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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
0
10
20
30
40
50
60
70
80
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
0
10
20
30
40
50
60
70
80
90
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
0
10
20
30
40
50
60
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
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
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
0
10
20
30
40
50
60
70
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
2
1
3
4
C
5
6
0
10
20
30
40
50
60
70
80
TV1 TV2 TV3 TV4 TV5 TV6 Control
Perc
ent i
nhib
ition
ove
r co
ntro
l
Isolates
Development of bioformulations
0
50
100
150
200
250
300
0 15 30 45 60 75 90 105 120
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
0102030405060708090
Per
cent
red
uctio
n ov
er c
ontr
ol
Treatments
Different bioformulations on the incidence of noni anthracnose under field conditions
0
10
20
30
40
50
60
70
80
90
Trial I Per cent reduction overcontrol
Trail II Per cent reduction overcontrol
Treatments
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