Evaluation of biocontrol agents of soil borne plant pathogens for promoting plant growth in chilli

255 Pages·2013·10.76 MB·English

EVALUATION OF NON-PATHOGENIC FUSARIUM
SPP. FOR THEIR BIOLOGICAL CONTROL
EFFICACY AGAINST FUSARIUM WILT OF
WATERMELON [Citrullus lanatus (Thunb.) Matsum
and Nakai]

RAGHUNANDAN, B. L.
PAL 0024

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU
2013
EVALUATION OF NON-PATHOGENIC FUSARIUM
SPP. FOR THEIR BIOLOGICAL CONTROL
EFFICACY AGAINST FUSARIUM WILT OF
WATERMELON [Citrullus lanatus (Thunb.) Matsum
and Nakai]

RAGHUNANDAN, B. L.
PAL 0024

Thesis submitted to the
University of Agricultural Sciences, Bengaluru
in partial fulfillment of the requirements
for the award of the degree of

Doctor of Philosophy
in
AGRICULTURAL MICROBIOLOGY

BENGALURU JULY, 2013
Affectionately
Dedicated to My
Beloved Mother
DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
UNIVERSITY OF AGRICULTURAL SCIENCES
BENGALURU
CERTIFICATE
This is to certify that the thesis entitled "EVALUATION OF NON-
PATHOGENIC FUSARIUM SPP. FOR THEIR BIOLOGICAL CONTROL
EFFICACY AGAINST FUSARIUM WILT OF WATERMELON" [Citrullus
lanatus (Thunb.) Matsum and Nakai] submitted by Mr. RAGHUNANDAN, B.
L., ID No. PAL 0024 in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in AGRICULTURAL MICROBIOLOGY to
the University of Agricultural Sciences, Bengaluru, is a record of research work
carried out by him during the period of study in this University under my guidance
and supervision and thesis has not previously formed the basis for the award of
any degree, diploma, associateship, fellowship or any other similar titles.

BENGALURU Dr. L. KRISHNA NAIK
JULY, 2013 Associate Professor
Dept. of Agricultural Microbiology
Major Advisor

APPROVED BY:
Chairman : ___________________________
(L. KRISHNA NAIK)
Members : 1. ___________________________
(K. R. SREERAMULU)
2. ___________________________
(M. K. SHIVAPRAKASH)
3. ___________________________
(A. N. BALAKRISHNA)
4. __________________________
(S. SRIRAM)

____________________
(M. N. SREENIVASA) 5. ___________________________
(P. H. RAMANJINI GOWDA)
(External Examiner)
ACKNOWLEDGEMENT

At the outset, I wish to express with utmost sincerity, my deep sense of
gratitude to the Chairman of my Advisory Committee, Dr. L. Krishna Naik,
Associate Professor, Dept. of Agricultural Microbiology, UAS, GKVK, Bengaluru,
for his guidance, constant encouragement, support and helpful criticism during
the course of my study and preparation of the thesis.

I express my profound sense of gratitude to Dr. S. Sriram, Senior Scientist,
Dept. of Plant Pathology, IIHR, Hesaraghatta, Bengaluru, Dr. K. R. Sreeramulu,
Dean of Student Welfare (DSW), Professor, Dept of Agril. Microbiology,
Dr. M. K. Shivaprakash, Professor and Head, Dept of Agril. Microbiology,
Dr. A. N. Balakrishna, Professor, Dept of Agril. Microbiology,
Dr. P. H. Ramanjini Gowda, Professor, Dept. of Plant Biotechnology, as
members of my Advisory Committee they supported and motivated me whenever I
was in need of their suggestions.

I am also thankful to teaching staff and non-teaching staff of Dept of
Agril. Microbiology for their cooperation throughout the study.

It’s my esteemed duty to reserve my high regards to my family members for
showing boundless love, inspiration and financial support during my studies.

A special word of thanks to my friends and senior friends (Shashidhar,
Divya, Raghavendra Kumar, Narotham Prasad and Aravind) for their valuable
timely help, suggestions and encouragement. My wholehearted thanks to
Manikanta, for his help and co-operation throughout my study. I wish to express
my heartiest thanks to my junior friends Sudheer, Sudha, Manoj, Shivabasu and
Ajay for their help during my studies.
I owe my grateful thanks to Ms. M. J. Savitha (NBAII) for her help during
molecular characterization work and Mr. N. Mohanraju, Sr. Lab assistant, Dept.
of Agril. Microbiology for his kind help and guidance during the course of
investigation.

I would like to acknowledge the financial assistance (INSPIRE
Fellowship) by Department of Science and Technology (DST), Govt. of India. I
am also thankful to National Bureau of Agriculturally Important Insects
(NBAII), Hebbal, Bengaluru, for providing laboratory facilities and Dr. Wade
Elmer, Connecticut Agricultural Experiment Station, USA, & USDA, ARS
Culture Collection, USA, for providing standard non-pathogenic Fusarium
cultures for my study.

Any omission in this brief acknowledgement does not mean lack of
gratitude.

Place: Bengaluru
July 2013 (Raghunandan, B.L.)
“Evaluation of non-pathogenic Fusarium spp. for their biological control
efficacy against Fusarium wilt of watermelon" [Citrullus lanatus
(Thunb.) Matsum and Nakai]

Raghunandan, B. L.
Thesis Abstract
An investigation was carried out to evaluate the efficacy of non-pathogenic
Fusarium spp. in the management of Fusarium wilt disease of watermelon. Out of 68
Fusarium cultures isolated from rhizosphere soil of watermelon crop, six Fusarium
cultures proved to be non-pathogenic to watermelon and other non-host crops.
Morphological, cultural and molecular characterization of non-pathogenic Fusarium
cultures was done and among six isolates, five were identified as Fusarium
oxysporum and one was Fusarium solani.
Significant inhibition in the growth of the wilt pathogen was showed by non-
pathogenic Fusarium cultures UAS NPFu2, UAS NPFu6 and USDA Fo47 in dual
culture study. The culture filtrate extracts and volatile compounds of non-pathogenic
cultures did not show any significant results on inhibition of the pathogen in vitro.
Increased levels of total protein, phenols and defense enzymes such as peroxidase,
polyphenol oxidase, phenylalanine ammonia lyase, β-1,3-glucanase and chitinase
were observed in the plants treated with non-pathogenic Fusarium cultures. Higher
levels of defense enzymes got induced in the plants inoculated with non-pathogenic
Fusarium cultures followed by challenged inoculation of pathogen. All the non-
pathogenic Fusarium cultures were positive for phosphate solubilization and
production of IAA and GA.
Under greenhouse conditions significant increase in plant growth parameters,
reduced wilt disease incidence and better root colonization by non-pathogenic
Fusarium cultures was observed. The treatments which received non-pathogenic
Fusarium + pathogenic Fusarium showed a slight increase in the plant growth
parameters compared to inoculation of non-pathogenic culture alone. The isolate UAS
NPFu6 performed better than other isolates under field conditions. Seed treatment
method gave best results compared to drenching the culture broth of non-pathogenic
Fusarium isolates under field study.

July, 2013 Dr. L. Krishna Naik
Department of Agril. Microbiology (Major Advisor)
UAS, GKVK, Bengaluru.
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CONTENTS

Sl. No Title Page No.
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-40
III MATERIAL AND METHODS 41-65
IV EXPERIMENTAL RESULTS 66-132
V DISCUSSION 133-149
VI SUMMARY 150-153
VII REFERENCES 154-186
APPENDIX
LIST OF TABLES
Table Page
TITLE
No. No.
Soil samples of watermelon fields collected from southern parts of
1 67
Karnataka and Fusarium cultures isolated.
Screening of Fusarium isolates for the non-pathogenicity by
2a 69
different methods.
Screening of Fusarium isolates for the non-pathogenicity by
2b 70
different methods.
3 Morphological characterization of non-pathogenic Fusarium isolates. 72
Colony morphology of non-pathogenic Fusarium isolates on
4a 74
different culture media.
Colony morphology of non-pathogenic Fusarium isolates on
4b 75
different culture media.
Non-pathogenic isolates of Fusarium and related isolates with their
5 85
accession numbers on NCBI blast programme for ITS region.
Non-pathogenic isolates of Fusarium and related isolates with their
6 86
accession numbers on NCBI blast programme for TEF-1α region.
Effect of non-pathogenic Fusarium on the growth of F. oxysporum f.
7 88
sp. niveum under in vitro.
Effect of cell free culture filtrate of non-pathogenic Fusarium on the
8 90
growth of F. oxysporum f. sp. niveum under in vitro.
Effect of volatile compounds of non-pathogenic Fusarium on the
9 91
growth of F. oxysporum f. sp. niveum under in vitro.
Protein content in watermelon seedlings treated with cultures of non-
10 93
pathogenic Fusarium spp.
Protein content in watermelon seedlings treated with cultures of non-
11 94
pathogenic Fusarium spp. and pathogenic Fusarium.
Phenol content in watermelon seedlings treated with cultures of non-
12 96
pathogenic Fusarium spp.
Phenol content in watermelon seedlings treated with cultures of non-
13 98
pathogenic Fusarium spp. and pathogenic Fusarium.
Peroxidase activity in watermelon seedlings treated with cultures of
14 100
non- pathogenic Fusarium spp.
Peroxidase activity in watermelon seedlings treated with cultures of
15 101
non-pathogenic Fusarium spp. and pathogenic Fusarium.