SCREENING AND ISOLATION OF ANTIMICROBIAL METABOLITES AND EVALUATION OF THE ...

306 Pages·2013·9.25 MB·English

SCREENING AND ISOLATION OF ANTIMICROBIAL METABOLITES AND
EVALUATION OF THE BIOCONTROL POTENTIAL OF Streptomyces sp. TC1
FOR THE MANAGEMENT OF BACTERIAL LEAF BLIGHT DISEASE IN RICE

By

N. JAIVEL, M.Sc. (Ag.)

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
AGRICULTURAL COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU AGRICULTURAL UNIVERSITY
COIMBATORE - 641003

2013
SCREENING AND ISOLATION OF ANTIMICROBIAL METABOLITES AND
EVALUATION OF THE BIOCONTROL POTENTIAL OF Streptomyces sp. TC1
FOR THE MANAGEMENT OF BACTERIAL LEAF BLIGHT DISEASE IN RICE

Thesis submitted in part fulfilment of the requirements for the award of the degree of
DOCTOR OF PHILOSOPHY (Agriculture) in AGRICULTURAL MICROBIOLOGY
to the Tamil Nadu Agricultural University, Coimbatore.

BY
N. JAIVEL
(I.D. No. 10-606-003)

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY
AGRICULTURAL COLLEGE AND RESEARCH INSTITUTE
TAMIL NADU AGRICULTURAL UNIVERSITY
COIMBATORE - 641003

2013
CERTIFICATE
This is to certify that the thesis entitled “SCREENING AND ISOLATION OF
ANTIMICROBIAL METABOLITES AND EVALUATION OF THE BIOCONTROL
POTENTIAL OF Streptomyces sp. TC1 FOR THE MANAGEMENT OF BACTERIAL
LEAF BLIGHT DISEASE IN RICE” submitted in part fulfilment of the requirements
for the award of the degree of DOCTOR OF PHILOSOPHY (Agriculture) in
AGRICULTURAL MICROBIOLOGY to the Tamil Nadu Agricultural University,
Coimbatore is a record of bonafide research work carried out by Mr. N. JAIVEL under
my supervision and guidance and that no part of this thesis has been submitted for the
award of any other degree, diploma, fellowship or other similar titles. However, part of
the thesis work has been published in peer reviewed scientific journal of national/
international repute (copy enclosed).

Place : Coimbatore (Dr. P. Marimuthu)
Date : Chairman

Approved by:

Chairman : (Dr. P. MARIMUTHU)

Members :

(Dr. S. KARTHIKEYAN)

(Dr. K. RAMARAJU)

(Dr. R. RABINDRAN)

(Dr. P. GOVINDARAJU)

Date : External Examiner:
Acknowledgement
ACKNOWLEDGEMENT
I wish to express very sincerely my heartfelt gratitude and indebtness to my chairman,
Dr.P. Marimuthu, Professor, Department of Agricultural Microbiology for his meticulous supervision,
benevolent and noble ideas, constructive criticism, surpassing guidance, sustained interest, mellifluous help
and care and unsurpassed encouragement rendered throughout the course of this investigation.
I would like to express my sincere thanks and profound gratitude to the members of the
advisory committee, Dr.S. Karthikeyan, Professor, Department of Agricultural Microbiology,
Dr.R. Rabindran, Registrar, Tamil Nadu Agricultural University, Dr.K. Ramaraju, Director, Centre
for Plant Protection studies and Dr.P. Govindaraju, Professor & Head, Department of Biochemistry
for their valid suggestions, expert guidance, keen interest and incessant help rendered throughout the
period of my study. My hearty thanks to Dr.S. Gunasekaran, Professor and Head, Department of
Agrl. Microbiology, for his constant inspiration throughout my course study. I am immensely grateful
to staff members of the Department of Agricultural Microbiology for their constant help in timely
needs. I wish to extend my thanks to Dr.D. Velmurugan, Madras University for helping me in
molecular docking studies and Dr.P.S. Mohan, Bharathiar University for his valuable suggestions in
identifying the compounds.
I am pleased to thank Dr.R. Rajesh, Dr.C. Uvarani, Mrs.S. Sangeetha, Mr. Chandraprakash
and Mrs.R. Umapriya for their wholehearted help. Words are insufficient to express my gratitude to
my friends Abi, Belli, Draj, Vinoth, Gopal, Arun and kicha for their moral support and helpful hand
at times of need. I owe a great deal to my classmates, who inspite of their busy schedule were always
willing to lend me a helping hand. The peerless help by the junior colleagues is gratefully
acknowledged.
I also take this opportunity to thank lab assistants Satheesh, Kanchana and Chitra for their
needful help.
My record of gratitude would be incomplete if I fail to mention my most affectionate
family members whose mellifluous love and prayers brought me to witness this day. I am indebted
greatly to them for everything that they had been to me.
I humbly dedicate my thesis at the feet of my family and God whose grace and blessings all
through has helped in completing my research successfully.

(N. Jaivel)
Abstract
ABSTRACT
SCREENING AND ISOLATION OF ANTIMICROBIAL METABOLITES AND
EVALUATION OF THE BIOCONTROL POTENTIAL OF Streptomyces sp. TC1
FOR THE MANAGEMENT OF BACTERIAL LEAF BLIGHT DISEASE IN RICE
By
N. JAIVEL
Degree : Ph. D. in Agricultural Microbiology
Chairman : Dr. P. Marimuthu,
Professor,
Department of Agricultural Microbiology,
Tamil Nadu Agricultural University,
Coimbatore - 641 003.
2013

An investigation was conducted to explore the antimicrobial potential of
Streptomyces sp. TC1 against Xanthomonas oryzae pv. oryzae (Xoo) which causes
bacterial leaf blight disease in rice. The TC1 strain was found to produce maximum
antimicrobial metabolites upto 7 days in starch casein broth. The starch and yeast extract
positively influences the antimicrobial metabolites production. The inoculum volume of
8 percent was considered to be an optimum cell load for increased antimicrobial
metabolite production. The produced antimicrobial metabolites in modified starch casein
broth were extracted using ethyl acetate and the pH of broth adjusted to 3 prior to
extraction process for the better recovery of antimicrobial metabolites. The ethyl acetate
crude extract possess an MIC value of 300 µg/mL and IC50 value of 125 µg/mL against
the test pathogen Xoo. The culture filtrate and crude extract of TC1 strain also showed
antimicrobial activity against various isolates of Xoo.
Thin layer chromatography (TLC) was performed for the purification and
separation of antimicrobial compounds from the ethyl acetate crude extract. Totally nine
bands were observed with closer R (relative front) values using chloroform: methanol in
f
the ratio of 24:1 as mobile phase. Accordingly, isolation and purification of antimicrobial
metabolites was done by silica gel column chromatography. The column eluted using
n-hexane and chloroform with increasing polarity yielded 27 different fractions. Among
27 different fractions, four fractions possessed antimicrobial activity against Xoo in agar
well diffusion assay and produced an inhibition zone of 1.9 to 2.5 cm. The isolated
antimicrobial compounds were extensively characterized by spectroscopic techniques like
FT-IR, NMR, GC-MS and their molecular formula was derived. The antimicrobial
metabolites includes three novel compounds which are 2-(1,1-diallyl-but-3-enyl)-5-
nonyl-phenol (compound 1), 3-(2,4-di-tert-butyl-5-fluoro-phenyl)-propionic acid (compound
2), 4, 5-diamino-2-hydroxy pentanoic acid-1 carbamoyl-3-methyl-butyl-amide (compound 3)
and a known compound 6-hydroxy-7-methoxycoumarin (compound 4). The isolated
antimicrobial compounds 1-4 contain phenol, carboxylic acid, amide and carbonyl
functionalities respectively. The existence of fluorine atom in compound 2 was confirmed
by X-ray crystallographic study and it was deposited at Cambridge Crystallographic Data
Centre, London, United Kingdom with the accession no. CCDC 923598. The compound 2 is
the first aromatic organofluorine secondary metabolite from natural sources.
The possible mode of action of the isolated antimicrobial metabolites was
analyzed by DNA binding, protein binding and molecular docking studies. The DNA
binding potential of isolated compounds was found to be low with a hypochromism effect
of 16.6-20.1%. Whereas, compounds showed substantial interactions with bovine serum
albumin (BSA) protein and the corresponding decrease in hypochromism of 34.3-88.5%
was observed by fluorescence quenching assay. Hence molecular docking studies were
performed for the isolated antimicrobial compounds against selected virulence proteins of
Xoo using Induced Fit Docking in Glide software. The isolated compounds were bind
well with Lip A (rice cell wall degrading protein) and Fab V (involved in cell wall
elongation) proteins of Xoo by producing a better docking score in molecular docking
studies. As the isolated compounds bind well with rice cell wall degrading protein, the
infection of rice plant by Xoo is eliminated in entry point itself and thus the systemic
infection can be prevented. Further the Xoo cells treated with ethyl acetate crude extract were
observed for deformation and reduced size under scanning electron microscopy study.
The isolated antimicrobial compounds exhibited moderate antioxidant activities in
ABTS+, DPPH, OH, O −•, FRAP and metal chelating assays. Whereas the reducing
2
power and total antioxidant activities of ethyl acetate crude extract was comparable to
that of standards rutin and butylated hydroxytoluene (BHT). The compound 2 exhibited
potent primary antioxidant activity compared to other compounds. The growth promoting
properties of the Streptomyces sp. TC1 was found to be insignificant. The TC1 culture was
analyzed for gibberellic acid production of 0.73 µg/mL and IAA production of 4.33 µg/mL.
But its siderophore producing capability was found to be moderate. The TC1 strain was
analyzed for production of both hydroxymate and catechol type of siderophores.
The Streptomyces sp. TC1, which capable of synthesizing antimicrobial
metabolites and siderophores developed into a biocontrol formulation for the
management of bacterial leaf blight disease in rice. The TC1 formulation was prepared
using the dried spores and mycelium of TC1 strain with soluble starch as carrier material.
The biocontrol potential of the developed formulation was assessed both under pot
culture and field trials. The effect of various concentrations of TC1 formulation on seed
infection, germination and vigour of TN1 variety paddy seeds were studied. The increase
in germination percentage was observed to be about 78.7% and the vigour of seedling to
the extent of 69.2% over the control. Application of TC1 formulation increased the
activity of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase
(PAL), superoxide dismutase (SOD) and phenol content of the rice plants. A 0.4 percent
TC1 formulation was found to be the optimum concentration for bacterial leaf blight
disease management in rice under pot culture and field conditions. The reduction in
disease incidence was observed to be about 56.6% and 37.4% over the control under pot
culture and field trials respectively.
CONTENTS
Chapter Page
Title
No. No.
I INTRODUCTION 1
II REVIEW OF LITERATURE 4
III MATERIALS AND METHODS 33
IV RESULTS 64
V DISCUSSION 204
VI SUMMARY 231
REFERENCES 234
APPENDICES