ByManjunath Hubballi

Department of Plant Pathology, Centre for Plant Protection Studies,TNAU,

Coimbatore - 641002, Tamil Nadu, India

MOLECULAR CHARACTERIZATION OF COLLETOTRICHUM GLOEOSPORIOIDES, THE INCITANT OF NONI ANTHRACNOSE AND EXPLOITING PGPR AND

FUNGAL ANTAGONISTS FOR ITS MANAGEMENT

Overview

Introduction

Distribution

Identification

Selection of elite strains of PGPR

Development of consortia

Evaluation of consortia

Understanding mode of action

Prelude• Morinda citrifolia – Noni - Indian mulberry

• 160+ nutraceuticals bundled in it - Wonder drug

• Native of Southeast Asia to Australia

• Fruit, leaves, seeds, bark, and roots – Medicinal

Value

• Simple cold to HIV infection

Noni to the length and breadth of India

Ubiquitous crop

Courtesy - Scot C. Nelson, 2003

Anthracnose

Fruit rot

Sooty mold

Algal leaf spot

Leaf blight

Stem canker and

Black flag

Stem blight

Foes

Brown necrotic spot Acervuli Shot hole

Journey towards endemic form

As a die back Fruit rot

Survey of the melody

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90Pe

rcen

t dis

ease

inde

x

District

Colletotrichum ?

14.71 - 11.25 m X 7.1 -5.00 m (100 X )

Eight day old culture on PDA Acervulus

Erroneous identification based on morphology

Pathogenicity Test

Ten days after inoculation

Pin prick + Spore suspension spray 5105 spores /ml

Fungal nutrition

Preference to PDA and Walks man’s agar

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Water agar

Oat meal agar

Richards agar

PDA

MRB

Czpek Dox

Host leaf extract

Walksman's agar

Myc

elia

l gro

wth

(mm

)

Media

ETS 18S ETS5.8S 28SITS 1 ITS 2

ITS 4

ITS 5

ITS4 (5’ TCC TCC GCT TAT TGA TAT GC 3’)ITS5 (5’ GGA AGT AAA AGT CGT AAC AAG G 3’)

496 bp

496 bp

1000 bp Marker (Joon Tae etal., 2008)

ITS4 (5’ TCC TCC GCT TAT TGA TAT GC 3’)ITS5 (5’ GGA AGT AAA AGT CGT AAC AAG G 3’)

Amplification of ITS region of C. coffeanum

Sequence of Colletotrichum spp. from GenBank data base for species confirmation

Accession No: GQ166975

Cont.,

100% homology with C. gloeosporioides

Management

Selection of Pseudomonas isolate

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PRO

C

Isolates

Why TDK‐1 is an effective antagonists?...

C. gloeosporioides -control

TDK1

TDK‐1 an effective antagonists

Pf-5T

DK

-11kb ladder

1050bp1000bp

DAPG

TDK1

PCA Biosynthetic gene

Phz1/phz2

Neg.ck

2-79TD

K-1

1kb ladder

1400bp

• Used specific primers developed from antibiotic‐biosynthetic genes

AU

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0.80

1.00

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1.60

1.80

2.00

Minutes10.00 20.00 30.00 40.00

HPLC based detection of  Phenazines

(RT= 20.3 and 22.8 min) 1: 2-OH-PHZ, 2: PCA

In vitro assay of antibiotics of Pseudomonas

against radial growth of C. gloeosporioides

TDK1 - DAPG TDk1 - Phenazine

Con

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Percen

t inh

ibition

 over con

trol

Isolates

In vitro selection of Bacillus isolates against radial growth of C. gloeosporioides

B.subtilis (SVPR‐ 4)  an effective antagonists

C. gloeosporioides - Control

In vitro assay of antibiotics of Bacillus against radial growth of C. gloeosporioides

1.SVPR4 2.SVPR 2

3.EPC8 4.TMV2

C.Control

21

3 4

C

Isolate Mycelial growth (mm)*

% Inhibition over control

EPC8 46.00 b 48.88

SVPR4 38.00 a 57.77

TMV2 50.00 d 44.44

SVPR2 44.50 c 50.55

Control 90.00 e -

In vitro screening of T. viride against  C. gloeosporioides

1.Tv1 2.TV2 3.TV3

4.TV4 5.Tv5 6.Tv6

C. Control

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1

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4

C

5

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TV1 TV2 TV3 TV4 TV5 TV6 Control

Perc

ent i

nhib

ition

ove

r co

ntro

l

Isolates

Development of bioformulations

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CFU x 10 

6

Different days after storage

Pf1 TDK1 EPC8 SVPR4 TV1

Bioformulations on the incidence of noni anthracnose disease under glass house conditions

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Per

cent

red

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ontr

ol

Treatments

Different bioformulations on the incidence of noni anthracnose under field conditions

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Trial I Per cent reduction overcontrol

Trail II Per cent reduction overcontrol

Treatments