plants in the
following growing season were observed. The effect of lectins on non-target soil
organisms and the processes in which they are involved, like nitrification and
decomposition of organic matter. Hevein lectin gene carrying Brassica juncea
plants showed increased resistance to Alternaria brassicae infection (Kanrar et al.,
2002).

Constitutive expression of a snowdrop lectin (GNA) directed by CaMV35S


promoter has been used to assess anti-nematode activity in oilseed rape, potato
and Arabidopsis (Ripoll et al., 2003). Transgenics expression of lectins had showed
potential as an anti-nematode defense. Lectin genes are induced in response to a
number of pathogens, including plant parasitic nematodes (Jammes et al., 2005;
Fuller et al., 2007). The lectin may exert its effect either through interaction with
glycoproteins present in the intestine or through binding to glycoproteins present
externally on the nematode, M. incognita and H. schachtii.
24

The expression of Gastrodia elata lectins in the vascular cells of roots and
stems was strongly induced by the fungus Trichoderma viride, indicating that lectin
is an important defense protein in plants (Sa et al., 2009b).

Developed transgenic plants expressing heterologous proteins with


nematicidal activity is considered important for reducing crop losses. A recent study
also indicated the role of taro cystain in sex determination of root-knot nematode
when expressed in tomato. A much lower proportion of female root-knot
nematodes without growth retardation were observed in the transgenic tomato. A
decrease of egg mass in transgenic plants indicated seriously impaired fecundity
(Chan et al., 2010; Bhanu Priya et al., 2011).

Bhagat (2010) found that rvl1 and srl1 gene carrying transgenic tomato
plants showed moderately resistance to Meloidogyne incognita. rvl1-transgenic
tomato plants had significantly reduced gall formation due to M. incognita infection
compared to srl1-transgenic tomato plants. srl1 expressed SRL1-T0(7), SRL1-
T0(10), SRL1-T0(21) transgenic tomato line reduced the root galling as 33.6, 43.2
and 40.8 galls/plant compared to controlled plants (Patil, 2011). rvl1 expressed
RVL1-T0(11) and RVL1-T0(28) transgenic tomato plants showed moderate
resistance to Meloidogyne incognita reduced 40.00 and 34.48 per cent of root
galling compared to control (Kolekar, 2012).

2.8 Mechanism of action of lectins in the suppression of soil- borne


pathogens

2.8.1 Fungi

Since plant lectins cannot bind to glycoconjugates on the fungal membranes


or penetrate the cytoplasm of the cells because of the presence of a thick and rigid
cell wall, a direct interference with the growth and development of these organisms
(i.e. through an alteration of the structure and/or permeability of the membrane or a
disturbance of the normal intracellular processes) seems unlikely. However,
indirect effects based on the binding of lectins to carbohydrates exposed on the
surface of the fungal cell wall are possible. By virtue of their specificity, chitin-
binding lectins seemed likely to have a role in the plant's defense against fungi.
25

Mirelman et al. (1975) found that wheat germ agglutinin (WGA), a lectin
specific for chitin oligosaccharides binds to hyphal tips and hyphal septa of
Trichoderma viride and inhibits hyphal growth and spore germination of this chitin
fungus. Lectins recognise and bind to sugar residues on fungal membranes or cell
walls (Horisberger and Vonlanthen, 1977).

Galun et al. (1976) examined the binding of FITC labelled WGA, ConA,
PNA, SBA, LFA and WBA with different sugar specificities, to the hyphal wall and
septum of three mycobionts isolated from lichens (Xanthoria parietina, Tornabenia
intricata and Sarcogyne sp.) and free living fungi (Trichoderma viride and
Phytophthora citrophthora). FITC-SBA (soybean agglutinin) and FITC-PNA (Peanut
agglutinin) bound to young conidiophores, metulae, vesicles, sterigmata and young
spores of the Penicillium and Aspergilli (Golan et al., 1978).

Several other chitin-binding plant proteins by definition are regarded as


lectins have antifungal properties. The first group is the chitin binding merolectins,
which are small proteins composed of a single chitin-binding domain. Hevein, a 43-
amino acid polypeptide from the latex of the rubber tree (Hevea brasrliensis) has
an antifungal activity comparable to that of the nettle lectin (Van Parijis et al.,
1991). The exact mechanism of the nettle lectin has not been elucidated yet, but it
is certainly not based on a chitinase activity and it does not affect the normal
metabolism of the fungal cells. Only the synthesis of the cell wall appears to be
affected as a result of disturbed chitin synthesis and/or deposition (Van Parijis et
al., 1992). Binding to cell wall constituents in fungi can interfere with their growth.
Fungal cell walls contain chitin, the β1-4 linked polymer of GlcNAc. It is therefore
likely that a fungicidal action is exerted by GlcNAc-binding lectins (Does et al.,
1999).

2.8.2 Bacteria

The bacterial cell wall prevents an interaction between the glycoconjugates


on their membrane and carbohydrate-binding proteins but also prevents these
proteins from penetrating the cytoplasm. Therefore the plant lectins cannot alter the
structure and permeability of the membrane or disturb the normal intercellular
processes of invading microbes. Therefore, if lectins play a role in the plant’s
defense against bacteria, it must be through an indirect mechanism that is based
26

on interaction with cell wall carbohydrate or extracellular glycans. It has been


suggested for instance, that the potato lectin (considered as a cell wall protein)
immobilized avirulent strains of Pseudomonas solanacearum in the cell wall
(Sequeira and Graham, 1977). By counteracting the chemotactic movement of soil
bacteria toward the germinating seed, the lectin may prevent invasion of the
seedling roots by potentially harmful bacteria. Since recent studies of the binding of
plant lectins to bacterial cell wall peptidoglycans indicated that several legume
seed lectins strongly interact with muramic acid, N-acetylmuramic acid, and
muramyl dipeptide, the involvement of lectins in the plant’s defense against
microbes may have been underestimated (Ayouba et al., 1994).

For bacteria and fungi the cell wall hampers any interaction of lectins with
glycoconjugates on the cell membrane as well as the entrance of lectins into the
cytoplasm. Through an indirect mechanism, such as interaction with sugar moieties
of the cell wall or extracellular glycans, lectins might have an influence on the
mobility of bacteria or cell-wall synthesis in fungi in the case of chitin-binding plant
lectins (Peumans et al., 1995). Araucaria angustifolia lectin promoted
morphological alterations including presence of pores in the Clavibacter
michiganensis membrane and bubbling on the Xanthomonas axanopodis cell wall
(Santi-Gadelha et al., 2006).

2.8.3 Nematode

The lectin may exert its effect either through interaction with glycoproteins
present in the intestine or through binding to glycoproteins present externally on
the nematode. Interaction with glycoproteins associated with chemoreception, on
the amphid sensory organs themselves or in their secretions, could disrupt sensory
perception and compromise the nematode’s ability to establish feeding sites
(Zuckerman, 1983). Utilizing a Concanavalin A (ConA) hemocyanin conjugate, the
majority of cuticular ConA binding sites were shown to be localized on the head
region of Caenorhabditis elegans and Meloidogyne incognita. Secretions which
apparently emanated from the amphids and inner labial papillae did not label
(McClure and Zuckerman, 1982) as well as specific localization of sialic acid
residues on the head region of Xiphinema index suggest a logical line of
speculation as the mode of action of Con A on M. incognita. Binding of the ligand
27

presumably could result in blocking or altering the conformation of the nematode


chemoreceptors (Spigel et al., 1982).

Aumann and Wyss (1989) detected specific binding sites of Con A, WGA
(Wheat germ agglutinin), PNA (Pean nut agglutinin), LFA (Limax flavus agglutinin)
and HPA (Helixa pomatia) in the region of the amphidial apertures, of Con A, WGA,
PNA and HPA on the spicule tips, and of HPA in the region of the excretory pore
opening of Heterodera schachtii male because the exudates of the main
chemoresceptors thus consist most like protein backbone to which oligosaccharide
chains are coupled superficially (Roberts and Goldstein, 1983; Aumann and Wyss,
1991).

Alternative mechanism involves interference in the sensory perception and


the ability to establish feeding cells by the binding of lectins to glycoproteins
localized on the surface of the nematodes or chemoreceptors present in the
amphids and amphidial secretions. A glycoprotein surface receptor has been
characterized from the nematode, Panagrellus redivivus (Borrebaeck et al., 1985).

Forrest and Robertson (1986) found specific binding sites of WGA on the
amphidial exudate of second stage juveniles (J2) of Globodera rostochiensis and
Forrest (1985) showed that lectin binding sites on this exudate can be reduced by
the protease pronase E. In another electron microscopic study McClure and Stynes
(1988) found UEA 1 binding sites on the amphidial exudate of Meloidogyne
incognita J2.

Davis et al. (1988) also report that the binding of ConA and WGA to the
amphidial secretions of secondary juveniles of Meloidogyne is not inhibited by the
specific sugars of either lectin. ConA and WGA have broader sugar specificity, or
their affinity for sugars in nematode secretions is too strong to be inhibited. A third
explanation is the affinity of certain lectins for other ligands than sugars. Binding of
WGA to the amphids, the amphidial secretions and the excretion pore of
Radopholus similis is specific. This means that the binding is inhibited by N-
acetylglucosamine oligomers. Non-specific binding of ConA and WGA observed at
vulval and anal region of Longidorus sp. (Robertson et al., 1989).

Lectins would also have opportunity to bind to the surface/cuticle and to the
primary chemosensory structures, the amphids. Lectin studies have revealed the
28

presence of various binding sites (carbohydrate residues) associated with the


amphidial secretions and the surface of plant nematodes (Spiegel et al., 1995;
Forrest et al., 1988).

ConA, contain a conserved hydrophobic binding site, and WGA and HPA
can also bind hydrophobic ligands. So non-specific interactions between lectins,
cell surfaces and secretions can occur results in binding to the vulva, spicules and
other body pores of R. similis (Wuyts et al., 2004). Trichoderma asperellum-203
and T. atroviride involved in M. javanica attachment and parasitic process which
mediated by chrbohydrate-lectin like interactions involved in attachment of conidia
to the nematodes (Sharon et al., 2007).

Fluorescent confocal microscopic studies demonstrated the binding of


Remusatia vivipara lectin to specific regions of the alimentary-tract and the binding
increased proportionally with time leading to the death of nematode (Bhat et al.,
2010a). MOA (Marasmium oreades agglutinin), fungal fruiting body chimerolectin
showed nematotocixity toward the model nematode C. elegans. Binding of MOA to
glycosphingolipds present in the nematode intestinal epithelium (Wohlschlager et
al., 2011).
3. MATERIAL AND METHODS
A systematic study was undertaken to test the efficacy of recombinant plant and
fungal lectins against soil-borne pathogens affecting tomato and fungal and bacterial
antagonists, in order to test their mechanisms of antimicrobial activity. Expressed srl1
and rvl1 genes encoding transgenic tomato were evaluated to study their biological
activity and plant growth promotion against soil-borne pathogens under glasshouse
conditions. The experiments were carried out from 2010-13 in the Department of Plant
Pathology and Department of Biotechnology, College of Agriculture, University of
Agricultural Sciences, Dharwad. Details of the materials used and the methods
followed for conducting the experiments are described hereunder.

3.1 Collection and isolation soil-borne pathogens

Tomato plants exhibiting symptoms due to Fusarium oxysporum f.sp.


lycopercisi, Sclerotium rolfsii, Rhizoctonia solani, Ralstonia solanacearum and
Meloidogyne incognita infections were collected from Main Agricultural Research
Station, UAS Dharwad field.

3.1.1 Isolation and purification of fungus

Tomato plants showing typical symptoms of Fusarium wilt, Sclerotium wilt and
Rhizactonia root rot were used for isolation of pure cultures of respective pathogens.
Standard tissue isolation procedure was followed for isolation of pathogen. The infected
parts were surface sterilized with 0.1 % sodium hypochlorite solution for 40 seconds and
washed serially in distilled water to remove the traces of sodium hypochlorite and then
transferred to sterilized Petri plate containing Potato Dextrose Agar. The Petri plates
were incubated at room temperature (27 ± 1oC) and observed periodically for the growth
of pathogens’ colonies. The colonies which developed from the bits were transferred to
PDA slants and incubated at 27 ± 1oC and they were purified by using hyphal tip
technique (Rangaswami, 1972). Such pure culture tubes were preserved in a refrigerator
at 4°C and used for further studies. Subculturing was performed once in a month.

.
30

3.1.2 Identification of the fungus

Based on morphological characters, the fungi were identified on the spore


morphology and colony characters of the fungus by referring to “Illustrated Genera of
Imperfect Fungi” (Barnett and Hunter, 1972). The characters were compared with those
described by Booth (1971) and the fungus was identified as Fusarium oxysorum.
Sclerotium rolfsii was identified based on the morphological and cultural characters and
formation of sclerotial bodies as described by Domsch et al. (1980). Similarly, the
morphological and cultural characters and formation of sclerotia were the principle
criteria to identify the pure cultures of Rhizoctonia solani. The characters were compared
with those described by Ashby (1927) and the fungus was identified as Rhizoctonia
solani.

3.1.3 Mass multiplication of R. solani and S. rolfsii

Sand-corn meal medium was prepared in the proportion of 90:10 in order to get
maximum inoculum of the fungus. About 400 g of sand-corn meal medium was taken in
1000 ml flasks and watered to 20 per cent of its weight and sterilized at 1.1 kg/cm²
pressure for one hour. The pure cultures of R. solani and S. rolfsii were inoculated
separately in different flasks under aseptic conditions and incubated at 27±1°C for 20
days. The flasks were shaken on alternate days to get uniform growth. The giant cultures
so obtained were used for further studies.

3.1.4 Mass multiplication of F. oxysporum

The multiplication of the pathogen on large scale was achieved by inoculating the
isolated pathogen into the flasks containing sterilized sorghum seeds. Sorghum grains
were soaked in water for overnight and excess of water was removed. Then about 200-
250 g seeds were placed in each 1000 ml conical flasks. These flasks were double
sterilized at 1.1 kg/cm2 pressure for an hour. The contents of the flasks were shaken
after sterilization to prevent clumping. A 5-mm colony disc of F. oxysporum was
aseptically inoculated to the cooled flasks. The flasks were incubated at 27 ± 1°C for 15
days. To obtain uniform growth, the contents of the flasks were shaken periodically.
Obtained giant culture was used for further studies.
31

3.1.5 Pathogenicity

Sterilized soil was taken in 30 cm earthen pots. Fifteen to twenty days old giant
culture was thoroughly mixed with soil at the rate of four per cent to get sick soil. Then
apparently healthy seedlings of tomato (cv. Pusa Ruby) were planted in pots filled with
sick soil. Seedlings planted in pots without inoculum served as control. Soil moisture
was maintained at 25 per cent moisture holding capacity of soil by adding sterilized
water on weight basis throughout the period. After 30 days of inoculation the plants
showing the typical wilting symptoms were observed. Reisolation was made from such
affected portion of the plant tissue and compared with that of original culture.

3.1.6 Isolation of R. solanacearum

Tomato plants showing typical symptom of wilting caused by R. solanacearum


were collected. Preliminary diagnosis of the disease was done by ooze test. The
bacterium was isolated by extracting the ooze in sterile distilled water taken in test tube,
followed by dilution plate technique on sucrose peptone agar containing TZC (triphenyl
tetrazolium chloride) medium. Small pieces of infected vascular tissue were aseptically
cut with a sterile surgical blade after peeling of epidermal layer. The bits of infected
tissue were surface sterilized in 0.1 per cent sodium hypochlorite for a minute and was
washed in two to three series of sterile water to remove traces of sodium hypochlorite.
The infected tissues were then suspended in sterile water taken in test tube which
became turbid due to oozing of bacterial cells from the infected tissue. The bacterial
suspension was serially diluted and 1000 l of the diluted bacterial suspension was
poured onto the surface of solidified sucrose peptone agar medium with a sterilized
spreader. The inoculated plates were incubated at 29 ± 1oC for 48 h. The plates were
observed for the development of well separated virulent colonies.

3.1.7 Purification, preservation and identification of R.solanacearum

The identification of the bacterium isolated from tomato plants was made based
on its morphological, physiological, cultural and biochemical characteristics besides
pathogenicity studies based on reports of Kelman (1954) and Schaad (1992). Well
separated virulent colonies of R. solanacearum on TZC medium were picked up and
streaked on the surface of TZC medium. The virulent colonies of R. solanacearum were
32

irregular, dull white, mucoidal, slimy with slight pink center. For the purpose of
purification, the well separated typical virulent colony was picked up and streaked on the
surface of TZC medium and plates were incubated at 29 ± 1oC for 48 h. Three to four
loopful of the bacterial culture picked up from the well separated colonies from streaked
TZC medium was suspended into 2 ml sterilized distilled water contained in eppendorf
tubes and stored at 4oC in refrigerator for further use as stock culture. For long term
preservation a loopful bacterium was suspended in 600 µl of sucrose peptone broth in
1.5 or 2 ml eppendorf tube to which 600 µl of glycerol (50 per cent) was added. The
ependorf tubes were stored at -80 oC for further use.

3.1.8 Growth phase

Sucrose peptone broth for R. solanacearum was used to study the growth phase
at different incubation period. Five ml of broth was dispensed in individual test tubes and
sterelized at 1.1 kg/cm2 for 15 min. The 48 h old inoculum was suspended in sterile
distilled water containing 2x106 cfu/ml used at the rate of 50 µl per test tube. The test
o
tubes were incubated at temperature 29±1 C. The growth was recorded
turbidometrically at 8h interval upto 168 h using Systronics PC based double beam
spectrophotometer 2202 at 600 nm. The increase in the turbidity of culture broth
indicated increase of microbial cell mass. The experiment was conducted with three
replications using Completely Randomized Design for statistical interpretations.

3.1.10 Pathogenicity

Inocula for pathogenicity tests were prepared with 48 h old cultures grown on
TZC medium at 29±1oC. Colonies were suspended in sterilized water to get 1x108
cfu/ml using a spectrophotometer transmission at A600=0.1 and were used for
inoculation immediately after preparation. The soil mixtures used in the research were
steam sterilized for 2 h. Fourteen days old seedlings of Pusa Ruby (Susceptible check
cultivar) were grown in the pot containing sterilized soil mixture as one plant per pot
and maintained under green house conditions. Root inoculation was done at
preferably third true leaf stage or slightly older root zone was damaged using a
sterilized scalpel and 50 ml of inoculum culture was introduced to each plant. Control
plant was applied with sterilized water.
33

3.1.11 Biochemical characterization of R. solanacearum

The biochemical characterization of R. solancearum was essentially done as per


the procedures outlined by Cappuccino and Sherman (1992). The tests conducted are
detailed below and the chemical composition of media used for the tests are listed in
Appendix I.

3.1.11.1 Gelatin liquefication

To the pre-sterilized nutrient gelatin deep tubes, the test culture was inoculated
and tubes were incubated at 29±1oC for 24 h. The tubes were later kept in a refrigerator
at 4 °C for 30 min. The tubes with cultures that remained liquefied were taken as positive
and those that solidified on refrigeration were taken as negative for the test (Blazevic and
Ederer, 1975).

3.1.11.2 Starch hydrolysis

Bacterial culture (24 h old) was spotted on the starch agar plates and incubated at
o
29±1 C for 24 h. After incubation the plates were flooded with Lugols Iodine solution.
Formation of clear zone around the colony was taken as positive for the test (Eckford,
1927).

3.1.11.3 Hydrogen sulfide (H2S) production

Pre-sterilized tubes containing Sulphur-indole-motility (SIM) agar medium was


stabbed with the test culture all along the walls of the test tubes. Inoculated tubes were
incubated for 48 h at 29±1oC. After incubation, the development of black colour along the
line of the stab was noted as positive for the test (Cappuccino and Sherman, 1992).

3.1.11.4 Three per cent KOH solubility test

A loopful of 24 h old bacterial culture was taken on a clean slide to which a drop of 3
per cent KOH (Potassium hydroxide) was added and stirred it circularly for 2-3 min. and
lifted the culture from inoculation loop and observed the thread form of bacteria.
34

3.1.11.5 Hypersensitive reaction on tobacco

The hypersensitive reaction test was carried out to know the pathogenic nature of
the R. solanacearum. A loopful of the bacterial culture from the stock culture stored at
4oC was streaked onto Petri plates with sterelized sucrose peptone agar. The plates
were incubated at 29±1oC for 48 h. Individual bacterial colonies were picked and
inoculated into conical flasks containing 100 ml of sucrose peptone broth and incubated
at 29±1oC for 48 h in a rotary shaker incubator at 120 rpm.

The bacterial suspension (2 x 106 cfu) was injected into the intercellular spaces of
the intact tobacco leaves (Nicotiana tabaccum L.) by holding the barrel of the
hypodermic syringe against the bottom of the leaf. A water soaked area developed as
the suspension infiltrated the mesophyll. The leaves were then washed with sterile
distilled water and tagged. Observations were made for development of chlorotic lesions
followed by confluent necrosis in the injected regions after 24 h incubation.

3.1.12 Collection of cultures and maintenance of root-knot nematodes

Root-knot nematode infected plants along with soil were collected from the tomato
infected field in polythene bags. Root portion was carefully removed from the soil and
washed gently under running tap water. Eggmasses were picked and kept for hatching in
water in a Petri plate. After 24 - 48 h, hatched juveniles were used to inoculate tomato
grown in sterilized soil: sand (1: 1) mixture in greenhouse. These plants served as
culture plants. After giving sufficient time so as to complete 3-4 generations of the
nematode, the plants were depotted carefully. The root system was washed free of soil
the galls containing eggmasses were used to get inoculum of the nematode for further
studies throughout.

3.1.13 Extraction of nematodes

Soil samples (200 g) were washed thoroughly and processed using combined
Cobb’s sieving and decanting and Baermann’s funnel method as given below.

• 200 g of soil was taken in 1000 ml beaker and sufficient quantity of water was
added to make a soil solution.
35

• This was stirred thoroughly and the heavier particles were allowed to settle down.

• Hard particles and stones if any, were removed and then the soil solution was
passed through a set of sieves of 60, 250 and 325 µm mesh size.

• The sievates from 325 µm mesh sieve were collected and poured over tissue
paper spread over a coarse mesh placed in a petri plate (Baermann’s funnel).

• The level of water in the Baermann’s funnel was maintained to keep the tissue
paper wet and kept undisturbed for 48 h.

• The contents from the Petri plate were emptied into a beaker, diluted to a suitable
volume and population counts were taken with the help of counting dish
(Fenwick’s), using research stereobinocular microscope.

The root-knot nematode present in the suspension was identified to genus level
by observing different morpho-anatomical characters. Root-knot nematodes were picked
separately to inoculate tomato grown in sterilized soil: sand (1: 1) mixture in green house
which served as culture plants.

3.1.14 Identification of root-knot nematode species

The galled root system was immersed in a beaker containing boiling 0.1 per cent
acid fuchsin in lactophenol and left overnight for clearing (Southey, 1986). The females
of root-knot nematode were dissected out from the well developed galls of the roots
under the stereobinocular microscope and were transferred to a drop of lactophenol
taken on a clean glass slide. The posterior portion of the females was cleaned. The
perineal region was trimmed and mounted for observation under oil immersion objective
of a compound microscope (100 X). The identification of the species was made on the
basis of characters of perineal pattern described by Eisenback et al. (1981).

3.1.15 Pathogenicity

The egg masses of M. incognita extracted from tomato plants were transferred
carefully to a wire gauze sieve containing two layers of facial tissue paper and kept in a
Petri dish holding sufficient water. After 24 h, the contents of a Petri dish were
transfered into a beaker diluted to a suitable volume and juveniles population counts
36

were made with the help of Fenwick’s multi chamber counting dish. Based on the
requirement, the suspension was diluted with sterile water and a known number of
nematodes were inoculated to tomato plants.

3.2 Expression of RVL1 and SRL1 in E. coli and its isolation

One plant tuber lectin (RVL1) from Remusatia vivipara and one fungal fruiting
body specific lectin (SRL1) from Sclerotium rolfsii (both previously characterized) were
selected. Both the lectin genes were cloned into pET vector (Invitrogen) under the
control of the isopropyl-b-D-thiogalactopyranoside (IPTG)-inducible T7-promoter.
Cloning of rvl1 and srl1 was described previously (Bhat et al., 2010; Neekhra et al.,
2011). E. coli strain BL21 (DE3) pLysS transformants containing rvl1 and srl1 were
cultivated in LB medium containing 50 µg⁄ml kanamycin at 37oC to OD600 =1, induced
with 1mM IPTG and incubated overnight at 23oC. Solubility of the recombinant lectins
was tested by disrupting the E. coli cells in PBS using a sonicator instrument and
checking the lectin content in the supernatant after centrifugation (16,000 g for 20 min)
by haemagglutination assay and SDS-PAGE.

3.2.1 Heamagglutination assay

The activity of both R. vivipara lectin and S. rolfsii lectin expressed in E. coli was
determined by haemagglutination assay (Lis and Sharon, 1998) using trypsinised
rabbit erythrocytes. Haemagglutination activity was assayed in an ELISA microtiter
plate by the serial two fold dilution technique of (Liener and Hill, 1953) with some
modifications. Phosphate buffer saline (PBS; 50 µl of 150 mM NaCl) was added to 12
wells in the first row and lectin sample (50 µl) was added only to the first well of the
assay plate. The contents of the first well (100 µl) were mixed well and 50 µl of it was
transferred to the second well and the process was repeated serially for the remaining
wells. Thus the lectin extract was serially two fold diluted to which 50 µl of trypsinized
erythrocytes suspension was added and gently mixed on a rotary shaker. After
incubation for 1 h at 37°C, the plates were visually examined for haemagglutination.
The highest dilution of the extract causing visible haemagglutination was regarded as
the 'titre'. The protein content in the highest dilution causing visible agglutination was
referred to as ‘one unit’ of haemagglutination activity. It was otherwise expressed as
37

MCA (minimum concentration of protein required for agglutination). The specific


haemagglutination activity was expressed as units of activity per mg of protein.

3.2.2 SDS-PAGE for E. coli expressed RVL1 and SRL1

Crude protein (50 µl containing 0.4 mg) was mixed with 2 X SDS gel loading
buffer (50 µl). The mixture was heated at 98°C for 5 min and thoroughly mixed. Protein
(20 µl) was loaded on 12 per cent polyacrylamide gel along with suitable control
protein. The gel was run at 60 V for 1 h and subsequently at 120 V for 3 h. After
staining with commassie brilliant blue, the excess dye was removed by repeated
washing every 2-3 h with de-staining solution till blue colour band appeared. The gel
was sealed in polyethylene bag and stored at 4°C.

3.2.3 Purification of E. coli expressed SRL1

Heterologous protein produced in E. coli required purification for all other


downstream experiments. Care was taken by selecting appropriate expression vectors
(pET- 32b (+) and pYES2/CT) and restriction sites to produce SRL which is His-tag
fused to enable Ni-NTA column based purification. To purify His-tag fused protein, E. coli
cells carrying respective recombinant expression vectors and plain expression vectors
(control) were induced for the production of heterologous protein, which was subjected to
Ni-NTA column based purification and run on the SDS-PAGE gel.

3.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)

The antiserum against SRL1 was produced by immunizing the six months old
two Newzealand white rabbit with purified SRL1. It was injected intramuscularly with
increasing dose of purified SRL1. An equimolar suspension of SRL1 and Freund’s
incomplete adjuvant (in 100 µl) containing different concentration of protein (25, 100,
500 and 1000 µg in PBS) was injected at an interval of one week. A booster dose with
complete Freund’s adjuvant containing 2 mg of protein was given at sixth week.
Antiserum was collected after 7 days of booster dose injection by bleeding the rabbit
ear vein.
38

Blood was allowed to clot in the refrigerator overnight and later serum was
collected. It was centrifuged at 1000 rpm for 10 min to separate serum from the blood
constituents. The antiserum thus obtained was stored in -20oC for further studies.

3.3.1 Slide agglutination test

A small drop of antiserum was kept on a microscopic cavity slide to which a


small drop of SRL1 was added and incubated at 37oC for 45 min. Appropriate controls
containing buffer were maintained. After incubation the mixture was observed under
microscope for the presence or absence of agglutination in microscopic cavity slide.

3.3.2 Partial purification of anti-SRL1 by ammonium sulphate precipitation

Antibodies for SRL1 were partially purified according to the method of Livingston
(1974). Serum was subjected to ammonium sulphate precipitation (50%), and the
precipitate was collected and dissolved in 10 mM potassium phosphate buffer of pH
6.8, the volume being equivalent to that of original serum sample prior to salt addition.
This solution was once again subjected to ammonium sulphate precipitation under
similar conditions and the resulting protein precipitate was again dissolved as earlier.
The solution was dialized against 10 mM potassium phosphate buffer of pH 6.8, stored
at 4oC and used as anti-SRL1 for further studies.

3.3.3 Determination of the titre of the antiserum

Microprecipitin test

To prepare lectin dilutions, ten microfuge tubes were placed in a rack, with the
first one containing purified lectin suspension. The tubes were marked with the lectins
dilution factors as 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512. Later 120 µl of saline
(0.85%) buffer was added to each of the tubes from 2 to 8, 120 µl of virus suspension
was added from tube one to tube two and mixed thoroughly. With a clean pipette tip,
120 µl suspension was transferred from tube two to tube three. This was repeated until
all tubes contained successive two-fold protein dilutions.

To prepare serum (anti-SRL1) dilutions, nine microfuge tubes were placed in a


rack and marked with the serum dilution factors 16 to 262174. 225 µl of saline (0.85%)
39

buffer was pipetted into tube one and 120 µl in the other six tubes. 15 µl of undiluted
serum was transferred into tube one and mixed thoroughly. 120 µl from tube one to
tube two was transferred. This was repeated until all tubes contained successive two-
fold serum dilutions. Ten vertical columns with the lectin dilution factors (1, 2, 4….) and
nine horizontal rows with the serum dilution factors (starting with 16) were labelled on a
drawing sheet. The last column and the last row were labelled B (buffer). The same
plan was drawn inside the clean lower lid of Petri plate with the help of a marker pen by
placing the lower lid of Petri plate on the marked drawing sheet (Fig. 1). 12 µl of saline
buffer was placed in the centre of the squares of the column labelled B. Similarly 12 µl
droplets of virus dilutions were placed in all squares of correspondingly labelled
columns. The same procedure was followed for antiserum dilutions in appropriate
rows. Finally the petri plate was incubated at 37oC for 2 h.

Antiserum Lectin (Antigen) dilutions


dilutions 1 2 4 8 16 128 256 512 1024 2048 4096 8192 16384 B
16
32
64
128
256
512
1024
2048
4096
8192
16384
32763
65536
131072
262144
B

Fig 1. Plan showing dilutions of virus and antiserum in microprecipitin test


40

The amount of precipitate was characterised according to the following scale,


upon observation in microscope.

++++ : Very heavy precipitate

+++ : Heavy precipitate

++ : Moderate precipitate

+ : Slight precipitate

± : Sparingly visible very slight precipitate

- : No precipitate

3.3.4 Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC ELISA)

Direct antigen coating (DAC) ELISA was performed as per the procedure of
Hobbs et al. (1987).

Standardized procedure for DAC-ELISA

• Purified SRL1 (antigen) was prepared in PBS using different dilutions of 1: 2048
and 1: 4096. The dilution was dispensed into each well of ELISA plate at the rate of
100 µl using microplate and the plate was incubated at 37oC for one hour.

• The contents of the plate was poured off and rinsed with PBS-Tween (Washing
buffer). Washing was carried out for three times allowing three minutes time interval
for each wash.

• Empty spaces in the wells were blocked with skimmed milk protein (blocking
buffer). 100 µl of this solution was dispensed into each well. The plate was then
incubated for one hour at 37oC.

• The contents of the plate was poured off and rinsed with PBS-Tween. Washing was
carried out for three times allowing three minutes time interval for each wash.
41

• Antibody (anti-SRL) at 1: 8192, 1: 16384, 1: 32768 and 1: 65536 was dispensed


into each well at the rate of 100 µl and the plate was incubated at 37oC for one
hour.

• The contents of the plate was poured off and rinsed with PBS-Tween. Washing was
carried out for three times allowing three minutes time interval for each wash.

• Alkaline phosphatase labelled antogoat IgG (Secondary antibody) at 1: 1000


dilution was then added to the wells at the rate of 100 µl and the plate was
incubated at 37oC for one hour.

• The contents of the plate was poured off and rinsed with PBS-Tween. Washing was
carried out for three times allowing three minutes time interval for each wash.

• Substrate p-nitrophenyl phosphate (PNP) was prepared in 10 per cent


diethanolamine. 0.05 mM magnesium chloride was added to this solution. 100 µl of
the substrate was added to each well and incubated for 15 to 30 minutes at 37oC

• Note: pH of diethanolamine was adjusted to 9.8 later PNP and magnesium chloride
was added to prepare the substrate.

• Change in the colour of the wells was observed.

• Absorbance values were recorded using ELISA reader (Compaq Imaging System)
at 405 nm.

3.4 Evaluation of plant and fungal lectins for the in vitro suppression of some
common soil-borne pathogens

3.4.1 In vitro antifungal assay

Biotoxicity assays of plant (RVL1) and fungal (SRL1) lectins expressed in


E. coli were performed against F. oxysporum, S. rolfsii and R. solani to know the
efficacy of lectins in inhibiting radial growth and spore/ sclerotia germination on suitable
medium using poisoned food, spread plate, inhibition zone assay and spore/ sclerotia
germination methods under in vitro condition. The crude proteins of RVL1 and SRL1
with three controls (bacterial control (BL21), treated with phosphate buffer saline
42

treated and untreated) were tested at different protein concentration viz. 1.2 mg/ml, 2.4
mg/ml, 3.6 mg/ml and 4.8 mg/ml.

3.4.1.1 Poisoned food technique

The poisoned food technique (Shravelle, 1961) was followed to evaluate the
efficacy of lectins in inhibiting the mycelial growth of F. oxysporum, S. rolfsii and R.
solani. These fungi were grown on PDA for 3 to 10 days prior to setting up the
experiment. Different lectins were added to the molten PDA taken in sterilized Petri
plates. Suitable check was maintained without addition of lectins. 5-mm mycelial disc
taken from the periphery of three to ten days-old colony was placed in the center of
Petri plates and incubated at 27±1o C for 3 to 12 days. Five replications were
maintained for each treatment. The diameter of the colony was measured in two
directions and average was recorded. Per cent inhibition of mycelial growth of the
fungus was calculated using the formula given by Vincent (1947).

(C-T)
I = -------------- X 100
C

Where,

I = Per cent inhibition

C = Radial growth in control

T = Radial growth in treatment

3.4.1.2 Spread plate method

Fifteen ml of PDA was poured into the sterile Petri plates and allowed to solidify.
One ml different concentrations of lectins and bacterial control BL21 and PBS were
spread uniformly on agar plate. A 5-mm mycelial disc of F. oxysporum, S. rolfsii and R.
solani taken from the periphery of 3 to10 days old colony was placed in the center of
petri plates and incubated at 27±1oC for 12 days and five replications were maintained
for each treatment. The diameter of the colony was measured in two directions and
43

average was recorded. Per cent inhibition mycelial growth of the fungus was calculated
by using the formula of Vincent (1947).

3.4.1.3 Inhibition zone assay

The assay for antifungal activity against F. oxysporum, S. rolfsii and R. solani
was executed using 90 mm Petri plates containing 10 ml of PDA. After the mycelial
colony had developed, sterile blank paper discs (6 mm in diameter) were placed
around and at a distance of 1 cm away from the rim of mycelial colony. An aliquot (40
µl) of different concentrations of the lectins in PBS buffer was introduced to a disc. The
plates were incubated at 27±1oC for 72 h until mycelial growth had enveloped
peripheral discs containing the control (buffer, sterile distilled water). Inhibition zones in
cm around the disc were recorded.

3.4.1.4 Disc diffusion assay

Fusarium spores were harvested from well sporulating colonies and suspended
in sterile water. The concentrations of the spore suspensions were determined in a
heamocytometer and adjusted to 1.0 to 2.5 x 106 spores per ml depending on the
fungus to be tested. The freshly prepared suspensions (0.5 ml for plates with dia. of 90
mm) were plated out on Petri plates containing the potato dextrose agar used for
maintenance of the test fungus. To allow for spore germination and initial vegetative
growth the plates were incubated for 20 to 24 h at room temperature. At this time
sterile filter paper discs (5 mm dia.) were laid on the agar surface, and 40 µl of the
different concentrations of lectins (1.2, 2.4, 3.6 and 4.8 mg/ml) to be tested were
applied to the discs. The plates were further incubated at room temperature for 72 h.
The observations for the production of inhibition zone around the filter paper discs was
calculated and analyzed statistically.

3.4.1.5 Spore germination inhibition

To get fresh spores of F. oxysporum was gown on Potato Dextrose Agar at


27±1oC for ten days. Spores were harvested after one week by flooding the agar plates
with sterile water and filtering through three layers of Miracloth (Calbiochem). Different
concentrations of lectins, bacterial control, PBS and sterile distilled water were added
to the 1.8 x 105 spores/ml suspension. Observations in respect of germination were
44

recorded at 6, 24 and 48 h under microscope and per cent inhibition of spore


germination was calculated (Does et al., 1999).

3.4.1.6 Inhibition of sclerotial germination

Mature and immature sclerotial bodies and sclerotia collected from a fresh
culture of Sclerotium rolfsii and Rhizoctonia solani were incubated for 30 min at 27±1oC
with different concentrations (1.2, 2.4, 3.6 and 4.8 mg/ml) of anti-SRL, SRL1, RVL1
and BL21 in screw-capped vials separetly. Treated sclerotial bodies were allowed to
germinate in a Petri plate containing Byrde’s (Appendix I) medium with 1.5 per cent
agar at 27±1oC for 72 h. For control, sclerotial bodies that was treated with normal
rabbit serum, PBS, and sterile distilled water was inserted in another well of the plate.
Experiment was conducted with five replications. Germination of the sclerotial bodies in
the plate was observed and inhibition of mature and immature sclerotia germination
and radial growth were recorded.

3.4.2 In vitro antibacterial assay

Biotoxicity assays of plant (RVL1) and fungal (SRL1) lectins expressed in


E. coli were performed against Ralstonia solanacearum to analyse the antimicrobial
properties of lectins under in vitro conditions.

Well diffusion assay was performed as described by Cole (1994) with some
modifications as follows. The bacterium was grown in Luria-Bertania agar (LBA) for
overnight. 50 µl of overnight culture were added to 5 ml of nutrient broth and incubated
at 29±1oC (with shaking at 120 rpm until reaching the mid-exponential phase). Then one
ml of appropriate dilution of this culture was added to 5 ml warm melted LBA. After
mixing, the overlay gel was poured over sterile 90 mm glass Petri plates containing 15 ml
LBA. 5 mm well was made using sterile cork borer on medium. 20 µl of crude protein
extract of RVL1 (6 mg/ml), SRL1 (6 mg/ml), Positive control BL21 (6 mg/ml) and a
negative control (1 mM PBS and sterile distilled water) were applied onto each different
wells. 10 µl of streptocycline (500 ppm/ml) were also loaded onto a disc and used as
antibacterial positive control. The plates were incubated for 48 h at 29±1oC. After
incubation the diameters of the inhibition zone surrounding the well were measured.
Three replications were maintained.
45

3.4.2.1 Determination of the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC)

Minimum inhibitory concentration corresponded to the minimum lectin


concentration that inhibited visible bacterial growth: MIC was determined by the dilution
tube test (Courvalin et al., 1985). Serial dilutions of SRL1 and RVL1 in PBS were
prepared and added to the bacterial cultures (in NB) containing 1 x 105 cells/ ml in the
exponential growth phase. The samples were incubated for 24 h at room temperature
(29±1oC). Afterwards, cultures were seeded onto NA and incubated for 24 h at room
temperature (29±1oC). The minimum bactericidal concentration (MBC) corresponded to
the minimum concentration of the lectin that reduced the number of CFU to 0.1 per cent
of the initial concentration.

3.4.2.2 Bacterial agglutination test

For quantitative determination of the agglutinating activity, the minimum


agglutinating concentration (MAC) corresponding to the minimum concentration of lectin
that promoted bacterial agglutination was recorded. Bacterial cultures which were grown
for 16 h, were diluted at a ratio of 1: 100 with NB. Agglutination assay was performed in
96-well microtitre plates with two-fold serial dilutions of lectin in 1 mM PBS. To each well,
100 µl of diluted bacterial suspension was added to a final volume of 200 µl. MAC was
determined by visual agglutination after 16 h incubation of plates at 29±1oC.

3.4.2.3 Inhibition zone assay/disc diffusion method

Antibacterial activity of crude protein of SRL1 and RVL1 was investigated by the
disc diffusion method (Cole, 1994). R. solanacearum was obtained from stock culture in
nutrient agar. One-hundred millilitres of warm NA and 0.5 ml of bacterial suspension
(1 x 105 cfu/ml) were spread on sterile Petri plates and allowed to solidify. Sterile blank
paper discs (6 mm dia.) impregnated with 40 µl of solution of SRL1, RVL1 and positive
control BL21 (0.6, 1.2, 2.4 and 3.6 mg/ml) and negative control 1 mM PBS and sterile
distilled water, were added on the agar plates. Plates were incubated at 29±1oC for 48 h.
A transparent ring around the paper disc revealed antimicrobial activity. Zones of growth
inhibition around discs were measured in centimetres.
46

3.4.2.4 Plant and fungal lectins’ activity

Luria Bertani’s broth was used for the lectins’ sensitivity test against R.
solanacearum. Five ml of LB broth was dispensed in individual test tubes and sterelized
at 1.1 kg/cm2 pressure for 15 min. The inoculum consisted of 48 h old culture,
suspended in sterile distilled water containing 1 x 105 cfu/ml used at the rate of 50 µl per
test tube. Then, the test tubes were inoculated with different concentrations (0.6, 1.2, 2.4
and 3.6 mg/ml) of SRL1, RVL1 and positive control Bl21) and negative control 1 mM
PBS and one untreated check, incubated at 29±1oC. The growth was recorded
turbidometrically after 48 h of inubation using Systronics PC based double beam
spectrophotometer 2202 at 600 nm.

3.4.3 Anti-nematode activity

3.4.3.1 In vitro egg hatching test

Egg masses were collected from culture plants maintained in Nematology


nethouse, Department of Plant Pathology, Agricultural College, Dharwad. Egg masses
were picked from host and were treated with NaOCl (0.5%) to dissolve the egg matrix
and to separate the individual eggs. After washing with sterile PBS, 50 eggs were
transferred to eppendorf tubes containing different dilutions of SRL1, RVL1 and bacterial
control BL21 (1: 25, 1: 50, 1: 75 and 1: 100) in PBS 1 mM, pH 7.2 and incubated at
27±1oC. Negative control (without lectin) PBS and sterile distilled water was used for
comparison. Inoculated vials were incubated at room temperature (27±1ºC) for 48 h.
Number of juveniles hatched at different intervals was counted from each lectin
concentration (6, 12, 18, 24, 36 and 48 h). Percentage inhibition of egg hatching was
computed from the three replications.

3.4.3.2 Juveniles inhibition test

Fifty freshly hatched nematodes (Meloidogyne incognita) were counted in a


counting dish using a stereo-binocular microscope and were washed with sterile PBS.
The juveniles were next transferred to eppendorf tubes containing different dilutions of
SRL1, RVL1 and bacterial control BL21 (1: 25, 1: 50, 1: 75 and 1: 100) in (PBS) and
incubated at 27±1oC. Negative control PBS and sterile distilled water was used for
comparison. Inoculated vials are incubated at room temperature (27±1ºC) for 48 h.
47

Number of live and dead nematodes from each lectin concentration set was counted at
different time intervals (6, 12, 18, 24, 36 and 48 h) under the stereobinocular
microscope. Each treatment was replicated three times and percentage mortality of
nematodes was calculated.

3.5 Influence of lectins on biocontrol agents in the suppression of soil borne


pathogens in vitro

3.5.1 Collection and isolation of different biocontrol agents

Collection of six bacterial and four fungal antagonists from different sources

Bioagents Source

Trichoderma viride Institute of Organic Farming, UAS, Dharwad

Trichoderma harzianum Institute of Organic Farming, UAS, Dharwad

Trichoderma virens Institute of Organic Farming, UAS, Dharwad

Trichoderma koningii Institute of Organic Farming, UAS, Dharwad

Department of Plant Pathology, UAS,


Pseudomonas fluorescens strain 30
Dharwad

Department of Plant Pathology, UAS,


Pseudomonas fluorescens strain 50
Dharwad

Pseudomonas fluorescens strain


NBAII, Banglore
HM439968

Pseudomonas fluorescens strain TNAU TNAU, Coimbatore

Department of Plant Pathology, UAS,


Bacillus subtilis
Dharwad

Bacillus subtilis strain HQ162493 NBAII, Banglore


48

3.5.1.1 Isolation of Paecilomyces lilacinus

Tomato roots and rhizosphere soils were collected from tomato field in UAS
Dharwad Campus, where root-knot infestation was prevalent. Root pieces were
washed in gentle running tap water for 5 min. The standard tissue isolation procedure
was followed for isolation of the fungal pathogen. The infected parts were surface
sterilized with 0.1% sodium hypochlorite solution for 40 seconds and washed serially in
distilled water to remove the traces of sodium hypochlorite and then transferred to
sterilized Petri plate containing PDA, before transfering to Petri plate PDA was
amended with 0.01 per cent chloramphenicol and 3 per cent sodium chloride.

For isolation of fungus from soil, serial dilution and pour plate technique was
employed (Johnson and Curl, 1972). After thorough mixing 10 g of soil sample was
transfered aseptically to 100 ml sterile distilled water and mixed thoroughly by shaking
for 30 min. 1 ml sample was drawn from the suspension and transferred to 9 ml sterile
distilled water blank. The suspension thus obtained in dilution process was shaken for
one minute before it was further diluted. 1 ml suspension from each of the appropriate
dilution of 102 and 103 was transferred aseptically into sterile Petri plates.15 ml of
appropriate molten medium (and cooled to 45°C) was transferred to Petri plates and
gently rotated clockwise and anticlockwise direction to get uniform mixing. The plates
were incubated at room temperature (27±1°C) for 7 days. Axenic (Pure) cultures were
obtained by single spore isolation. These were maintained on PDA slants.

3.5.1.2 Identification of Paecilomyces lilacinus

P. lilacinus was identified based on the cultural and morphology charcters as


described by Samson (1975).

3.5.2 Growth phase studies for bacterial antagonists

Growth phase studies for Pseudomonas fluorescens and Bacillus subtillis strains
were done by following the procedure described in 3.1.8
49

3.5.3 In vitro screening of plant fungal lectins against bacterial and fungal
antagonists

Plant (RVL1) and fungal (SRL1) lectins expressed in E. coli were evaluated
against bacterial and fungal biocontrol agents to analyse the antimicrobial properties of
a lectins in vitro.

3.5.4.1 In vitro screening of bacterial and fungal antagonists against F. oxysporum,


S. rolfsii and R. solani

Six bacterial (Pseudomonas fluorescens and Bacillus subtilis strains) and five
fungal antagonists (Trichoderma viride, T. virens, T. koningii, T. harzianum and
Paecilomyces lilacinus) were evaluated against Fusarium oxysporum, Sclerotium rolfsii
and Rhizoctonia solani for their efficacy employing dual culture technique. The bioagents
and the test fungus were inoculated side by side on a single Petri plate containing
solidified PDA. Five replications were maintained for each treatment with one control by
maintaining only pathogen separately. They were incubated for 3 to 10 days. The
diameter of the colony of both bioagents and the pathogen was measured in two
directions and average was recorded. Per cent inhibition of growth of the test fungus was
calculated by using the formula of Vincent (1947).

3.5.4.2 In vitro screening of plant and fungal lectins against fungal antagonists

3.5.4.2.1 Disc diffusion assay

Bio-efficacy of SRL1 and RVL1 tested against P. lilacinus and T. viride by


employing disc diffusion assay as described in 3.4.1.4.

3.5.4.2.2 Inhibition of Spore germination

SRL1 and RVL1 evaluated against 2 x 105 spores/ml spore suspension of


Paecilomyces lilacinus and selected one Trichoderma sp. by using same procedure as
described in 3.4.1.5.
50

3.5.5.1 In vitro screening of plant fungal lectins against bacterial antagonists

P. fluorescens and B.subtilis strains were collected from different sources as


mentioned above. All bacteria were maintained in Nutrient Agar (NA) and stored at 4
o
C. For agglutination studies and evaluation of antimicrobial activity of SRL1 and RVL1,
bacterial growth was obtained using shake flasks (250 ml), containing Nutrient Broth
(NB) incubated overnight in a shaker at 29±1oC. Biomass concentration was
determined by measuring suspension turbidity at 600 nm and converted to colony
forming units (cfu/ml) using appropriate calibration curves. All the experiments were
carried out with a biomass concentration of 105–106 cfu/ ml.

3.5.5.2 Disc diffusion assay

P. fluorescens and B. subtilis strains were grown in Luria Bertani’s agar medium
for overnight. One-hundred millilitres of warm LB agar and 0.5 ml of bacterial suspension
(1 x 106 cfu/ml) were spread on sterile Petri plates and allowed to solidify. The sterile
discs (6 mm in diameter) were then placed on plates. 40 µl of crude protein extract of
RVL1 (6 mg/ml), SRL1 (6mg/ml), Positive control BL21 (6 mg/ml) and a negative control
(1 mM PBS and sterile distilled water) were applied onto each different disc. 20 µl of
streptocycline (500 ppm/ml) were also loaded onto a disc and used as antibacterial
positive control. The plates were incubated for 48 h at 29±1oC. After incubation, the
diameters of the inhibition zone surrounding the disc were measured. The experiments
were carried out in triplicate.

3.5.5.3 Determination of the minimum inhibitory concentration (MIC) and the


minimum bactericidal concentration (MBC)

Minimum inhibitory concentration corresponded to the minimum lectin


concentration that inhibited visible bacterial growth, bacterial agglutination, lectins’
sensitivity against P. fluorescens and B. subtilis were determined using the procedure as
explained in 3.4.2.
51

3.5.5.4 In vitro screening of bacterial and fungal antagonists against Ralstonia


solanacearum

Four P. fluorescens strains, two B. subtilis strains and four Trichoderma spp.
were evaluated for their efficacy against the growth of Ralstonia solanacearum by
inhibition zone assay method. A heavy suspension of R. solanacearum multiplied in
nutrient broth (20 ml) was mixed with luke warm nutrient agar medium (100 ml). The
inoculated flasks were incubated at 29±1oC for 48 h. The bacterial suspension was
then seeded to the luke warm nutrient agar medium (100 ml). The seeded medium was
poured into the sterilized Petri plates and plates were allowed to solidify. Loopful
culture of the antagonistic organism was dissolved in sterile distilled water to make
suspension. In case of fungal antagonists, culture filtrates of antagonists were taken
from fresh antagonists culture. Then, 20-40 µl bacterial suspension and fungal culture
filtrates were spotted on the medium. The inoculated plates were then incubated at
29±1oC for 48 h. The observations for the production of inhibition zone around the
antagonistic microorganisms was calculated and analyzed statistically.

3.5.6 In vitro screening of bacterial and fungal antagonists in combination with lectins
against R. solanaceaarum

Six bacterial and four fungal antagonists in combination with different


concentrations of RVL1, SRL1, bacterial control BL21 (0.6, 1.2, 2.4 and 3.6 mg/ml) and
negative control PBS and sterile distilled water evaluated against R. solanacearum
through inhibition zone assay in vitro.

Overnight grown R. solanacearum growth was inoculated to nutrient broth and


incubated for overnight in shaker at 29±1oC. 100 ml of warm NA and 0.5 ml of bacterial
suspension (1x105cfu/ml) were spread on sterile Petri plates and allowed to solidify.
Sterile blank paper discs (6 mm dia.) impregnated with solution of SRL1, RVL1 and
positive control BL21 (0.6, 1.2, 2.4 and 3.6 mg/ml) and negative control 1mM PBS and
sterile distilled water containing bacterial antagonist suspensions and culture filtrates of
fungal antagonist were added on the agar plates. Plates were incubated at 29±1oC for 48
h. Each treatment was replicated thrice. A transparent ring around the paper disc
revealed antimicrobial activity. Zones of growth inhibition around discs were measured in
centi meter.
52

3.5.7 In vitro screening of bacterial and fungal antagonists against nematode with
lectins

3.5.7.1 Evaluation of bacterial antagonists in combination with lectins against


nematode

3.5.7.1.1 Preparation of cell-free extract

A single colony of each bacterial strain was cultured in a screw-capped test tubes
containing 10 ml of sterilized nutrient broth incubated at 29±1oC in shaker at 150 rpm for
48 h. The culture was subsequently passed through sterilized Whatman filter papers
No.1 and 42 and concentrated by centrifugation at 10,000 rpm for 10 min. The
supernatant was collected and finally passed through Millipore filter of 0.22 µm. This was
designated as undiluted standard cell free filtrate of cent per cent concentration. The cell
free extract was further diluted to 75, 50 and 25 per cent, respectively and these dilutions
were added with lectins (3.6 mg/ml) and appropriate positive and negative control
maintained to study their effect on nematodes. In vitro evaluation of P. fluorescens and
B. subtilis strains against root-knot nematode was carried out on egg hatching and
juveniles mortality.

3.5.7.1.2 In vitro egg hatching test

Egg masses were collected from culture plants maintained in Nematology glass
house, Department of Plant Pathology, College of Agriculture, University of Agricultural
Sciences, Dharwad. Egg masses were picked up and treated with NaOCl (0.5%) to
dissolve the egg matrix and to separate the individual eggs. A known number of eggs
(50) was carefully transferred to each vial containing lectins and bacterial control with cell
free culture filtrate of P. fluorescens and B. subtilis strains of 100, 75, 50 and 25 per cent
concentrations. Inoculated vials are incubated at room temperature (27±1ºC) for 48 h.
Four controls were maintained by transferring 50 eggs to a vial containing cell free
extracts of strains without lectins, sterilized nutrient broth, Phosphate buffer saline and
water. After 12, 24 and 48 h the number of juveniles hatched was counted under stereo
binocular microscope and per cent inhibition of egg hatching in different dilutions in each
vial was calculated.
53

3.5.7.1.3 In vitro mortality test of juveniles

Freshly hatched 50 active juveniles were counted in a counting dish using a


stereo binocular microscope and were carefully transferred to individual vials containing
5 ml of each of the bacterial cell free filtrates of different concentrations (100, 75, 50 and
25 per cent) with and without lectins. Each treatment was replicated three times and
incubated at 27±1ºC. Observations were recorded at 12 h, 24 h and 48 h after exposure
period and per cent mortality was calculated.

3.5.7.2 In vitro screening of fungal antagonists combination with lectins against


nematode

3.5.7.2.1 Egg hatching inhibition

A known number (50) of eggs was carefully transferred to each vial containing
different concentrations of lectins and bacterial control (1.2, 2.4, 3.6 and 4.8 mg/ml) with
1 x 105 spore suspension of P. lilacinus and T. viride. Inoculated vials were incubated at
room temperature (27±1ºC) for 48 h. Three controls were maintained by transferring 50
eggs to a vial containing, spore suspension without lectins, Phosphate buffer saline and
water. After 12, 24 and 48 h, the number of juveniles hatched was counted under stereo
binocular microscope and per cent inhibition of egg hatching in different dilutions in each
vial was calculated.

3.5.7.2.2 Juvenile mortality test

Freshly hatched fifty juveniles were transferred to vials containing different


concentrations of lectins and bacterial control (1.2, 2.4, 3.6 and 4.8 mg/ml) with 1 x 105
spore suspension of Paecilomyces lilacinus and Trichoderma viride. Inoculated vials
were incubated at room temperature (27±1ºC) for 48 h. Three controls were maintained
by transferring 50 juveniles to a vial containing, spore suspension without lectins,
Phosphate buffer saline and water. After 12, 24 and 48 h, the number of dead and live
nematodes was counted under stereo-binocular microscope and per cent inhibition of
egg hatching in different dilutions in each vial was calculated.
54

3.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the
suppression of tomato soil-borne pathogens
3.6.1 Analysis of transgenic and nontransgenic plants

3.6.1.1 PCR confirmation

Genomic DNA was extracted from the leaf tissues by following a modified CTAB
method (Sambrook and Russell, 2001). A forward primer (5' CTAGTCTAGA
ATGGCCAAGCTGCTCCTCTT 3') and a reverse primer (5' GGCGGATCCGC
CCATCATCTTCTCCTA 3') were used to amplify rvl1 gene. Similarly, a forward primer
(5' ATGGATCCATGACTTATAAGATTACCGT 3') and a reverse primer (5'
CGAGCTCTCACCCGATAATGACGTT 3') were used to amplify srl1 gene. A forward
primer (5' ATGGATCCATGACTTATAAGATTACCGT3') and a reverse primer
(5'CGAGCTCTCACCCGATAATGACGTT3') were used to amplify a 430 bp uidA gene.
PCR products were electrophoresed on a 1.2 per cent agarose gel.

3.6.1.2 Expression analysis

3.6.1.2.1 Protein extraction

Young leaves (5 g) of T3 plants were homogenized in an extraction buffer (0.05


M sodium phosphate buffer pH 7.4). The extract was centrifuged at 10,000 rpm for 10
min until clear supernatant was obtained. This crude extract was stored at -20oC for
further use.

3.6.1.2.2 Protein content estimation

Protein concentration in the crude as well as partially purified sample was


estimated by the method of Lowry et al. (1951) using BSA (1 mg/ml) as the standard.

3.6.1.3 SDS-PAGE

SDS-PAGE was done for both crude protein and partially purified protein
isolated from rvl1- and srl1-transgenic plants. SDS-PAGE was performed under
alkaline conditions (pH 8.3) in 12% (w/v) polyacrylamide gel according to the method
55

described by Laemmli (1970) and electrophoresis was carried out at constant voltage
(100 V) for 4-5 h. After the run, gel was stained with 0.1 per cent Coomassie Brilliant
Blue (R250, HIMEDIA) prepared in destaining solution (1: 3: 6 acetic acid: methanol:
water v/v).

3.6.1.4 Heamagglutination assay

Haemagglutination assay was carried out by following the protocol of Lis and
Sharon (1998). Sodium citrate treated rabbit erythrocytes were washed extensively
with phosphate buffer saline (PBS, pH 7.4) and finally resuspended in the required
volume of PBS to generate 1 per cent (v/v) erythrocyte suspension. Agglutination
assays were performed in microtiter plates in a total volume of 150 µl containing 50 µl
of erythrocyte suspension per well with addition of serially diluted measured amount of
protein samples. The agglutination reaction was monitored visually after one hour
incubation at 37oC temperature. The highest dilution of the extract, causing visible
haemagglutination was regarded as the 'titre'. The protein content in the highest
dilution causing visible agglutination was referred to as ‘one unit’ of haemagglutination
activity or expressed as Minimum concentration of protein required for agglutination
(MCA). Based on MCA, total activity of lectin (in units) in the protein sample was
calculated. The specific haemagglutination activity was expressed as units of activity
per mg of protein.

3.6.2 Evaluation of rvl1 and srl1 transgenic and non transgenic tomato lines
against individual and combination of vascular pathogens of tomato under
glasshouse conditions

A glasshouse study was conducted to test the efficacy of srl1 and rvl1 genes
expessed homozygous T3 generation transgenic tomato plants, non transgenics
without lectin and control tomato plants against F. oxysporum, R. solanacearum and
M. incognita individually and in combinations of pathogens. RVL1 and SRL1
transgenics seeds were collected from the Department of Biotechnology, UAS,
Dharwad.
56

Three weeks-old PCR confirmed transgenic plants and non-transformed (in vitro
regenerated) control seedlings were transplanted in 30 cm earthen pots, containing
sterile pot mixture (soil: sand in 1: 1). After ten days of their establishment, the above
said pathogens were inoculated into the individual pots in the root zone (at 50 g
sorghum grain medium of F. oxysporum, 50 ml NB of 48 h R. solanaceanum (1.00 OD
at 600 nm) and 200 Meloidogyne juveniles/pot), by making three holes around the
plant at root zone and covered with sterilized soil.

The following treatments were maintained with five replications and the pots
were arranged in completely randomized design.

Treatment
Transgenic lines Treatment details
no.
SRL1-T0(3) SRL1-T0(3)-T1(9)-T2(7) F R M F+R F+M R+M F+R+M
SRL1-T0(7) SRL1-T0(7)-T1(3)-T2(6)
SRL1-T0(21) SRL1-T0(21)-T1(3)-T2(7)
SRL1-T0(32) SRL1-T0(32)-T1(7)-T2(9)
SRL1-T0(10) SRL1-T0(10)-T1(2)-T2(9)
SRL1-T0(90) SRL1-T0(90)-T1(2)-T2(3)
SRL1-T0(95) SRL1-T0(95)-T1(7)-T2(4)
SRL1- SRL1-T0(116)-T1(5)-
T0(116) T2(8)
RVL1-T0(12) RVL1-T0(12)-T1(8)-T2(2)
RVL1-T0(11)-T1(4)- -do-
RVL1-T0(11)
T2(10)
RVL1-T0(28) RVL1-T0(28)-T1(5)-T2(7)
RVL1-T0(2) RVL1-T0(2)-T1(2)-T2(9)
RVL1-T0(33) RVL1-T0(33)-T1(2)-T2(6)
RVL1-T0(48) RVL1-T0(48)-T1(3)-T2(7)
RVL1-T0(20)-T1(9)-
RVL1-T0(20)
T2(11)
RVL1-T0(50) RVL1-T0(50)-T1(3)-T2(6)
Control Non-transgenic
Control Non-transgenic No pathogens
57

Where,

F- Fusarium-alone M- Meloidogyne-alone R- Ralstonia-alone

F+R – Fusarium + Ralstonia M+F – Meloidogyne + Fusarium

M+R - Meloidogyne + Ralstonia M+F+R - Meloidogyne + Fusarium + Ralstonia

Observations were recorded on plant growth parameters like shoot length, root
length, fresh shoot weight, fresh root weight, dry shoot weight, dry root weight, gall
index, disease incidence, recovery of Ralstonia and Fusarium propagules from soil,
final nematode population in soil, number of egg masses per root, female fecundity and
reproduction factor at 90 days after inoculation. Whereas rating of root-knot index was
done following the scale suggested by Bridge and Page (1980) as given below:

Gall index % of galls per plant

0 No galls

1 10% root with galls

2 20% root with galls

3 30% root with galls

4 40% root with galls

5 50% root with galls

6 60% root with galls

7 70% root with galls

8 80% root with galls

9 90% root with galls

10 All root with galls

The disease incidence was recorded by 0-4 scale as described by Weitang et al.
(2004) where zero represents no infection and four denotes complete infection. Five
58

replications were maintained for each treatment in two separate experiments. The 0–4 scale of the
disease severity was classified as follows:

0 - No infection.

1 - Slight infection, which is about 25% of leaves wilted.

2 - Moderate infection, two or three leaves turned yellow, 50% of leaves wilted.

3 - Extensive infection, all the leaves turned yellow, 75% of leaves wilted and growth is
inhibited

4 - Complete infection, the leaves of the whole plant turned yellow, 100% of leaves
wilted, and the plants died.

The percentage of disease severity was determined using the formula given by
Weitang et al. (2004)

Disease severity (%) = Σ Scale x number of plants infected


× 100
Highest scale x number of plants

3.6.2.1 Enumeration of pathogen population

3.6.2.1.1 Serial dilution method

The soil sample collected from treated pots was subjected to serial dilution with
following steps. After thorough mixing, 10 g of soil sample from each treatment was
transferred aseptically to 250 ml flask containing 100 ml sterile distilled water
separately and mixed thoroughly by hand shaking for 30 min. 1ml sample was drawn
from the suspension and transferred to 9 ml of sterile distilled water blank. The
suspension thus obtained in dilution process was shaken for one minute before it was
further diluted. One ml suspension from each of the appropriate dilution was
transferred aseptically into sterile Petri plates. Fifteen ml of specific medium for
Fusarium FSM (Fusarium specific medium) and for Ralstonia used sucrose peptone
agar melted and cooled to 45°C was transferred to Petri plates and gently rotated
clockwise and anticlockise direction to get uniform mixing. The plates were incubated
59

at room temperature. Microbial counts were assessed on the second day for bacteria
and seven days for fungi and counted colony forming units.

3.6.2.2 Nematodes extraction

Cobb’s sieving and decanting technique was followed to extract nematodes from
soil for which 200 cc of the samples was collected from treated pots and the sample
taken in a container and mixed thoroughly with water. Hard particles and stones if any,
were removed by stirring the suspension and were then passed through a set of sieves
of 60, 250 and 325 µm pore size. The sievates were collected on a tissue paper spread
over a coarse mesh, which was then placed in a Petri plate containing enough water so
as to keep the tissue paper just wet. The nematode suspension collected in the Petri
plate was examined using stereo binocular microscope. The root-knot nematodes
present in the suspension were identified by observing different morphological
characters.

3.6.2.3 Egg mass production and determination of reproduction factor of


nematodes

At 90 dpi, the root systems were washed free of soil and weighed. Egg masses
were stained with a 0.05% of acid fuchsin and recorded. Eggs from the entire root
system were extracted by maceration in a blender containing a 0.5% NaOCl solution
for 10 min (Hussey and Barker, 1973) and counted to determine final population (Pf).
Both non-hatched eggs and empty eggs (egg shells) were recorded and the hatching
rate was estimated. The fecundity of the females and reproduction factor was
calculated by using formula,

Final population (Pf)


Female fecundity =
Egg masses

Final population (Pf)


Reproduction factor (Rf) =
Initial population (Pi)
60

3.6.2.4 Assay of defense related enzymes and compounds

3.6.2.4.1 Enzyme extract

The leaf samples collected from the inoculated tomato plants were immediately
homogenized with liquid nitrogen. One g of powdered sample was extracted with 2 ml
of sodium phosphate buffer, 0.1 M (pH 7.0) at 4ºC. The homogenate was centrifuged
for 20 min at 10,000 rpm. Protein extracts prepared from tomato leaf tissues were used
for estimation of defense enzymes like peroxidase (PO), polyphenol oxidase (PPO),
phenylalanine ammonia lyase (PAL) and total phenols.

3.6.2.4.2 Assay of peroxidase (PO)

Assay of PO activity was carried out as per the procedure described by


Hammerschmidt et al. (1982). The reaction mixture consisted of 2.5 ml of a mixture
containing 0.25 per cent (v/v) guaiacol in 0.01 M. Sodium phosphate buffer, pH 6.0 and
0.1 M hydrogen peroxide. Enzyme extract (0.1 ml) was added to initiate the reaction
which was followed colorimetrically at 470 nm. Crude enzyme preparations were
diluted to give changes in absorbance at 470 nm of 0.1 to 0.2 absorbance units/min.
Boiled enzyme was used as blank. Activity was expressed as the increase in
absorbance at 470 nm min/mg of protein.

3.6.2.4.3 Assay of polyphenoloxidase (PPO)

A sample of one g was homogenized in 2 ml of 0.1 M sodium phosphate buffer


(pH 6.5) at 4ºC. The homogenate was centrifuged at 20,000 rpm for 15 min at 4ºC. The
supernatant served as enzyme source and polyphenoloxidase activity was determined
as per the procedure given by Mayer et al. (1965). The reaction mixture consisted of
1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5) and 200 µl of the enzyme extract. To
start the reaction, 200 µl of 0.1M catechol was added and the activity was expressed
as change in absorbance min/mg of protein.

3.6.2.4.4 Total phenol content

Phenol content was estimated as per the procedure given by Zieslin and Ben-
Zaken (1993). One g of sample was homogenized in 10 ml of 80% methanol with
61

pestle and mortar and agitated for 15 min. One ml of the methanolic extract was added
to 5 ml of distilled water and 250 µl of Folin-Ciocalteau reagent (1 N) and the solution
was kept at 25±1oC. After 3 min one ml of saturated solution of sodium carbonate and
one ml of distilled water was added and the reaction mixture was incubated for 1 h at
25±1oC. The absorption of the developed blue colour was measured using UV-visible
spectrophotometer at 725 nm. The content of the total soluble phenols was calculated
according to a standard curve obtained from a Folin-Ciocalteau reagent with a phenol
solution (C6H6OH) and expressed as catechol equivalents mg/g tissue weight.

3.7 Evaluation srl1 and rvl1 transgenics and non-transgenics lines for root
infection by Meloidogyne incognita and its development

3.7.1 Inoculation process

The PCR-confirmed eight srl1 and rvl1 homozygous T3 transgenic plants and
non-transformed control were screened to compare penetration and reproduction of
root-knot nematode on lectins carrying transgenic tomato lines by following the method
of Gaofu et al. (2008). Three-week old seedlings were transplanted in earthen pots,
surface sterilized with 70% alcohol and filled with sterile potting mixture (soil: sand in 1:
1). After a week, the pots were inoculated with 200 freshly hatched J2 (less than 72 h-
old) 5 ml of water. The pots were maintained in the greenhouse and regularly watered
with tap water.

The following treatments were maintained with 24 replications and the pots were
arranged in completely randomized design.

Treatments details

Treatment No. Transgenic lines Treatment details


SRL1-T0(3) SRL1-T0(3)-T1(9)-T2(7)
SRL1-T0(7) SRL1-T0(7)-T1(3)-T2(6)
SRL1-T0(21) SRL1-T0(21)-T1(3)-T2(7)
Nematode-alone
SRL1-T0(32) SRL1-T0(32)-T1(7)-T2(9)
SRL1-T0(10) SRL1-T0(10)-T1(2)-T2(9)
SRL1-T0(90) SRL1-T0(90)-T1(2)-T2(3)
62

SRL1-T0(95) SRL1-T0(95)-T1(7)-T2(4)
SRL1-T0(116) SRL1-T0(116)-T1(5)-T2(8)
RVL1-T0(12) RVL1-T0(12)-T1(8)-T2(2)
RVL1-T0(11) RVL1-T0(11)-T1(4)-T2(10)
RVL1-T0(28) RVL1-T0(28)-T1(5)-T2(7)
RVL1-T0(2) RVL1-T0(2)-T1(2)-T2(9) Nematode-alone
RVL1-T0(33) RVL1-T0(33)-T1(2)-T2(6)
RVL1-T0(48) RVL1-T0(48)-T1(3)-T2(7)
RVL1-T0(20) RVL1-T0(20)-T1(9)-T2(11)
RVL1-T0(50) RVL1-T0(50)-T1(3)-T2(6)
Control Non-trnagenic
Control Non-transgenic Uninoculated

3.7.2 Nematode root infection

A time course experiment was conducted to compare root infection by


M. incognita on eight srl1 and rvl1 transgenic lines and control. Each plants
combination was replicated 24 times. Tests were run separately and each seedling
was grouped into eight sets, one set per harvest date at 1, 3, 5, 7, 11, 15, 21, 25, 30
and 45 days after inoculation (dai).

At each harvesting time (1, 3, 5, 7, 11, 15, 21, 25, 30 and 45 dai), three plants/
treatment were carefully removed from the pots and the root system washed free of
soil. Roots were stained with acid fuchsin 0.05 per cent (Bridge and Page, 1982), and
examined under a stereo binocular microscope to determine infection percentage
Nematodes were categorized according to their developmental stages as J2
vermiform, J2 sausage-like) and adults (Taylor and Sasser, 1978).

3.7.3 Nematode reproduction

At 45 dpi, the root systems were washed free of soil and fresh shoot and root
length, fresh shoot and root weight and dry shoot and root weight were recorded. Egg
masses were stained with a 0.1 g/ml acid fuchsin for 2 h (Byrd et al., 1983) and
recorded. Eggs from the entire root system were extracted by maceration in a blender
containing a 0.5% NaOCl solution for 10 min (Hussey and Barker, 1973) and counted
63

to determine Pf. Both non-hatched eggs and empty eggs (egg shells) were recorded
and the hatching rate was estimated. The fecundity of the females and Reproduction
factor was calculated using the formula as mentioned above.

3.7.4 Histopathological studies

Histopathological and histochemical changes in nematode inoculated


trangnsgenic and nontransgenic lectins plants at 21 days after inoculation were studied
by employing standard microtchniques procedures.

3.7.4.1 Sampling

Lateral root samples of tomato were collected from following treatments

• Inoculated and uninoculated eight transgenic srl1 lines

• Inoculated and uninoculated eight transgenic rvl1 lines

• Inoculated and uninoculated control plants

The root samples of the above treatments were taken 21 days after inoculation.

3.7.4.2 Fixation and dehydration

Lateral or feeder infected (galled) roots from different treatments were cut into 1-
1.5 cm. They were then washed with tap water to remove soil particles and were
sepeartely fixed in formalin acetic acid-alcohol fixative (two parts of ethyl alcohol 70% +
one parts of formaldehyde +one part of acetic acid) for overnight.

Further, they were washed in ethyl alcohol and subjected to dehydration using
graded series of ethyl alcohol (50, 70, 80, 90 and 100%) with 3 h in each step. Further,
samples were subjected to ethyl alcohol: butanol mixture (75: 25, 50: 50, 25: 75) and
100% butanol with 3 h in each step. This was followed it with one change in butanol for
2-3 h.

3.7.4.3 Infiltration and embedding

Transfer the specimens to small glass vials containing little butanol to which
molten paraffin wax was added later. Which were incubated at room temperature for
overnight after that transfer the vials to oven for 12 h at 62-65oC. Finally the specimens
64

were given changes with fresh molten, pure paraffin wax at 60oC in oven then replacing
the last traces of butanol with paraffin. The specimens were subsequently embedded in
paraffin wax taken in glass lids of coplin jar which were previously smeared with
glycerol. The specimens in paraffin wax was properly oriented before it got solidified.
The lids with solidified wax were plunged into iced water to seprate the solidified wax.

3.7.4.4 Microtoming and affixing the specimen sections

Rectangular blocks were then cut, trimmed and attached to the block holder of a
rotary Microtome (Leica). The block holder along with mounted wax square/block were
kept in cold water in fridge overnight. Then the block were fixed to microtome, to cut
uniform thin sections of 10-12 µm. Then the paraffin ribbons were carefully taken from
the microtome using foreceps. The paraffin ribbons were cut into convenient lengths
with the help of a blade and placed on a clean glass slide previously flodded with
gelatin adhesive (1 g gelatin dissolved in 90 ml water to which 10 ml formaldehyde
added later). Further, ensure proper expansion of sections by keeping the slides for a
little while on hot plate at 50-55oC. Drain off the excessive adhesive keep the slides
overnight at 40 oC for drying in oven.

3.7.4.5 Staining and Mounting

Paraffin wax was removed from the sections by placing the slide in xylene
followed by one change in it for 5 min.

Procedure

• The xylene and all other solutions were kept in coplin jars in which slides were
held.

• The sections were partially dehydrated by passing through a series of ethyl


alcohol of decreasing concentrations from absolute alcohol to 50 per cent (100,
90, 70 and 50%), the sections were kept for 2-5 min in each step

• The sections were stained in safranin for 12hr.


65

• The sections were washed in distilled water for 5 min then rapidly passed
through graded alcohol series (30, 50, 70, 90 and 100%) with 5-10 min at each
step.

• Sections were counter stained with fast green (in clove oil) for 1 min.

• Excess stain was cleared by palcing it in a carbol-xylene for 10 min before the
sections are brought to xylene (10 min).One more bathing in xylene for 10 min.

• Mount the sections DPX mountant upon carefully slides to be left for a couple of
days before they are examined under compound microscope.

3.8 Mechanism of action of lectins in the suppression of soil- borne pathogens

3.8.1 FITC conjugation of lectins

Fluorescein isothiocyanate-conjugated SRL1, RVL1 and anti-SRL1 were


prepared according to the procedure described by Goldman (1968) where the proteins,
10 mg/ml were taken in 50 mM carbonate buffer, pH 9.5 containing 150 mM NaCl.
FITC dissolve in the carbonate buffered saline was added (25 µg/mg of protein) to
chilled protein solution, and with gentle stirring the solution was kept overnight in cold.
Untreated FITC was removed by dialyzing the reaction mixture against repeated
changes with water and finally with 10 mM phosphate buffered saline, pH 7.2. This
FITC conjugated SRL1, RVL1 and anti-SRL1 were used for binding studies and FITC
anti-SRL1 for localization studies.

3.8.2 Lectin-binding assay for fungus

Lectin-binding assay for mycelium vegetative of R. solani, S. rolfsii,


F. oxysporum, P. lilacinus and T. viride from fresh culture broth of two-seven days
depending on fungus were washed repeatedly by centrifugation at 8000 rpm at 4oC.
Washed mycelial filaments were again washed with phosphate buffered saline (PBS).
Subsequently the filaments were incubated with FITC-SRL1, FITC-RVL1, bacterial
control FITC-BL21 (10 mg/ml) and inhibitor mucin in PBS for 30 min with gentle
shaking. Excess FITC stain was removed by washing with PBS on centrifugation.
Essentially the same procedure was adopted for lectin binding on mature and
immature sclerotial bodies of S. rolfsii and sclerotia of R. solani. Interaction of FITC
66

labelled lectins and appropriate controls on fungal mycelium was observed and
photographed under fluorescence microscope (Zeiss Axioscope A1) using excitation
filter 450-490 nm in the path of excitation light and barrier filter 515 nm in the path of
emitted light.

Young colonies of F. oxysporum, P. lilacinus and T. viride were obtained by


growth for 40-48 h at 27±1oC on microscope slides coated with minimal medium
(Galun, 1972), which had been inoculated 50 µl of 105 spores/ml suspension in distilled
water. Labelling of colonies by FITC labeled RVL1 and SRL1 lectins (0.5- 2 mg/ml) in
buffered saline pH 7.0 with the control FITC-BL21 and mucin sugar (inhibitor) were
according to the procedure of Mirelman et al. (1975). Parallel preparations were treated
by FITC-conjugated lectins preincubated for 30 min at 27±1oC. After 30-40 h grown in
dark FITC lectins 50 µg in 0.1 ml PBS was spread over half of the colony allowed
interacting for 30 min at room temperature. Slides were then washed with saline and
observed with a standard Zeiss Axioscope A1 fluorescence microscope using a BG-12
exciter filter and No. 53 barrier filter.

3.8.3 Immunolocalization of anti-SRL1 in S.rolfsii and R.solani

3.8.3.1 Preparation of periodate-BSA (p-BSA)

BSA was subjected to periodate oxidation, to get itself freed from associated
glycoproteins and converted to p-BSA. Periodate treated BSA was prepared according
to the method of Glass et al. (1981). BSA in 0.1M sodium acetate buffer, pH 4.5 (4
g/100 ml) was treated with 10 mM periodic acid for 6 hours at room temperature. Then
excess of periodate was eliminated by adding glycerol to afinal concentration of 10 mM
and later solution was dialyzed extensively against 10 mM PBS and lastly against
water and freeze dried.

3.8.3.2 Immunolocalization of lectins

Localization of lectin in vegetative mycelium and immature and mature sclerotial

bodies of S. rolfsii during development was carried out by immunolabeling the lectin
sites with FITC anti-SRL1 (Swamy et al., 2004). Vegetative mycelial mass from the S.
rolfsii culture broth of 10 days was washed repeatedly by centrifugation at 8000 rpm.
Washed mycelial filaments were suspended in phosphate buffered saline (PBS)
67

containing p-BSA (3%) and incubated for 30 min at 37oC to block nonspecific binding
by lectin antibodies, followed by extensive washing with PBS. Subsequently the
filaments were incubated with FITC anti-SRL1 in PBS (50 mg/ml) for 30 min with gentle
shaking. Excess unbound antibodies were removed by washing with PBS on
centrifugation. Essentially the same procedure was adopted for lectin localization on
sclerotial bodies. Interaction of FITC anti-SRL1 on vegetative mycelia and sclerotial
bodies was observed and photographed under fluorescence microscope (Zeiss
Axioscope A1) with an excitation filter 450-490 nm in the path of excitation light and
barrier filter 515 nm in the path of emitted light.

The above same procedure used for anti-SRL1 lectin binding assay for
mycelium and sclerotia of R.solani.

3.8.4 Staining of nematode secretions

Secretions of M. incognita juveniles were stained according to the method of


Premachandran et al. (1988). Nematode secretions were studied by using following
staining solutions.

1. 1% CBB-R dissolved in water,

2. 0.2% CBB-R dissolved in 20% methanol,

3. 0.2% CBB-R dissolved in 40% methanol and 10% acetic acid

4. 0.1% CBB-R dissolved in 40% ethanol and 10% acetic acid

Nematodes were concentrated to one ml in a eppendorff tube and 200 µl of a


different staining solution containing Coomassie Brilliant Blue R (CBB-R) was added. A
drop of the suspension was put on a glass microscope slide under a coverslip. Pieces
of glass wool were placed on the slide to support the coverslip which was slowly
lowered onto the nematode sample, then sealed with clear nail polish. Samples were
incubated in the dark at room temperature for 24 h, followed by observation and
analysis under a Differential interference contrast microscope (Zeiss Axio Scope.A1).

3.8.5 Lectin-binding assay for M.incognita

Interaction of RVL1 and BL21 with M. incognita was investigated using FITC-
RVL1 and FITC-BL21 by fluorescent microscopy. Nematodes (10) were suspended in
68

1.0 ml solution of RVL1 (4.8 mg/ml) and BL21 (4.8 mg/ml) incubated at 27±1°C in the
dark. At different intervals of time (6 and 12 h), 200 µl aliquots were drawn and washed
thrice by centrifugation (1000 rpm for 2 min) with PBS to remove excess FITC-RVL1.
Nematodes were finally collected on a membrane sieve (25 µm), mounted on glass
slides and observed under a fluorescent microscope (Zeiss, Axioscope A1) with an
excitation filter 450-490 nm in the path of excitation light and barrier filter 515 nm in the
path of emitted light. Receptor-mediated RVL1 binding to nematode was confirmed by
using FITC-RVL1 complexed with mucin.

FITC-RVL1 (4.8 mg/ml) was incubated with mucin (125 µg) in 1.0 ml of PBS for
1 h at room temperature. To this lectin-sugar complex solution, 10 nematodes were
added and incubated as described earlier. After 48 h nematodes were observed for the
fluorescence label under the florescent microscope.

For the binding experiment with SRL1, nematodes were washed twice in
phosphate-buffered saline (PBS), pH 7. Fluorescein isothiocyanate (FITC) conjugated
lectin of pure SRL1 (30 µg/ml PBS) was added to the nematode suspension (10
juveniles) and incubated in the dark at room temperature for 4 h. Nematodes were
washed with PBS, followed by an extensive rinse with distilled water. Nematodes were
then collected with distilled water and mounted on glass microscope slides for
observation under a fluorescent microscope (Zeiss, Axioscope A1) with an excitation
filter 450-490 nm in the path of excitation light and barrier filter 515 nm in the path of
emitted light.

3.8.6 Statistical analysis

The experimental data collected were analyzed statistically for its significance of
difference by the normal statistical procedure adopted for Completely Randomized
Design and Factorial Completely Randomized Design (two-way and three way anova)
and interpretation of data was carried out in accordance with Walter (1997). The level
of significance used in ‘F’ and ‘T’ test was P=0.01. Critical differences were calculated
wherever ‘F’ test was significant. The values of per cent disease incidence were
subjected to angular transformation according to the table given by Sundarraj et al.
(1974) before they were analyzed.
4. EXPERIMENTAL RESULTS
Experiments were conducted on various suppression activities of plant and fungal lectins on
soil-borne pathogens during the period 2010 to 2013. Expression of rvl1 and srl1 in transgenic
tomato was checked. Toxicity of transgenic tomato plants on soil borne pathogens was assayed. An
attempt was also made to elucidate the mechanism of action of lectins. The results obtained with
various experiments are presented here.

4.1 Collection and isolation of soil-borne pathogens

Tomato plant samples affected by different soil-borne pathogens were collected from fields of
Main Agricultural Resarch station, UAS, Dharwad. From the wilted plants, pathogens like Sclerotium
rolfsii, Rhizoctonia solani, Fusarium oxysporum and Ralstonia solanacearum were isolated (Plate 1).

Galled tomato roots and rhizosphere soil were also collected to isolate Meloidogyne
incognita. These cultures were used for further investigations.

4.1.1 Isolation, purification, preservation of the soil-borne pathogens from


tomato infected plants and soil

4.1.1.1 Isolation, purification, preservation of soil-borne fungal pathogens

Standard tissue isolation technique was followed to obtain causal agents from the diseased
tomato plants showing typical symptoms. Repeated isolations yielded F. oxysporum, S. rolfsii, R.
solani. The infected specimens were cut into small bits and washed in running water. These bits
were surface sterilized with one per cent sodium hypochlorite solution for one minute, then
aseptically transferred to PDA slants and were incubated at 27±1oC for three to ten days. Same
procedure was followed to obtain the pure culture of F. oxysporum, S. rolfsii and R. solani. Such
pure culture tubes were preserved in a refrigerator at 4°C and used for further studies. They were
sub-cultured every month.
70

Plant 1. Tomato root diseases from which the pathogens were isolated
71

4.1.1.2 Identification of pathogen

On the basis of morphological and cultural characteristics, the pathogen were identified as
Fusarium oxysporum, Sclerotium rolfsii and Rhizoctonia solani (Plate 2).

4.1.1.3 Fusarium oxysporum

The fungus was identified based on colony morphology, mycelial character, spore production
and pigmentation on PDA: Fusarium on Potato Dextrose Agar put-forth rapid growth covering the
Petri plate in 7-10 days. The mycelium was sparse to dense, white to light pinkish in colour. It
produced both micro- and macroconidia. The microconidia formed abundantly on the aerial
mycelium from elongated phialides and were abundant hyaline and cylindrical to fulcate. They
measured 10.8 to 13.8 x 3.3 to 6.6 µm. The macroconidia developed in 6 days from well developed
conidiophores. They were sparse to abundant, hyaline, fusoid-subulate with 2-5 septa and measured
29.7 to 43.8 x 3.8 to 5.5 µm. Apex of macroconidia were pointed and beaked. Chlamydospores
formed 12 to 14 days after incubation in cultures. They were globose to oval, single celled, smooth to
rough walled, measured 8.25 to 11.5 x 6.60 to 9.90 µm and seen terminally or intercalary in the
hyphae.

4.1.1.3.4 Sclerotium rolfsii

The fungus produced white, dense, radiating mycelial growth on PDA. In the early stages, the
fungus produced silky white mycelium and gradually lost its luster and became dull in appearance.
Sclerotial initials were observed from seventh day onwards. Initially, the sclerotial bodies were
spherical and white, later became chocolate brown to dark brown at maturity. Matured sclerotia were
spherical to ellipsoidal.

4.1.1.5 Rhizoctonia solani

Fungus produced dull white, sparse, radiating mycelium on PDA. Later, mycelium became
raised, light brown and powdery. Irregular to round, brown colored sclerotia were seen in the plates
after seven days of incubation. Microscopic observations revealed hyphae were branched at right
angles, were septate and hyaline.
72

Plate 2a. Root pathogens and the growth in vitro

Plate 2b. Their hyphae and spores


73

4.1. 2 Pathogenicity of fungal pathogens

The pathogenicity of F. oxysporum, S. rolfsii and R. solani conducted on tomato revealed that
the fungi were pathogenic.

4.1.3 Isolation, purification, preservation of Ralstonia solanacearum

Isolation of R. solanacearum was made from infected tomato plants showing typical wilt
symptoms on Sucrose Peptone Agar containing tetrazolium chloride. The bacterial colonies
appeared fluidal, irregular to round, convex, smooth, dull white with light pink center.

The virulent colonies which appeared as dull white with light pink centre after 36 h incubation
at 27±1oC were picked and purified by single colony isolation technique on Sucrose Peptone Agar
medium and suspended in polypropylene tubes containing sterile distilled water and preserved at
4oC. These served as stock culture for further studies.

4.1.3.1 Growth phase study of R. solanacearum

There was a gradual increase in the growth of bacterium at different interval (Table 1, Fig. 2).
The growth started after 16 h (0.119 OD) and maximum growth was observed at 112 and 120 h after
inoculation with OD value with 1.847. After 120 h, there was gradual decrease in growth. The
dynamics of the bacterial growth was studied by plotting the cell growth (absorbance) versus the
incubation time. The curve thus obtained was a sigmoid curve and was taken as a standard growth
curve of the bacterium.

4.1.3.2 Morphological and biochemical characteristics R. solanacearum

The results of the various morphological, physiological and biochemical tests are given in
Table 2, Plate 3. The bacterium is a rod shaped, strictly aerobic, gram negative, oxidase positive and
amphitrichously flagellated. It was positive for starch hydrolysis, liquefaction of gelatin and produced
hydrogen sulphide.
74

Table 1. Growth phase studies of Ralstonia solanacearum

Hours after inoculation OD (absorbance) at 600 nm

8 0.082
16 0.119
24 0.458
32 0.634
40 0.854
48 1.007
56 1.176
64 1.220
72 1.394
80 1.489
88 1.517
96 1.514
104 1.740
112 1.817
120 1.847
128 1.840
136 1.840
144 1.840
152 1.840
160 1.840
168 1.840
Mean 1.328
S.Em ± 0.004
CD at 1% 0.14
75
76

4.1.3.3 Hyper-sensitive reaction

The pure culture of Ralstonia solanacearum injected into intercellular spaces of


tobacco leaves (Nicotiana tabaccum var. samsun) produced characteristic water soaked
lesions within 16 to 20 h of inoculation. Further after 24 h, the light yellow area started
collapsing and formed a desiccated light brown necrotic area.

4.1.3.4 Pathogenicity

The pathogenicity of R. solanacearum was determined by inoculating a bacterial


suspension (1x108 cfu/ ml) into susceptible tomato (Pusa Ruby) plants at preferably the
third true leaf stage. Artificially inoculated plants started expressing wilt symptoms within 15
days. Reisolation yielded colonies which were similar to the ones isolated from diseased
tomato plants.

4.1.4 Collection, identification and maintenance of root-knot nematode

The collection, maintenance and identification of root-knot was carried out as


explained in ‘Material and Methods’. The affected root samples and soil of tomato were
used for the extraction of M. incognita. The morphology of perineal pattern was used for
species identification. Important diagnostic characters of the perineal pattern of thus
identified species as M. incognita are summarized in Table 3, Plate 4. The observed
characters were compared with the descriptions given by Eisenback et al. (1981).

4.1.4.1 Pathogenicity

Pathogenicity of M. incognita was proved by inoculating tomato seedlings using


fresh nematode suspension. The artificially inoculated plants showed stunted growth with
yellow coloured leaves. When infected plants were uprooted, deformed roots with
prominent galls of varying sizes were noticed.

4.2 Expression of RVL1 and SRL1 in E. coli

Expression of RVL1 and SRL1 was checked with total protein isolated from
induced E. coli BL21(DE3)pLysS. Total protein induced from cell mass of E. coli
recombinant clones was compared with their respective controls (clones without srl1 and
rvl1 gene) upon induction. Recombinant E. coli clones had a total protein content of
77

12 mg/ml compared to 10.00 mg/ml in control. The isolated protein was subjected for
haemagglutination assay and SDS–PAGE.

4.2.1 Haemagglutination

The crude extracts of plant tuber lectin Remusatia vivipara lectin (RVL1) and
fungal lectin Sclerotium rolfsii lectin (SRL1) were prepared from E. coli BL21(DE3)pLysS
cells carrying respective recombinant expression vectors and plain expression vectors
(control) and the presence of lectin was confirmed by haemagglutination assay (Table 4,
Plate 6). It strongly agglutinated trypsinised rabbit erythocytes, indicating blood specificity.
Minimum concentration required for agglutination (MCA) was 1.37 µg and 2.73 µg for SRL1
and RVL1, respectively. For SRL1, the total activity was 0.87 × 104 and the specific activity
was 7.29 × 102. Likewise, for RVL1, the total activity was 0.43 ×104 and the specific activity
was 3.66 × 102 with rabbit erythocytes. In general, the concentration of lectin was more in
partially purified protein.

4.2.2 SDS-PAGE

Expression of SRL1and RVL1 was checked with total protein isolated from induced
E. coli BL21(DE3)pLysS cells carrying respective recombinant expression vectors and plain
expression vectors (control). The induced protein was subjected to SDS–PAGE. The gels
stained with Commassie brilliant blue showed an extra protein band corresponding to
molecular weight of 28.2 kDa for RVL1 and 19.5 kDa for SRL1 (Plate 7).

4.2.3 Purification of SRL

Heterologous protein produced in E. coli requires purification for all


other downstream experiments. Care was taken by selecting appropriate expression
vector pET-32b(+) and restriction sites to produce SRL which is His-tag fused to enable
Ni-NTA column based purification. To purify His-tag fused protein, E. coli cells carrying
respective recombinant expression vectors and plain expression vectors (control)
were Induced for the production of heterologous protein, which was then subjected to
Ni-NTA column based purification and run on the SDS-PAGE gel. The purified protein
78

Table 2. Morphological and biochemical characterization of Ralstonia solanacearum

Test Inference
Colony morphology on sucrose peptone agar with TZC medium
Configuration Round
Margin Entire
Elevation Convex
Surface Smooth
Pigment Pink at the centre
Biochemical characterization
Shape Rod
Arrangement Single
Gram reaction Negative
Starch hydrolysis Positive
Gelatin liquefaction Positive
H2S production Positive
Tobacco hypersensitivity Positive

Table 3. Important characters of perineal pattern of tomato root-knot nematode

Original description
Feature Characters observed
(Eisenback et al., 1981)

Dorsal arch High squarish High squarish

Lateral field Lateral ridges absent, marked Lateral ridges absent, marked by
by breaks and forks in striae breaks and forks in striae

Striae Coarse, smooth to wavy Coarse, smooth to wavy and zigzag

Tail terminus Often with distinct whorl Often with distinct whorl

Table 4. Heamagglutination assay for E. coli-expressed SRL1 and RVL1

Total activity Specific activity


Treatment MCA (µg)
(unit) (unit/mg)
SRL1 1.37 0.87×104 7.29×102
RVL1 2.73 0.43×104 3.66×102
79

Hypersensitive reaction on tobacco


Plate 3. Ralstonia solanacearum: growth in vitro, morphology, biochemical characterization
and hypersensitivity
80

Plate 4. Meloidogyne incognita: Eggmass and perineal pattern

Plate 5 Mass multiplication (Giant culture)


81

Plate 6 Heamagglutination assay of E. coli-expressed RVL1 and SRL1

Plate 7 SDS-PAGE analysis of E. coli-expressed RVL1 and SRL1


82

showed a single band with molecular weight of 19.5 kDa, which was in agreement with the
calculated molecular weight of DP-SRL1.

4.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)

Antiserum against SRL1 was prepared using 3-month old female albino rabbit by
giving four (weekly) intramuscular injections with purified preparation of the SRL1
suspended at different concentrations of 25, 100, 500 and 1000 µg in PBS. Finally blood
was collected and processed by following the procedure described in “Material and
Methods”. The antiserum thus produced was used for further studies.

4.3.1 Slide agglutination test

Slide agglutination test was performed to know the efficacy of antibodies raised
against SRL1 by mixing a drop of antiserum with SRL1 and PBS. The results revealed that
there was agglutination in SRL1. No agglutination was observed in PBS which indicated
that antiserum was active.

4.3.2 Determination of titre of antiserum

After production and partial purification of antisera by ammonium precipitation test


from SRL1 antigen, they were tested for titre value determination by micro precipitation
test. The highest dilution at which reaction of antigen and antibody could be demonstrated
was considered the titre of the serum. The sera possessed a reaction up to 1: 65536
dilutions (Table 5, Plate 8).

4.3.3 Direct antigen coating ELISA

Titre of antisera was determined by two-fold serial dilutions of antiserum from 100 to
204800 and purified SRL1 used as an antigen. The titre of antisera against antigen was
determined by DAC- ELISA. Antisera was detectable up to 262174 dilution and this was
found out based on colour changes in the substrates at optical density value of 405 nm
wave length, using ELISA reader.

Reactivity of SRL1 antiserum (anti-SRL1) to the antigen purified SRL1, E. coli


expressed SRL1 was determined using DAC - ELISA. The antigen was diluted with
phosphate buffer to adjust the dilutions of 1: 2048 and 1: 4096 and antiserum at1: 8192
83
84

1: 16384, 1: 32768 and 1: 65536 dilutions. The results showed that the antigens at
1: 2048 and antiserum 1: 8192 (and above) showed positive reaction (Table 6). The buffer
and BL21 controls gave negative results.

4.4 Evaluation of plant and fungal lectins for the in vitro suppression of some common
soil-borne pathogens

4.4.1 In vitro antifungal assay

Antifungal activity of different amounts of plant and fungal lectins from Remusatia
vivipara and Sclerotium rolfsii was determined for soil borne fungal pathogens like F.
oxysporum, S. rolfsii and R. solani as described in “Material and Methods”. Screening of
different concentrations of RVL1 and SRL1 Lectins with bacterial control (BL21) were
evaluated against F. oxysporum, S. rolfsii and R.solani in laboratory each at four
concentrations (1.2, 2.4, 3.6 and 4.8 mg/ml) by poisoned food technique, spread plate,
inhibition zone assay and spore or sclerotia germination methods. In order to get double
confirmation of results used different methods for evaluation of these lectins.

4.4.1.1 Poisoned food technique

Data with respect to inhibition of mycelial growth of F. oxysporum, S. rolfsii and R.


solani at four concentrations of RVL1, SRL1 and BL21 (Bacterial control) were recorded
and are presented (Table 7, Plate 9).

4.4.1.1a F. oxysporum

The efficacy of the two lectins, concentrations and their interaction on per cent
inhibition of mycelial growth of F. oxysporum differed significantly (Table 7a). Maximum per
cent inhibition (25.28%) of F. oxysporum was recorded in RVL1 plant lectin which was
significantly superior to SRL1 (22.52%). Least per cent inhibition of 15.56 per cent was
noticed in SRL1 at a low concentration (1.2 mg/ml). However, the maximum per cent
inhibition of mycelial growth was observed at 4.8 mg/ml concentration of lectins.
85

Table 6. Anti-SRL1 titre determination by DAC-ELISA

Optical Density at 405 nm


Antigen
Sample Antiserum dilution
dilution
1:8192 1:16384 1:32763 1:65536

1:2048 1.635 1.569 1.432 0.951


Total protein SRL1
1:4096 1.521 1.237 0.751 0.597

1:2048 0.952 0.880 0.771 0.436


Pure SRL1
1:4096 0.855 1.014 0.902 0.421

1:2048 0.339 0.227 0.165 0.148


Positive control
(BL21)
1:4096 0.189 0.106 0.100 0.095

Negative control
1:2048 0.089 0.083 0.080 0.080
(PBS)

Table 7. Evaluation of plant and fungal lectins against F. oxysporum and S. rolfsii
through poison food technique

Table 7a. Fusarium oxysporum

Per cent inhibition


Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 15.56 (23.24)* 22.78 (28.52) 25.00 (30.02) 26.73 (31.15) 22.52 (28.34)

RVL1 21.11 (27.37) 22.22 (28.14) 27.78 (31.82) 30.00 (33.23) 25.28 (30.20)
Control (BL21) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Control 0.00 (0.00)


Treatments Concentration TxC

S.Em± 0.64 0.55 0.32

CD at 1% 2.03 1.84 1.07

*Figures in the parenthesis are arc sine transformed values


86

Plate 8. Titre of polyclonal antibody against SRL1antigen determined by DAC-ELISA


87

At all tested concentrations of 4.8, 3.6, 2.4 and 1.2 mg/ml RVL1 recorded highest
per cent inhibition of mycelial growth of 30.00, 27.78, 22.22, and 21.11 per cent
respectively which was significantly superior to SRL1. SRL1 recorded 26.73, 25.00, 22.78
and 21.11 per cent inhibition of the mycelial growth. There was no inhibition in mycelia
growth of fungus which is treated with bacterial control (BL21 extract). At all the tested
concentrations among plant and fungal lectin, plant lectin recorded highest inhibition at all
concentrations.

4.4.1.1b S. rolfsii

Differences in mycelial growth in different treatments were seen two days after
inoculation of the pathogen on PDA. Both RVL1 and SRL1 treated plates showed smooth,
pure white, sparse radiating mycelium. But in respective controls, luxurious radiating and
aerial growth of the fungus was noticed. All lectin treated PDA plates as well as control
plates without lectins, covered the petriplate on 3rd day.

In SRL1 treated plates, it was found that pre-sclerotial bodies formation started on
second day of inoculation. On fourth day, white pre-sclerotial bodies formation was seen in
all lectin treated plates except that of control. In both control PDA plates, sclerotial bodies
started forming on 7th day of inoculation. On 6th day of inoculation, colour of sclerotial
bodies turned to dark brown in PDA plate treated with 3.6 mg/ml and 4.8 mg/ml of E. coli
protein containing SRL1. Formation of dark brown sclerotial bodies was observed on 7th
day of inoculation in PDA plate treated with 1.2 mg/ml and 2.4 mg/ml of E. coli protein
containing SRL1 and PDA plate treated with 1.2 mg/ml, 2.4 mg/ml, 3.6 mg/ml and 4.2
mg/ml of E. coli protein containing RVL1. Compared to SRL1 and RVL1 treated PDA
plates, control plates took 12 days for formation of dark brown sclerotial bodies (Table 7b,
Plate 9).

Rate of formation of sclerotial bodies increased with increasing concentration of both


lectins. SRL1 treated PDA plates produced bigger, heavier and more sclerotial bodies with
increasing concentrations of SRL1. Compared to SRL1 treated plates, RVL1 treated plates
produced small, lighter and less sclerotial bodies. Both control PDA plates containing
bacterial protein without lectin, and PDA plate without protein showed small sized and less
number of sclerotial bodies.
88
89

Plate 9. In vitro evaluation of SRL1 and RVL1 against F. oxysporum, S. rolfsii and
. R.Solani using poison food technique
90

Sclerotium rolfsii: Sclerotial germination

Plate 9. contd….
91

Germination of sclerotial bodies is another event in the development of fungus.


Sclerotia were plated on water agar to know the effect of both the lectins on their
germination. Results revealed that there was no germination of sclerotial bodies in all
concentrations of RVL1 treatment compared to SRL1 and control. In both the controls,
(bacterial protein without lectin and untreated sclerotial bodies), sclerotia started
germinating after two days of inoculation. Pure white, smooth, radiating mycelial growth
was seen after four days. At the same time, both SRL1 and RVL1 treated PDA plates
failed to show germination of sclerotial bodies. But after five days of inoculation, slight
germination of sclerotial bodies and dull white, sparse thead-like mycelial growth (5 to 12
mm length) was observed in all concentrations of SRL1. There was no sclerotial
germination in all concentrations of RVL1.

4.4.1.1c R. solani

Data with respect to inhibition of mycelial growth of R. solani at four concentrations


of SRL1, RVL1 and BL21 (bacterial control) were recorded by incorporating the protein on
growing medium. There was no inhibition of mycelial growth of R. solani by incorporating
lectins into the growing medium (Plate 9).

4.4.1.2 Spread plate method

The antifungal activity of lectins was assayed at four concentrations in the


laboratory for their efficacy against F. oxysporum, R. solani and S. rolfsii using spread
plate method as described in “Material and Methods”.

Data with respect to inhibition of mycelial growth of fungus at four concentrations of


RVL1, SRL1, anti-SRL1 and BL21 (bacterial control) were recorded and per cent inhibition
is presented in Table 8. It was observed that lectins’ concentrations and their interaction
differed significantly with respect to inhibition of the mycelial growth of fungus. Between
the concentrations of lectins, efficacy was significant from lower to higher concentrations
with greater efficacy at higher concentration.

4.4.1.2a F. oxysporum

Effect of RVL1, SRL1 and anti - SRL1 on the fungal growth was
significant (Table 8a, Plate 10). Among the lectins, maximum per cent inhibition of growth
92

of F. oxysporum was observed in RVL1 (40.69%) which was significantly superior to SRL1
(29.35%) and anti-SRL1 (0.00%). Among the four tested concentrations, 4.8 mg/ml
concentration was significantly superior to 3.6, 2.4 and 1.2 mg/ml. Maximum per cent
inhibition of mycelial growth (54.81%) of the fungus was recorded in RVL1 at 4.8 mg/ml
followed by SRL1 (42.96%). There was no inhibition of mycelial growth of the fungus by
anti-SRL1 and BL21. At 3.6 mg/ml, maximum per cent inhibition of mycelial growth
(43.48%) of the fungus was recorded in RVL1. Further, RVL1 of 2.4 mg/ml (36.30%) and
3.6 mg/ml of SRL1 (31.11%) were effective in inhibiting the pathogen. At 1.2 mg/ml
concentration, maximum per cent inhibition of mycelial growth of the fungus was recorded
in RVL1 (28.15%). Similarly SRL1 at 2.4 and 1.2 mg/ml showed 22.96 and 20.37 per cent
inhibition of the mycelial growth respectively.

4.4.1.2b S. rolfsii

Maximum per cent inhibition of growth of the fungus was recoreded in anti-SRL1
(37.81 %) than RVL1 (32.78%) and presented in Table 8b, Plate 10. Among tested lectins,
anti-SRL1 at 4.8 and 3.6 mg/ml showed maximum per cent inhibition of 44.44 and 44.07
per cent against S. rolfsii and they remained onpar with each other. Further, 43.33 and
40.00 per cent inhibition of mycelial growth was recorded in RVL1 at 4.8 and 3.6 mg/ml
concentration. At 2.4 mg/ml, maximum percent inhibition of mycelial growth was recorded
in anti-SRL1 (35.37%). Further, 28.15 and 27.59 per cent inhibition was recorded in RVL1
and anti-SRL1 at 2.4 and 1.2 mg/ml concentrations respectively, and they remained on par
with each other. Least per cent inhibition was recorded at 1.2 mg/ml of RVL1 (19.63%).
There was no inhibition of mycelial growth of fungus in SRL1 and controls.

4.4.1.2c R. solani

Maximum per cent inhibition of mycelia growth was recoded in anti-SRL1 (49.63%)
followed by RVL1 (27.31%). There was no inhibition of mycelial growth in SRL1 and BL21
treatments. Between the concentrations of lectins, efficacy was significant from lower to
higher concentrations with greater efficacy at higher concentration (Table 8c, Plate 10).
Anti-SRL1 at 4.8, 3.6, 2.4 and1.2 mg/ml concentrations gave maximum per cent inhibition
of mycelial growth of 55.55, 50.37,
93

Table 8. Evaluation of plant and fungal lectin against F. oxysporum, S. rolfsii and R.
solani through spread plate technique
Table 8a. Fusarium oxysporum

Per cent inhibition

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 20.37 (26.84)* 22.96 (28.65) 31.11 (33.92) 42.96 (40.98) 29.35(32.60)

Anti-SRL1 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

RVL1 28.15 (32.06) 36.30 (37.07) 43.48 (41.28) 54.81 (47.79) 40.69 (39.55)

Control 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)
(BL21)

Control 0.00 (0.00)

Treatments Concentration TxC

S.Em± 0.37 0.32 0.18

CD at 1% 1.20 1.16 0.70

*Figures in the parenthesis are arc sine transformed values

Table 8b. Sclerotium rolfsii

Per cent inhibition

Treatment Concentration (mg/ml)


1.2 2.4 3.6 4.8 Mean

SRL1 0.00 (0.00)* 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Anti-SRL1 27.59 (31.47) 35.37 (36.51) 44.07 (41.62) 44.44 (41.83) 37.87 (37.69)

RVL1 19.63 (26.18) 28.15 (32.06) 40.00 (39.25) 43.33 (41.18) 32.78 (34.72)
BL21 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Control 0.00 (0.00)

Treatments Concentration TxC


S.Em± 0.21s 0.21 0.11

CD at 1% 0.77 0.77 0.68


*Figures in the parenthesis are arc sine transformed values
94

Table 8c. Rhizoctonia solani

Per cent inhibition

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 0.00 (0.00)* 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Anti-SRL1 44.81 (42.04 ) 47.77 (43.74) 50.37 (45.23) 55.55 (48.21) 49.63 (44.81)

RVL1 21.85 (27.88) 24.81 (29.89) 28.51 (32.29) 34.07 (35.73) 27.31 (31.45)

Control (BL21) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00)

Control 0.00 (0.00)

Treatments Concentration TxC

S.Em± 0.36 0.36 0.18

CD at 1% 1.08 1.08 0.70

*Figures in the parenthesis are arc sine transformed values


95

Plate10. In vitro evaluation of SRL1 and RVL1 against F. oxysporum, S. rolfsii and
R.solani using spread plate menthod
96

47.77 and 44.81 per cent, respectively. Further, RVL1 showed 34.07, 28.51, 24.81, and
21.85 per cent inhibition of mycelial growth of the fungus.

4.4.1.3 Inhibition zone assay

Antifungal activity of the plant and fungal lectins was tested under in vitro condition
by inhibition zone assay as described in “Material and Methods”. Lectins, concentrations
and their interaction differed significantly with respect to inhibition of the mycelial growth of
fungus. Between the concentrations of lectins, efficacy was significant from lower to higher
concentrations with greater efficacy at higher concentration.

4.4.1.3a F. oxysporum

Results of inhibition zone assay against F.oxysporum were recorded looking to


inhibition line forming around the disc (Table 9a Plate 11). Maximum inhibition zone of
mycelial growth was recorded in RVL1 (1.64 cm) followed by SRL1 (1.25 cm). At 4.8
mg/ml maximum inhibition zone of mycelial growth of 2.24 cm was recorded in RVL1
followed by 1.94 and 1.93 cm of SRL1 and RVL1 at 4.8 and 3.6 mg/ml concentrations,
respectively. Further, 1.64 and 1.61 cm inhibition zone of SRL1 and RVL1 were recorded
at 2.4 mg/ml and they remained on par with each other followed by RVL1 at 1.2mg/ml
(1.41 cm). Least mycelial growth inhibition was found in RVL1 (1.16 cm) at 1.2 mg/ml.
BL21 (bacterial control), buffer and sterile distilled water controls did not show inhibition
zone.

4.4.1.3b S. rolfsii

Antifungal activity of lectins was evident at higher concentrations and results are
presented in Table 9b, Plate 11. Anti-SRL1 recorded highest mean inhibitory zone of 0.98
cm which is significantly higher than that of RVL1 (0.84 cm). Anti-SRL1 recorded maximum
inhibition zone of mycelial growth of 1.60 and 1.36 cm at 4.8 and 3.6 mg/ml concentrations
and it was followed by 1.20 and 1.06 cm inhibition zone in RVL1 (4.8 and 3.6 mg/ml),
respectively. Further, at 2.4 and 1.2 mg/ml concentrations of RVL1 recorded 0.69 and 0.48
cm inhibition zone. Least inhibition zone was recorded at 2.4
97

Table 9. Evaluation of plant and fungal lectin against F. oxysporum and S. rolfsii
through inhibition zone assay

Table 9a. Fusarium oxysporum

Inhibition zone (cm)

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 1.41 (1.55)* 1.64 (1.62) 1.74 (1.65) 1.94 (1.71) 1.68 (1.64)

RVL1 1.16 (1.47 ) 1.61(1.62) 1.93 (1.71) 2.24 (1.80) 1.73 (1.65)

Anti-SRL1 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)

Control (BL21) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)

Control (PBS) 0.00 (1.00)

Control (S.W) 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values

Table 9b. Sclerotium rolfsii

Inhibition zone (cm)

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 0.00 (1.00)* 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)

RVL1 0.48 (1.22) 0.61 (1.27) 1.06 (1.44) 1.2 (1.48) 0.84 (1.36)

Anti-SRL1 0.38 (1.17) 0.56 (1.25) 1.36 (1.54) 1.6 (1.61) 0.98 (1.41)

Control (BL21) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)

Control (PBS) 0.00 (1.00)

Control (S.W) 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values


98

and 1.2 mg/ml concentrations of anti-SRL1 (0.56 and 0.38 cm). SRL1, BL21, buffer and
sterile distilled water controls did not show inhibition zone.

4.4.1.3c R. solani

Maximum inhibition around the disc in RVL1 (1.02 cm) was observed at higher
concentration than anti-SRL1 (0.74 cm). SRL1 and controls did not show inhibition (Table
9c, Plate 11). At 4.8 mg/ml, highest zone of inhibition of mycelial growth of 1.45 cm was
observed in RVL1 followed by 1.40 cm of inhibition zone in anti-SRL1 treatment. Further,
RVL1 and SRL1 recorded 1.08 cm and 0.93 cm at 3.6 mg/ml and 0.88 and 0.63 cm at 2.4
mg/ml, respectively. At 1.2 mg/ml, 0.69 cm inhibition zone of mycelial growth of the fungus
recorded in RVL1 treatment. But anti-SRL did not show inhibition zone of mycelia growth
at 1.2 mg/ml.

4.4.1.4 Disc diffusion assay of F. oxysporum

Data presented in Table 9d, Plate 11 indicated that strong antifungal activity was
shown by protein towards F. oxysporm spore suspension (1.0 to 2.5 x 106 spores /ml)
spread on medium as explained in “Material and Methods”. Results revealed that among
the tested lectins and bacterial control, RVL1 recorded highest mean inhibitory zone of
1.64 cm which was significantly superior to SRL1 (1.25 cm). RVL1 recorded maximum
inhibition zone of 2.45 cm at 4.8 mg/ml, 1.95 cm at 3.6 mg/ml, 1.25 cm at 2.4 mg/ml and
0.92 cm at 1.2 mg/ml concentrations, respectively. Further, SRL1 recorded 1.76, 1.59,
1.10 and 0.55 cm inhibition zone at 4.8, 3.6, 2.4 and 1.2 mg/ml concentrations. BL21
extract (bacterial control), buffer and sterile distilled water controls did not show inhibition
zone.

4.4.1.5 Spore germination inhibition

Spore germination inhibition was measured at varying concentrations of SRL1,


RVL1 and BL21 using F. oxyporum spores. Relative spore germination is expressed as
percentage of the spore germination of control. Lectins inhibited the spore germination
even at 1.2 mg/ml concentration (Table 10). Per cent inhibition of spore germination
increased with decrease in concentration of plant and fungal lectins. However, spore
germination was significantly reduced at all concentrations of lectins. The interaction
99

Table 9c. Rhizoctonia solani

Inhibition zone (cm)

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 0.00 (1.00)* 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00(1.00)

RVL1 0.69 (1.30) 0.88 (1.37) 1.08 (1.44) 1.45 (1.56) 1.02 (1.42)

Anti-SRL1 0.00 (1.00) 0.63 (1.27) 0.93 (1.39) 1.40 (1.54) 0.74 (1.30)

Control (BL21) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00(1.00)

Control (PBS) 0.00 (1.00)

Control (S.W) 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values

Table 9d. Evaluation of SRL1 and RVL1 against F. oxysporum (disc diffusion assay)

Inhibition zone (cm)

Treatment Concentration (mg/ml)

1.2 2.4 3.6 4.8 Mean

SRL1 0.55 (1.24)* 1.10 (1.45) 1.59 (1.61) 1.76 (1.66) 1.25 (1.49)

RVL1 0.92 (1.38) 1.25 (1.50) 1.95 (1.72) 2.45 (1.86) 1.64 (1.61)

Control (BL21) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)

Control (PBS) 0.00 (1.00)

Control (S.W) 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values


100

Plate11. Evaluation of SRL1 and RVL1 against F. oxysporum, S. rolfsii and R.solani
employing inhibition zone assay
101

between treatment and concentration was significant which indicated that an increase in
concentration tended to modify the effect of other in a significant manner. RVL1 and
SRL1 at 4.8, 3.6, 2.4 and 1.2 mg/ml concentrations significantly inhibited the
germination of macro and microconidia. The greatest decrease in inhibition of spore
germination was noticed with RVL1 in treatment concentration interaction (55.26%)
followed by SRL1 (42.26%) at 4.8 mg /ml.

Maximum inhibition of spore germination of was recorded in RVL1 (47.66%) at


3.6 and 2.4 mg/ml concentrations followed by SRL1 (35.31%). Further, 19.30 per cent
inhibition of spore germination was recorded in RVL1 at 1.2 mg/ml. Least per cent
inhibition of spore germination was recorded at 1.2 mg/ml in SRL1 treatment (9.65 %).

4.4.1.6 Inhibition of sclerotial germination of S. rolfsii

In order to investigate the role of lectin in inhibition of sclerotial germination


of S. rolfsii after capping lectin sites in sclerotial bodies was examined by SRL1, anti-
SRL1, RVL1 and BL21 (control) it was tested and calculated for inhibition of the hyphal
growth (Table 11, Plate 12).

Interstingly sclerotia treated with SRL, Bl21, buffer and normal rabbit serum
germinated with lavish growth. Not only anti-SRL1, but also RVL1 inhibited the
germination of sclerotial bodies. SRL1 did not inhibit the sclerotial bodies germination.
Cent per cent inhibition of mature and immature sclerotial germination was found at all
concentrations (4.8, 3.6, 2.4 and 1.2 mg/ml) of anti-SRL1 treatment. In all, 51.01, 45.84,
21.03 and 15.73 per cent inhibition of mature sclerotial bodies and 74.61, 63.82, 55.54
and 51.69 per cent inhibition of immature sclerotial bodies was noticed by RVL1 at
sequential concentration in dcreasing order.

4.4.1.7 Inhibition of germination of sclerotia of R. solani

Different concentrations of lectins inhibited the germination of sclerotial bodies


of R. solani. SRL1, BL21 and the controls did not inhibit the germination. anti-SRL1 and
RVL1 treated sclerotia inhibited its growth (Table 12, Plate 13).

Maximum inhibition of sclerotia germination of 89.08 Per cent was recorded in


anti-SRL1 at 4.8 mg/ml followed by RVL1 (85.17%. Further, 80.44 and 80.42 per cent
102
103
104

Plate 12. Effect of lectins on sclerotial (mature and immature) germination inhibition
on Byrde’s medium

Plate 13. Effect of lectins on micro-sclerotia germination inhibition on Byrde’s medium


105

inhibition was recorded in anti-SRL1 and RVL1 at 3.6 mg/ml and they remained on par
with each other. This was followed by 67.08 and 78.82 per cent inhibition of anti-SRL and
RVL1 at 2.4 mg/ml. Least per cent inhibition was recorded at 1.2 mg/ml of 60.05 and 48.93
per cent in anti-SRL1 and RVL1.

4.4.2 In vitro antibacterial assay

4.4.2.1 Well diffusion assay

In vitro antibacterial assays demonstrated that the plant lectin (RVL1) and fungal
lectin (SRL1) exhibited antibacterial activity against the bacterium R.solanacearum as
shown by inhibition haloes using well method (Table 13a, Plate 14).

Both SRL1 and RVL1 exhibited dose dependent antimicrobial activity against R.
solanacearum which was found to be more sensitive to RVL1 (2.85 cm) followed by
Streptocycline control (2.59) and SRL1 (2.35 cm). RVL1 was the most potent against the
bacterium.

4.4.2.2 Determination of the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC)

Antibacterial activity of SRL1 and RVL1 against R. soalnacearum, MIC and MBC
values are presented in Table 13b, Plate 15.

The antibacterial efficacy of the protein was found to be moderate against the
bacterium. No antibacterial activity was noticed in the control set (BL21, PBS and sterile
distilled water). It was significant that the MIC which corresponds to the minimum lectin
concentration capable to inhibit the visible growth of the R. solanacearum was 0.625
mg/ml for both SRL1 and RVL1 with 1.87 and 1.56 mg/ml of MBC which corresponds to
the minimum concentration of the lectin capable to reduce the number of cfu for 0.1% of
the initial inoculum respectively MBCs determined by agar diffusion were higher than the
ones obtained by MICs.

Lectins which exhibited good antibacterial activity on R. solanacearum by disc


diffusion test demonstrated a good activity at MBC of 1.33 and 1.65 cm in RVL1 and
SRL1, respectively.
106

4.4.2.3 Inhibition zone assay

Minimum protein concentration required for inhibition of bacterial growth were


determined by MIC and MBC values. RVL1 and SRL1 showed inhibitory activity against R.
solanacearum at different concentrations and inhibition line around the disc measured
(Table 13c, Plate 16). R. solanacearum was found to be more sensitive to RVL1 (1.83 cm)
than SRL1 (1.44 cm). BL21 and buffer and water control did not show any inhibition.

Interaction effect of lectins and their concentrations indicated that RVL1 (3.6
mg/ml) was significantly superior to SRL1 with an inhibition zone of 2.62 cm. The effect of
SRL1 and RVL1 were on par with each other at 3.6 and 2.4 mg/ml concentrations,
respectively. They showed maximum inhibition zone of 2.07 cm (3.6 mg/ml) and 2.35 cm
(2.4 mg/ml) concentrations respectively. Inhibition zones of 1.33 and 1.23 cm produced in
RVL1 and SRL1 at 1.2 and 2.4 mg/ml respectively, followed by 1.00 cm inhibition zone in
RVL1 at 0.6 mg/ml. SRL1 at 0.6 mg/ml showed least inhibition zone (0.85 cm).

4.4.2.4 Plant and fungal lectin activity against R. solanacearum

RVL1 and SRL1 gradually reduced the growth R. solanacearum as their


concentration increased from 1.225 and 1.146 to 0.865 and 0.849 OD, respectively (Table
13d, Fig. 3, Plate 17). It also reduced the bacterial colonies on medium from 27x106 to 10
x106 and 19x106 to 7x106 cfu/ml in SRL1 and RVL1 after 96 h incubation respectively
(Table 13e). There was no reduction in growth of the bacterium in BL21, buffer controls.
The cfu also increased (33 x 106 cfu/ml).

4.4.3 In vitro antinematode activity

4.4.3.1 In vitro bioassay

In order to determine the growth inhibitory role of E. coli expressed RVL1


and SRL1 on Meloidogyne incognita, an in vitro bioassay was conducted at different
dilutions (1: 25, 1: 50, 1: 75 and 1: 100). Protein extract from E. coli BL21, PBS and
distilled water were used as the negative controls. Inhibition of egg hatching and
mortality of juvenile was observed periodically at 3, 6, 12, 24 and 48 h. Eggs were not
107

Table 13. In vitro evaluation of plant and fungal lectins against R. solanacearum

Table 13a. Well diffusion assay

Treatment Concentration Inhibition zone (cm)

SRL1 6 mg/ml 2.35 (1.83)*


RVL1 6 mg/ml 2.85 (1.96)
Streptocycline control 500 ppm/ml 2.59 (1.89)
Control (BL21) 6 mg/ml 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values

Table 13b. Determination of minimum inhibitory concentration for Ralstonia solanacearum

Lectin MIC mg/ml MBC mg/ml Inhibition zone

SRL1 0.625 1.87 1.33


RVL1 0.625 1.56 1.65

MIC- Minimum inhibitory concentration


MBC- Minimum bactericidal concentration

Table 13c. Evaluation of SRL1 and RVL1 against R. solanacearum

Inhibition zone (cm)


Treatment Concentration (mg/ml)
3.6 2.4 1.2 0.6 Mean

SRL1 2.07 (1.75)* 1.60 (1.61) 1.23 (1.49) 0.85 (1.36) 1.44 (1.55)
RVL1 2.62 (1.90) 2.35 (1.83) 1.33 (1.53) 1.00 (1.41) 1.83 (1.67)
Control (BL21) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00) 0.00 (1.00)
Control (PBS) 0.00 (1.00)
Control (S.W.) 0.00 (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values


108

Plate14. In vitro evaluation of plant and fungal lectins against R. solanacearum


through well diffusion assay

Minimum bactericidal concentration

Plate15. Determination of Minimum inhibitory concentration of SRL1 and RVL1


against R. solanacearum

Plate16. Evaluation of SRL1 and RVL1 against R. solanacearum


109

Table 13d. Activity of SRL1 and RVL1 lectin against R. solanacearum

OD (absorbance) at 600 nm
Concentration (mg/ml)
Treatment
0.6 1.2 2.4 3.6
48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean
SRL1 1.000 1.450 1.225 0.896 1.280 1.088 0.800 1.200 1.000 0.730 1.000 0.865
RVL1 1.000 1.291 1.146 0.830 1.130 0.980 0.750 0.803 0.777 0.715 0.983 0.849
Control
1.350 1.650 1.500 1.350 1.650 1.500 1.350 1.650 1.500 1.350 1.650 1.500
(BL21)
Control
1.000 1.590 1.295
(PBS)
Control 1.200 1.590 1.395

Table 13e. Activity of SRL1 and RVL1 lectin against R. solanacearum

Colony forming units (106cfu/ml)


Treatment Concentration (mg/ml)
0.6 1.2 2.4 3.6
SRL1 10 16 20 27
RVL1 7 9 16 19
Control (BL21) 25 27 30 30
Control (PBS) 33
Control 33
110

1.7

0.6 mg/ml 1.2 mg/ml 2.4 mg/ml 3.6 mg/ml

1.5

1.3
OD (Absorbance) at 600 nm

1.1

0.9

0.7

0.5
SRL1 RVL1 BL21
Treatments

Fig. 3: Activity of different concentrations of SRL1 and RVL1 on Ralstonia solanacearum


111

hatching and juveniles were not regaining activity after moving them in to water for 2 h
and results of survival of the nematodes as function of time are presented in Table 14-15.

4.4.3.1.1 Inhibition of egg hatching

Hatching of M. incognita juveniles increased with decrease in concentration of


lectins However, hatching was significantly reduced at all concentrations of SRL1 and
RVL1. The interaction between treatment and concentration was significant which
indicated that an increase in concentration tended to modify the effect of other in a
significant manner (Table 14, Fig. 4).

Both SRL1 and RVL1 showed nematicidal activity against root-knot nematode and
the egg hatching inhibition was observed periodically at 12, 24 and 48 h. Inhibition of egg
hatching was noticed after 12 h of incubation, it increased with time and concentration.
Maximum inhibition of 86 and 82 per cent was noticed with 1: 25 dilution after 48 h in
RVL1 and SRL1. Maximum of more than 80% inhibition of egg hatching was seen in 1: 25
dilution after 48 h and the eggs placed in BL21, water and PBS did not show any
inhibition.

4.4.3.1.2 Juvenile mortality

Nematicidal activity of SRL1 and RVL1 at different concentrations against


Meloidogyne incognita juveniles was evaluated and the results of survival of the
nematodes as function of time are presented in Table 15, Fig. 5.

These results were the average values of triplicate sets; each containing second
stage 50 juveniles. Lectins showed concentration dependent toxicity on M. incognita. Even
at a dilution of 1: 100, more than 25 per cent mortality was observed after 48 h. Juvenile
mortality was observed periodically at 6, 12, 18, 24, 36 and 48 h. The mortality was found
later than 3 h (at 6 h) and from then on it increased with increase in time and lectin
concentration.

Highest mortality of 92.67 and 82.67 per cent was found at 1: 25 dilution in SRL1
and RVL1 after 48 h incubation demonstrating the potential nematicidal activity
112

Table 14. Effect of SRL1 and RVL1 on inhibition of egg hatching of M. incognita

% Egg hatching inhibition


Treat Dilution
ment 1:100 1:75 1:50 1:25
12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24h 48 h Mean 12 h 24 h 48 h Mean
57.78* 65.83 68.67 64.09 61.11 68.33 74.00 67.81 71.11 78.33 79.33 76.26 74.44 78.33 82.00 78.26
SRL1
(49.50) (54.26) (55.99) (53.21) (51.45) (55.78) (59.37) (55.46) (57.52) (62.29) (62.99) (60.87) (59.66) (62.29) (64.93) (62.24)

71.11 68.33 74.00 71.15 74.44 78.33 78.00 76.93 74.44 80.83 84.00 79.76 81.11 83.33 86.00 83.48
RVL1
(57.52) (55.78) (59.37) (57.40) (59.66) (62.29) (62.06) (61.32) (59.66) (64.07) (66.46) (63.30) (64.27) (65.94) (68.06) (66.05)

Contro 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
l
(BL21) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)

Contro 0.00 0.00 0.00 0.00


l (PBS) (0.00) (0.00) (0.00) (0.00)

Contro 0.00 0.00 0.00 0.00


l (S.W) (0.00) (0.00) (0.00) (0.00)

S.Em± CD at 1%

Treatments (T) 0.62 2.23

Dilutions (D) 0.53 1.91

TxD 0.31 1.02

Hours (H) 0.44 1.15

TxH 0.36 1.08

DxH 0.31 1.02

TxDxH 0.18 0.70


*Figures in the parenthesis arc sine transformed values
113

90

1:100 1:75 1:50 1:25

80

70
% Egg hatching inhibition

60

50

40

30

20

10

0
SRL1 RVL1 BL21 PBS S.W
Treatments

Fig. 4: Effect of SRL1 ad RVL1 on inhibition of egg hatching of Meloidogyne incognita


114

Plate 17. Activity of SRL1 and RVL1 on R. Solanacearum

Plate 18. Paecilomyces lilacinus : growth in vitro and spores and sporophores
115

Table 15. Effect of RVL1 and SRL1 on juvenile mortality of M. incognita


Per cent Mortality
Dilution
Treatment
1:100 1:75
6h 12 h 18 h 24 h 36 h 48 h Mean 6h 12 h 18 h 24 h 36 h 48 h Mean
3.67 6.67 14.67 18.67 27.67 34.67 17.67 5.67 14.67 21.33 31.67 50.67 53.67 29.61
SRL1
(11.05)* (14.97) (22.53) (25.61) (31.75) (36.09) (23.67) (13.78) (22.53) (27.52) (34.26) (45.41) (47.13) (31.77)
0.00 3.67 9.67 12.67 24.33 29.67 13.33 4.67 11.67 19.67 24.67 39.67 47.67 24.67
RVL1
(0.00) (11.05) (18.12) (20.86) (29.57) (33.02) (18.77) (12.48) (19.98) (26.34) (29.79) (39.06) (43.68) (28.56)
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Control (BL21)
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
0.00 0.00 0.00 0.00 0.00 0.00 0.00
Control (PBS)
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
0.00 0.00 0.00 0.00 0.00 0.00 0.00
Control (S.W)
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)
Per cent Mortality
Dilution
Treatment
1:50 1:25
6h 12 h 18 h 24 h 36 h 48 h Mean 6h 12 h 18 h 24 h 36 h 48 h Mean
41.67 74.67 13.67
7.67 19.67 29.67 52.67 37.67 34.67 54.67 66.67 77.67 92.67
SRL1 (40.22) (59.81) (21.71) 56.67 (49.40)
(16.08) (26.34) (33.02) (46.55) (37.00) (36.09) (47.70) (54.76) (61.83) (74.33)
59.67 10.67
6.67 17.67 25.67 34.67 42.67 31.17 25.67 39.67 54.67 69.67 82.67
RVL1 (50.60) (19.07) 47.17 (43.05)
(14.97) (24.87) (30.45) (36.09) (40.80) (32.96) (30.45) (39.06) (47.70) (56.61) (65.43)
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Control (BL21)
(0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00)

S.Em± CD at 1%
Treatment (T) 0.31 1.02
Dilutions (D) 0.27 0.91
TxD 0.16 0.56
Hour (H) 0.22 0.74
TxH 0.13 0.33
DxH 0.11 0.29
TxDxH 0.06 0.17

*Figures in the parenthesis arc sine transformed values


116

60

1:100 1:75 1:50 1:25

50

40
Per cent mortality

30

20

10

0
SRL1 RVL1 BL21 PBS S.W
Treatments

Fig. 5: Effect of RVL1 and SRL1 on juvenile mortality of Meloidogyne incognita


117

of lectins. However, other dilutions (1: 50, 1: 75 and 1: 100) also showed high mortality in
SRL1 (74.67, 53.67 and 34.67%) followed by RVL1 (59.67, 47.67 and 29.67%)
respectively, after 48 h.

4.5 Influence of lectins on biocontrol agents in the


suppression of soil borne pathogens in vitro

4.5.1 Collection and isolation of fungal and bacterial antagonists

As mentioned in “Material and Methods”, four purified fungal bioagents,


Trichoderma viride, Trichoderma harzianum, Trichoderma virens, Trichoderma koningii,
and six bacterial antagonists, Pseudomonas fluorescens strain 30, 50, HM439968, TNAU,
and Bacillus subtilis, Bacillus subtilis strain HQ162493 collected from different sources
were evaluated for their efficacy against lectins and soil-borne pathogens and in
combination of those lectins and pathogens. Along with these bioagents Paecilomyces
lilacinus isolate also used (Plate 18).

4.5.2 In vitro screening of bacterial and fungal antagonists against F.


oxysporum,

S. rolfsii and R. solani (without lectin)

Efficacy of six bacterial and five fungal antagonists was screened against F.
oxysporum, S. rolfsii and R. solani by dual culture technique as described in ‘Material and
Methods’. The per cent inhibition over control was worked out based on the fungal growth in
control plate.

4.5.2 .1 F. oxysporum

Antagonists significantly reduced the growth of F. oxysporum. Maximum reduction


in colony growth was observed in T. viride (61.30%) which was significantly superior to all
other bioagents tested (Table 16, Fig. 6, Plate 19). Next best was T. harzianum (55.19%)
followed by T. virens (43.70%) and T. koningii (35.19%). Further, P. lilacinus showed
27.41 per cent inhibition followed by P. fluorescens strains 30, 50, HM439968, TNAU and
B. subtilis HQ162493 (>20%). Least inhibition was noticed in B. subtilis (16.67%).
118

4.5.2 .2 S. rolfsii

There were significant differences between the fungal bioagents tested with respect
to per cent inhibition of mycelial growth of S. roflsii. T. viride showed the maximum
inhibition of S. rolfsii (52.78%) and it was significantly superior to rest of the bioagents
tested (Table 16, Fig. 6, Plate 19). This was followed by T. harzianum (49.44%), P.
fluorescens strain HM439968 (45.00%), P. fluorescens 50 (43.89%), P. fluorescens TN
(42.78%), T. virens (40.56%), T. koningiii (39.44%), P. fluorescens 30 (38.33%), B. subtilis
HQ162493 (29.44%) and B. subtilis (27.22%). P. lilacinus showed least per cent inhibition
(23.33%).

4.5.2.3 R. solani

There were significant differences among all the tested bioagents. T. koningii
(46.30%) was found to be significantly superior in inhibiting the mycelial growth of R.
solani followed by T. viride (42.96%), T. harzianum (38.52%). Further T. virens and P.
lilacinus (28.52%) were on par with each other. The least inhibition of mycelial growth of
(5.19%) was recorded in all P. fluorescens strains and B. subtilis strains they were on par
with each other (Table 16, Fig. 6, Plate 19).

4.5.3 In vitro screening of plant fungal lectins against fungal antagonists

4.5.3.1 Disc diffusion assay

Different concentrations of SRL1 and RVL1 with bacterial and buffer control were
used against T. viride and P. liacinus in vitro condition by inhibition zone or disc diffusion
method. Inhibition zone produced across the antagonistic microorganisms was recorded
(Table 17, Plate 20). The results indicated that SRL1 resulted in maximum inhibition of the
P. lilacinus with an inhibition zone of 1.90 cm which was found significantly higher than
other treatments followed by RVL1 (1.82 cm) at 4.8 mg/ml. The efficacy of SRL1 and
RVL1 decreased with decrease in concentrations. RVL1 did not show any inhibition at 1.2
mg/ml concentration against P. lilacinus. SRL1 and RVL1 were less and moderately
effective among concentrations against T. viride with slight inhibition zone of 0.47 to 0.65
and 0.50 to 0.98 cm, respectively.
119

Table 16. In vitro evaluation of antagonists against S. rolfsii, R. solani and


F. oxysporum (without lectins)

Per cent inhibition


Biocontrol agent
R. solani S. rolfsii F. oxysporum

T. viride 52.78 (46.62)* 42.96 (4.98) 61.30 (51.55)

T. harzianum 49.44 (44.70) 38.52 (38.38) 55.19 (48.00)

T. virens 40.56 (39.58) 28.52 (32.29) 43.70 (41.40)

T. koningii 39.44 (38.93) 46.30 (42.90) 35.19 (36.40)

P. lilacinus 23.33 (28.90) 28.52 (32.29) 28.15 (32.06)

P. fluorescens 30 38.33 (38.27) 5.19 (13.15) 27.41 (31.58)

P. fluorescens 50 43.89 (41.51) 5.19 (13.15) 21.11 (27.35)

P. fluorescens HM439968 45.00 (42.15) 5.19 (13.15) 21.48 (27.62)

P. fluorescens TNAU 42.78 (40.87) 5.19 (13.15) 24.07 (29.39)

B. subtilis 27.22 (31.46) 5.19 (13.15) 16.67 (24.10)

B. subtilis HQ162493 29.44 (32.88) 5.19 (13.15) 22.22 (28.14)

S.Em± 0.33 0.39 0.47

CD at 1% 1.17 1.19 1.27

*Figures in the parenthesis are arc sine transformed values


120
70
R. solani S. rolfsii F. oxysporum

60

50
Per cent inhibition

40

30

20

10

0
T. viride T. harzianum T. virens T. koningii P. lilacinus P. fluorescens P. fluorescens P. fluorescens P. fluorescens B. subtilis B. subtilis
30 50 HM439968 TNAU HQ162493
Biocontrol agents
Fig. 6: Evaluation of antagonists' against S. rolfsii, R. solani and F. oxysporum (without lectins)
121

Plate 19. In vitro evaluation of antagonists against S. rolfsii, R. solani and F. oxysporum
(without lectins)
122

Table 17. In vitro evaluation of SRL1 and RVL1 lectin on T. viride and P. lilacinus

Inhibition zone (cm)

Concentration (mg/ml)
Treatment
4.8 3.6 2.4 1.2 Mean

P. lilacinus T. viride P. lilacinus T. viride P. lilacinus T. viride P. lilacinus T. viride P. lilacinus T. viride

SRL1 1.90 0.65 1.37 0.58 1.05 0.50 0.63 0.47 1.24 0.55

RVL1 1.82 0..98 1.43 0.95 0.95 0.85 0.00 0.50 1.05 0.82

Control
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(BL21)

Control
0.00 0.00
(PBS)

Control
0.00 0.00
(S.W)
123

Paecilomyces lilacinus

a. 4.8 mg/ml b. 3.6 mg/ml

c. 2.4 mg/ml d. 1.2 mg/ml

Plate 20. In vitro evaluation of SRL1 and RVL1 on T. viride and P. lilacinus
124

4.5.3.2 Inhibition of fungal spore germination

Antifungal activity of different amounts of RVL1 and SRL1 on spore germination of


T. viride, and P. lilacinus was compared. Growth of the fungi was examined by light
microscopy at different time intervals. Relative spore germination was expressed as
percentage of the spore germination over control. Between the concentrations of lectins,
efficacy was significant from lower to higher concentrations with greater efficacy at higher
concentration with respect to inhibition of the spore germination after 48 h. BL21 and
buffer control did not show any inhibition. RVL1 at 4.8 mg/ml showed maximum per cent
inhibition of T. viride spore germination 43.52 per cent after 48 h which was significantly
superior to others followed by P. lilacinus (25.19%). Whereas, SRL1 inhibited 16.56 and
6.96 per cent of T. viride and P. licinaus spores germination at 4.8 mg/ml. Further, RVL1
showed inhibition of T. viride and P. lilacinus spore germination (6.15 to 27.22 and 1.37 to
14.07 per cent at 1.2, 2.4 and 3.6 mg/ml, respectively).

Least per cent inhibition of 4.35 to 11.19 per cent of T. viride spore germination in
SRL1 at 2.4 and 3.6 mg/ml followed by no inhibition of T. viride spore germination at1.2
mg/ml. SRL1 showed no inhibition of P. lilacinus spore germination at 1.2 2.4 and 3.6
mg/ml concentration (Table 18, Fig. 7).

4.5.4. In vitro screening of plant fungal lectins against bacterial antagonists

4.5.4.1 Growth phase studies

P. fluorescens and B. subtilis growth gradually increased at different time intervals


without lectins (Table 19, Fig. 8). The growth started after 8 and 16 h i.e., 0.667 and 0.110
OD in P. fluorescens and B. subtilis respectively. Maximum growth of P. fluorescens and B.
subtilis was observed at 80 and 104 h after inoculatiom with OD value with 1.968 and 1.963
i.e., stationary phase after 80 and 104 h there was gradual decrease in growth i.e., decline
phase respectively. The dynamics of the bacterial growth was studied by plotting the cell
growth (absorbance) versus the incubation time. The curve thus obtained is a sigmoid curve
and is known as a standard growth curve of the P. fluorescens and B. subtilis.
125

Table 18. Effect of SRL1 and RVL1 on inhibition spore germination of P. lilacinus and T. viride

% Spore germination inhibition

Concentration (mg/ml)
Treatment
4.8 3.6 2.4 1.2

6h 12 h 24 h 48 h Mean 6h 12 h 24 h 48 h Mean 6h 12 h 24 h 48 h Mean 6h 12 h 24 h 48 h Mean

SRL1+ P. lilacinus 0.00 15.97 9.40 2.23 6.90 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

SRL1+ T. viride 23.33 19.23 13.68 10.00 16.56 15.56 13.85 10.00 7.74 11.79 6.67 3.85 5.26 1.61 4.35 0.00 0.00 0.00 0.00 0.00

RVL1+ P. lilacinus 0.00 43.28 41.28 16.20 25.19 0.00 28.99 19.46 7.82 14.07 0.00 21.01 13.09 2.51 9.15 0.00 5.46 0.00 0.00 1.37

RVL1+ T. viride 66.67 53.85 34.21 19.35 43.52 50.00 30.77 18.42 9.68 27.22 30.00 16.92 8.95 3.87 14.94 15.56 6.92 2.11 0.00 6.15

Control (BL21) + P. lilacinus 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Control (BL21) + T. viride 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Control (PBS)+ P. lilacinus 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Control (PBS)+ T. viride 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Control (S.W)+ P. lilacinus 0.00 0.00 0.00 0.00 0.00

Control (S.W)+ T. viride 0.00 0.00 0.00 0.00 0.00


126
127

4.5.4.2 In vitro screening of bacterial and fungal antagonists against Ralstonia


solanacearum

The antagonistic microorganisms viz., Trichoderma harzianum, T. viride,


T. koningii, T. virens, Pseudomonas fluorescens strains and Bacillus subtilis strains were
evaluated against R. solanacerum under in vitro condition by inhibition zone method as
explained in the “Material and Methods”. Inhibition zone produced across the antagonistic
microorganisms was recorded and presented in Table 20, Plate 21.

The results indicated that the antagonistic microorganism P. fluorescens 30,


P. fluorescens 50, P. fluorescens TNAU and P. fluorescens HM439968 resulted in maximum
inhibition of the R. solanacearum with an inhibition zone of 1.82 to 1.87 cm which was found
significantly superior over other treatments followed by B. subtilis and B. subtilis strain
HQ162493 (1.22 to 1.27 cm) remained on par with each other. Whereas, the fungal
antagonists like T. viride (1.02 cm), T. koningii (0.97 cm), T. virens (0.91 cm) and
T.harzianum (0.87 cm) were moderately effective with slight inhibition zone produced
around them.

4.5.4.3 Determination of the minimum inhibitory concentration (MIC) and the minimum
bactericidal concentration (MBC)

Antibacterial activity of SRL1 and RVL1 against P. fluorescens strains 30, 50,
HM439968 and TNAU and B. subtilis, B. subtilis strain HQ162493 and the MIC (minimum
inhibitory concentration), MBC (minimum bactericidal concentration) values as described
in “Material and Methods” are presented in Table 21, Plate 22.

No antibacterial activity was noticed in the control set (BL21, PBS and Sterile
distilled water. It was significant to find that the MIC which corresponds to the minimum
lectin concentration capable to inhibit the visible growth of the bacteria ranges from 0.63 to
2.50 and 0.63 to 1.25 mg/ml of SRL1 and RVL1 respectively with 1.94 to 3.14 and 1.78 to
3.08 mg/ml of MBC which corresponds to the minimum concentration of the lectin capable
to reduce the number of cfu for 0.1% of the initial inoculums respectively and MBCs
determined by agar diffusion were higher than the ones obtained by MICs.
128

Table 19. Growth phase studies of Pseudomonas fluorescens and Bacillus subtilis

OD (absorbance) at 600 nm
Hours after inoculation
P. fluorescens B. subtilis
8 0.667 0.110
16 0.857 0.728
24 1.267 0.925
32 1.755 1.065
40 1.841 1.225
48 1.907 1.522
56 1.912 1.715
64 1.942 1.772
72 1.962 1.860
80 1.968 1.963
88 1.967 1.960
96 1.967 1.960
104 1.967 1.960
112 1.967 1.960
120 1.967 1.960
128 1.960 1.960
Mean 1.742 1.540
S.Em ± 0.02 0.03
CD at 1% 0.08 0.12

Table 20. In vitro evaluation of bacterial and fungal bioagents against Ralstonia
solanacearum

Biocontrol agents Inhibition zone (cm)


P. fluorescens 30 1.87 (1.69)*
P. fluorescens 50 1.82 (1.68)
P. fluorescens TNAU 1.87 (1.69)
P. fluorescens HM439968 1.82 (1.68)
B. subtilis 1.27 (1.51)
B. subtilis HQ162493 1.22 (1.49)
T. harzianum 0.87 (1.37)
T. koningii 0.97 (1.40)
T. virens 0.91 (1.38)
T. viride 1.02 (1.42)
S.Em ± 0.02
CD at 1% 0.07

*Figures in the parenthesis are square root (√X+1) transformed values


129
130
131

Table 21. Antimicrobial activity, MIC, MBC and MAC of SRL1 and RVL1 against bacterial biocontrol agents

MIC mg/ml MBC mg/ml MAC mg/ml Inhibition zone (cm)


Biocontrol agent
SRL1 RVL1 SRL1 RVL1 SRL1 RVL1 SRL1 RVL1

P. fluorescens 30 1.25 0.63 2.87 1.78 0.15 0.30 1.67 1.69

P. fluorescens 50 1.25 0.63 2.50 2.04 0.15 0.30 1.67 1.79

P. fluorescens TNAU 1.25 1.25 3.08 2.88 0.15 0.30 1.69 1.72

P. fluorescens HM439968 0.63 0.63 1.94 2.71 0.15 0.30 0.87 1.63

B. subtilis 1.25 1.25 2.56 3.08 NT NT 1.43 1.63

B. subtilis HQ162493 2.50 0.63 3.14 1.94 NT NT 1.67 1.72

NT- Not determined MIC - Minimum inhibitory concentration MBC - Minimum bactericidal concentration

MAC - Minimum agglutination concentration


132

Plate 21 In vitro evaluation of bacterial and fungal bioagents against Ralstonia solanacearum
(without lectins)

Plate 22. Determination of Minimum inhibitory concentration for bacterial biocontrol


agents
133

SRL1 and RVL1 showed good activity on P. fluorescens strains (MIC values: 0.63 to
1.25 mg/ml and MBC values: 1.94 to 3.08 and 1.78 to 2.78 mg/ml) followed by B. subtillis
strains (MIC values: 1.25 to 2.25 and 0.63 to 1.25 mg/ml and MBC values: 2.56 to 3.14 and
1.94 to 3.08 mg/ml), respectively.

4.5.4.4 Bacterial agglutination

Quantitative determination of the agglutinating activity, the minimum agglutinating


concentration (MAC) corresponding to the minimum concentration of RVL1 and SRL1 that
promoted bacterial agglutination was recorded upto 0.15 and 0.30 mg/ml concentrations
among all P. fluorescens strains, respectively, but for B. subtillis strains agglutination was not
determined.

4.5.4.5 Inhibition zone assay

Minimum protein concentrations required for inhibition of bacterial growth were


determined by MIC and MBC values. RVL1 and SRL1 showed inhibitory activity against P.
fluorescens strains 30, 50, HM439968 and TNAU and B. subtilis, B. subtilis strain HQ162493
at different concentrations (Table 22, Fig. 9 and Plate 23). The antibacterial efficacy of the
protein was found to be moderate and more or less same against the tested bacteria.

SRL1 and RVl1 demonstrated broad spectrum activity against all tested bacterial
strains with inhibition zones in the range of 0.87 to 1.79 cm. However, the lectins showed
selective activity against tested bacteria compared to the controls. The most potent lectin was
RVL1 (1.63 to 1.79 cm inhibition zone) followed by SRL1 (0.87 to 1.67 cm). RVL1 showed
maximum inhibition zone of 1.79 cm against P. fluorescens 50, followed by P. fluorescens
TNAU (1.72 cm), B. subtillis HQ162493 (1.72 cm) P. fluorescens 30 (1.69), P. fluorescens
HM439968 (1.63) and B. subtilis (1.63 cm) which remained on par with each other.
Correspondingly, less inhibition zone recorded in SRL1.

4.5.4.6 Plant and fungal lectin activity against P. fluorescens and B. subtilis

P. fluorescens’s and B. subtilis’s sensitivity against different concentrations of SRL1 and


RVL1 was evaluated and measured in terms of turbidity (absorbance) and colony forming units
(Table 23).
134

Table 22. Effect of SRL1 and RVL1 lectin on P. fluorescens and B. subtilis strains

Inhibition zone (cm)

Biocontrol agent SRL1 RVL1 Control (BL21) Control Control

3.6 2.4 1.2 0.6 Mean 3.6 2.4 1.2 0.6 Mean 3.6 2.4 1.2 0.6 Mean (PBS) (S.W)

2.37 2.00 1.83 0.00 1.55 2.47 2.30 2.03 1.10 1.98 0.00 0.00 0.00 0.00 0.00 0.00 0.00
P. fluorescens 30 (1.83)* (1.73) (1.68) (1.00) (1.56) (1.86) (1.82) (1.74) (1.45) (1.72) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

2.22 2.05 1.38 0.60 1.56 2.42 2.35 1.98 1.30 2.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00
P. fluorescens 50 (1.79) (1.75) (1.54) (1.26) (1.59) (1.85) (1.83) (1.73) (1.52) (1.73) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

2.17 2.10 2.08 1.10 1.86 2.57 2.20 2.03 0.70 1.88 0.00 0.00 0.00 0.00 0.00 0.00 0.00
P. fluorescens TNAU (1.78) (1.76) (1.76) (1.45) (1.69) (1.89) (1.79) (1.74) (1.30) (1.68) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

2.12 1.75 1.18 0.60 1.41 1.82 1.80 1.23 0.00 1.21 0.00 0.00 0.00 0.00 0.00 0.00 0.00
P. fluorescens HM439968 (1.77) (1.66) (1.48) (1.26) (1.54) (1.68) (1.67) (1.49) (1.00) (1.46) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

1.92 1.90 1.23 0.75 1.45 2.52 2.25 1.68 0.95 1.85 0.00 0.00 0.00 0.00 0.00 0.00 0.00
B. subtilis (1.71) (1.70) (1.49) (1.32) (1.56) (1.88) (1.80) (1.64) (1.40) (1.68) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

2.17 1.92 1.68 0.90 1.67 2.33 2.03 1.48 1.00 1.71 0.00 0.00 0.00 0.00 0.00 0.00 0.00
B. subtilis HQ162493 (1.78) (1.71) (1.64) (1.38) (1.63) (1.82) (1.74) (1.58) (1.41) (1.64) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)

*Figures in the parenthesis are square root (√X+1) transformed values


135

Plate 23 Effect of SRL1 and RVL1 on P. fluorescens and B. subtilis strains


136

As concentration of lectins increased the turbidity of the P. fluorescens growth


reduced (Table 23a). Maximum OD of 1.587 and 1.409 was recorded at 0.6 mg/ml
concentrations of SRL1 and RVL1 followed by OD of 1.454 and 1.245, 1.362 and 1.109 and
1.227 and 1.018 at 1.2, 2.4 and 3.6 mg/ml concentrations, respectively.

Similar results were found for B. subtilis (Table 23b). Maximum OD values were
recorded in 0.6 mg/ml concentration of SRL1 and RVL1 (1.635 and 1.452 OD) respectively.
Further, 1.329 and 1.169, 1.236 and 1.082 and 1.160 and 0.943 OD values were recorded in
SRL1 and RVL1 at 1.2, 2.4 and 3.6 mg/ml concentrations respectively.

Further, growth of the bacteria measured in terms of colony forming units (Table 23c).
Compared to control, SRL1 and RVL1 lectins reduced the colonies of P. fluorescens and B.
subtilis as concentration of the lectins increased the number of colony forming units (CFU)
reduced from 23 to 11x106 and 17 to 4 and 22 to 7 and 20 to 3x106 respectively.

Whereas, BL21 increased the growth of bacterium as concentrations increased and


there was no inhibition of growth and cfu of the bacterium in controls. Finally as compared to
SRL1, RVL1 reduced growth of the bacterium to a greater extent.

4.5.4.7 In vitro screening of bacterial and fungal antagonists in combination with


lectins against R. solanacearum

Evaluation of different concentrations of SRL1 and RVL1 in combination with


bacterial (P. fluorescens and B. subtilis strains) and fungal (Trichoderma species)
antagonists against R. solancearum in vitro was done by inhibition zone assay (Table 24,
Fig. 10, Plate 24).

Inhibition of R. solancearum was maximum in RVL1 with bacterial antagonists


ranging from 1.50 to 1.89 cm inhibition followed by SRL1 (0.75 to 1.65 cm). Whereas, fungal
antagonists in combination with SRL1 exhibited inhibition zone of 0.85 to 1.37 cm. The least
inhibition zone of 0.74 to 0.86 cm was recorded in fungal antagonists in combination with
RVL1, which was less inhibitory compared to SRL1
137

Table 23. Activity of SRL1 and RVL1 lectin on P. flourescens and B. subtilis

Table 23a. Activity of SRL1 and RVL1 on P. fluorescens

OD (absorbance) @ 600 nm
Concentration (mg/ml)
Treatment
0.6 1.2 2.4 3.6
48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean
SRL1 1.360 1.815 1.587 1.263 1.644 1.454 1.163 1.561 1.362 1.091 1.363 1.227
RVL1 1.265 1.553 1.409 1.094 1.395 1.245 1.014 1.204 1.109 0.971 1.064 1.018
Control (BL21) 1.712 1.855 1.785 1.716 1.870 1.791 1.870 1.866 1.869 1.712 1.915 1.813
Control (PBS) 1.505 1.953 1.729
Control 1.564 1.952 1.758

Table 23b. Activity of SRL1 and RVL1 on B. subtilis

OD (absorbance) @ 600 nm
Concentration (mg/ml)
Treatment
0.6 1.2 2.4 3.6
48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean 48 h 96 h Mean
SRL1 1.410 1.860 1.635 1.306 1.356 1.329 1.178 1.295 1.236 1.160 1.160 1.160
RVL1 1.306 1.597 1.452 1.136 1.186 1.161 1.056 1.109 1.082 0.933 0.954 0.943
Control (BL21) 1.760 1.805 1.785 1.760 1.832 1.796 1.760 1.842 1.801 1.795 1.946 1.870
Control (PBS) 1.550 1.923 1.736
Control 1.590 1.923 1.756
138

Table 23c. Activity of SRL1 and RVL1 on P. fluorescens and B. subtilis

6
Colony forming units (10 /ml)

Concentration (mg/ml)
Treatment
0.6 1.2 2.4 3.6

P. fluorescens B. subtilis P. fluorescens B. subtilis P. fluorescens B. subtilis P. fluorescens B. subtilis

SRL1 23 22 20 17 16 12 11 7

RVL1 17 20 12 15 9 11 4 3

Control
25 23 27 29 28 31 32 36
(BL21)

Control (PBS) 32 35

Control 33 36
139

Table 24. Influence of bacterial and fungal antagonists’ in combination with lectins on R. solanacearum

Inhibition zone (cm)


SRL 1 (mg/ml) RVL 1 (mg/ml) BL21 (mg/ml)
Treatment PBS S.W
0.6 1.2 2.4 3.6 Mean 0.6 1.2 2.4 3.6 Mean 0.6 1.2 2.4 3.6
1.03 1.35 1.67 2.55 1.65 1.22 1.52 1.92 2.90 1.89 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + P. flourescens 30
(1.43)* (1.53) (1.63) (1.88) (1.62) (1.49) (1.59) (1.71) (1.98) (1.69) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.93 1.27 1.52 2.00 1.43 1.02 1.57 1.67 2.55 1.70 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + P. flourescens 50
(1.39) (1.51) (1.59) (1.73) (1.55) (1.42) (1.60) (1.63) (1.88) (1.64) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
Ralstonia + P. flourescens 0.82 1.14 1.52 1.85 1.33 0.92 1.57 1.67 2.50 1.66 0.00 0.00 0.00 0.00 0.00 0.00
HM439968 (1.35) (1.46) (1.59) (1.69) (1.52) (1.38) (1.60) (1.63) (1.87) (1.62) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
Ralstonia + P. flourescens 0.64 1.01 1.22 1.65 1.13 1.02 1.52 1.92 2.65 1.78 0.00 0.00 0.00 0.00 0.00 0.00
TNAU (1.28) (1.42) (1.49) (1.63) (1.45) (1.42) (1.59) (1.71) (1.91) (1.66) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.82 0.97 1.12 1.40 1.08 0.87 1.22 1.52 2.40 1.50 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia +B. subtilis
(1.35) (1.40) (1.45) (1.55) (1.44) (1.37) (1.49) (1.59) (1.84) (1.57) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
Ralstonia + B. subtilis 0.52 0.68 0.82 1.00 0.75 1.02 1.32 1.52 2.60 1.61 0.00 0.00 0.00 0.00 0.00 0.00
HQ162493 (1.23) (1.30) (1.35) (1.42) (1.32) (1.42) (1.52) (1.59) (1.90) (1.61) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.73 1.02 1.12 1.35 1.06 0.67 0.77 0.87 0.90 0.80 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + T. viride
(1.32) (1.42) (1.45) (1.53) (1.43) (1.29) (1.33) (1.37) (1.38) (1.34) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.52 0.72 0.92 1.00 0.79 0.77 0.77 0.77 0.75 0.76 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + T. harzianum
(1.23) (1.31) (1.38) (1.42) (1.34) (1.33) (1.33) (1.33) (1.32) (1.33) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.62 0.72 0.72 0.85 0.73 0.72 0.77 0.87 0.85 0.80 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + T. koningii
(1.27) (1.31) (1.31) (1.36) (1.31) (1.31) (1.33) (1.37) (1.36) (1.34) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
0.57 0.67 0.77 0.85 0.71 0.67 0.72 0.77 0.80 0.74 0.00 0.00 0.00 0.00 0.00 0.00
Ralstonia + T. virens
(1.25) (1.29) (1.33) (1.36) (1.31) (1.29) (1.31) (1.33) (1.34) (1.32) (1.00) (1.00) (1.00) (1.00) (1.00) (1.00)
Concentration Concentration
Treatment (T) TxC Treatment (T) TxC
(C) (C)
S.Em ± 0.005 0.008 0.002 0.004 0.007 0.002
CD at 1% 0.013 0.020 0.006 0.012 0.019 0.006
*Figures in the parenthesis are square root (√X+1) transformed values
140
141

Plate 24 Influence of bacterial and fungal antagonists in combination with SRL1 And RVL1 on R. solanacearum
142

4.5.5 In vitro screening of bacterial and fungal antagonists against nematode with lectins

4.5.5.1 Evaluation of culture filtrates of bacterial antagonists in combination with


lectins against nematode

4.5.5.1a In vitro egg hatching test

Hatching of M. incognita eggs decreased with increase in concentration of lectins.


However, hatching was significantly reduced at all concentrations of
P. fluorescens and B. subtilis strains culture filtrates combined with SRL1 and RVL1. The
interaction between lectins and concentration was significant which indicated that an
increase in concentration tended to modify the effect of other significantly. Egg hatching
inhibition was observed periodically at 12, 24 and 48 h. The egg hatching inhibition was
noticed after 12 h of incubation, it increased with time and concentration (Table 25).

The culture filtrates of P. fluorescens and B. subtilis strains at highest 100, 75, 50 and
25 per cent significantly inhibited the hatching of eggs. The decrease in egg hatching was
recorded with the P. fluorescens strains 30, 50, HM439968 and TN in treatment
concentration interaction (71.69, 69.07, 63.76 and 61.24 per cent), respectively. Whereas,
B. subtillis strains HQ162493 and B. subtilis inhibited per cent egg hatching, to the tune of
50.80 and 45.57 respectively.

Maximum inhibition of egg hatching of 94.17, 84.17, 80.17 and 78.17 per cent was
recorded in SRL1 when combined with P. fluorescens 30, 50, HM439968, and TNAU
followed by B. subtilis HQ162493 and B. subtilis (70.17 and 66.17 per cent) at cent per cent
concentration after 48 h. In case of RVL1 comibined with P. fluorescens and B. subtilis
strains culture filtrtates, cent per cent egg hatching inhibition was recorded in P. fluorescens
30 at cent per cent concentration followed by P. fluorescens 50 (96.56%), P. fluorescens
HM439968 (90.17%) and P. fluorescens TNAU (88.17%) respectively. Further, 80.17 and
76.17 per cent inhibition was recorded in B. subtilis HQ162493 and B. subtilis at cent
percent concentration. There was no inhibition in bacterial control (BL21) alone with respect
to egg hatching but in combination with
143

Table 25. Effect of bacterial antagonists’ culture filtrates with lectins on eggs hatching of M. incognita
% Egg hatching inhibition
Bacterial concentration
Treatment 100% 75% 50% 25%
12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean
33.22 47.33 56.17 45.57 30.17 44.83 53.67 42.89 19.83 37.33 48.33 35.17 16.44 35.33 46.00 32.59
B. subtilis
(35.22)* (43.49) (48.57) (42.48) (33.33) (42.06) (47.13) (40.93) (26.46) (37.68) (44.07) (36.39) (23.93) (36.49) (42.73) (34.83)
B. subtilis 39.89 52.33 60.17 50.80 43.50 54.83 61.67 53.33 33.17 47.33 56.33 45.61 29.78 45.33 53.83 42.98
HQ162493 (39.19) (46.36) (50.89) (45.48) (41.29) (47.80) (51.77) (46.94) (35.18) (43.49) (48.66) (42.50) (33.09) (42.34) (47.22) (40.99)
66.56 72.33 76.17 71.69 63.50 69.83 73.67 69.00 49.83 59.67 66.33 58.61 36.44 50.33 58.33 48.37
P. fluorescens 30
(54.70) (58.29) (60.81) (57.88) (52.86) (56.71) (59.16) (56.20) (44.93) (50.60) (54.56) (49.98) (37.15) (45.21) (49.82) (44.09)
63.22 69.83 74.17 69.07 56.83 64.83 69.67 63.78 46.50 57.33 64.33 56.06 36.44 50.33 57.67 48.15
P. fluorescens 50
(52.69) (56.71) (9.48) (56.24) (48.95) (53.66) (56.61) (53.02) (43.01) (49.24) (53.36) (48.50) (37.15) (45.21) (49.44) (43.96)
P. fluorescens 53.22 62.33 68.17 61.24 43.50 54.83 61.67 53.33 29.83 44.83 54.33 43.00 23.11 40.33 50.00 37.81
TNAU (46.87) (2.17) (55.68) (51.52) (41.29) (47.80) (51.77) (46.94) (33.12) (42.06) (47.51) (41.00) (28.75) (39.45) 45.02) (37.97)
P. fluorescens 56.44 64.67 70.17 63.76 53.50 62.33 67.67 61.17 43.17 54.67 62.33 53.39 33.11 47.83 56.33 45.76
HM439968 (48.73) (53.56) (56.92) (53.01) (47.03) (52.17) (55.37) (51.48) (41.09) (47.70) (52.17) (46.97) (35.15) (43.78) (48.6) (42.59)
49.89 59.83 66.17 58.63 46.83 57.50 63.67 56.00 36.50 49.67 58.33 48.17 33.11 47.67 55.67 45.48
SRL1+ B. subtilis
(44.96) (50.70) (54.46) (49.99) (43.21) (49.34) (52.96) (48.47) (37.19) (44.83) (49.82) (43.97) (35.15) (43.68) (48.28) (42.43)
SRL1+ B. subtilis 56.56 64.83 70.17 63.85 60.17 67.33 71.67 66.39 49.83 59.67 66.33 58.61 46.44 57.67 63.67 55.93
PDBC (48.79) (53.66) (56.92) (53.07) (0.89) (55.17) (57.87) (54.59) (44.93) (50.60) (54.56) (49.98) (42.98) (49.44) (52.96) (48.43)
SRL1+ P. 90.33 92.33 94.17 92.28 86.84 89.83 91.67 89.45 66.50 72.33 76.33 71.72 53.06 62.83 68.00 61.30
fluorescens 30 (71.92) (73.96) (76.06) (73.90) (68.76) (71.44) (73.26) (71.08) (54.66) (58.29) (60.92) (57.90) (46.78) (52.46) (55.58) (51.55)
SRL1+ P. 79.67 82.33 84.17 82.06 73.50 77.3 3 79.67 76.83 63.17 69.67 74.33 69.06 53.11 62.33 67.67 61.04
fluorescens 50 (63.23) (65.18) (66.59) (64.97) (59.05) (61.60) (63.23) (61.26) (52.66) (56.61) (59.59) (56.23) (46.81) (52.17) (55.37) (51.40)
SRL1+ P. 69.89 74.83 78.17 74.30 60.17 67.33 71.67 66.39 46.50 57.33 64.33 56.06 39.67 52.67 59.67 50.67
fluorescens TNAU (56.75) (59.92) (62.17) (59.57) (50.89) (55.17) (57.87) (54.59) (43.01) (49.24) (53.36) (48.50) (39.06) (46.55) (50.60) (45.41)
SRL1+ P.
73.22 77.33 80.17 76.91 70.17 74.83 77.67 74.22 59.83 67.33 72.33 66.50 49.67 60.27 66.33 58.76
fluorescens
(58.87) (61.60) (63.59) (61.31) (56.92) (59.92) (61.83) (59.52) (50.70) (55.17) (58.29) (54.66) (44.83) (50.95) (54.56) (50.07)
HM439968

Contd…
144

66.56 72.33 76.17 71.69 63.50 69.83 73.67 69.00 53.17 62.33 68.33 61.28 49.67 60.00 65.67 58.44
RVL1+ B. subtilis
(54.70) (58.29) (60.81) (57.88) (52.86) (56.71) (59.16) (56.20) (46.84) (52.17) (55.78) (51.54) (44.83) (50.79) (54.16) (49.89)
RVL1+ B. subtilis 73.22 77.33 80.17 76.91 76.83 79.83 81.67 79.44 66.50 72.33 76.33 71.72 63.11 70.33 73.67 69.04
HQ162493 (58.87) (61.60) (63.59) (61.31) (61.26) (63.35) (64.68) (63.07) (54.66) (58.29) (60.92) (57.90) (52.63) (57.03) (59.16) (56.22)
100.00 100.00 100.00 100.00 99.67 99.83 100.00 99.83 83.17 84.67 86.33 84.72 69.78 75.17 78.33 74.43
RVL1+ P. fluorescens 30
(90.05) (90.05) (90.05) (90.05) (86.73) (87.70) (90.05) (87.70) (65.81) (66.98) (68.34) (67.03) (56.68) (60.14) (62.29) (59.65)
94.17 94.83 96.56 95.19 90.17 89.83 89.67 89.89 79.83 82.33 84.33 82.17 69.67 75.33 78.00 74.33
RVL1+ P. fluorescens 50
(76.06) (76.90) (79.35) (77.36) (71.76) (71.44) (71.29) (71.50) (63.35) (65.18) (66.72) (65.05) (56.61) (60.25) (62.06) (59.59)
RVL1+ P.fluorescens 86.44 87.33 88.17 87.31 76.83 79.83 81.67 79.44 63.17 69.67 74.33 69.06 56.44 65.17 70.33 63.98
TNAU (68.43) (69.19) (69.91) (69.17) (61.26) (63.35) (64.68) (63.07) (52.66) (56.61) (59.59) (56.23) (48.73) (53.86) (57.03) (53.15)
RVL1+ P. fluorescens 89.67 89.83 90.17 89.89 86.83 87.33 87.67 87.28 76.50 79.67 82.33 79.50 66.44 72.33 76.33 71.70
HM439968 (71.29) (71.44) (71.76) (71.50) (68.76) (69.19) (69.48) (69.14) (61.03) (63.23) (65.18) (63.11) (54.63) (58.29) (60.92) (57.89)
39.89 52.33 60.17 50.80 36.83 49.83 57.67 48.11 26.50 42.33 52.33 40.39 23.06 40.33 49.83 37.74
BL21+ B. subtilis
(39.19) (46.36) (50.89) (45.48) (37.38) (44.93) (49.44) (43.94) (31.00) (40.61) (46.36) (39.48) (28.71) (39.45) (44.93) (37.92)
BL21+B. subtilis 46.56 57.33 64.17 56.02 50.17 59.83 65.67 58.56 39.83 52.33 60.33 50.83 36.44 50.30 57.67 48.14
HQ162493 (43.05) (49.24) (53.26) (48.48) (45.12) (50.70) (54.16) (49.95) (39.15) (46.36) (50.99) (45.50) (37.15) (45.19) (49.44) (43.95)
73.22 79.83 84.17 79.07 70.17 74.83 77.67 74.22 56.50 64.67 70.33 63.83 43.22 55.17 62.33 53.57
BL21+ P. fluorescens 30
(58.87) (63.35) (66.59) (62.81) (56.92) (59.92) (61.83) (59.52) (48.76) (53.56) (57.03) (53.06) (41.13) (47.99) (52.17) (47.07)
70.33 74.83 78.17 74.44 63.50 69.83 73.67 69.00 53.17 62.33 68.33 61.28 43.11 55.33 62.00 53.48
BL21+ P. fluorescens 50
(57.03) (59.92) (62.17) (59.66) (52.86) (56.71) (59.16) (56.20) (46.84) (52.17) (55.78) (51.54) (41.06) (48.09) (51.69) (47.02)
BL21+ P. fluorescens 59.67 67.33 72.17 66.39 50.17 59.83 65.67 58.56 36.50 49.67 58.33 48.17 29.67 45.17 54.33 43.06
TNAU (50.60) (55.17) (58.19) (54.59) (45.12) (50.70) (54.16) (49.95) (37.19) (44.83) (49.82) (43.97) (33.02) (42.25) (47.51) (41.03)
BL21+ P. fluorescens 63.22 69.83 74.17 69.07 60.17 67.33 71.67 66.39 49.83 59.67 66.33 58.61 39.83 52.67 60.33 50.94
HM439968 (52.69) (56.71) (59.48) (56.24) (50.89) (55.17) (57.87) (54.59) (44.93) (50.60) (54.56) (49.98) (39.15) (46.55) (50.99) (45.56)
0.00 0.00 0.00 0.00
Control (PBS)
(0.00) (0.00) (0.00) (0.00)
0.00 0.00 0.00 0.00
Control (S.W)
(0.00) (0.00) (0.00) (0.00)

*Figures in the parenthesis are square root (√X+1) transformed values

S.Em± CD at 1%
Treatment (T) 0.40 1.04
Concentration (C ) 0.99 2.45
TxC 0.20 0.72
Hour (H) 1.14 2.95
TxH 0.23 0.79
CxH 0.57 1.96
TxCxH 0.12 0.30
145

culture filtrates of antagonist, egg hatching inhibition ranged from 60.17 to 84.17 per cent at
cent per cent concentration after 48 h.

4.5.5.1b Juvenile mortality

Cell-free culture filtrates of four P. fluorescens strains and two B. subtilis strains and
also in combination with SRL1, RVL1 and BL21 (bacterial control) were tested in vitro for
their nematicidal action on M. incognita (Table 26). Data indicated that combination of
lectins to various concentrations of P. fluorescens, and B. subtilis strains culture filtrates
were highly deleterious to the nematode. In general, juvenile mortality increased with
increase in exposure period and increase in concentration of antagonoists strains. No
nematode mortality was recorded in control (BL21, Buffer and distilled water). Cent per cent
nematode mortality was observed in 100 per cent concentration of all the strains which are
combined with SRL1 and RVL1. The lowest juvenile mortality was recorded in SRL1 with B.
subtillis (40.33 %) at 25 per cent concentration.

Interaction between concentration and time at 100 per cent concentration with 12, 24
and 48 h exposure time recorded the 100 per cent mortality of juveniles in RVL 1 with P.
fluorescens 30 followed by 24 and 48 h exposure time in SRL and RVL1 combinations with
P. fluorescens strains 30, 50, TNAU and HM439968 and B. subtilis HQ162493. The lowest
mortality of 39.67 per cent was observed in 25 per cent concentration with 12 and 24 h
exposure period in SRL1 with B. subtillis.

In case of BL21, combination with culture filtrates of P. fluorescens and B. subtilis


strains showed 92 per cent juveniles mortality in P. fluorescens 30 followed by P.
fluorescens HM439968, TNAU B. subtilis HQ162493, P. fluorescens 50 and B. subtilis at
cent per cent concentration after 48 h exposure (86.00, 82.00, 76.00, 72.17 and 66.00 %),
respectively.

A maximum nematode mortality (60% and above) were observed in 100 per cent
concentration of strains P. fluorescens 30, 50, HM439968, TNAU, B. subtilis HQ162493,
and B. subtilis without any lectins. The lowest juvenile mortality was recorded in B. subtilis
HQ162493 (9.67%) at 25 per cent concentration and B. subtilis showed no inhibition at 25
per cent concentration.
146

Table 26. Effect of bacterial antagonists’ culture filtrates with lectins on juvenile mortality of M. incognita
Per cent mortality
Bacterial concentration
Treatment 100% 75% 50% 25%
12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean
15.67 50.33 60.00 42.00 10.17 36.17 39.80 28.71 0.00 5.67 5.67 3.78 0.00 0.00 0.00 0.00
B. subtilis
(23.33)* (45.21) (50.79) (40.42) (18.60) (36.99) (39.13) (32.42) (0.00) (13.78) (13.78) (11.21) (0.00) (0.00) (0.00) (0.00)
B. subtilis 21.67 59.67 70.00 50.44 14.17 46.17 49.80 36.71 5.67 15.67 16.33 12.56 0.00 8.00 9.67 5.89
HQ162493 (27.76) (50.60) (56.82) (45.28) (22.12) (42.82) (44.91) (37.31) (13.78) (23.33) (23.85) (20.76) (0.00) (16.44) (18.12) (14.05)
50.33 80.17 86.00 72.17 36.17 70.17 83.80 63.38 27.67 49.67 63.67 47.00 14.33 35.67 44.33 31.44
P .fluorescens 30
(45.21) (63.59) (68.06) (58.19) (36.99) (56.92) (66.30) (52.79) (31.75) (44.83) (52.96) (43.30) (22.26) (36.69) (41.77) (34.13)
42.17 65.67 66.00 57.94 26.17 16.17 25.80 22.71 11.67 20.33 19.67 17.22 0.00 6.33 10.33 5.56
P. fluorescens 50
(40.51) (54.16) (54.36) (49.60) (30.78) (23.72) (30.54) (28.48) (19.98) (26.82) (26.34) (24.53) (0.00) (14.58) (18.76) (13.64)
P. fluorescens 39.67 65.83 76.00 60.50 20.17 52.17 55.80 42.71 13.67 21.67 22.33 19.22 6.33 14.33 16.33 12.33
TNAU (39.06) (54.26) (60.70) (51.09) (26.70) (46.27) (48.36) (40.83) (21.71) (27.76) (28.22) (26.02) (14.58) (22.26) (23.85) (20.57)
P. fluorescens 44.17 70.33 80.00 64.83 28.17 60.17 69.80 52.71 24.33 35.67 45.67 35.22 18.33 28.33 30.33 25.67
HM439968 (41.67) (57.03) (63.47) (53.66) (32.07) (50.89) (56.69) (46.58) (29.57) (36.69) (42.54) (36.42) (25.36) (32.18) (33.44) (30.45)
55.67 90.00 100.00 81.89 50.17 76.17 79.80 68.71 43.67 49.67 50.33 47.89 39.67 39.67 40.33 39.89
SRL1+ B. subtilis
(48.28) (71.60) (90.05) (64.85) (45.12) (60.81) (63.32) (56.02) (41.38) (44.83) (45.21) (43.81) (39.06) (39.06) (39.45) (39.19)
SRL1+ B. subtilis 61.67 95.67 100.00 85.78 54.17 86.17 89.80 76.71 50.33 59.83 60.33 56.83 40.33 47.67 49.67 45.89
HM439968 (51.77) (78.02) (90.05) (67.88) (47.41) (68.20) (71.41) (61.18) (45.21) (50.70) (50.99) (48.95) (39.45) (43.68) (44.83) (42.66)
SRL1+ P. 89.67 100.00 100.00 96.56 76.17 100.00 100.00 92.06 72.33 90.33 95.67 86.11 54.33 76.33 84.33 71.67
fluorescens 30 (71.29) (90.05) (90.05) (79.34) (60.81) (90.05) (90.05) (73.67) (58.29) (71.92) (78.02) (68.15) (47.51) (60.92) (66.72) (57.87)
SRL1+ P. 81.67 100.00 100.00 93.89 66.17 76.17 85.80 76.04 55.67 64.07 64.33 61.36 40.33 45.67 50.33 45.44
fluorescens 50 (64.68) (90.05) (90.05) (75.73) (54.46) (60.81) (67.90) (60.73) (48.28) (53.20) (53.36) (51.59) (39.45) (42.54) (45.21) (42.41)
SRL1+ P. 79.67 100.00 100.00 93.22 60.17 92.17 95.80 82.71 58.33 66.00 66.33 63.56 46.33 54.33 56.33 52.33
fluorescens TNAU (63.23) (90.05) (90.05) (74.95) (50.89) (73.78) (78.21) ) (65.46) (49.82) (54.36) (54.56) (52.89) (42.92) (47.51) (48.6) (46.36)
SRL1+ P.
83.67 100.00 100.00 94.56 68.17 100.00 99.80 89.32 65.00 80.17 90.33 78.50 58.33 68.00 70.33 65.56
fluorescens
(66.20) (90.05) (90.05) (76.55) (55.68) (90.05) (87.48) (70.96) (53.76) (63.59) (71.92) (62.41) (49.82) (55.58) (57.03) (54.09)
HM439968
61.67 100.00 100.00 87.22 56.17 82.17 85.80 74.71 50.33 55.67 56.33 54.11 45.67 45.67 46.33 45.89
RVL1+ B. subtilis
(51.77) (90.05) (90.05) (69.09) (48.57) (65.05) (67.90) (59.84) (45.21) (48.28) (48.66) (47.38) (42.54) (42.54) (42.92) (42.66)

Contd…
147

RVL1+ B. subtilis 67.67 100.00 100.00 89.22 60.17 92.17 95.80 82.71 56.33 65.67 66.33 62.78 46.33 54.33 55.67 52.11
HQ162493 (55.37) (90.05) (90.05) (70.87) (50.89) (73.78) (78.21) (65.46) (48.66) (54.16) (54.56) (52.43) (42.92) (47.51) (48.28) (46.23)

RVL1+ P. fluorescens 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00 78.33 96.17 100.00 91.50 60.33 82.33 90.33 77.6761.
30 (90.05) (90.05) (90.05) (90.05) (90.05) (90.05) (90.05) (90.05) (62.29) (78.75) (90.05) (73.09) (50.99) (65.18) (71.92) 83)

RVL1+ P. fluorescens 87.67 100.00 100.00 95.89 72.17 82.17 91.80 82.04 62.33 70.00 70.07 67.47 46.33 52.33 56.33 51.67
50 (69.48) (90.05) (90.05) (78.34) (58.19) (65.05) (73.40) (64.96) (52.17) (56.82) (56.86) (55.25) (42.92) (46.36) (48.66) (45.98)

RVL1+ P. fluorescens 85.67 100.00 100.00 95.22 66.17 98.17 100.00 88.11 63.67 71.93 72.00 69.20 52.33 60.33 62.33 58.33
TNAU (67.79) (90.05) (90.05) (77.41) (54.46) (82.26) (90.05) (69.87) (52.96) (58.04) (58.08) (56.32) (46.36) (62.33) (52.17) (49.82)
RVL1+ P. fluorescens 89.67 100.00 100.00 96.56 74.17 100.00 100.00 91.39 73.67 86.30 96.00 85.32 64.33 74.33 76.33 71.67
HM439968 (71.29) (90.05) (90.05) (79.34) (59.48) (90.05) (90.05) (72.97) (59.16) (68.31) (78.50) (67.51) (53.36) (59.59) (60.92) (57.87)
21.67 55.67 66.00 47.78 16.17 42.17 45.80 34.71 5.67 11.83 12.33 9.94 5.67 5.67 6.33 5.89
BL21+ B. subtilis
(27.76) (48.28) (54.36) (43.75) (23.72) (40.51) (42.61) (36.12) (13.78) (20.13) (20.57) (18.39) (13.78) (13.78) (14.58) (14.05)

BL21+B. subtilis 27.67 65.67 76.00 56.44 20.17 52.17 55.80 42.71 11.67 22.30 22.33 18.77 6.33 14.33 15.33 12.00
HQ162493 (31.75) (54.16) (60.70) (48.73) (26.70) (46.27) (48.36) (40.83) (19.98) (28.19) (28.22) (25.68) (14.58) (22.26) (23.06) (20.28)
BL21+ P.fluorescens 55.67 85.67 92.00 77.78 42.17 76.17 89.80 69.38 33.67 55.97 70.33 53.32 20.33 42.33 50.33 37.67
30 (48.28) (67.79) (73.61) (61.91) (40.51) (60.81) (71.41) (56.43) (35.48) (48.45) (70.33) (46.93) (26.82) (40.61) (45.21) (37.88)
BL21+ P.fluorescens 47.67 72.00 72.17 63.94 32.17 22.17 31.80 28.71 17.67 26.27 26.33 23.42 6.33 12.33 16.33 11.67
50 (43.68) (58.08) (58.19) (53.12) (34.57) (28.10) (34.34) (32.42) (24.87) (30.85) (30.89) (28.96) (14.58) (20.57) (23.85) (19.98)
BL21+ P. fluorescens 45.67 71.67 82.00 66.44 26.17 58.17 61.80 48.71 19.67 28.33 28.33 25.44 12.33 20.33 21.67 18.11
TNAU (42.54) (57.87) (64.93) (54.63) (30.78) (49.73) (51.85) (44.28) (26.34) (32.18) (32.18) (30.31) (20.57) (26.82) (27.76) (25.20)
BL21+ P. fluorescens 49.67 75.67 86.00 70.44 34.17 66.17 75.80 58.71 29.67( 41.83(4 52.33 41.28 24.33 34.33 35.67 31.44
HM439968 (44.83) (60.47) (68.06) (57.10) (35.79) (54.46) (60.56) (50.04) 33.02) 0.32) (46.36) (40.00) (29.57) (35.89) (36.69) (34.13)
0.00 0.00 0.00 0.00
Control (PBS)
(0.00) (0.00) (0.00) (0.00)
0.00 0.00 0.00 0.00
Control (S.W)
(0.00) (0.00) (0.00) (0.00)
*Figures in the parenthesis are square root (√X+1) transformed values
S.Em± CD at 1%
Treatment (T) 0.27 0.91
Concentraion (C ) 0.66 2.09
TxC 0.13 0.37
Hour (H) 0.76 2.24
TxH 0.15 0.40
CxH 0.38 1.08
T x C xH 0.08 0.20
148

The immobile/inactive nematodes were randomly picked and placed in sterile water.
None of the juveniles regained their activity, indicating that the nematicidal action of the
SRL1, RVL1 and P. fluorescens and B. subtilis strains was long lasting.

4.5.5.2 In vitro screening of fungal antagonists combination with lectins against


nematode

In order to determine the growth inhibitory role of E. coli expressed RVL1 and SRL1
and also eggs and juvenile parasites of T. viride and P. lilacinus on the juveniles and eggs of
Meloidogyne incognita, an in vitro bioassay was conducted using different concentrations
(4.8, 3.6, 2.4 and 1.2 mg/ml) of lectins with 1 x 105 spores/ml spores suspension of T. viride
and P. lilacinus with BL21 (bacterial control), buffer and sterile water control.

Both the lectins (SRL1 and RVL1) showed varying degrees in colonizing eggs and
juveniles with P. lilacinus and T. viride spores as observed periodically at 12, 24 and 48 h.

4.5.5.2a Inhibition of egg hatching

Maximum per cent inhibition of egg hatching of 93.00 and 89.33 per cent was
recorded in P. lilacinus with SRL1 followed by SRL1 and RVL1 alone (89.00 and 85.33%) at
4.8 mg/ml concentration after 48 h (Table 27, Fig. 11, Plate 25). Further, with T. viride with
SRL1 and RVL1, 77.33 and 71.00 per cent egg hatching inhibition was recorded at 4.8
mg/ml.

There was no egg hatching inhibition in bacterial control (BL21) alone, PBS and
sterile water control but in combination with BL21 and P. lilacinus and T. viride were
recorded 69.33 and 57.33 per cent inhibition. Similar results were obtained in P. lilacinus
and T. viride-alone treatments.

4.5.5.2b Juvenile mortality

Mortality was found later than 6 h and from then on it increased with increase in time
and lectins and concentration in all combinations (Table 28, Fig. 12). Maximum mortality
observed was 92.33 and 88.33 per cent with in SRL1 and RVL1-alone,
149

Table 27. Effect of lectins and fungal antagonists’ on inhibition of egg hatching of M. incognita

% Eggs hatching inhibition

Concentration (mg/ml)
Treatment
4.8 3.6 2.4 1.2

12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean

SRL1 + P. lilacinus 88.89 91.67 93.00 91.18 82.22 84.17 85.33 83.91 65.56 71.67 75.33 70.85 48.89 59.17 65.33 57.80

RVL1 + P. lilacinus 88.89 89.17 89.33 89.13 75.56 79.17 81.33 78.69 55.56 64.17 69.33 63.02 38.89 51.67 59.33 49.96
SRL1 + T. viride 68.89 74.17 77.33 73.46 58.89 66.67 71.33 65.63 38.89 51.67 59.33 49.96 15.56 34.17 45.33 31.69

RVL1 + T. viride 58.89 66.67 71.00 65.52 48.89 59.17 65.33 57.80 25.56 41.67 51.33 39.52 12.22 24.17 37.33 21.24

Control (BL21) + P. lilacinus 55.56 64.17 69.33 63.02 55.56 64.17 69.33 63.02 55.56 64.17 69.33 63.02 55.56 64.17 69.33 63.02
Control (BL21) + T. viride 35.56 49.17 57.33 47.35 35.56 49.17 57.33 47.35 35.56 49.17 57.33 47.35 35.56 49.17 57.33 47.35

SRL1 85.56 86.67 89.00 87.07 75.56 79.17 81.33 78.69 45.56 56.67 63.33 55.19 32.22 46.67 55.33 44.74

RVL1 82.22 84.17 85.33 83.91 68.89 74.17 77.33 73.46 35.56 49.17 57.33 47.35 15.56 36.67 45.33 32.52
Control (BL21) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

P. lilacinus 55.56 64.17 69.33 63.02

T. viride 35.56 49.17 57.33 47.35


Control (PBS) 0.00 0.00 0.00 0.00

Control (S.W) 0.00 0.00 0.00 0.00


150
151

Plate 25 Swollen invaded egg bearing a conidiophore and chains of conidia (400 X)
152

Table 28. Effect of lectins and fungal antagonists on juvenile mortality of M. incognita

Juvenile mortality (%)

Concentration (mg/ml)
Treatment
4.8 3.6 2.4 1.2

12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean 12 h 24 h 48 h Mean

SRL1 + P. lilacinus 72.22 79.17 83.33 78.24 65.56 71.67 75.33 70.85 48.89 59.17 65.33 57.80 32.22 46.67 55.33 44.74

RVL1 + P. lilacinus 72.22 76.67 79.33 76.07 58.89 66.67 71.33 65.63 38.89 51.67 59.33 49.96 22.22 39.17 49.33 36.91
SRL1 + T. viride 48.89 59.17 65.33 57.80 35.56 49.17 57.33 47.35 25.56 41.67 51.33 39.52 18.89 36.67 47.33 34.30

RVL1 + T. viride 62.22 69.17 73.33 68.24 48.89 59.17 65.33 57.80 35.56 49.17 57.33 47.35 32.22 46.67 55.33 44.74

Control (BL21) + P. lilacinus 38.89 51.67 69.00 53.19 38.89 51.67 69.00 53.19 38.89 51.67 69.00 53.19 38.89 51.67 69.00 53.19
Control (BL21) + T. viride 18.89 36.67 47.33 34.30 18.89 36.67 47.33 34.30 18.89 36.67 47.33 34.30 18.89 36.67 47.33 34.30

SRL1 82.56 88.67 92.33 87.85 68.89 74.17 79.33 74.13 28.89 44.17 53.33 42.13 15.56 34.17 45.33 31.69

RVL1 77.89 83.17 88.33 83.13 65.56 71.67 75.33 70.85 18.89 36.67 47.33 34.30 0.00 24.17 35.33 19.83
Control (BL21) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

P. lilacinus 38.89 51.67 69.00 53.19

T. viride 18.89 36.67 47.33 34.30


Control (PBS) 0.00 0.00 0.00 0.00

Control (S.W) 0.00 0.00 0.00 0.00


153
154

followed by P. lilacinus in combination with SRL1 (83.33 %) and RVL1 (76.07%) at


4.8 mg/ml concentration after 48 h respectively. Whereas, T. viride with SRL1 and RVL1
recorded 68.24 and 57.80 per cent mortality compared to control without lectins and with
BL21, P. lilacinus and T. viride recorded 53.19 and 34.30 per cent mortality after 48 h
respectively. Lowest mortality percentage recorded in lower concentrations of lectins as
concetration decreased, mortality per cent decreased. 47.33 and 49.33 per cent mortality
were recorded at 1.2 mg/ml concentration in T. viride with SRL1 and RVL1 treatments
respectively. There was no mortality of nematode juveniles in BL21, PBS and sterile water
control.

Bioassay with SRL1 and RVL1 at different concentrations revealed an increased


juvenile mortality and egg hatching inhibition with increase in time or exposure period as
well as concentration M. incognita eggs and juveniles were more vulnerable to P. liacinus
and T. viride.

4.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the suppression of tomato
soil-borne pathogens

4.6.1 Analysis of transgenic and nontransgenic plants

Eight srl1 and rvl1 transgenic events grown in controlled condition were used.
Further, for confirmation PCR was employed for srl1 and rvl1 transgenic events. All srl1 and
rvl1 transgenic events showed 410 and 412 bp amplicons (Palte 26).

4.6.1.1 Expression of RVL1 and SRL1

4.6.1.1a Protein extraction and quantification

The protein content in the crude extract from leaves of non-transformed control
tomato plant was 5.4 mg/10 ml. The protein content in SRL1 transgenic lines varied from
5.00 to 7.0 mg/10 ml. Similarly, RVL1 transgenic lines contained 4.50 to 7.00 mg/10 ml
protein (Table 29).

4.6.1.1b SDS-PAGE

Crude proteins of srl1 and rvl1-transgenic tomato were run on SDS-PAGE. Both
these samples showed 16 and 26.2 kDa bands, whereas the control plants failed to show
155

Table 29. Heamagglutination activity of srl1 and rvl1 expressed transgenic events

Protein concentration
Transgenic lines MCA (µg) Total activity (unit) Specific activity (unit/mg)
(mg/10 ml)

Non-transformed 5.40 0 0 0
3 2
RVL1-T0(50)-T1(3)-T2(6) 5.00 27.50 0.80×10 0.36×10

RVL1-T0(28)-T1(5)-T2(7) 7.00 15.00 1.6×103 0.66×102


3 2
RVL1-T0(2)-T1(2)-T2(9) 6.00 28.75 0.80×10 0.34×10
3 2
RVL1-T0(11)-T1(4)-T2(10) 6.00 25.00 0.80×10 0.40×10
3 2
RVL1-T0(12)-T1(8)-T2(2) 4.50 26.25 0.80×10 0.38×10

RVL1-T0(20)-T1(9)-T2(11) 4.50 47.50 0.40×103 0.21×102


3 2
RVL1-T0(48)-T1(3)-T2(7) 4.50 50.25 0.40×10 0.19×10
3 2
RVL1-T0(33)-T1(2)-T2(6) 5.00 55.00 0.40×10 0.18×10

SRL1-T0(3)-T1(9)-T2(7) 6.60 13.12 1.61×103 0.76×102


3 2
SRL1-T0(7)-T1(3)-T2(6) 6.60 13.75 1.60×10 0.72×10
3 2
SRL1-T0(10)-T1(2)-T2(9) 5.50 28.75 0.87×10 0.37×10
3 2
SRL1-T0(21)-T1(3)-T2(7) 7.20 12.50 1.60×10 0.80×10

SRL1-T0(32)-T1(7)-T2(9) 5.50 47.50 0.40×103 0.21×102


30 2
SRL1-T0(90)-T1(2)-T2(3) 5.00 55.00 0.40×10 0.18×10
3 2
SRL1-T0(95)-T1(7)-T2(4) 5.00 55.00 0.40×10 0.18×10
3 2
SRL1-T0(116)-T1(5)-T2(8) 5.00 52.50 0.40×10 0.19×10
MCA- Minimum concentration required for agglutination
156

the band. However, SDS-PAGE with crude and partially purified protein from srl1-transgenic
plant did not reveal the expected band of 16 kDa (Plate 28 and 28a).

4.6.1.2 Haemagglutination activity for srl1 and rvl1 expressed in transgenic tomato

Crude protein extracts of eight rvl1 and srl1-transgenic tomato plants showed
haemagglutination with rabbit erythrocytes indicating the expression of rvl1 and srl1.
Whereas control plants did not show any activity, minimum concentration of protein required
for agglutination (MCA) varied from 15 to 55.0 µg and 12.5 to 55.0 µg rvl1 and srl1
transgenic lines respectively. The crude extract of protein obtained from rvl1 and srl1
transgenic tomato plants contained a total lectin activity of 0.40 to 1.60 x 103, whereas srl1-
transgenic plant contained 0.40 to 1.61 x 103. The specific activity was 0.18 to 0.40 x 102
units for rvl1 and 0.18 to 0.76 x 102 units for srl1 (Table 29, Plate 27).

4.6.2 Evaluation of rvl1 and srl1 transgenic and non transgenic tomato lines against
individual and combination of vascular pathogens of tomato under glasshouse conditions

The pathogenic potential of F. oxysporum, R. solanacearum and M. incognita, on


lectins gene carrying tomato was determined. In glass house, pot culture experiments were
conducted to investigate the efficacy of eight PCR confirmed rvl1 and srl1 homozygous T3
transgenic events for suppression of vascular pathogens and their interactions. Significant
reduction in plant growth parameters such as plant height, number of branches, fresh and
dry weight observed 90 days after inoculation whereas, more disease severity and root-knot
index was observed in control treatments where only individual pathogens and their
combinations were inoculated to control tomato plants. RVL1-T0(28) and SRL1-T0(21)
recorded maximum plant height, number of branches, fresh and dry weight of the plants with
less disease severity and root-knot index in individual as well as interactions of different
pathogens.

The perusal of data (Table 30-g, Plate 29) indicated that disease appeared only in
those treatments where F. oxysporum, R. solanacearum were inoculated either singly or in
combinations with root- knot nematode.
157

Plate 26 PCR confirmation of srl1 and rvl1 expressed transgenic events

Plate 27 Haemagglutination assay with rvl1 and srl1 expressed in transgenic tomato
lines
158

Plate 28 SDS-PAGE of crude protein from srl1 transgenic events

Plate 28a SDS-PAGE of crude protein from rvl1 transgenic events


159

There was a significant increase in root-knot index in plants inoculated with


nematode alone in control. Less number of galls, with no disease severity was observed
in transgenic plants treated with nematode.

4.6.2.1 Plant height

Significant differences in plant height were observed among the treatments and
transgenic lines. Highest reduction of plant height of 76.20 and 63.83 cm was observed
in treatments transgenic SRL1-T0(116) and RVL1-T0(20) which were inoculated with
Meloidogyne + Fusarium+ Ralstonia. This was followed by Meloidogyne + Fusarium,
Ralstonia alone, Meloidogyne + Ralstonia, and Fusarium-alone in the same events.
Correspondingly highest plant height of 140.25 and 124.33 cm recorded in transgenic
events of SRL1-T0(21) and RVL1-T0(28) inoculated with Meloidogyne-alone.

4.6.2.2 Plant biomass

There was a significant reduction in fresh (fresh shoot root weight) and dry (dry
shoot and root weight) biomass in inoculated plants compared to uninoculted control in
general. The highest percentage reduction in fresh (23.20 and 18.73 g) and dry (16.87
and 8.42 g) biomass was observed with the treatment which had all the three pathogens
inoculated to transgenic lines of SRL1-T0(116) (94.06 and 29.70 g) and RVL1-T0(20)
(85.10 and 27.57 g), respectively.

4.6.2.3 Disease severity

Maximum disease severity of 50.07, 40.31, 36.60, 32.80, 30.22, 27.75 and
21.58 per cent were observed in SRL1-T0(116) (Meloidogyne + Fusarium+ Ralstonia),
SRL1-T0(116) (Fusarium + Ralstonia), SRL1-T0(32) Meloidogyne + Fusarium, SRL1-
T0(116) (Meloidogyne + Ralstonia), SRL1-T0(116) (Ralstonia alone) and SRL1-T0(116)
(Fusarium alone), in srl1 transgenics whereas, in case of rvl1 transgenics, maximum
disease severity (53.97, 41.54, 37.83, 34.03 30.22 and 27.75%) were recorded in
RVL1-T0(20) (Meloidogyne + Fusarium+ Ralstonia), RVL1-T0(20) (Fusarium +
Ralstonia), RVL1-T0(20) Meloidogyne + Fusarium, RVL1-T0(20) (Meloidogyne +
Ralstonia), RVL1-T0(20) (Ralstonia alone) and RVL1-T0(20) (Fusarium alone) followed
by RVL1-T0(33) and RVL1-T0(48), i.e., in control, disease severity was more (>60%).
160

Correspondingly minimum disease severity was recorded in transgenic plants of RVL1-


T0(28), RVL1-T0(2) and SRL1-T0(21) and SRL1-T0(7).

4.6.2.4 Gall index

There was significant increase in root-knot index in plants inoculated with


nematode alone and in combination with Fusarium and Ralstonia. Less root-knot index
was observed in plants treated with Meloidogyne + Fusarium + Ralstonia (4.55) in
SRL1-T0(21) and RVL1-T0(28). Highest Root-knot index was recorded in SRL1-T0(116)
and RVL1-T0(20) plants treated with Meloidogyne alone (7.22 and 8.00) followed by
Meloidogyne + Fusarium (7.33 and 7.20) and Meloidogyne + Ralstonia (6.22 and 3.77),
respectively. Whereas compared to transgenic lines, maximum root-knot index was
observed in control inoculated with pathogens.

4.6.2.5 Recovery of Fusarium units

Maximum recovery of Fusarium population /g of soil were recorded in SRL1-


T0(116) and RVL1-T0(20) plants treated with Fusarium (635.72 and 658 x 106 )-alone
followed by Meloidogyne + Fusarium + Ralstonia (619.80 and 591.67 x 106),
Meloidogyne + Fusarium (576.90 and 599.67 x 106), and Fusarium + Ralstonia (547.37
and 562.31 x 106) respectively. Correspondingly minimum recovery of Fusarium
population recorded in RVL1-T0(28) and SRL1-T0(21) plants.

4.6.2.6 Recovery of Ralstonia units

Maximum recovery of Ralstonia population /g of soil were recorded in SRL1-


T0(116) and RVL1-T0(20) plants treated with Ralstonia (16.60 and 17.83 x 106 )-alone
followed by Meloidogyne + Ralstonia (14.30 and 15.53 x 106), Fusarium + Ralstonia
(14.00 and 15.23 x 106), and Meloidogyne + Fusarium + Ralstonia (13.01 and 14.24 x
106) respectively. Minimum recovery of Fusarium population recorded in RVl1-T0(28)
and SRL1-T0(21) plants.

4.6.2.7 Root-knot juveniles population

Maximum nematode population recorded were Meloidogyne + Fusarium


(444.40 and 547.67) and Meloidogyne + Fusarium + Ralstonia (444.33 and 546.50)
161

followed by Meloidogyne-alone (411.90 and 373.33) and Meloidogyne + Ralstonia


(379.00 and 483.00) in SRL1-T0(116) and RVL1-T0(20) plants. Events RVL1-T0(28) and
SRL1-T0(21) plants supported less population.

4.6.2.8 Egg masses per root system, eggs per root system, female fecundity, final
population and reproduction factor of M. incognita

Significant differences were observed in respect of eggmass per root, eggs per
root, female fecundity, final population and reproduction factor of the M. incognita in
different transgenic lines treated with Meloidogyne-alone and in combination of
Fusarium and Ralstonia. Maximum eggmass per root, eggs per root, female fecundity,
final population and reproduction factor was noticed in SRL1-T0(116) and RVL1-T0(20)
plants. Correspondingly minimum values were recorded in SRL1-T0(21) and RVL1-
T0(28) events.

In the present study, increased plant height, fresh and dry weight of the plant as
well as less disease severity, root-knot index, less recovery of Fusarium, Ralstoinia and
nematode populations were observed in transgenic plants compared to control
(inoculated). Protection of the plants from infection depended on lectin gene carrying
tomato plants. Even in individual or combinations of pathogens treated in transgenics
SRL1-T0 (21) and RVL1-T0 (28) recorded maximum plant height, more fresh and dry
weight with least disease severity, as well as root-knot indices. It was clear that the
disease severity was more when the nematode, M. incognita interacted with other
pathogens Fusarium and Ralstonia in combination.

4.6.2.9 Assay of defense related enzymes and compounds

Induced systemic resistance through biochemical analysis revealed the increased


activities of the enzymes, viz. peroxidase (PO), polyphenol oxidase (PPO), and also
phenolic compounds in the SRL1 and RVL1 transgenic lines challenged with individual
and combinations of other pathogens. 90 days after inoculation, PO and PPO activity
was compared between leaf extracts from transgenic and control plants (Table 30 h).
162

4.6.2.9.1 Peroxidase (PO)

Assay of peroxidase activity in RVL1 and SRL1 transgenic plants inoculated with
different pathogens showed differences among the various treatments. Increased
activity of PO was observed with the treatment RVL1-T0(28) and SRL1-T0(21) plants
upon challenge inoculation with individual and combination of pathogens, when
compared to control.

In RVL1-T0(28) and SRL1T0 (21) plants, PO activity varied from 0.151 to 0.331
and 0.134 to 0.254, respectively. Lowest PO activity was observed in RVL1-T0(20) and
SRL1-T0 (116) over control plants.

4.6.2.9.2 Polyphenol oxidase (PPO)

All the SRL1 and RVL1 events tested indicated significant increase in the activity
of PPO when compared to control plants. However, accumulation of polyphenol oxidase
was higher in RVL1-T0(28) and SRL1-T0 (21) plants challenged with Fusarium,
Ralstonia and Meloidogyne individually and also in combinations.

PPO activity varied from 0.011 to 0.032. Maximum PPO activity was noticed in
RVL1-T0(28) with Meloidogyne+Fusarium and Meloidogyne alone (0.035) over control
followed by SRL1-T0(21) challenged with Meloidgyne + Ralstonia (0.031). Lowest PPO
activity was observed in RVL1-T0(20) and SRL1-T0(116) when challenged with
Meloidgyne and Ralstonia alone.

4.6.2.9.3 Total phenols

Increase in accumulation of total phenols was observed in all the SRL1 and
RVL1 transgenic events (Table). Phenol accumulation varied from 0.28 to 2.72. Highest
phenol accumulation was noticed in SRL1 transgenic plants challenged with
Meloidogyne (2.72 mg/g) alone followed by Fusarium alone (2.35 mg/g) and with
Ralstonia (1.94 mg/g), respectively. Lowest phenol accumulation recorded in
Meloidogyne+ Fusarium+ Ralstonia (0.57) in SRL1-T0(116) and RVL1-T0(20)
Meloidogyne + Ralstonia (0.28 mg/g) respectively.
163

Table 30. Biocontrol potentiality of srl1 and rvl1 expressed transgenic events against different pathogens (alone and in combinations)

Table 30a. Meloidogyne-alone

Fresh Dry Egg Final


Female Reproduction Nematodes/200
Transgenic lines Plant height (cm) biomass biomass Gall index mass/root Eggs/root population
fecundity factor (Rf) cc
(g) (g) system (Pf)
SRL1-T0(3) 134.90 90.20 25.94 5.44 90.33 311.67 19659.00 14659.00 73.30 314.80
SRL1-T0(7) 138.10 92.80 30.14 5.22 89.33 305.67 18811.33 13811.33 69.06 311.57
SRL1-T0(21) 140.25 94.06 29.70 5.00 89.00 299.67 18170.67 13170.67 65.85 305.23
SRL1-T0(32) 124.83 75.10 23.77 7.22 92.33 361.67 24899.00 19899.00 99.50 340.57
SRL1-T0(10) 128.52 80.55 25.03 6.44 91.33 343.67 22893.33 17893.33 89.47 326.90
SRL1-T0(90) 130.57 82.90 25.63 6.00 90.33 321.67 20562.33 15562.33 77.81 286.23
SRL1-T0(95) 126.07 76.60 24.77 7.00 92.33 349.67 23791.00 18791.00 93.96 326.90
SRL1-T0(116) 122.57 70.10 23.90 7.22 92.33 371.67 25822.33 20822.33 104.11 411.90
RVL1-T0(12) 114.20 75.40 24.83 7.00 110.00 366.67 25840.00 20840.00 104.20 395.73
RVL1-T0(11) 119.60 79.80 26.43 6.22 107.00 344.67 22386.00 17386.00 86.93 383.73
RVL1-T0(28) 124.33 85.10 27.57 5.44 103.00 322.67 18739.67 13739.67 68.70 373.73
RVL1-T0(2) 122.37 83.30 27.43 6.00 119.00 328.67 24611.33 19611.33 98.06 378.73
RVL1-T0(33) 109.50 63.50 20.63 7.44 118.33 379.67 30427.67 25427.67 127.14 408.73
RVL1-T0(48) 112.17 69.40 21.33 7.22 117.67 372.67 29352.67 24352.67 121.76 395.73
RVL1-T0(20) 106.87 63.10 19.93 8.00 123.00 394.67 34042.67 29042.67 145.21 513.73
RVL1-T0(50) 116.47 77.60 25.23 6.22 113.33 344.67 24563.33 19563.33 97.82 388.73
Control (Inoculated) 74.73 45.97 14.30 9.67 126.33 467.67 39586.67 26586.67 132.93 730.33
Control (Uninoculted) 157.56 105.85 33.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00
S.Em± 0.58 0.41 0.39 0.15
CD at 1% 1.66 1.57 1.48 0.58
164

Table 30b. Fusarium alone

Disease severity (0-9 Fusarium population


Transgenic lines Plant height (cm) Fresh biomass(g) Dry biomass (g) 6
scale) (x10 cfu /g soil)
SRL1-T0(3) 127.75 71.97 24.33 20.87 429.62
SRL1-T0(7) 130.30 79.17 25.73 20.17 403.69
SRL1-T0(21) 133.67 82.72 25.48 19.37 389.79
SRL1-T0(32) 116.78 50.17 20.10 21.37 585.66
SRL1-T0(10) 122.27 61.42 21.28 21.17 686.86
SRL1-T0(90) 125.43 66.77 22.03 20.87 684.62
SRL1-T0(95) 118.20 55.17 20.73 21.37 504.76
SRL1-T0(116) 116.60 46.27 19.87 21.58 635.72
RVL1-T0(12) 106.43 63.37 19.73 21.17 509.79
RVL1-T0(11) 111.22 70.57 21.73 21.12 487.61
RVL1-T0(28) 118.73 75.27 23.07 19.67 462.57
RVL1-T0(2) 113.30 73.67 22.93 20.37 476.58
RVL1-T0(33) 101.33 55.83 15.43 21.58 538.62
RVL1-T0(48) 103.90 59.27 16.13 21.58 518.65
RVL1-T0(20) 96.83 53.67 14.63 27.75 658.50
RVL1-T0(50) 109.87 66.57 20.33 21.12 507.50
Control (Inoculated) 70.30 39.83 10.50 64.79 814.84
Control (Uninoculated) 157.56 105.85 33.61 0.00 0.00
S.Em± 0.60 0.65 0.35 0.35
CD at 1% 1.73 1.86 1.35 1.33
165

Table 30c. Ralstonia-alone

Dry biomass Disease severity Ralstonia population


Transgenic lines Plant height (cm) Fresh biomass(g) 6
(g) (0-9 scale) (x10 cfu/g soil)
SRL1-T0(3) 101.07 59.97 22.17 22.87 12.17
SRL1-T0(7) 103.85 62.27 23.60 21.67 9.57
SRL1-T0(21) 104.37 63.52 23.45 21.37 8.90
SRL1-T0(32) 88.17 42.87 18.50 27.49 16.57
SRL1-T0(10) 94.63 52.82 19.33 25.37 13.07
SRL1-T0(90) 98.00 55.97 19.90 22.87 12.17
SRL1-T0(95) 90.18 45.67 19.33 27.37 13.37
SRL1-T0(116) 86.93 37.97 18.33 27.75 16.60
RVL1-T0(12) 55.97 50.97 17.43 26.87 14.13
RVL1-T0(11) 90.53 56.00 18.67 23.37 12.90
RVL1-T0(28) 101.03 59.20 20.97 21.87 10.42
RVL1-T0(2) 95.70 58.30 20.47 22.87 10.42
RVL1-T0(33) 81.97 44.20 14.84 27.75 16.60
RVL1-T0(48) 83.30 47.60 15.44 27.75 14.13
RVL1-T0(20) 80.27 40.90 13.84 30.22 17.83
RVL1-T0(50) 88.93 53.80 17.67 23.75 12.90
Control (Inoculated) 62.80 22.87 9.20 68.50 29.00
Control (Uninoculted) 157.56 105.85 33.61 0.00 0.00
S.Em± 0.34 0.19 0.32 0.32
CD at 1% 1.32 0.72 1.24 1.22
166

Table 30d. Meloidogyne + Fusarium

Egg Reprod Fusarium


Dry Gall index Final Nematode population
Plant height Fresh Disease mass/ro Female uction
Transgenic lines biomass (0-10 Eggs/root population density/20 6
(cm) biomass(g)
(g) severity scale)
ot fecundity
(Pf)
factor
0 cc soil (x10 cfu/g
(0-9 scale) system (Rf) soil)
SRL1-T0(3) 111.37 65.63 26.27 32.17 5.00 89.00 337.33 21530.00 14530.00 72.65 324.13 520.80
SRL1-T0(7) 118.37 69.83 27.77 29.57 4.77 86.33 331.33 20114.67 15114.67 75.57 318.13 514.87
SRL1-T0(21) 131.30 73.38 27.42 28.90 4.55 83.00 317.33 17847.33 12847.33 64.24 315.07 500.97
SRL1-T0(32) 101.02 48.63 21.70 36.57 6.44 92.00 373.33 25851.33 20851.33 104.26 355.13 556.83
SRL1-T0(10) 106.50 59.18 23.12 33.07 6.00 91.33 344.33 22954.67 17954.67 89.77 350.13 528.03
SRL1-T0(90) 107.70 61.93 23.77 32.17 5.22 91.33 342.33 22770.67 17770.67 88.85 327.33 525.80
SRL1-T0(95) 102.76 55.23 22.80 33.37 6.11 93.00 352.33 24270.33 19270.33 96.35 350.33 535.93
SRL1-T0(116) 99.42 45.93 21.63 36.60 7.33 96.00 393.33 29263.33 24263.33 121.32 444.40 576.90
RVL1-T0(12) 100.57 63.93 22.47 34.13 6.22 112.00 367.33 26650.00 16650.00 83.25 419.33 551.07
RVL1-T0(11) 104.57 69.13 24.37 32.90 5.22 111.00 365.33 26060.67 16060.67 80.30 393.33 548.88
RVL1-T0(28) 109.87 73.13 26.13 30.42 4.55 110.00 340.33 22945.33 12945.33 64.73 384.33 523.85
RVL1-T0(2) 106.50 70.23 25.47 30.42 4.77 110.00 354.33 24485.33 14485.33 72.43 399.00 537.85
RVL1-T0(33) 96.60 56.93 17.77 36.60 6.44 110.00 396.33 29105.33 19105.33 95.53 429.00 579.90
RVL1-T0(48) 98.27 58.73 18.47 34.13 6.44 113.00 376.33 28034.33 18034.33 90.17 422.67 559.93
RVL1-T0(20) 91.97 52.43 16.97 37.83 7.20 115.00 416.33 33387.00 23387.00 116.94 547.67 599.67
RVL1-T0(50) 102.73 65.63 23.27 32.90 5.77 112.00 365.33 26426.00 16426.00 82.13 396.33 548.67
Control (Inoculated) 68.87 38.67 12.83 78.57 10.00 132.67 469.33 42764.67 29764.67 148.82 689.33 811.50

Control (Uninoculted) 157.56 105.85 33.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
SE.m± 0.50 0.25 0.35 0.29 0.18
CD at 1% 1.14 0.95 1.35 1.12 0.67
167

Table 30e. Meloidogyne + Ralstonia

Plant Dry Disease Egg Final Nematode Ralstonia


Fresh Gall index (0- Female Reproductio
Transgenic lines height bioma severity mass/root Eggs/root populatio density / population
biomass(g) 10 scale) fecundity n factor (Rf)
(cm) ss (g) (0-9 system n (Pf) 200 cc soil (x 106cfu/g
scale) soil)
SRL1-T0(3) 95.73 51.13 23.07 26.37 4.55 71.33 344.67 16085.67 11085.67 55.43 270.33 9.87

SRL1-T0(7) 100.43 54.63 24.37 24.84 4.33 70.33 338.67 15319.67 10319.67 51.60 267.33 7.27

SRL1-T0(21) 102.23 59.88 24.32 24.14 3.77 68.00 324.67 13578.33 8578.33 42.89 255.00 6.60

SRL1-T0(32) 84.10 35.73 18.50 31.47 6.00 74.33 380.67 19795.00 14795.00 73.98 295.33 14.27

SRL1-T0(10) 90.73 40.58 19.92 28.07 5.22 73.67 351.67 17405.33 12405.33 62.03 295.33 10.77

SRL1-T0(90) 95.90 44.83 20.57 26.37 5.00 73.00 349.67 17025.33 12025.33 60.13 286.33 9.87

SRL1-T0(95) 85.60 37.53 19.60 27.82 5.44 70.67 359.67 16916.33 11916.33 59.58 295.00 11.07

SRL1-T0(116) 81.80 34.03 18.53 32.80 6.22 75.00 400.67 21548.33 18350.67 91.75 379.00 14.30

RVL1-T0(12) 79.40 55.13 17.97 28.73 5.40 90.67 374.67 19469.67 14469.67 72.35 362.33 11.83

RVL1-T0(11) 85.32 62.73 19.77 27.32 5.00 90.67 372.67 19288.33 14288.33 71.44 340.33 10.60

RVL1-T0(28) 91.40 66.23 21.73 24.77 4.33 88.67 347.67 16326.33 11326.33 56.63 324.33 8.12

RVL1-T0(2) 89.30 64.43 20.97 25.97 4.33 90.67 361.67 18291.00 13291.00 66.46 337.33 8.12

RVL1-T0(33) 75.17 47.83 13.77 32.80 6.22 90.67 403.67 22099.00 17099.00 85.50 364.33 14.30

RVL1-T0(48) 76.53 50.33 14.37 28.73 6.00 90.67 383.67 20285.67 15285.67 76.43 368.33 11.83

RVL1-T0(20) 74.20 46.03 12.97 34.03 6.44 90.67 423.67 23912.33 18912.33 94.56 483.00 15.53

RVL1-T0(50) 82.23 57.53 18.57 27.85 5.22 90.67 372.67 19288.33 14288.33 71.44 356.33 10.60

Control (Inoculated) 59.27 27.80 9.13 74.97 8.33 92.67 474.67 24485.67 19485.67 97.43 619.33 26.70

Control (Uninoculted) 157.56 105.85 33.61 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
SE.m± 0.20 0.46 0.35 0.27 0.19
CD at 1% 0.78 1.78 1.35 1.05 0.74
168

Table 30f. Fusarium+Ralstonia

Dry biomass Disease severity Fusarium population Ralstonia population


Transgenic lines Plant height (cm) Fresh biomass(g) 6 6
(g) (0-9 scale) (x10 cfu /g soil) (x10 cfu/g soil)

SRL1-T0(3) 91.20 39.50 20.90 36.60 407.20 9.57


SRL1-T0(7) 93.37 44.30 22.70 35.83 396.63 6.97
SRL1-T0(21) 96.25 46.45 22.35 35.57 379.17 6.30
SRL1-T0(32) 78.77 24.40 16.70 38.60 520.70 13.97
SRL1-T0(10) 79.33 34.55 18.25 36.60 428.47 10.47
SRL1-T0(90) 88.07 35.30 19.00 36.60 413.93 9.57
SRL1-T0(95) 82.10 29.90 17.90 37.83 472.57 10.77
SRL1-T0(116) 75.93 25.40 16.30 40.31 547.37 14.00
RVL1-T0(12) 70.30 32.20 13.60 38.60 477.22 11.53
RVL1-T0(11) 76.17 35.63 15.20 36.60 449.18 10.30
RVL1-T0(28) 82.03 41.63 17.00 36.60 434.52 7.82
RVL1-T0(2) 78.77 37.83 16.30 36.60 438.57 7.82
RVL1-T0(33) 67.30 26.73 11.02 40.31 485.93 14.00
RVL1-T0(48) 68.70 29.33 11.72 40.42 481.00 11.53
RVL1-T0(20) 64.57 23.93 10.22 41.54 562.33 15.23
RVL1-T0(50) 70.23 34.53 13.40 37.83 462.33 10.30
Control (Inoculated) 48.57 13.60 7.54 84.75 677.67 26.40
Control (Uninoculted) 157.56 105.85 33.61 0.00 0.00 0.00
SE.m± 0.28 0.37 0.30 0.23
CD at 1% 0.81 1.43 1.14 0.89
169

Table 30g. Meloidogyne + Fusarium + Ralstonia

Egg Reprodu Nematode Fusarium


Fresh Dry Disease Gall Final
Plant height mass/ro Female ction density population Ralstonia
Transgenic lines biomass bioma severity index (0- Eggs/root populatio 6
(cm) ot fecundity factor /200 cc (x10 cfu/g population
(g) ss (g) (0-9 10 scale) n (Pf) 6
system (Rf) soil soil) (x 10 cfu/g
scale)
soil
SRL1-T0(3) 88.30 37.30 18.83 42.10 4.77 65.33 342.33 9007.00 9428.33 30.04 313.50 370.37 8.58
SRL1-T0(7) 90.60 42.10 20.61 41.60 4.55 64.33 339.33 8801.00 8721.33 29.01 309.33 316.77 5.98
SRL1-T0(21) 96.83 44.25 20.46 40.60 4.55 59.67 324.33 7122.33 6894.00 20.61 302.50 297.87 5.31
SRL1-T0(32) 79.57 22.20 15.70 48.38 5.89 70.00 326.33 7908.33 14425.00 24.54 345.67 579.73 12.98
SRL1-T0(10) 82.63 32.35 16.33 45.10 5.44 66.67 334.33 8457.67 11988.33 27.29 323.50 441.93 9.48
SRL1-T0(90) 86.77 33.10 17.01 42.81 5.00 66.00 339.33 9213.00 11552.00 29.01 317.67 479.70 8.58
SRL1-T0(95) 77.90 27.70 15.92 45.30 5.67 64.33 345.33 8801.00 10301.00 31.07 323.50 507.83 9.78
SRL1-T0(116) 76.20 23.20 16.87 50.07 6.11 68.33 352.33 9693.67 14490.00 33.47 444.33 619.80 13.01
RVL1-T0(12) 71.13 22.60 10.01 47.34 6.00 68.67 320.33 12428.33 6007.00 47.14 392.50 481.97 10.54
RVL1-T0(11) 74.37 28.53 12.23 42.86 5.33 68.67 314.33 11721.33 5801.00 43.61 382.50 458.78 9.31
RVL1-T0(28) 82.40 33.33 14.13 41.63 4.55 66.67 308.33 9894.00 4122.33 34.47 371.63 443.75 6.83
RVL1-T0(2) 79.63 30.73 13.43 41.63 4.77 68.67 370.33 17425.00 4908.33 72.13 378.50 445.75 6.83
RVL1-T0(33) 66.17 20.13 9.22 50.07 6.44 68.67 352.33 14988.33 5457.67 59.94 414.33 473.80 13.01
RVL1-T0(48) 67.43 21.13 9.82 48.97 6.44 68.67 358.33 14552.00 5801.00 57.76 392.67 484.83 10.54
RVL1-T0(20) 63.83 18.73 8.42 53.97 6.87 68.67 380.33 17490.00 18841.00 94.21 546.50 591.67 14.24
RVL1-T0(50) 72.57 26.03 11.87 44.63 5.77 68.67 330.33 13301.00 6213.00 51.51 386.67 478.67 9.31
Control (Inoculated) 50.40 10.40 2.87 99.60 8.55 96.67 342.33 32803.00 9428.33 30.04 652.33 809.03 25.41
Control (Uninoculted) 157.56 105.85 33.61
S.Em± 0.18 0.46 0.36 0.23 0.27
CD at 1% 0.69 1.75 1.38 0.90 1.02
170

Table 30h. Activity of defense enzymes in srl1 and rvl1 transgenic events inoculated with different pathogens (alone and in combinations)

Total Phenols (mg/g of leaf) Peroxidase (unit/mg protein/g of leaf) Polyphenol oxidase (unit/mg protein)
Transgenic
lines M+F+ M+F+
M F R M+F M+R F+R M+F+R M F R M+F M+R F+R M F R M+F M+R F+R
R R
SRL1-T0(3) 2.54 2.17 1.84 1.72 1.55 1.79 1.43 0.210 0.117 0.153 0.166 0.105 0.151 0.144 0.037 0.016 0.037 0.022 0.022 0.022 0.023

SRL1-T0(7) 2.36 2.08 1.85 1.63 1.46 1.84 1.48 0.231 0.150 0.146 0.161 0.099 0.187 0.125 0.027 0.020 0.027 0.020 0.020 0.020 0.024

SRL1-T0(21) 2.72 2.35 1.94 1.72 1.64 1.93 1.57 0.254 0.238 0.134 0.231 0.158 0.240 0.184 0.025 0.016 0.025 0.029 0.031 0.029 0.028

SRL1-T0(32) 1.91 1.71 1.21 1.18 0.82 0.93 0.60 0.142 0.116 0.138 0.154 0.062 0.102 0.118 0.028 0.022 0.028 0.024 0.022 0.024 0.027

SRL1-T0(10) 2.09 1.89 1.57 1.36 0.91 1.29 0.93 0.192 0.075 0.223 0.146 0.072 0.126 0.118 0.005 0.023 0.005 0.020 0.022 0.020 0.025

SRL1-T0(90) 2.18 1.98 1.75 1.54 1.00 1.57 1.20 0.127 0.172 0.125 0.133 0.065 0.098 0.099 0.028 0.021 0.028 0.029 0.022 0.029 0.026

SRL1-T0(95) 1.91 1.80 1.66 1.27 0.82 1.02 0.66 0.086 0.074 0.082 0.090 0.068 0.091 0.095 0.011 0.019 0.011 0.021 0.019 0.021 0.025

SRL1-T0(116) 1.91 1.44 1.12 0.82 0.82 0.84 0.57 0.083 0.071 0.079 0.087 0.065 0.088 0.092 0.008 0.016 0.008 0.018 0.016 0.018 0.022

RVL1-T0(12) 1.72 1.44 0.85 1.03 0.74 1.12 0.76 0.200 0.180 0.188 0.210 0.190 0.223 0.125 0.029 0.021 0.029 0.030 0.024 0.030 0.022

RVL1-T0(11) 1.82 1.62 1.21 1.03 0.92 1.21 0.85 0.160 0.188 0.202 0.209 0.160 0.185 0.144 0.031 0.019 0.031 0.029 0.047 0.029 0.024

RVL1-T0(28) 2.00 1.80 1.39 1.12 1.10 1.58 1.21 0.308 0.331 0.201 0.196 0.153 0.151 0.184 0.035 0.030 0.035 0.033 0.031 0.033 0.027

RVL1-T0(2) 1.91 1.71 1.30 1.21 1.01 1.39 1.03 0.209 0.187 0.154 0.162 0.281 0.142 0.099 0.028 0.023 0.028 0.029 0.027 0.029 0.022

RVL1-T0(33) 1.45 1.17 0.66 0.57 0.56 0.85 0.48 0.273 0.193 0.129 0.137 0.222 0.120 0.118 0.013 0.024 0.013 0.027 0.028 0.027 0.025

RVL1-T0(48) 1.63 1.26 0.75 0.75 0.47 1.03 0.67 0.213 0.180 0.171 0.137 0.135 0.140 0.130 0.011 0.021 0.011 0.028 0.026 0.028 0.029

RVL1-T0(20) 1.36 1.08 0.57 0.48 0.28 0.85 0.48 0.151 0.158 0.144 0.172 0.165 0.079 0.095 0.029 0.024 0.029 0.030 0.028 0.030 0.023

RVL1-T0(50) 1.82 1.53 0.94 0.94 0.92 1.21 0.85 0.151 0.158 0.144 0.172 0.165 0.079 0.095 0.026 0.021 0.0263 0.027 0.0254 0.027 0.020
Control
0.25 0.14 0.15 0.13 0.11 0.14 0.13 0.077 0.065 0.073 0.081 0.059 0.082 0.086 0.016 0.011 0.0163 0.017 0.0154 0.017 0.010
(Inoculated)

M - Melodoiogyne-alone F- Fusarium-alone R – Ralstonia-alone M+F – Melodoiogyne + Fusarium


M+ R - Melodoiogyne + Ralstonia F+ R - Fusarium + Ralstonia M+F+R - Melodoiogyne + Fusarium + Ralstonia
171

Plate 29 Performance of srl1 and rvl1 transgenic events against


combined inoculation of tomato root pathogens
172

Meloidogyne + Fusarium +Ralstonia

0-10 Scale for root gall index

Meloidogyne-alone
Plate 29. Contd…
173

Ralstonia- alone

Plate 29 contd…
174

Meloidogyne + Ralstonia

Meloidogyne + Fusarium

Fusarium +Ralstonia

Plate 29 contd…
175

4.7 Evaluation of rvl1 and srl1 transgenics and non-transgenics for root infection
by M. incognita and its development and reproduction

Biocontrol potentiality of srl1 and rvl1 T3 generation transgenic lines was


determined by inoculating M. incognita. M. inocgnita infection sites, juveniles
development and the nematode reproduction was recorded from the day after
incoculation to 45 days. Finally, the effect on plant growth parameters and gall index
were recorded 45 days after inoculation and presented in Table 31, Plate 30.

4.7.1 M. inocognita root infection

Significantly, more infection sites were found on RVL1-T0(20) and SRL1-T0(116)


infected by M. incognita at 7 dai (days after inoculation) than SRL1-T0(21) and RVL1-
T0(28). The number of juveniles invading roots or infection sites increased in numbers
(5 to 71.33) significantly from the day after inoculation to 7 dai (Table 31, Plate 30).

Root sections of both rvl1 and srl1 transgnics lines and control were observed
one day after inoculation and root infection followed a similar pattern on all transgenic
lines and control. J2 invaded RVL1-T0(20) (12.33, 24.67, 50.6 and 70.67) and SRL1-
T0(116) (8.33, 18.33, 41.00 and 61.67) rapidly and highest in number at one, three, five
and seven days after inoculatrion than other events. Infective second stage juveniles
were observed only in the cortex of tomato roots and some of the roots showed that
juveniles had entered only partially into the root tissue. Several juveniles had penetrated
the root tips and oriented in various directions vertical or parallel to the longitudinal axis
of the root.

4.7.2 Nematode development

Post-embryonic development of M. incognita (200 J2) was observed at 7, 11, 15,


21 and 25 dai. Highest number of sausage shape juveniles significantly varied from 9.67
to 25.67 in RVL1-T0(20) and 9.33 to 24.67 in SRL1-T0(116) after 7, 11 and 15 dai over
the control. Correspondingly less number of sausage stage juveniles were recorded in
SRL1-T0(21) and RVL-1T0 (28). Whereas, number of female nematodes or adults varied
176

from 7.33 to 44.33 and 5.33 to 41.67 in SRL1-T0(116) and RVL1-T0(20) at 21, 25, 30
and 45 dai. Main results from this comparative study were; high root penetration and
reproduction of nematode in control plants and reduced invasion and delay in post-
embryonic development in transgenic plants.

4.7.3 Histopathological studies

Infection by and development of root-knot nematode juveniles inside host tissue


result in histopathological changes in the form of giant cells. The giant cells formed
around the developing nematode in the vascular region of the plant were large,
multinucleate and had cytoplasm and highly invaginated cell walls (Plate 31).

Histopathological changes were observed in both transgenics and control plant


root tissue at 21 dai. Groups of 3 to 9 giant cells around the developing nematode were
observed. The size as well as number of giant cells varied. In some sections, more than
one group of giant cells were observed. This is due to the presence of several
nematodes at the same locus. Less number and size of the giant cells were observed in
SRL1-T0(21) and RVL1-T0(28) compared to control (inoculated).

In a normal secondary control healthy plant, the undamaged phloem and


protoxylem was observed in the vascular region. In infected roots, clusters of giant cells
and various juvenile stages of the nematodes were recorded in vascular region. The
formation of giant cells in the proximity of each nematode caused abberation of the
vascular region. Consequently, the vascular region of smaller lateral root was badly
damaged due to the giant cell formation.

4.7.4 Nematode reproduction

At 45 dai, plants were uprooted. The egg masses per root, eggs per root system,
final population, female fecundity and reproduction factor were estimated (Table 31a).

4.7.4.1 Egg masses /root

Among the transgenic lines, number of egg masses/root was significantly lowest
in SRL1-T0(21) (39.00) and RVL1-T0(28) (49.00) followed by 42.00 and 51.00 of SRL1-
T0(7) and RVL1-T0(2). However, highest number of egg masses per root 59.00
177

was recorded in RVL1-T0(20) and 52.33 in SRL1-T0(116). These treatments differed


significantly over inoculated control.

4.7.4.2 Eggs/root

Number of eggs per root system was significantly higher in RVL1-T0(20)


(22872.33) and SRL1-T0(116) (19084.33) over control. However less number of eggs
per root was recorded in SRL1-T0(21) and RVL1-T0(28) events.

4.7.4.3 Final nematode population (Pf)

RVL1-T0(20) (17872.33) and SRL1-T0(116) (14084.33) recorded highest final


nematode population over the control. Correspondingly less nematode population were
recorded in transgenic events like SRL1-T0(21) and RVL1-T0(28).

4.7.4.4 Female fecundity

The fecundity of the females was estimated by dividing final population by egg
masses. SRL1-T0(21) and RVL1-T0(28) significantly reduced the fecundity of the
females 280.67 and 315.67, followed by SRL1-T0(7) (298.67) and RVL1-T0(2) (321.67)
respectively. Whereas, highest female fecundity of 387.67 and 364.67 was recorded in
RVL1-T0(20) and SRL1-T0(116), respectively over the control.

4.7.4.5 Reproduction factor (Rf)

Reproduction factor of M. incognita was estimated by dividing the final population


by initial population. Reproduction factor was reduced significantly in SRL1-T0(21) and
RVL1-T0(28) over control.

4.7.4.6 Plant growth parameters

Plant growth parameters increased significantly in SRL1-T0(21) and RVL1-T0(28)


compared control (Table 31a) .

4.7.4.7 Shoot and root length

Maximum shoot and root length were recorded in SRL1-T0(21) (60.20 and 21.25
cm) and RVL1-T0(28) (48.63 and 19.20 cm) plants followed by SRL1-T0 (7) (59.73 and
20.70 cm) and RVL1-T0(2) (47.73 and 18.00 cm) respectively. Whereas, highest
178

reduction in shoot and root length was recorded in SRL1-T0(116) and RVL1-T0(20)
plants.

4.7.4.8 Total biomass

There was significant difference in total biomass (fresh and dry) of the plants.
Maximum fresh biomass (fresh shoot and root weight) and dry biomass (dry shoot and
root weight) was recorded in SRL1-T0(21) (37.88 and 20.00 g) and RVL1-T0 (28) (20.33
and 8.80 g) plants. Correspondingly, less biomass of the plants recorded in SRL1-
T0(116) (35.33 and 20.33 g) and RVL1-T020) (17.43 and 5.07 g).

4.7.5 Nematode incidence

4.7.5.1 Gall index

rvl1 and srl1 transgenic events had significantly reduced root-knot index which
was accounted to be 3.67 and 4.33 in SRL1-T0(21) and RVL1-T0(28) event followed by
SRL1-T0(7)(4.33) and RVL1-T0(2) and RVL1-T0(11) of 5.33 over control (9.67).
Correspondingly, highest root-knot index were recorded in RVL1-T0(20) (7.33) and
SRL1-T0(116) (6.33).

4.7.5.2 Root-knot juveniles population (per 200 cc soil)

With respect to all transgenic lines and control, significantly lowest number of
149.67 and 176.23 juveniles were recorded in SRL1-T0(21) and RVL1-T0 (28)
respectively, followed by SRL1-T0(7) (155.00) and RVL1-T0(2) (181.13) these
treatments differed among themselves. However RVL1-T0(20) and SRL1T0(116)
recorded higher number of juveniles 215.10 and 189.40 per 200 cc of soil over the
control respectively.

4.8 Mechanism of action of lectins in the suppression of soil- borne pathogens

4.8.1 Lectin-binding assay for fungus

SRL1, anti-SRL1 and RVL1 interact mostly with chitin oligomers. So using FITC-
SRL1, FITC- RVL1 with appropriate controls to locate lectin sites for soil-borne fungal
pathogens was done and summarized in Table 33.
179

Table 31. Infection and development of M. incognita in srl1 and rvl1 expressed transgenic events

No. of females / root system No. of Gaint


No. of J2 per root system (dai) J2 development (dai) Gaint cells
Transgenic lines (dai) cells
dia. (µm)
1 3 5 7 7 11 15 21 25 30 45 21 dai
SRL1-T0(3) 5.33 14.67 37.67 56.33 7.33 15.67 22.00 6.33 12.67 27.67 42.33 3 1330.90
SRL1-T0(7) 6.00 17.00 35.67 55.67 8.33 16.67 23.00 5.33 12.00 26.67 41.33 3 1687.80
SRL1-T0(21) 5.00 15.33 31.33 51.00 7.00 15.00 21.33 5.00 11.67 26.67 41.33 3 1212.40
SRL1-T0(32) 7.33 18.33 41.00 60.33 9.00 18.00 24.33 6.33 14.00 28.67 43.33 4 4632.00
SRL1-T0(10) 6.67 18.33 40.33 59.67 8.33 16.67 23.00 5.33 12.00 26.67 41.33 4 3041.60
SRL1-T0(90) 6.00 18.33 40.33 58.67 8.33 16.67 23.00 6.33 13.00 27.67 42.33 4 2580.20
SRL1-T0(95) 7.33 18.67 40.33 60.33 8.67 17.33 23.67 5.33 13.33 28.67 43.33 4 2195.40
SRL1-T0(116) 8.33 18.33 41.00 61.67 9.33 18.67 24.67 7.33 14.67 29.67 44.33 5 9618.30
RVL1-T0(12) 8.67 21.67 46.67 66.67 10.00 19.00 25.33 5.33 11.67 27.00 41.33 5 10197.80
RVL1-T0(11) 6.67 19.67 45.33 65.33 8.67 17.33 23.67 7.00 14.00 28.67 43.00 4 1986.40
RVL1-T0(28) 4.33 17.67 42.33 62.00 8.33 16.67 23.00 5.33 11.67 27.33 41.67 3 3571.10
RVL1-T0(2) 5.67 18.67 44.33 64.33 10.00 19.00 25.33 10.67 16.33 32.33 46.67 4 4632.00
RVL1-T0(33) 10.33 23.33 48.67 69.00 8.67 17.33 23.67 12.33 18.33 34.33 48.67 4 8482.70
RVL1-T0(48) 9.67 22.67 47.00 67.00 9.67 18.33 24.67 14.67 20.00 35.33 49.67 4 1366.50
RVL1-T0(20) 12.33 24.67 50.67 71.67 9.67 19.33 25.67 13.33 19.00 34.33 48.67 5 7914.30
RVL1-T0(50) 7.67 20.67 46.33 66.33 9.00 18.00 24.33 15.67 21.67 37.00 51.33 4 11640.50
Control (Inoculated) 18.00 41.00 78.33 107.67 13.67 24.33 30.67 19.33 32.67 48.33 62.67 9 12475.80

Day after inoculation - dai


180

Table 31a. Plant growth and nematode parameters as influenced by M. incognita infection in srl1 and rvl1 expressed transgenic events

Shoot weight Root weight Total biomass (g) Egg Reprodu


Nematode Final
Treatments Shoot Root (g) (g) Gall mass Female ction
population/ Eggs /root populatio
length length index /root Fecundity factor
Fresh Dry Fresh Dry Fresh Dry 200 cc soil n (Pf)
(cm) (cm) system (Rf)
SRL1-T0(3) 57.82 19.50 20.77 9.90 12.70 7.53 33.47 17.43 4.67 160.30 43.00 304.67 13101.67 8101.67 40.51
SRL1-T0(7) 59.73 20.70 23.10 10.33 13.90 8.28 37.00 18.62 4.33 155.00 42.00 298.67 12545.00 7545.00 37.73
SRL1-T0(21) 60.02 21.25 23.43 11.63 14.45 8.37 37.88 20.00 3.67 149.67 39.00 280.67 10945.00 5945.00 29.73
SRL1-T0(32) 51.40 14.20 15.10 6.17 7.40 3.77 22.50 9.93 6.33 183.83 49.00 354.67 17379.33 12379.33 61.90
SRL1-T0(10) 52.73 16.75 17.10 8.20 9.95 4.78 27.05 12.98 5.33 172.33 45.33 336.67 15262.67 10262.67 51.31
SRL1-T0(90) 54.43 17.30 17.83 8.80 10.50 5.33 28.33 14.13 5.33 164.83 44.00 314.67 13846.67 8846.67 44.23
SRL1-T0(95) 52.40 14.60 15.77 6.90 7.80 4.67 23.57 11.57 6.33 172.33 45.00 342.67 15421.00 10421.00 52.11
SRL1-T0(116) 50.40 13.30 13.83 4.80 6.50 4.00 20.33 8.80 6.33 189.40 52.33 364.67 19084.33 14084.33 70.42
RVL1-T0(12) 42.57 15.00 13.17 5.83 13.90 6.73 27.07 12.57 6.33 198.27 55.33 359.67 19901.67 14901.67 74.51
RVL1-T0(11) 45.67 17.50 116.43 8.80 15.20 8.03 31.43 16.83 5.33 186.33 52.33 337.67 17671.00 12671.00 63.36
RVL1-T0(28) 48.63 19.20 18.83 10.90 16.50 9.33 35.33 20.23 4.33 176.23 49.00 315.67 15468.00 10468.00 52.34
RVL1-T0(2) 47.73 18.00 18.17 9.10 16.10 8.93 34.27 18.03 5.33 181.13 51.00 321.67 16405.00 11405.00 57.03
RVL1-T0(33) 39.77 13.30 9.90 2.80 10.00 2.83 19.90 5.63 6.33 210.83 56.67 372.67 21118.00 16118.00 80.59
RVL1-T0(48) 41.87 14.00 11.83 4.83 10.50 3.33 22.33 8.17 6.33 198.40 56.00 365.67 20477.33 15477.33 77.39
RVL1-T0(20) 37.77 12.40 8.03 2.83 9.40 2.23 17.43 5.07 7.33 215.10 59.00 387.67 22872.33 17872.33 89.36
RVL1-T0(50) 43.67 16.30 14.10 6.87 14.20 7.03 28.30 13.90 5.33 191.67 52.67 337.67 17783.67 12783.67 63.92
Control
31.82 10.50 9.83 2.97 7.57 1.90 17.40 4.87 9.67 530.67 69.00 490.33 33832.00 28832.00 144.16
(Inoculated)

Control
62.82 23.90 25.97 11.97 14.45 8.28 40.42 20.25 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(Uninoculated)

S.Em± 0.34 0.16 0.22 0.14 0.34 0.21 0.54 0.26 0.9

CD at 1% 1.31 0.61 0.86 0.53 1.31 0.81 1.54 1.01 1.2


181

Plate 30 Meloidogyne incognita infection and development in srl1 and rvl1


transgenic events
182

Nematode infection one day after inoculation

Nematode development seven day after inoculation

Nematode development 11 Days after inoculation

Nematode development 15 Days after inoculation

Fecundity of Meloidogyne incognita in transgenic events as against control

Plate 30. Contd…


183

srl1- transgenic events

wild type

rvl1- transgenic events

G-Giant cell

Plate 31. Histopathology of Meloidogyne infected transgenic and non-transgenic root


system
184

In order to get an insight in to the mode of SRL1, anti-SRL1 and RVL1 toxicity effect on
S. rolfsii, R. solani, F. oxysporum, P. lilacinus and T. viride, determined the binding of lectins
after incubating the fungus in FITC-lectin solution. Binding pattern of lectins differed between
the fungus observed by fluorescent microscopy. SRL1 and RVL1 bound to all fungus,
primarily to the hyphal wall and septa and to a lesser extent lined to the hyphal tips and weak
fluorescence was exhibited in T. viride spores. Details of anti-SRL binding to the S. rolfsii and
R. solani are explained in immunolocalization studies.

FITC-RVL1 strongly bound to all five pathogens, but its binding pattern differed vastly
from that FITC-SRL1, in that it was more or less even along the hyphal wall with little binding
to hyphal tips and strong binding to the branching points, septa and spores of the fungus.

S. rolfsii with extensively branched filaments was seen with densly localized
fluorescence at branching points and hyphal wall. Binding sites were also seen on mature and
immature sclerotial bodies that indicated the strong binding FITC-RVL1 on sclerotia and
mycelial filaments (Plate 32).

FITC-RVL1 exhibited uniform strong flouresence at hyphal wall, branching point,


septum and hyphal tip of R. solani and also around the sclerotia (Plate 33).

The pattern of FITC-RVL1 binding to F.oxysporum was similar to some extent to the
FITC-SRL1 and anti-SRL1, i.e. hyphal wall but to the lesser extent RVL1 bound to
chlamydospores, micro and macroconidia (Plate 34).

The binding pattern of RVL1 to hyphal wall, septum, spore wall and phialides wall of
T.viride was similar to SRL1 but differed with pattern of binding. RVL1 strongly bound over the
entire surface of the spores and phialides on the other hand, bound in descrete patches over
the whole mycelium (Plate 35).

RVL1 conjugated with FITC exhibited intermediate fluorescence on hyphal wall of P.


lilacinus, but to the lesser extent SRL1 and anti-SRL1 also bounded to the hyphal wall.
Binding was not observed in P. lilacinus spores incubated with FITC-RVL1, SRL1 and anti-
SRL (Plate 36).
185

Plate 32. FITC -conjugated SRL1, anti SRL1 and RVL1 labelling of Sclerotium rolfsii
186

FITC –SRL1 (400X)

FITC –anti SRL1 (400X)

Plate 33. FITC -conjugated SRL1, anti-SRL1 and RVL1 labelling of Rhizoctonia solani
187

Septa

FITC-RVL1 (400X)

Plate 33. Contd…


188

FITC anti SRL1

FITC SRL1

FITC RVL1 (400X)

Plate 34 FITC -conjugated SRL1, anti SRL1 and RVL1 labelling of Fusarium oxysporum
189

FITC-RVL1

Plate 35. FITC -conjugated SRL1, anti SRL1 and RVL1 labeling of Trichoderma viride
190

Plate 36. FITC -conjugated SRL1, anti SRL1 and RVL1 labelling of Paecilomyces
lilacinus
191

4.8.2 Immunolocalziation studies of anti-SRL1 in S. rolfsii and R. solani

The results of immunolocalization studies of FITC anti-SRL1 with vegetative mycelia,


immature and mature sclerotial boides of S. rolfsii and R. solani are shown in Table 33 that
reveals the occurrence of lectin as seen under fluorescence microscope (Plate 37).

S. rolfsii mycelia with extensively branched hyphal filaments were seen with descrete
intense spots of fluorescence densely localized at the branching regions and strong
fluorescence along the mycelia indicating the occurrence of high levels of lectin at the
branching points. However uniformly distributed fluorescent binding pattern was found on
young hyphal filaments suggesting high levels of lectin. Immature sclerotial bodies in the final
stage of formation still associated with highly branched mycelia around showed dense mass
of congregated lectin sites arising due to aggregation of mycelium, whereas in the completely
matured sclerotial bodies intense fluorescent label was seen indicating the uniform distribution
of lectin at very high levels when compared to mycelia.

Anti-SRL1 conjugated with FITC, showed extensive binding at branches and septum of
the R. solani and also to wall and tip of the hyphae seen with uniform fluorescence densely
localized inside the hyphae and strong fluorescence at branching point and septum indicating
the occurrence of high levels of lectins in mycelium. Lectin binding to sclerotia showed strong
fluorescence around the sclerotia indicating high level of lectin with strong and uniform
binding.

4.8.3 Staining of nematode secretions

Different solutions of CBB-R were tested: (a) 1% CBB-R dissolved in water (b) 0.2%
CBB-R dissolved in 20% methanol (c) 0.2% CBB-R dissolved in 40% methanol and 10%
acetic acid, and (d) 0.1% CBB-R dissolved in 40% ethanol and 10% acetic acid. Solutions (c)
and (d) gave clear and reproducible staining of the secretions of M.incognita juveniles. It was
decided to proceed with solution (d). The secretion in the head region of juveniles was stained
as one big spot or as two distinct rays. These represent the secretions of the amphids and the
secretions of the excretory pore were
192

Immature sclerotia

Mature Sclerotia

Immature micro-sclerotia

Mature micro-sclerrotia

Plate 37. Immunolocalization of anti SRL1 in sclerotia and microsclerotia of


sclerotium rolfsii and Rhizoctonia solani respectively
193

Table 32. Binding of histochemical dyes to exudates of Meloidogyne incognita juveniles

Nematode secretions
Dye
Amphids Excretory pore Phasmids

1% CBB-R dissolved in water, + + -

0.2% CBB-R dissolved in 20%


+ - -
methanol,

0.2% CBB-R dissolved in 40%


++ + -
methanol and 10% acetic acid

0.1% CBB-R dissolved in 40% ethanol


++ ++ ++
and 10% acetic acid

++ : Strong binding

+: Intermediate binding

+/- : Less frequent binding

- : No binding
194

0.1% CBB-R dissolved in 40% ethanol and 10% acetic acid

Plate 38 Staining of amphidial and excretory secretions of M. Incognita (400x)


195

also visible as one big spot or as a thin ray. Additionally, secretions were stained at the
phasmids region (Table 32, Plate 38).

4.8.4 Lectin-binding assay for M. incognita

In order to get an insight in to the mode of SRL1 and RVL1 toxicity effect on M.
incognita, the binding of lectins after incubating the nematodes in FITC-RVL solution was
observed. Binding of RVL in nematodes was observed by fluorescent microscopy (Table 33,
Plate 39).

Purified SRL1 and E. coli expressed RVL1 and lectins binding were found at many
regions of the nematode body and it was very clear that lectins were ingested by nematodes.
FITC-SRL1 and FITC-RVL1 treated juveniles exhibited epiflourescence in the head region,
mid gut of the alimentary-tract, excretory pore and extended to the posterior regions
(phasmids) but the principal lectin-binding sites were the pores or the secretions of the
amphids and excretory pore. Binding was not observed in nematode gut incubated with FITC-
RVL1 complexed with mucin and FITC-BL21 (bacterial control).
196

Table 33. Binding of FITC conjugated lectins

Binding sites Pure SRL1 Anti-SRL1 Crude RVL1 BL21 Mucin


Meloidogyne incognita
Excretory pore ++ ++ + - -
Phasmids ++ ++ ++ - -
Alimentary-tract ++ ++ ++ - -
Amphids ++ ++ + - -
Rhizoctonia solani
Septum +/- ++ ++ - -
Hyphal tip - ++ ++ - -
Hyaphal wall +/- ++ ++ - -
Hyphal branch - ++ ++ - -
Sclerotia - ++ ++ - -
Sclerotium rolfsii
Hyphal wall +/- ++ ++ - -
Septum - ++ ++ - -
Hyphal branch - ++ ++ - -
Mature sclerotia - ++ ++ - -
Immature sclerotia - ++ ++ - -
Fusaium oxysporum
Hyphal wall + + ++ - -
Spore wall +/- +/- + - -
Macroconidia - - +/- - -
Microconidia - - +/- - -
Chlamydospores - - +/- - -
Trichoderma viride
Hyphal wall ++ ++ ++ - -
Spores + + ++ - -
Phialides + + ++ - -
Paecilomyces lilacinus
Hyphal wall +/- +/- + - -
Spores - - - - -

++ : Strong binding

+ : Intermediate binding

+/- : Less frequent binding

- : No binding
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Plate 39 Binding of FITC conjugated SRL1, anti SRL1 and RVL1 to the Meloidogyne incognita juveniles
5. DISCUSSION

Due to increased use of chemicals worldwide, it has now become necessary to


balance agricultural needs with environmental and health issues. So, efforts are being
made to reduce broad spectrum toxicants added to the environment and to use plant
defense proteins. In recent years, several investigations focused on plant defense
proteins such as lectins, ribosome-inactivating proteins, protease inhibitors and α-
amylase inhibitors as these have been shown to possess fungicidal, bactericidal and
nematicidal activity towards a range of economically important plant pathogens
(Gatehouse and Gatehouse, 1998; Vasconcelos and Oliveira, 2004). Lectins play an
important role in host-pathogen interactions and plant’s defense against various
pathogens, cell to cell interaction, serum glycoprotein turnover and innate immune
response (Vijayan and Chandra, 1999).

Currently, some lectins of plant and fungal origin have been found toxic to a wide
range of important pathogens (Lam and Ng, 2011). Lectins have been engineered
successfully into a variety of crops including wheat, rice, tobacco, potato and tomato.
This approach could be used as a part of integrated disease management strategies
and caveat pathogens’ attack. In general, it seems that large-scale use of transgenic
fungicidal, nematicidal, insecticidal and herbicide-tolerant plants do not display
considerable negative effects on the environment. Moreover, some transgenic plants
can improve the corresponding environments and human health because their
production considerably reduces the load of chemical fungicides, bactericides and
nematicides (Velkov et al., 2005; Bhagat, 2010). For effective management of the soil
borne pathogens, viz. Sclerotium rolfsii, Rhizoctonia solani, Fusarium oxysporum,
Ralstonia solanacearum and Meloidogyne incognita in tomato, there is a need to use
environmentally safe management practices. Hence, in the present study, an attempt is
made to screen E. coli-expressed RVL1 and SRL1 lectins to soil borne pathogens.

Remusatia vivipara, the tubers which are cooked and eaten worldwide contains a
lectin (RVL1) (Bhat et al., 2010a) that can be classified under monocot mannose-
binding lectins with tetrameric proteins. Further, studies also showed E. coli expressed
RVL1 lectin is nematicidal against a common root-knot nematode, M. incognita (Bhat et
al., 2010a). Fungal lectin SRL1 from Sclerotium rolfsii showed nematicidal
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properties (Bhat et al., 2010b). S. rolfsii lectin (SRL1) is also involved in development
and morphogenesis of the fungus, rather than host parasite interactions (Swamy et al.,
2004) With this background, an effort was made to evaluate the E. coli-expressed SRL1
and RVL1 against common soil-borne pathogens of tomato for antimicrobial activity
against fungal and bacterial antagonists. Selected PCR positive T3 generation RVL1
and SRL1 transgenic lines were tested against Fusarium, Ralstonia and Meloidogyne to
study their plant growth effects and to elucidate their mechanism of biocontrol. The
results obtained during the experimentation are discussed here.

5.1 Collection and isolation of soil-borne pathogens affecting tomato

Soil-borne pathogens affecting tomato (Fusarium oxysporum, Sclerotium rolfsii,


Rhizoctonia solani, Ralstonia solancearum and Meloidogyne incognita) were collected
from fields of Main Agricultural Resarch station, UAS, Dharwad and were isolated.
Cultural and morphological characters of established the isolated soil-borne pathogens
as Fusarium oxysporum, Sclerotium rolfsii, Rhizoctonia solani, Ralstonia solancearum
and Meloidogyne incognita (Kelman, 1954; Taylor, 1971; Fargette et al., 1996;
Kamalakannan et al., 2003; Sachidananda, 2005).

5.2 Expression of RVL1 and SRL1 in E. coli

Crude protein induced from RVL1 and SRL1 expressed E. coli could agglutinate
trypsinized rabbit blood, indicating the presence of lectin (Chao et al., 1994). Minimum
concentration required for agglutination (MCA) was 1.37 µg and 2.73 µg for SRL1 and
RVL1, respectively. For SRL1, the total activity was 0.87×104 and the specific activity
was 7.29×102. Likewise, for RVL1, the total activity was 0.43×104 and the specific
activity was 3.66×102 with rabbit erythocytes. In general, the concentration of lectin was
enhanced in partially purified protein. Both SRL1 and RVL1 showed an expected band
of 19.5 and 28.2 kDa. The present results are conformity with Wu et al. (2001) who
showed that 17 kDa S. rolfsii lectin (SRL) agglutinated trypsinized rabbit and human
erythrocytes, and displayed a strong binding to disaccharide Gal β1-3 GalNAc-α-
(Thomsen Friedenreich antigen) that was over-expressed in human tumor cells. SRL
shared a common structural topology, glycan specificity, and carbohydrate-binding sites
(Leonidas et al., 2007; Sathisha et al., 2008) with XCL.
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5.2.1 Purification of SRL1

From the crude protein, SRL1 was purified by using His-tag purification kit. Upon
induction of the T7 polymerase by IPTG, fusion protein produced in various hosts allows
easy purification based on the His-tag affinity to Ni-NTA columns (Terpe, 2003).
Numerous studies in the past could successfully recover from hetereologous hosts the
His-tag fused functional lectins of GNA (Trung et al., 2006), Lablab purpureus (Kim et
al., 2007), Ricinus communis (Reed et al., 2005), Robinia pseudoacacia (Nishiguchi et
al., 1997), Zingiber officinale (Chen et al., 2005), Arisaema heterophyllum (Zhao et al.,
2006) and Phlebodium aureum (Tateno et al., 2003).

5.3 Raising polyclonal antibodies against SRL1 (anti-SRL1)

PAbs were raised in (six month old) white albino rabbit, using Puified SRL1 as
antigen similar method was conducted by Charudattan and De Vay, (1974); Swamy et
al. (2004). Because the antiserum obtained in this study appears to be specific for SRL1
and did not cross react with other proteins The micro-precipitation results indicated that
the antiserum produced against SRL1 was working (1: 65536 dilutions). Further
confirmation was done by DAC-ELISA technique. Titre of antisera was determined by
two-fold serial dilutions of antiserum from 100 to 65536. The titre of antisera against
antigen was determined by DAC - ELISA. It was sensitive enough to detect the antigen
up to 4096 dilutions.

5.4 Evaluation of plant and fungal lectins for the in vitro suppression of some
common soil-borne pathogens

5.4.1 In vitro antifungal assay

5.4.1.1 Evaluation of lectins against fungi by poison food method

5.4.1.1.1 F. oxysporum

With the insecticidal action of plant lectins, the spectrum of external functions is
certainly not completely covered yet. Binding to cell wall constituents in fungi can
interfere with their growth. Fungal cell walls contain chitin, the β1-4 linked polymer of
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GlcNAc. It is, therefore, likely that a fungicidal action is exerted by GlcNAc-binding


lectins (Sharon, 1984). SRL1 and RVL1 lectin showed inhibitory activity of 25.28 and
22.52 per cent against F. oxysporum as demonstrated by the growth of mycelia when
cultured on medium containing variable concentrations (1.2, 2.4, 3.6 mg/ml) of protein
solution. It has been shown previously that some plant lectins do exhibit fungicidal
activity against F. oxysporum, e.g. lectins isolated from Talisia esculenta (Freire et al.,
2002) and Pouteria torta (Boleti et al., 2007) were active at a concentration of 280
µg/ml.

5.4.1.1.2 S. rolfsii

In vitro studies demonstrated that both RVL1 and SRL1 played a role in early
formation of sclerotial bodies rather than simply serving as reserve storage protein.
Germination of sclerotial bodies is another event in development of the fungus, which
was strongly inhibited by SRL1 and RVL1 lectins. In contrast to an endogenous
function, the cytoplasmic localization of fruiting body lectins is ideally suited for the
proposed role of these proteins in the defence of fungi against pathogen.

5.4.1.1.3 R. solani

Both SRL1 and RVL1 lectins showed no inhibitory activity against R. solani as
demonstrated by the growth of mycelia when cultured on medium containing variable
concentrations (1.2 to 4.8 mg/ml) of protein solution. It has been shown previously that
some plant lectins do exhibit fungicidal activity against R. solani, e.g. lectins isolated
from Phaseolus coccineus seeds (Chen et al., 2009) and Phaseolus vulgaris cv “red
kidney bean” seeds (Ye et al., 2001) both of which were active at a concentration of 330
µg/ml.

5.4.1.2 Evaluation of lectins against fungi by spread plate method

In the present investigation, different concentrations of SRL1 anti-SRL1 and


RVL1 were evaluated against F. oxysporum, S. rolfsii and R. solani. This spread plate
method gives direct contact for lectin to act on pathogens. Results revealed that among
the lectins, RVL1 inhibited the mycelial growth of the F. oxysporum of 42.96 per cent,
whereas, anti-SRL1 inhibited the mycelia growth of S. rolfsii (44.44%) and R. solani
202

(55.55%). Comparison of different concentration of lectins revealed significant


differences and showed increased efficacy or increased sensitivity by the fungus to
RVL1 and anti-SRL1 with increased concentration. These findings are similar with those
of Lis and Sharon (1981) who showed inhibition of fungal growth could occur through
lectin binding to hyphae resulting in poor absorption of nutrients as well as by
interference on spore germination process.

5.4.1.3 Evaluation of lectins against fungi by inhibition zone assay

SRL1, RVL1 and anti SRL inhibited the growth of the fungi R. solani, S. rolfsii
and F. oxysporum at different concentrations (1.2 to 4.8 mg/ml) as seen by employing
the disc plate diffusion assay of Roberts and Selitrenikoff (1988). This technique
deirectly act on hyphal growth and does not indicate whether there is inhibition of spore
germination.

The antifungal properties of chitin-binding lectins have been a matter of


controversy ever since the report of Mirelman et al. (1975) on the inhibitory effect of
WGA on fungal growth. Schlumbaum et al. (1986) have presented convincing evidence
that chitinase-free preparations of WGA or potato lectin are devoid of any antifungal
activity (Broekaert et al., 1989). But, another chitin-binding lectin, UDA, is a potent
inhibitor of chitin-containing fungi, and that its antifungal effect is different from that of
chitinases (Broekaert et al., 1989). It is seen in the present study that SRL1, RVL1 and
anti-SRL also inhibit several chitin-containing fungi, though to a lesser extent than other
chitin binding lectins like, UDA WGA. However, RVL1 and SRL1 are more potent
inhibitors than chitinase, which excludes the possibility that the antifungal effects were
merely caused by the presence of contaminating chitinase. Most fungi are not inhibited
by the chitinase is consistent with that of Mauch et al. (1988) who showed that in most
cases chitinase must act in coordination with β-1,3-glucanase in order to exert notable
antifungal characteristics.

However, there was no such report about antifungal activity of RVL1 and SRL1
lectins. In the present investigation, these lectins displayed antifungal activity in broad
fungal species including F. oxysporum, S. rolfsii and R. solani.
203

5.4.1.4 Evaluation of lectins against F. oxysporum by disc diffusion assay

E. coli expressed SRL1 and RVL1 lectins showed in vitro antifungal activity
against F. oxysporum. It strongly inhibited the growth of Fusarium at the higher
concentration of 4.8 mg/ml. Antifungal activity has been observed in other lectins where,
for example, Astragalus mongholicus root lectin revealed antifungal activity against
various species of phytopathogenic fungi (Yan et al., 2005). Similarly, the lectin from
Curcuma amarissima Roscoe rhizome inhibited the growth of F. oxysporum,
Colletotrichum cassiicola and Exserohilum turicicum (Kheere et al., 2010). In vitro
studies of two novel chitin-binding lectins from the seeds of Artocarpus integrifolia
inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae (Trindade
et al., 2006). Many studies on plant and fungal lectins as antifungal protein have
showed that they are implicated in the host defense mechanism. However, to date, only
a small number of lectins have been reported to have actual antifungal activity, including
those from potato tubers, Amarnthus caudatus seeds, stinging nettle rhizomes, wheat
germ, and P. vulgaris seeds.

5.4.1.5 Spore germination

Growth inhibition by the plant lectin RVL1 and fungal lectin SRL1 was indicated
by their effect on fungal spore germination. Both the lectins caused a marked inhibition
at 1.2 mg/ml and more. It seems that the inhibition of spore germination occurs in a very
early stage of the germination process, after spores have been allowed to swollen and
before initiation of the germ tubes. Inhibition of germination was mainly expressed by
the prolongation of the latent period which precedes germination. However once
initiated, the rate of germination appeared quite normal and after 48 h of incubation, the
percent of lectin treated spores that germinated was almost the same as that of the
untreated ones. Inhibition of F. oxysporum spore germination by RVL1 and SRL1 clearly
indicated their inference with normal growth. These findings are in conformity with the
Mirelman et al. (1975); Golan et al. (1978); Broekaert et al. (1989) that lectins in plants
are part of their protection system, helping them to combat attack by fungal pathogens.
These results indicate that the macroconidia were able to adapt to chitinase (Sela-
Buurlage, 1996). It is possible that RVL1 and SRL1 altered the hyphal cell wall
architecture of F. oxysporum as exemplified in Phycomyces blakesleeanus, B.
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cinerea, T. viride, and C. lindemuthianum germlings treated with UDA (Van Parijs et al.,
1992; Does et al., 1999). A change in cell wall composition might result in the resistance
of fungi to RVL1 and SRL1.

5.4.1.6 Inhibition of sclerotial germination

Formation of sclerotia in R. solani and S. rolfsii is closely related to the nutrient


stress, such as depletion of carbohydrates in the medium (Townsend, 1957). Recently,
it was reported that the lectin activity was found in Rhizopus stolanifer only under spore
forming condition (Oda et al., 2003), which further strengthens the view that the fungal
lectins play key role in spore formation. In R. solani, lectin occurring in small amounts
on young hypahe increased dramatically at the time of maturation, and accumulated in
mature scleotinia (Kellens and Peumans, 1990). Lectin accumulated in the sclerotia of
Sclerotinia sclerotiarum represented as high as 40 per cent of the total sclerotial protein,
hence they concluded that the lectin serve as reserve storage protein as in sclerotia. In
contrast Cooper et al. (1997) suggested that the small, saline-soluble galactose-binding
lectins (fungal galectins) secreted by many fungi are developmentally regulated with
high expression in fruiting bodies.

To further investigate the involvement of lectin in fungal growth, we examined the


germination of sclerotial bodies after capping the lectin sites by anti-SRL. Interestingly,
sclerotial bodies treated with anti-SRL1 did not germinate even after seven days but the
sclerotial bodies treated with normal rabbit serum and BL21 germinated normally and
lavish growth was observed. Not only anti-SRL but also RVL1 inhibited the germination
at higher concentration. These findings revealed the necessity of free form of lectin for
the germination and when it bound to extraneous lectin binding molecules, such as
capping of the lectin sites by anti-SRL, germination of the mature and immature
sclerotial bodies of S. rolfsii and R. solani was inhibited. These results are in confirmity
with Swamy et al. (2004) where they have shown that capping of the lectin sites by anti-
SRL strongly inhibited the germination of sclerotial bodies. Similar inhibition was found
by treating these bodies with mucin or fetuin, with which SRL binds strongly (Swamy et
al., 2001).

For the onset of sclerotial body germination, lectin–receptor complex could be


mediating a critical signaling pathway. Probably this signaling event was interrupted
205

when the lectin is complexed with anti-SRL. Recently it was shown that the disruption of
the glucosyl ceramide synthesis using inhibitors of UDP-Glc, ceramide
glucosyltransferase, lead to inhibition of spore germination, cell cycle, and hyphal
growth in Aspergillus nidulans and Aspergillus fumigatus (Levery et al., 2002).
Membrane bound glycosyl ceramides are reported to be widely distributed in many fungi
during their syntheses, sugar moieties are added directly to ceramides, which are
referred to as glycosylinositol phosphorylceramides (GIPCs). This class of glycosyl
ceramides occurring in fungi (Lester and Dickson, 1993; Dickson, 1998) are mostly
glucosylceramides. However, there are also reports of galactosylceramides occurring in
some fungi (Levery et al., 2000; Toledo et al., 1999, 2000). GIPCs are believed to act as
signaling molecules and are implicated for their diversified roles in viability (Zhong et al.,
2000) and cell growth (Chung et al., 2001). Inhibition of spore germination by disrupting
the ceramide synthesis using inhibitors of glycosyl transferases in A. nidulans and A.
fumigates (Levery et al., 2002) is analogous to present study, inhibition of sclerotial
body germination by capping the lectin sites on sclerotial bodies.

5.4.2 Antibacterial activity

Antibacterial activity of the SRL1 and RVL1 was assayed in vitro by the well
diffusion method. The E. coli expressed lectins SRL1 and RVL1 showed antibacterial
activity against R. solanacerum. The diameters of growth inhibition areas were in the
range of 2.35 and 2.85 cm. In view of the results obtained by the well diffusion method,
the MIC values of the SRL1 and RVL1 were determined. The MIC values obtained
confirmed the existence of a significant activity against the bacterium, which ranged
from 0.625 mg/ml. Simple radial diffusion assay is a method used for the qualitative
detection of lectin interactions with saccharide compounds being used to determine the
saccharide residues present in glycoproteins (Lima et al., 1997). RVL1 and SRL1
showed remarkable antibacterial activity against R. solanacearum. RVL1 has a high
antibacterial activity as demonstrated by the inhibition zone (1.65 cm).

The results describing the effect of RVL1 and SRL1 on the bacterial agglutination
was confirmed the interaction between the lectin and the bacterium. Ralstonia seemed
to be most sensitive to the RVL1. It is known that lectin binds not only with non-
reducing terminal sugars of glycoproteins, but some also react with internal components
of the carbohydrate chains or with non-carbohydrate ligands (Goldstein and Poretz,
206

1986). Inhibition of bacterial agglutination by RVL1 and SRL1 showed that lectin binding
occurs by interaction with bacterial surface carbohydrates. Similar to RVL1 and SRL1,
the lectin of Araucaria angustifolia exhibited antibacterial activity against Clavibacter
michiganensis and Xanthomonas axonopodis pv. passiflorae (Santi-Gadelha et al.,
2006). Although the mechanism of action of the lectins has not yet been elucidated in
detail, the presented data confirm the in vitro antibacterial activity of RVL1 and SRL1
against R. solanacearum. It has been proposed that the proteins with antibacterial
action form a channel on cell membrane and the cell dies as a result of the out-flowing
of cellular contents, this mechanism being different from that of antibiotics (Talas-Ogras
et al., 2005). The complex interaction between RVL1 and SRL1 and the bacterial
surface is further suggested by glycoprotein inhibition. This is the first report on the
antibacterial activity of RVL1 and SRL1 against R. solanacearum which needs
confirmation further against different hosts affected by same pathogen.

5.4.2.1 Plant and fungal lectins’ activity against R. solanacearum

The antibacterial activity of RVL1 and SRL1 was very similar to the lectin of Morus
alba exhibited antibacterial activity against Pseudomonas syringae pv. mori. The bacterial
agglutination was also inhibited by the glycoproteins, fetuin and tyroglobulin (Ratanapo et
al., 2001). The sugar residues which serve as lectin binding site on surface of R.
solanacearum apparently exist for a limited period during exponential phase of bacterial
growth 1.225 and 1.146 to 0.865 and 0.849 OD of RVL1and SRL1 respectively. Similar
dynamic changes of bacterial surface during growth also have been demonstrated in the
case of Jackfruit, Bacillus thuringeinsis interaction (Prapanpoj, 1986) and monodin-Vibrio
vulnificus interaction (Ratanapo et al., 1992).

5.4.3 In vitro antinematode activity

Production of functional RVL1 and SRL1 in E. coli allows preparation of pure and
homogeneous protein required for structural, biological and mechanistic studies.
E. coli expressed RVL1 and SRL1 was used for nematode bioassay. Since the RVL1
and SRL1 was not purified, total protein produced in E. coli was used for checking
nematicidal activity against Meloidogyne incognita. Total protein used for the bioassay
was quantified to be 12.00 mg and it was diluted to 1: 25, 1: 50, 1: 75 and 1: 100. Egg
hatching inhibition and mortality of juvenile nematodes was observed periodically at 3,
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6, 12, 24 and 48 h. Egg hatching inhibition was found at 12 h and juvenile mortality was
found only after 3 h (at 6 h) and from then on it increased with increase in time and
lectins concentration.

Maximum egg hatching inhibition (86 and 82%) and juvenile mortality (92.67 and
82.67%) at 1: 25 dilution was observed in SRL1 and RVL1 after 12 and 48 h incubation,
respectively. However, other dilutions (1: 50, 1: 75 and 1: 100) also showed high
mortality in SRL1 followed by RVL1 after 48 h. It was evident that the less concentration
of SRL1 and RVL1 (based on the activity) caused fairly high egg hatching inhibition and
juvenile mortality. The mechanism of action of lectins has been ascribed to the ability of
lectins to bind glycoproteins and proteins localized on the surface coat of nematodes
(Marban-Mendoza et al., 1987; Ripoll et al., 2003). By and large, this was confirmed by
Bhat et al., 2010a; Bhat et al., 2010b, suggesting that binding of lectin to glycoprotein
receptor sites, local or systemic deleterious effects lead to mortality or reductions in
growth of the victims (Peumans and Van Damme, 1995). The receptor sites found along
with the digestive tract of the nematodes and as such, their guts directly exposed to the
content of the diet. Thus the luminal side of the gut has potential binding sites for dietary
lectin.

5.5 Influence of lectins on biocontrol agents in the suppression of soil borne


pathogens in vitro

5.5.1 In vitro screening of bacterial and fungal antagonists against F. oxysporum S.


rolfsii and R. solani (without lectin)

In the light of present day constraints in plant disease management practices


especially those on the use of pesticides, biological control is increasingly occupying the
minds of scientists all over the world as they are eco-friendly and cost effective. In
recent years, the use of Trichoderma and Pseudomonas has gained more importance.
These antagonistic organisms act on the pathogen by different mechanisms, viz.
competition, lysis, antibiosis, siderophore production and hyperparasitism
(Vidyasekaran, 1999). Formulations of antagonistic organisms are available at cheaper

rate and these organisms once introduced into the soil survive for a longer period.
There is also circumstantial report that native antagonists are more efficient than
208

introduced antagonists (Kulkarni and Sagar, 2006). Use of bioagents, now a days, is
best and has been most emphasized and widely accepted practice as it is
environmentally safe and can overcome the residual problems associated with heavy
use of fungicides for the management of diseases. Hence, the present investigation was
taken up to screen the bioagents for effective management of root pathogens of tomato.

In the present study, maximum reduction in colony growth of S. rolfsii, R. solani


and F. oxysporum was observed in T. viride and P. fluorescens strains that were
significantly superior to all the bioagents tested. Species of Trichoderma, viz.
T. harzianum, T. viride, T. koningii, T. virens and P. liacinus showed higher mycelial
inhibition of organisms compared to bacterial antagonists. This can be attributed to
higher competitive ability. Similar results have also been reported by Sivan and Chet
(1986), Kavitha et al. (2004), Ramprasad (2005) and Shwetha (2011).

5.5.2 In vitro screening of SRL1 and RVL1 against fungal antagonists

5.5.2.1 Disc diffusion assay

RVL1 and SRL1 exerted antifungal action against fungal antagonists T. viride and
P. lilacinus. SRL1 strongly inhibited the growth of P. liacinus at the higher concentration of
4.8 mg/ml followed by RVL1. SRL1 and RVL1 were less and moderately effective against
T. viride with slight inhibition zone around the disc and was concentration dependent. The
specificity of the effect was indicated by the normal growth of the fungus in controls, BL21
or buffer. The antifungal properties of chitin-binding lectins have been a matter of
controversy eversince the report of Mirelman et al. (1975) on the inhibitory effect of WGA
on fungal growth of T. viride. It is possible that SRL1 and RVL1 protect the plants against
chitin containing phytopathogens. Lectins with sugar specificities which differ from that of
SRL1 and RVL1 may inhibit the growth of other soil microorganisms like Trichoderma or
P. lialcinus, the surface of which are covered by polysaccharides. SRL1 and RVL1 may
be small enough to penetrate through the fungal cell wall and reach the plasma
membrane, where they may have an effect on the active sites involved in cell-wall
morphogenesis or these lectins

might interfere with growth by binding or cross-linking newly synthesized chitin chains. In
this way the "steady-state" model of hyphal growth proposed by Wessels (1988) is
209

disturbed. Alternatively, the delicate balance between chitin synthesis and selective
hydrolysis of preformed chitin chains might be interrupted (Cabib 1987). The evidence
presented above indicates the binding of SRL1 and RVL1 to hyphal tips inhibits chitin
synthesis, hyphal growth and spore germination. Although the mechanism of apical
growth is poorly understood, it has been suggested that the addition of newly synthesized
chitin for extension of the hyphal cell wall and the onset of germination in spores, require
the cleavage of pre-existing chitin in the hyphal tip by selective lysis (Bartnicki-Garcia,
1969 and Mirelman et al., 1975).

5.5.2.2 Inhibition of fungal spore germination

Different concentrations of RVL1 and SRL1 displayed identical antifungal


activities on spores of T. viride and P. lialcinus. T. viride showed maximal inhibition of
spore germination by RVL1 between 24 and 48 h. Similarly, P. lilacinus showed less
inhibition by RVL1 followed by SRL1. Little is known about the mechanism of fungus-
growth inhibition by lectins. Unlike WGA, UDA does not display hydrolase activity and is
not able to lyse the cell walls of fungi that contain chitin (Mirelman et al., 1975;
Schlumbaum et al., 1986; Mauch et al., 1988; Broekaert et al., 1989; Sela- Buurlage et
al., 1996). It is suggested SRL1 and RVL1 can penetrate the cell wall and affect cell wall
synthesis because of their small size.

5.5.3 In vitro screening of plant fungal lectins against bacterial antagonists

5.5.3.1 Growth phase study of P. fluorescens and B. subtilis (without lectin)

There was a gradual increase in the growth of P. fluorescens and B. subtilis at


different intervals. Maximum growth was observed at 80 and 104 h after inoculatiom
with OD value with 1.968 and 1.963, after 80 and 104 h there was gradual decrease in
the growth of the bacterium. The bacterium starts utilizing the components of the media
and it will increase in its size and cellular mass. Manod (1949) reviewed the growth of
bacterial culture and mentioned quantitative characteristics of the growth cycle,

essentially the three growth constants i.e. total growth, exponential growth rate
and growth lag.
210

The study indicated that further increase in the incubation period resulted in
decrease in the growth of bacterium because of cell death or reduction in bacterial
multiplication due to exhaustion of nutrients in the medium after incubation for optimum
number of days.

5.5.3.2 In vitro screening of bacterial and fungal antagonists against Ralstonia


solanacearum

Native antagonists’ like T. harzianum, T. viride, T. koningii, T. virens, P.


fluorescens strains and B. subtilis strains were screened for their efficacy against the
growth of R. solanacearum with in vitro disc diffusion method. Among the different
antagonistic organisms, P. fluorescens strains were found superior over other
antagonists followed by B. subtilis. All other antagonists, viz. T. viride, T. koningii, T.
virens and T. harzianum, were found to have moderate efficacy. This was in conformity
with the results of Mallesh (2008) and Raghu (2011).

5.5.3.3 Determination of the minimum inhibitory concentration (MIC) and the


minimum bactericidal concentration (MBC)

SRL1 and RVL1 proteins showed interesting antibacterial activities which could
inhibit all the tested antagonistic bacterial strains. The bacterial inhibition activities
depended on bacterial strain. SRL1 and RVL1 lectin showed antibacterial activities
against different P. fluorescens strains (MIC 0.63 mg/ml) and B. subtilis strains (1.25
and 0.63 mg/ml), respectively, and MBC values: 2.56 to 3.14 and 1.94 to 3.08 mg/ml.
MBC which corresponds to the minimum concentration of the lectin capable to reduce
number of cfu for 0.1% of the initial inoculum: MBCs determined by agar diffusion were
higher than the ones obtained by MICs. A previous report (Sa et al., 2009a) has shown
that GlcNAc specific lectin from Myracrodruon urundeuva had a MIC and MBC of 0.58
and 8.1 µg/ml respectively for B. subtilis. These MIC and MBC values of SRL1 and
RVL1 are about four to five times higher than those MIC and MBC values of M.
urundeuva lectin, which shows a better antibacterial ability for B. subtilis. Similarly,
purified lectin EuniSL from seeds of Eugenia uniflora strongly inhibited the growth of

Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a MIC of


1.5 µg/ml: it moderately inhibited the growth of B. subtilis Streptococcus sp. and
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Escherichia coli with a MIC of 16.5 µg/ml (Oliveira et al., 2008). From the MIC and MBC
values obtained, it can be concluded that the ability to inhibit P. fluorescens and B.
subtilis strains was similar to Eugenia uniflora and M. urundeuva lectins. However,
crude extracts of SRL1 and RVL1 could inhibit P. fluorescens and B. subtilis better than
other lectins. In all cases, the MBC was always found to be similar or two-fold higher
than MIC values.

A good MIC and MBC value against the tested bacteria indicated that lectins
have a high potential for widespread applications. These lectins might be used for
control of plant pathogenic bacteria that are reported to be resistant to antagonists.

5.5.3.4 Inhibition zone assay

Inhibition zone with SRL1 and RVL1 against P. fluorescens and B. subtilis strains
confirmed the reports on utilization of EuniSL against B. subtilis, Pseudomonas
aeruginosa and Klebsiella sp. (Oliveira et al., 2008). In the diffusion test, SRL1 and
RVL1 showed an inhibition zone of 0.87 to 1.79 cm. Plant lectin RVL1 showed
maximum inhibition zone than fungal lectin SRL1. As reported in the literature,
variations can occur due to different concentration differences of antimicrobial proteins.
The results of the present study showed that SRL1 and RVL1 have similar antimicrobial
activity for P. fluorescens and B. subtilis strains which can be used for the biological
control of plant pathogenic bacterium.

5.5.3.5 Plant and fungal lectin activity against P. fluorescens and B. subtilis

The activity RVL1 and SRL1 was very similar to ethonolic extract of Zuccagnia
punctata which exhibited antibacterial activity against 47 strains of Gram positive and
Gram negative bacteria (Iris et al., 2005). This time-kill assay was performed to
determine the lectins are bactericidal. SRL1 and RVL1 exhibited bactericidal activities at
against P. fluorescens and B. subtilis for a limited period during exponential phase (96
h) of bacterial growth. Similar dynamic changes of bacterial surface during growth also
have been demonstrated by Alcaraz et al. (2000).

5.5.4 In vitro screening of bacterial and fungal antagonists in combination with lectins
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against R. solanacearum

Combination of antagonists with SRL1 and RVL1 lectins showed remarkable


antimicrobial activity on R. solanacearum. Results from the present study showed that
maximum inhibition of R. solancearum was recorded in RVL in combination with
bacterial antagonists (1.50 to 1.89 cm inhibition) followed by SRL1 (0.75 to 1.65 cm).

5.5.5 In vitro screening of bacterial and fungal antagonists in combination with lectins
against nematode

5.5.5.1 Evaluation of culture filtrates of bacterial antagonists in combination with


lectins against nematode

In the present study, in vitro bioassay with cell-free culture filtrates of four
P. fluorescens and two B. subtilis strains at different concentrations revealed an
increased juvenile mortality and egg hatching inhibition with increase in exposure period
as well as concentration. M. incognita juveniles and eggs were highly vulnerable to the
P. fluorescens and B. subtilis along with this combination of culture filtrates of bioagents
and lectins SRL1 and RVL1 at 4.8 mg/ml showed higher larvicidal and ovicidal action on
M. incognita juveniles and eggs. Reduction in mobility and viability of juveniles and eggs
of M. incognita are induced by secondary metabolites such as 2, 4-diacetylphlorglucinol,
pyrolnitrin, tropolone, pyocyanin, phenazines and lytic enzymes which are produced in
culture filtrates of P. fluorescens (Elsherif and Grossmann, 1996; Dunne et al., 1998).
Similar toxic property of P. fluorescens culture filterates was also reported on the
juvenile mortality of M. incognita and Heterodera cajani (Gokte and Swarup, 1988). The
juvenile mortality and egg hatching inhibition of M. incognita observed in the present
study might be due to antibiosis.

5.5.5.2 In vitro screening of fungal antagonists combination with lectins against


nematode

To infect J2 stage of the nematode, P. lilacinus and T. viride need to overcome


the cuticle of nematode. The cuticle is a non-cellular layer of the hypodermis, which
consists of keratin, collagen and fibers (Huang et al., 2004). Once the cuticle is
penetrated by fungal hyphae, the nematode gets paralyzed, invaded and digested
(Tunlid and Jansson, 1991). In this study, spore suspension of P. lilacinus and T. viride
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in combination of SRL1 and RVL1 lectins demonstrated high mortality effect on


juveniles and inhibition of egg hatching compared to P. lilacinus-alone and T. viride-
alone showed less egg hatching inhibition and juvenile mortality. P. lilacinus an egg
parasitic fungus and Trichoderma produces chitinase and proteolytic enzymes which
dissolves the egg shell of the nematode and macerates inner content. This confirmed
the findings of Sun et al. (2006) who reported a low average J2 mortality percentage of
16% for their 186 P. lilacinus isolates. It also agrees with the reports of Jatala (1986),
Bonants et al. (1995), Singh and Mathur (2010) that P. lilacinus primarily colonized eggs
but not juveniles. Similarly, Al-Kader (2008) reported a high nematicidal effect of their P.
lilacinus culture filtrate on J2 of M. incognita, with 99% of J2 immobilized after 2 days of
treatment. It is suggested that P. lilacinus and T. viride in combination of SRL1 and
RVL1 exhibit nematicidal effect on eggs and juveniles.

5.6 Evaluation srl1 and rvl1 transgenics and non-transgenics for the suppression of
tomato soil-borne pathogens

5.6.1 Expression analysis of SRL1 and RVL1

Expression of srl1 and rvl1 in transgenic tomato was checked by


haemagglutination assay. The lectin-induced agglutination of cells has originally served
as the most common assay to detect and quantify lectin activity in a variety of
organisms (Vlodavsky and Sachs, 1975; Doyle et al., 1984; Goldhar, 1995). Protein
from the crude extract of both the events could agglutinate rabbit erythrocytes,
indicating lectin activity in transgenic plants. Non-transformed control plants did not
show haemagglitnation. Minimum concentration of protein required for agglutination
(MCA) was varied from 15 to 55.0 µg and 12.5 to 55.0 µg rvl1 and srl1 transgenic lines
respectively. The crude extract of protein obtained from rvl1 and srl1 transgenic tomato
plants contained a total lectin activity of 0.40 to 1.60 x 103, whereas srl1-transgenic
plant contained 0.40 to 1.61 x 103. The specific activity was 0.18 to 0.40 x 102 units for
RVL1 and 0.18 to 0.76 x 102 units for SRL1. Both srl1 and rvl1 transgenic lines showed
an expected band of 16 and 26.2 kDa corresponding to the molecular mass of srl1 and
rvl1, which was absent in the control sample.
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5.6.2 Evaluation of rvl1 and srl1 transgenic and non-transgenic tomato lines
against individual and combination of vascular pathogens of tomato under
glasshouse conditions

Synthetic chemical fungicides and bacteriacides are widely used against fungus
and bacteria. However, such chemicals can cause a series of problems including
environment pollution and long-term residue issues. As a result present attention has
been to develop biological control approaches (Siddiqui and Mahmood, 1996).
Developing transgenic plants expressing heterologous proteins with fungicdal and
bactericidal activity is considered important for reducing crop losses.

Canola plants carrying the 35S-chitinase-gene exhibit increased resistance to


infection by the root and stem rot pathogen, Rhizoctonia solani Seedlings constitutively
expressing the bean chitinase gene showed delayed development and progression of
disease symptom (Broglie et al., 1991). Constitutive expression of tomato chitinase
gene under CaMV 35S promoter provides field tolerance to canola against
Cylindrosporium concentricum, Phoma lingam and Sclerotinia sclerotiorum (Grison et
al., 1996). Hevein lectin gene carrying Brassica juncea plants showed increased
resistance to Alternaria brassicae infection (Kanrar et al., 2002). However there are no
reports so far, on the use of transgenic technology to control wilt complex. In present
investigation srl1 and rvl1 genes expressed tomato plants evaluated to determine the
effectiveness of lectins against F. oxysporum, R. solanacearum and M. incognita

Pot culture tests were conducted to know the interaction among the pathogens
using srl1 and rvl1 transgenic tomato plants representing different events. In the present
investigation, eight homozygous T3 generation transgenic events of tomato expressing
SRL1 and RVL1 were evaluated for resistance to tomato wilt pathogens. F. oxysporum,
R. solanacearum and M. incognita are potential pathogens. Due to lectin activity in
transgenic plants severity of the disease of fore mentioned pathogens was reduced.
The plants inoculated simultaneously with the pathogens F. oxysporum + R.
solanacearum + M. incognita recorded high disease severity and pathogen population in
soil when compared with other combinations. Interaction of Meloidogne with Fusarium
and Ralstonia lead to synergistic effect. All the organisms were pathogenic in
independent inoculations. But combined infection resulted in rapid
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drying of the shoot followed by root in control, which was further protected for long time
from infection and disease development in transgenic plants.

The reduction in number of root-galls, decrease in soil nematode population, egg


masses per root system female fecundity, Pf and Rf in SRl1 and RVl1 where Fusarium
and Ralstonia were also present along with nematode, suggests that Fusarium and
Ralstonia were inhibitory to nematode multiplication. Similar type of reduced root-knot
index in the presence of wilt fungi had been reported by Powell (1971) and Pathak et al.
(1999). This is probably due to the adverse effect on nematode penetration and direct
invasion of giant cells by the fungi and bacteria disrupting nematode feeding and
subsequent reproduction. Disease severity was significantly higher in simultaneous
inoculation with nematode and fungi or bacteria. This might be due to the predisposition
of plants by the nematode to the fungus and bacterium. It was evident that the
nematode acted as a predisposing factor for entry of the pathogen by causing injury on
the root surface as well as weakened the root tissues by causing rotting or lesions,
thereby the soil-borne pathogens had easy access for causing greater damage as
reported by Schindler et al. (1960).

Aggrevation of disease was due to pre-infection by nematode which predisposes


the crop for secondary infection. This is in conformity with the findings of Mani and Sethi
(1987) who suggested that the nematode provide a congenial atmosphere either by
facilitating the entry of fungi or bacteria, into roots through the openings or modifying the
substrate or by producing stimulants in the form of secretions which help the
multiplication of fungi. Sumer Jan and Khan (2002) reported that the high incidence in
the presence of nematode might be due to the physiological changes in the host
induced by the nematode infection and resulting in accumulation of carbohydrates and
amino acids. Such an environment would be favourable to fungal and bacterial growth
(Borchers and Wyss, 1981).

There are no reports so far, however, of the use of lectin gene-expressed


transgenic plants to control Fusarium, Ralstonia and Meloidogyne causing wilt complex.
Present study attempted the effectiveness of SRL1 and RVL1 transgenic tomato plants
against individual combined infections by Fusarium, Ralstonia and Meloidogyne. There
is a need to develop lectins mediated resistance against other vascular plant pathogens
216

besides Fusarium and Ralstonia due to higher plant growth parameters and lesser
disease severity observed in srl1 and rvl1 transgenic plants.

5.6.3 Assay of defense enzymes

Plants exhibit a variety of responses during infection by pathogens, which involve


the activation of host defense genes. Activation of these genes leads to physical and
biochemical changes in plant cells which are not favorable for damage progress in
plant. Among the major biochemical changes, is biosynthesis and the accumulation of
inducible defense related proteins. Most of these proteins correspond to pathogenesis-
related proteins. These proteins are mostly of low molecular weight, preferentially
extracted at low pH, resistant to proteolysis, and localized predominantly in the
intercellular spaces of leaves. The defense protection afforded by augmentation of
PPO-based phenolic oxidation, in combination with other pathogen recognition-
independent resistance genes, may provide valuable enhancement of plant quantitative
defense resistance.

Expression of defense related enzymes in srl1 and rvl1 transgenic plants


challenged with Fusarium, Ralstonia and Meloidogyne. This study was primarily focused
for the defense related proteins, viz. PO, PPO and phenols. The results revealed that
there was significant increase in the activity of PO, PPO and total phenolic contents in
SRL1 and RVL1 transgenic. Similar studies which showed an over expression PPO
activity nptII gene expressed transgenic tomato leads to Pseudomonas syringae pv.
tomato resistance were reported by John (2002). Although the transgenic plants
expressing defense enzymes dramatically altered both the developmental profile and
the levels of leaf PO and PPO, the expression and response patterns of endogenous
PO and PPOs in tomato suggest the involvement of these enzymes in disease
resistance responses.

5.7 Evaluation of rvl1 and srl1 transgenics and non-transgenics for root infection
by M. incognita and its development and reproduction

Ever since Marban-Mendoza et al. (1987) provided the first indication that ConA
may have anti-nematode activity on root-knot nematode M. incognita, several
217

reports have shown that lectins could be used to develop transgenics with the
objective of building resistance to nematodes. In fact, Leal-Bertioli (2003) observed that
transgenic tobacco plants that were expressing tarin1 a mannose-binding lectin from
Colocasia esculenta were on par with the control plants in inhibiting the reproduction
and root damage caused by M. javanica. However, Arabidopsis plants expressing GNA
were highly tolerant to root-knot nematode (M. incognita). This variable toxicity of
different lectins is attributable to amino acid differences leading to altered sugar
specificity, the differences in the receptors found in different species of Meloidogyne
(incognita or javanica) and the transformation event itself. Constitutive expression of a
snowdrop lectin (GNA) directed by the CaMV 35S promoter has been used to assess
antinematode activity in oilseed rape, potato and Arabidopsis (Burrows and de Waele,
1997; Burrows et al., 1998 and Ripoll et al., 2003). In the present investigation, eight
transgenic tomato lines expressing rvl1 and srl1 and non-transformed control plants
were evaluated for nematode resistance. Toxicity effects of transgenic tomato on
infection and development of J2 of M. incognita was checked. J2 invaded RVL1-T0 (20)
(12.33, 24.67, 50.6 and 70.67) and SRL1-T0(116) (8.33, 18.33, 41.00 and 61.67) rapidly
and significantly highest at one, three, five and seven dai than other events. Infective
second stage juveniles were observed only in the cortex of tomato roots and some of
the roots showed that juveniles had entered only partially into the root tissue. Several
juveniles had penetrated the root tips and oriented in various directions vertical or
parallel to the longitudinal axis of the root.

Post-embryonic development of M. incognita (200 J2) was observed at 7, 11, 15,


21 and 25 dai. Highest number of sausage shape juveniles significantly varied from 9.67
to 25.67 in RVL1-T0(20) and 9.33 to 24.67 in SRL1-T0(116) after 7, 11 and 15 days after
inoculation over the control. Whereas, number of female nematodes or adults varied
from 7.33 to 44.33 and 5.33 to 41.67 in SRL1-T0(116) and RVL1-T0(20) at 21, 25, 30,
and 45 dai.

Rate of infection may not be influenced by toxicity of rvl1 and srl1, however, the
development and gall formation could be determined by srl1 and rvl1. Hence, gall index
was scored in transgenics after 45 days of nematode inoculation. Based on the 0 to 10
scale (Bridge and Page, 1980), average root gall index was 3 to 5 (30-50% moderately
resistant) for all the transgenic srl1 and rvl1 transgenic events as compared to gall index
218

of (% susceptible) for the control plants. Transformed plants showed significantly


reduced root-knot index which was accounted to be 3.67 and 4.33 in SRL1-T0(21) and
RVL1-T0(28) event followed by SRL1-T0(7) (4.33) and RVL1-T0(2) and (11) of 5.33. In
the first effort to develop root-knot resistant transgenic tomato with srl1 and rvl1, Bhagat
(2010) reported 30% gall formation in transgenics as against 68% in control plants.
Complete mortality (100%) of banana root-knot nematode was reported with crude and
purified lectins from mature Jatropha curcas seed embryos (Abd-ElMaksoud et al.,
2005). A previous study indicated that more than 65% of the transgenic plum (Prunus
domestica var. ‘Stanley’) lines expressing Gastrodia elata anti-fungal protein (GAFP), a
monocot mannose-binding lectin (MMBL) had less than 10% of gall formation, against
>35% plants showing 26-50% gall formation in the non-transformed control (Nagel,
2011).

Generally, nematode infection and gall formation leads to stunted plant growth
and reduced biomass. In the present study, significantly higher shoot and root biomass
(fresh and dry weight) was observed for SRL1-T0(21) (60.20 and 21.25 cm) and RVL1-
T0(28) (48.63 and 19.20 cm) compared to control plant. SRL1-T0(21) and RVL1-T0(28)
showed only marginally higher biomass compared to control plant and also these
events significantly reduced the nematode population in soil.

Interestingly, it was found that srl1 and rvl1 gene expressed plants did not affect
J2 penetration into roots and there was no evidence of early HR. Significantly, more
infection sites were found on RVL1-T0(20) and SRL1-T0(116) events infected by M.
incognita at 7 dai (days after inoculation) than in SRL1-T0(21) and RVL1-T0(28).

In this study, giant cell formation was well marked in the vascular region of both
SRL1 and RVl1 transgenic lines rather than in the cortex. 3 to 9 Giant cells observed at
25 dai in all transformed and control plants were thick-walled and had dense cytoplasm.
Vacuolated giant cells also were observed in the same region. Taylor (1976) observed
giant cells mostly in the region of phloem and few in the xylem and cortical tissues and
noted thin walls in the giant cells. Observations of this study were in agreement with
those of Siddiqui and Taylor (1970), who observed thick walled giant cells n the
vascular region of wheat and oat roots infected by M. naasi. Tandon and Praveen
219

Kumar (1979) observed clusters of giant cells, only in the vascular region and not in the
cortical tissue of tomato parasitized by M. lucknowica. In fact, the nematodes were able
to initiate and maintain healthy giant cells in resistant roots for about 21 days before
visible signs of deterioration occurred especially vacuolation and cell wall thinning,
leading to giant cell collapse. Tracking the movement of nematodes and formation of
giant cells within such plant is expected to reveal the nature/mechanism of rest to
nematode. The formation of giant cells in the proximity of each nematode causes
aberration of the vascular region. Consequently, the vascular region of smaller roots
were badly damaged due to the giant cell formation.

Though lectins-based resistance has been studied genetically and has been
used extensively in breeding, little was known about mechanism of lectin mediated
resistance. Mechanism of SRL1 and RVL1 gene mediated resistance in plants have
been reported (Bhagat 2010; Patil, 2011; Kolekar, 2012), post infection resistance in
which nematodes are able to penetrate roots but fail to develop. Post infection
resistance is often associated with an early hypersensitive reaction (HR) meadiated cell
death, in which rapid localized cell death in root tissue around the nematode prevents
formation of a developed feding site, leading to resistance. Tomato (Dropkin, 1969;
Williamson, 1999) plants that are resistant to show typical HR upon avirulent M.
incognita infection observed as early as 24 h post inoculation. Whereas in soybean,
pepper and coffee the HR was visible at one to six day after inoculation (Kaplan et al.,
1979; Pegard et al., 2005; Anthony et al., 2005).

M. incognita showed a remarkable interaction with SRL1 transgenic events with


significantly greater numbers of penetrating J2, similar J2 development, eggmass
production and lower Pf and Rf than RVL1 transgenic events. The specificity of this
interaction for transgenics and root-knot nematode is not presently known but deserves
further exploration since it resulted in increased J2 penetration, reduced giant cell
formation, and reduced female fecundity and Pf. This is an interesting combination of
effects that might be of utility for the sustainable management of nematode infestation.
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5.8 Mechanism of action of lectins in the suppression of soil- borne pathogens

5.8.1 Lectin-binding assay for fungus

Plant lectins can neither bind to glycoconjugates on the fungal membranes nor
penetrate the cytoplasm owing to the cell wall barrier (Horisberger and Vonlanthen,
1977). However, there may be indirect effects produced by the binding of lectins to
carbohydrates on the fungal cell wall surface of S. rolfsii, R. solani, F. oxysporum, T.
viride and P. lilacinus. Chitinase-free chitin-binding stinging nettle (Urtica dioica lectin)
impeded fungal growth. Cell wall synthesis was interrupted because of attenuated chitin
synthesis and/or deposition (Van Parijs et al., 1991; Broekaert et al., 1992).
Selitrennikoff (2001), reported that polysaccharide chitin is constituent of fungi cell wall
and chitin-binding lectins showed antifungal activity; impairment of synthesis and/or
deposition of chitin in cell wall may be the reasons of antifungal action. Inhibition of
fungal growth by the action of lectins appears to be due to inhibition of spore
germination as well as the growth of the mycelium. The exact mechanism of action has
not yet been elucidated.

In present investigation, it was observed that SRL1 and RVL1 lectins bound to
fungal cell, possibly due to modification of fungal membrane structure. It was found that
RVL1 and SRL1 lectin bound primarily to hyphal tips and septa. Moreover, exposure of
fungal mycelium to FITC-RVL1 and SRL1 inhibited hyphal extention, leading to the
suggestion that lectins from plant tuber and fungi have a role in protecting the soil
pathogens (Mirelman et al., 1975).

FITC-RVL1 was found to bind to the hyphal walls of S. rolfsii, R. solani, F.


oxysporum, T. viride and P. lilacinus and the pattern of binding was similar in all cases-a
rather uniform coverage of the hyphal walls was observed with little or no binding to
septa and a conspicuous low binding at the hyphal tips. A significant finding was the
strong binding of SRL1 and RVL1 lectins in braching and septa R. solani and S. rolfsii.
The pattern of FITC-RVL1 binding to the hyphal wall F. oxysporum was similar to some
extent to the FITC-SRL1 and anti-SRL1. RVL1 also bound to chlamydospores, micro
and macroconidia. Similarly, RVL1 and SRL1 binding to hyphal wall, septum,
221

spore and phialides wall of T. viride. RVL1 strongly bound over the entire surface of the
spores and phialides on the other hand, bound in descrete patches over the whole
mycelium than SRL1. The observations are in agreement with the specific carbohydrate
metabolism of lichens revealed by Galun et al. (1976) indicating that the use of FITC-
conjugated lectins is a useful tool for probing hyphal wall components. The lack of WGA
binding to the chitin-less Phytophthora strongly supports the above indication.

The interpretation of binding patterns obtained to all fungi with FITC-RVL1 and
SRL1 is somewhat similar. But it also indicated that there were obvious differences
among the fungi tested in respect to hyphal wall structure. These lectins directly inhibit
fungal growth by modifying fungal membrane structure and or permeability. The lack of
RVL1 and SRL1 binding to any of the fungi serves as a negative control, indicating that
a lectin which binds specifically to a certain charbohydrates complex, sugar monomers.
It is therefore hoped that in future the availability of additional lectins with restricted
binding specificities will permit further characterization of the hyphal walls of pathogenic
fungi.

5.8.2 Immunolocaliztion studies

Expression of lectin activity at different stages of growth by S. rolfsii, was similar


to the observations (studied by immunolocalization) of Barak et al., 1985; Barak and
Chet, 1990; Swamy et al., 2001. These results together revealed that SRL1 is formed
initially on the young hyphae and very high levels accumulated rapidly at the time of
sclerotial body formation. Thus, SRL1 is developmental stage specific lectin secreted by
S. rolfsii and it is well known that S. rolfsii produces sclerotial bodies under stress,
hence it may be concluded that SRL1 is expressed in response to stress. Probably the
lectin secreted will help the mycelial filaments to cross link and to form sclerotial bodies.

Germination of sclerotial bodies is another event in the development of the


fungus, which also involves the role of the lectin as shown by lectin capping studies. In
order to investigate the involvement of lectin in growth of the fungus, sclerotia of R.
solani and S. rolfsii were examined after capping the lectin sites by anti-SRL under
fluorescent microscopy. Immature sclerotial bodies in the final stage of formation was
associated with highly branched mycelia around showing dense mass of congregated
222

lectin sites arising due to aggregation of mycelium in addition to weaker fluorescence on


non aggregated regions. In the completely matured sclerotial bodies and
microscleroatia, intense uniform fluorescent label was seen over the entire surface of
the mature sclerotial bodies, revealing uniform distribution of lectin at very high levels
compared to vegetative mycelia. These findings are in confirmity with Swamy et al.
(2004) about the binding of anti-SRL1 on vegetative hyphae and sclerotial bodies.

5.8.3 Staining of nematode secretions

Secretions of plant parasitic nematodes have many functions. In sedentary


species they take part in the infection of roots and the induction and maintenance of
feeding structures (Coomans and De Grisse, 1981). Amphidial secretions wash and
protect the dendrites of the sensillae and are believed to be responsible for the capture
of chemotactic compounds and their transport to the membranes of the sensillae
(Zuckerman, 1983; Zuckerman and Jansson, 1984). The secretory system is often
thought to be involved in the production of the glycocalyx. This surface layer is a
lubricant when nematodes move through the soil, and supplies the nematode body with
recognition domains, important in the interactions between nematodes and parasitic
micro-organisms and between nematodes and host plants. Moreover, the glycocalyx is
regularly renewed to avoid recognition and the induction of resistance in the host plant
(Bird et al., 1988). In the experiments with CBB-R, only localised staining at the different
body pores was observed, instead of all over the body surface. These findings confirm
with Spiegel and McClure, 1995; Spiegel, et al., 1997; Wuyts, et al., 2009 about the
involvement of the secretory system in the secretion of the glycocalyx.

5.8.4 Lectin-binding assay for M. incognita

Lectins are generally used in nematology to identify sugar moieties on the


surface or in secretions of nematodes. For the past twenty years, research has been
focused on the free-living nematode, C. elegans and the sedentary endoparasitic
nematodes of plants, Meloidogyne spp., Globodera spp. and Heterodera spp. Only
Robertson et al. (1989) included migratory nematodes, such as Radopholus similis, in
lectin-binding experiments. They observed no binding of ConA to R. similis, in contrast
to the results demonstrated. Binding of SRL1 and RVL1 in nematode was found at head
region (amphids), mid gut of the alimentary-tract, excretory pore which extended to the
223

posterior region (phasmids). Wuyts, et al. (2009) showed binding of ConA and WGA in
the head region, at the excretion pore, the pores of the reproduction system and
phasmids of R. similis. WGA binds to the amphids or the head region in general, and to
the excretion pore. Aumann and Wyss (1989) mentioned the binding of ConA, WGA
and HPA to the spicule pores of H. schachtii. Coomans and De Grisse (1981) described
the spicules as chemoreceptors that produce secretions that can be labelled by lectins.
These chemoreceptors are, however, used only to test the surroundings of the vagina.
In long-distance migration towards a female nematode, sex pheromones are detected
by the amphids.

Binding of E. coli-expressed RVL1 and purified SRL1 in nematodes was


observed by fluorescent Microscopy. Both SRL1 and RVL1 lectin binding was found at a
specific region mostly in the mid portion of the alimentary-tract (either at anterior or
posterior regions of the gut), the amphids, excretory pore and posterior region. Binding
was not observed in nematode gut incubated with FITC-RVL complexed with mucin,
which confirmed carbohydrate receptor mediated binding of RVL on the gut epithelial
cells. Although nematicidal properties of few lectins are reported, there are no direct
evidences available for the mechanisms of their toxic effects. Earlier reports showed
cuticular binding of lectins specifically to the head region, and to the pores of excretion
and reproduction of different nematodes by plant lectins (McClure and Zuckerman,
1982; Jansson et al., 1986; Davis et al., 1988; Forrest et al., 1988 and Aumann et al.,
1991). It is argued that the lectin binding inhibits the chemoreception of host signals
disturbing the localization of hosts by nematodes. In contrast, lectin purified from the
tubers of R. vivapra could bind to a specific region in the mid portion of the alimentary-
tract and also at anterior or posterior regions of the gut while causing the toxicity effect
on M. incognita as determined by its binding after incubating the nematodes in FITC
conjugated lectin solution. Similar binding was observed with SRL1 and binding was not
observed in nematode gut incubated with FITC-lectin complexed with mucin, confirming
carbohydrate receptor mediated binding of the lectin on the gut epithelial cells (Bhat et
al., 2010a). This study for the first time, demonstrated that the lectin ingested by the
nematodes specifically interacted across the gut lining and the binding increased
proportionally with time leading to death of the nematode. However, the
224

present investigations could be mediated by complex processes involving signal


transduction or by causing abnormalities in the epithelial cells.

Future line of work

1. Identification of receptor sites for SRL1 and RVL1 involved in suppression of soil-
borne pathogens.

2. Detailed study for mode of toxicity of SRL1 and RVL1 on plant pathogenic fungi
and bacteria.

3. Screening of SRL1 and RVL1 against other plant pathogenic fungi, bacteria and
nematodes.

4. Detailed study on histopathological and histochemical changes in srl1 and rvl1


expressed transgenic tomato plants as influenced by root-knot nematode.

5. Evaluation of srl1 and rvl1 expressed transgenic tomato plants with other soil-
borne pathogens
6. SUMMARY AND CONCLUSIONS

The present investigation was undertaken during the period from 2010-2013 at
the Department of Plant Pathology as well as Department of Biotechnology,
University of Agricultural. Sciences, Dharwad, which included determining the efficacy
of E. coli-expressed SRL1 (a fungal lectin) and RVL1 (a plant lectin) against soil-
borne pathogens affecting tomato also against some fungal and bacterial antagonists.
The specific objectives were to screen the srl1 and rvl1 expressing transgenic tomato
plants of different events against species of Fusarium, Ralstonia and Meloidogyne
causing wilt complex in tomato and to elucidate their mechanisms of biocontrol. The
salient features of the findings are summarized below:

Soil-borne pathogens affected tomato samples were collected from fields of


Main Agricultural Resarch station, UAS, Dharwad and were isolated. Cultural and
morphological characters of soil-borne pathogens isolated such as Fusarium
oxysporum, Sclerotium rolfsii, Rhizoctonia solani, Ralstonia solancearum and
Meloidogyne incognita were established.

Expression of SRL1and RVL1 was checked with total protein isolated from
induced E. coli BL21(DE3)pLysS cells carrying respective recombinant expression
vectors. Recombinant E. coli clones had a total protein content of 12 mg/ml compared
to 10.00 mg/ml in control. Both RVL1 and SRL1 proteins showed 28.2 kDa and 19.5
kDa bands, respectively on SDS-PAGE.

RVL1 and SRL1 showed haemagglutination with rabbit erythrocytes, whereas


control did not. Minimum concentration of protein required for agglutination (MCA)
was 2.73 µg and 1.73 µg, respectively for RVL1 and SRL1. RVL1 contained a total
activity of 0.43 ×104, whereas SRL1 contained 0.87 × 104. The specific activity was
determined to be 3.66 × 102 units for RVL1 and 7.29 × 102 units for SRL1.

E. coli expressed SRl1 lectin was purified through His-tag purification kit.
Purified SRL1 showed 19.5 kDa amplicon on SDS-PAGE. A polyclonal antibody was
raised against SRL1 by immunizing rabbits with pure antigen (SRL1). The specificity
of antiserum produced against SRL1 was tested by DAC- ELISA. It was sensitive and
precise enough to detect the antigen up to 65536 dilutions.
226

Antifungal activity of SRL1, anti-SRL1 and RVL1 against F. oxysporum,


S. rolfsii and R. solani was tested by employing poisoned food technique, spread
plate, inhibition zone assays and spore or sclerotia germination method. Inhibition of
fungi was observed with increase in concentration of plant and fungal lectins.

RVL1 showed maximum per cent inhibition of mycelial growth of F. oxysporum


compared to SRL1 at 4.8 mg/ml in all three assays (poisoned food, spread plate and
inhibition zone). RVL1 also significantly inhibited (55.26%) the germination of macro
and microconidia.

Incorporating RVL1 and SRL1 to medium showed early formation of sclerotial


bodies rather than serving as reserve storage protein. Germination of sclerotial
bodies was strongly inhibited by SRL1 and RVL1 lectins. anti-SRLa showed
maximum inhibition of mycelial growth in spread plate and disc diffusion method as
compared to RVL1. No inhibition of mycelial growth of R. solani was observed when
lectins were incorporated into the growing medium. But in spread plate method and
disc diffusion method RVL1 showed maximum inhibition of mycelial growth compared
to SRL1 and anti-SRL1 because by in this method lectins directly act on hyphal
growth.

RVL1 and SRL1 were evaluated for antibacterial activity against Ralstonia
solanacearum by well diffusion and disc diffusion assay. RVL1 was found to be more
potent against the bacterium compared to SRL1. The antibacterial efficacy of the
RVL1 and SRL1 was found to be moderate against R. solanacearum. MIC which
corresponds to the minimum lectin concentration capable of inhibiting the visible
growth of the R. solanacearum was 0.625 mg/ml for both SRL1 and RVL1 with 1.87
and 1.56 mg/ml of MBC which corresponded to the minimum concentration of the
lectin capable of reducing the number of cfu for 0.1% of the initial inocula.

Interaction effect of lectins and concentrations showed RVL1 (3.6 mg/ml) was
significantly superior to SRL1 with an inhibition zone of 2.13 cm. RVL1 and SRL1
gradually reduced the growth of R. solanacearum from 1.225 and 1.146 to 0.865 and
0.849 OD, respectively.

E. coli-expressed RVL1 and SRL1 possessed growth inhibitory activity on


Meloidogyne incognita. Bioassay conducted using different dilutions of 12.00 mg total
227

protein, showed maximum egg hatching inhibition and juvenile mortality after 48 h of
incubation at 1: 25 dilution. However, other dilutions (1: 50, 1: 75 and 1: 100) also
showed high mortality and egg hatching inhibition.

Efficacy of six bacterial and five fungal antagonists was studied against
F. oxysporum, S. rolfsii and R. solani. Among eleven bioagents, T. viride and
P. fluorescens strains were found significantly superior in inhibiting the growth of
pathogens.

Trichoderma sp. and Paecilomyces lilacinus were more sensitive to SRL1


compared to RVL1. SRL1 and RVL1 showed maximum inhibition zone in case of P.
lilacinus and maximum inhibition of T. viride spore germination.

Among eleven bacterial and fungal antagonists evaluated against


R. solancearum, P. fluorescens and B. subtilis strains were found superior over other
fungal antagonists viz. T. viride, T. koningii, T. virens and T. harzianum.

RVL1 and SRL1 were evaluated against P. fluorescens and B. subtilis strains
by disc diffusion assay. MIC and MBC values were determined. SRL1 and RVL1
showed good activity on P. fluorescens and B. subtillis strains with MIC of 0.63 to
2.50 and 0.63 to 1.25 mg/ml and MBC of 1.94 to 3.14 and 1.78 to 3.08 mg/ml.

SRL1 and RVL1 were found to possess broad spectrum activity against all
tested bacterial strains with inhibition zones in the range of 0.87 to 1.79 cm. RVL1
showed maximum inihibition zone against P. fluorescens strains than B. subtilis
strains. Sensitivity of SRL1 and RVL1 on P. fluorescens (1.227 and 1.018 OD) and B.
subtilis (1.635 and 1.452 OD) growth was gradually reduced.

Combination of lectins and antagonists were evaluated against R.


solancearum. Results showed that inhibition of R. solancearum was maximum in
RVL1 with bacterial antagonists followed by SRL1 with bacterial antagonists.

Culture filtrates of bacterial antagonists, fungal antagonists and combination of


SRL1 and RVL1 showed greater egg hatching inhibition and juvenile mortality.
228

T3 genertaion of homozygous transgenic tomato events carrying srl1 and rvl1


genes were expressed and screened against F. oxysporum, R. solancearum and M.
incognita individually and in their combinations. The crude extract of lectin from
leaves of transgenic tomato plants carrying srl1 and rvl1 was 4.5 to 7.00 mg/10 ml
and 5.00 to 7.00 mg/10 ml respectively compared to 5.4 mg/10 ml in control plant.
Crude proteins of srl1 and rvl1-transgenic tomato showed 16 kDa and 26.2 kDa band
on SDS-PAGE.

Crude protein extracts of rvl1- and srl1-transgenic tomato plants showed


haemagglutination with rabbit erythrocytes, whereas control plants did not show any
activity. Minimum concentration of protein required (from crude sample) for
agglutination (MCA) was 15 to 55.0 µg and 12.5 to 55.0 µg, respectively for RVL1 and
SRL1.

The crude extract of protein obtained from rvl1 transgenic tomato plants
contained a total lectin activity of 0.40 to 1.60 x 103, whereas srl1-transgenic plant
contained 0.40 to 1.61 x 103. The specific activity was 0.18 to 0.40 x 102 units for rvl1
and 0.18 to 0.76 x 102 units for srl1.

Efficacy of eight PCR confirmed rvl1 and srl1 transgenic events for
suppression of vascular pathogens and their interactions were studied. Results
indicated a significant increase in plant height and biomass with less disease severity
and root-knot index in SRL1-T0(21) and RVL1-T0(28) events.

srl1 and rvl1 transgenic tomato plants, challenged with Fusarium, Ralstonia
and Meloidogyne individually and in combinations showed higher defense enzymes
activities of peroxidase, polyphenol oxidase, and total phenol.

Toxicity effects of RVL1 and SRL1 transgenic tomato lines on infection and
development of J2 of M. incognita was observed. Infective second stage juveniles
were observed only in the cortex of tomato roots, and some of the roots showed that
juveniles had entered only partially into the root tissue. Highest number of sausage
shaped juveniles varied significantly from 9.67 to 25.67 in RVL1-T0(20) and 9.33 to
24.67 in SRL1-T0(116) after 7, 11 and 15 dai observed in root tissue.

The histopathological and histochemical studies revealed that giant cells


induced by nematode originated in the pericycle and endodermis, adjacent to cortex.
229

It was found that giant cells wall fragments are thickend and had dense cytoplasm.
Also, multinucleate condition was observed.

Plant height and plant biomass significantly increased with less gall index in
SRL1-T0(21) and RVL1-T0(28) transgenic events. In general, control plants showed
the gall index of 9 which fell under the highly susceptible category, whereas
transgenic lines were classified as moderately resistant with a gall index of 3 to 5.

Elucidation of mode of action of SRL1, RVL1, anti-SRL1 on soil


microorganisms was attempted in order to get an insight into the mode of the lectins
toxicity effect on S. rolfsii, R. solani, F. oxysporum, T. viride and P. lilacinus and M.
incognita juvenile. Binding of lectins after incubating the fungus mycelium, spores and
juveniles in FITC-RVL1 solution was noticed. SRL1 and RVL1 bound to all fungi,
primarily to the hyphal wall and septa and to a lesser extent, lined to the hyphal tips
and weak fluorescence was exhibited in F. oxysporum and T.viride spores.

Localization studies of anti-SRL1 were made in mature and immature sclerotial


bodies of S. rolfsii and R. solani. Localization of lectins showed dense mass of
congregated lectin sites arising due to aggregation of mycelium in addition to weaker
fluorescence on non aggregated regions. And it also showed extensive binding at
branches and septum of the R. solani and also to wall and tip of the hyphae seen with
uniform fluorescence densely localized inside the hyphae.

Secretions of M. incognita were stained and visualised with the protein-specific


dye, Coomassie Brilliant Blue R. Secretions appeared at the amphids, the excretion
pore and the phasmids.

Binding of SRL1 and RVL1 lectins in nematode was found at head region, mid
gut of the alimentary-tract, excretory pore and extended to the posterior regions
(phasmids). But the principal lectin-binding sites were the pores or the secretions of
the amphids and excretory pore. Binding was not observed in nematode gut
incubated with FITC-RVL1 complexed with mucin and FITC-BL21 (bacterial control).

This is the first report on the antifungal and antibacterial activity of RVL1 and
SRL1. However, further study is required to evaluate the toxicity of these compounds.
230

Conclusions

 Soil-borne pathogens (F. oxysporum, S. rolfsii, R. solani, R. solancearum and


M. incognita) affected tomato samples were collected from MARS, UAS,
Dharwad and were isolated.

 Expression of SRL1 and RVL1 was checked with total protein isolated from
induced E. coli BL21(DE3) pLysS.

 E. coli expressed RVL1 and SRL1 showed 28.2 kDa and 19.5 kDa amplicons
on SDS-PAGE. SRL1 was purifeid through His-tag purification kit.

 RVL1 and SRL1 showed heamgglutination with trypsinized rabbit erythrocytes.

 Polyclonal antibody was raised against SRL1 by immunizing rabbits with pure
antigen (SRL1) and the specificity of the antiserum (anti-SRL1) was
determined by DAC-ELISA.

 Evaluation of different concentration of SRL1, anti-SRL1 and RVL1 against F.


oxysporum, S. rolfsii, R. solani, R. solancearum and M. incognita by employing
different methods showed maximum inhibition.

 SRL1 and RVL1 are more potent inhibitor for soil-borne pathogens.

 Among eleven fungal and bacterial antagonist evaluated against soil-borne


pathogens, Trichoderma spp. and P. fluorescens strains found to be effective
against all soil-borne pathogens.

 Combination of lectins and antagonists was found to be effective in


suppressing the growth of soil-borne pathogens.

 SRL1 and RVL1 also showed inhibition agaisnts fungal and bacterial
antagonists.

 T3 genertaion of homozygous eight transgenic tomato events carrying srl1 and


rvl1 genes showed moderately resistant reaction to Fusarium, Ralstonia and
Meloidogyne and to comibation of all these three pathogens. Significant
increase in plant height and biomass with less disease severity and root-knot
index recorded in SRL1-T0(21) and RVL1-T0(28) events
231

 Toxicity effects of rvl1 and srl1 transgenic tomato lines on infection and
development of J2 of M. incognita was studied.

 Histopathological and Histochemical changes studied in transgenic and non-


transgenic tomato plants. Reduced giant cell formation were recorded in srl1
and rvl1 transgenics than control.

 Mode of action of SRL1, RVL1 and anti-SRL1 on soil borne fungi was
elucidated. FITC-labelled lectins bound to all fungi, primarily to hyphal wall,
septum, and spores of respective fungus.

 Localization of anti-SRL1 on sclerotia of S. rolfsii and R. solani showed dense


mass of congregated lectin sites.

 Nematode secretions were observed at amphidial, excretory pore and


phasmidial region using CBB-R histochemical dye.

 Lectins bound to head, midgut and posterior regions of nematode juveniles.


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Appendix I : Composition of different growth media/reagents/indicators used

A. Potato dextrose agar (Okon et al., 1977)


Peeled potato 200.00 g
Dextrose 20.00 g
Agar 18.00 g
Distilled water 1000 ml

B. Nutrient agar (Anon, 1957)


Peptone 5.00 g
Beef extract 3.00 g
Sodium chloride 5.00 g
Agar 18.00 g
Distilled water 1000 ml
Adjust pH to 6.8-7.2 before adding agar.

C. Starch agar
Peptone 5.00 g
Beef extract 3.00 g
Starch solution 10.00 ml
Agar 18.00 g
Distilled water 1000 ml
Adjust pH to 7.2

D. Iodine solution (For starch hydrolysis)


Iodine 0.50 g
Potassium iodide 1.00 g
Distilled water 1000 ml

E. Nutrient gelatin (Cappuccino and Sherman, 1992)


Peptone 5.00 g
Beef extract 3.00 g
Gelatin 120.00 g
Distilled water 1000 ml
Adjust pH to 6.8-7.0
269

F. SIM agar (Cappuccino and Sherman, 1992)


Peptone 30.00 g
Beef extract 3.00 g
Ferrous ammonium Sulphate 0.20 g
Sodium thiosulphate 0.025 g
Distilled water 1000 ml
Agar 18.00 g
Adjust pH to 7.3 before adding agar

G. Sucrose peptone agar


Sucrose 10.00 g
Peptone 10.00 g
Casein hydrosylate enxyme 1.00 g
Agar 18.00 g
Distilled water 1000 ml
Adjust pH to 7.3
Just before pouring the medium into the plates 500 µ1 of stock solution of
sterilized 2, 3, 5- Triphenyl tetrazolium chloride (stock 10mg/ml) was added.
H. Luria-Bartani Agar (LBA)
Tryptone 10.00 g
Yeast Extract 5.00 g
Sodium Chloride 5.00 g
Agar 18 g
Distilled water 1000 ml
STUDIES ON LECTINS FOR SUPPRESSION OF SOIL-BORNE PATHOGENS
JAYALAKSHMI K. 2014 Dr. S. LINGARAJU
Major Advisor
ABSTRACT
Soil-borne pathogens affecting tomato (Fusarium oxysporum, Sclerotium
rolfsii, Rhizoctonia solani, Ralstonia solancearum and Meloidogyne incognita) were
collected from MARS, UAS, Dharwad. Expression of a fungal lectin SRL1 and a plant
tuber lectin RVL1 was checked with total protein isolated from induced E. coli
BL21(DE3) pLysS. E. coli induced RVL1 and SRL1 could agglutinate rabbit
erythrocytes. Polyclonal antibody was raised against SRL1 by immunizing rabbits
with pure antigen (SRL1) and the specificity of the antiserum (anti-SRL1) was
determined by DAC-ELISA. Different concentrations of SRL1, anti-SRL1 and RVL1
were efficacious against soil-borne pathogens as demonstrated by employing
different methods (poison food, spread plate, inhibition zone, spore germination
inhibition). Combination of lectins with antagonists (Trichoderma spp. and
Pseudomonas fluorescens strains) was found to be effective in suppressing the
growth of soil-borne pathogens. But SRL1 and RVL1 suppressed some of the fungal
and bacterial antagonists.

Eight transgenic tomato events carrying srl1 and rvl1 genes (T3 genertaion of
homozygous) showed moderately resistant reaction [SRL1-T0(21) and RVL1-T0(28)]
to Fusarium, Ralstonia and also Meloidogyne and to combination of all these three
pathogens. Histopathological and histochemical changes in transgenic and non-
transgenic tomato plants revealed reduced giant cell formation in srl1 and rvl1
transgenics. Elucidation of the mode of action of SRL1, RVL1 and anti-SRL1 on soil
borne pathogens and antagonists: FITC-labelled lectins bound to hyphal wall,
septum, and spores of respective fungi; localization of anti-SRL1 on sclerotia of S.
rolfsii and R. solani showed a dense mass of congregated lectin sites. Nematode
secretions were observed at amphidial, excretory pore and phasmidial region using
CBB-R histochemical dye. Binding of lectins in nematode was found at head region,
mid gut of the alimentary-tract, excretory pore which extended to the posterior region
(phasmids).

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