BIOCONTROL OF FUSARIUM UDUM BY CHITINOLYTIC BACTERIA IN PIGEON PEA
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Date
2017
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DIVISION OF MICROBIOLOGY ICAR- INDIAN AGRICULTURAL RESEARCH INSTITUTE
Abstract
Wilt disease caused by Fusarium udum Butler is one of the most important soil-borne
diseases of pigeon pea (Cajanus cajan (L) Millsp.) throughout India and world. It is the
most serious disease causing lethal damage to the crop and considerable economic losses.
Presently, management of pathogens by excessive use of synthetic chemicals is effective
and convenient but it has caused serious ecological, economic and social concerns. In
recent times, biological control of F. udum using microorganisms that can grow and
colonize in rhizosphere, has attracted attention throughout the world. In view of above
consideration, the present study was conducted to study/investigate in vitro and in vivo
biological control of F. udum by chitinolytic bacteria isolated from pigeon pea
rhizosphere.
53 bacterial chitinolytic morphotypes were isolated from pigeon pea rhizospheric
soil. In qualitative screening, thirteen isolates among the 53 evaluated, produced clear
chitin hydrolysis zone (between 2.6 mm and 8.8 mm) on colloidal chitin agar (CCA)
medium plates. Among these selected isolates, highest chitinase activity (798.35 U mg-1
protein) was detected in isolate NS-22, followed by isolate NS-1 (632.33 U mg-1
protein).
In vitro antifungal assay against F. udum, eleven isolates were able to cause significant
reduction in the mycelial growth of F. udum. Chitinolytic bacterial isolate NS-22
recorded highest fungal growth inhibition (67.57 %) followed by isolate NS-1 (62.16%).
Five most promising chitinolytic isolates were further selected for studying in vivo
antagonistic potential against F. udum under pot culture conditions and seed inoculation
with chitinolytic bacteria (NS-1 and NS-22) resulted in better seed germination, reduced
wilt incidence and improved plant biomass over all other treatments. Based on the in
vitro and in vivo screening for antagonistic activity against F. udum, two most promising
and effective chitinolytic bacterial isolates NS-1 and NS-22 were selected and
characterized. Based on by 16s rRNA gene sequencing, bacterial isolate NS-1 was
identified as Pseudomonas spp. while NS-22 was identified as Bacillus spp. The effect of
different growth and cultural conditions on activity of extracellular chitinase enzyme of
Pseudomonas spp. strain NS-1 and Bacillus spp. strain NS-22 was determined. Luria
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broth as growth media, neutral pH range, temperature (30°C for Pseudomonas spp. strain
NS-1 and 35°C for Bacillus spp. strain NS-22) was found optimum for induction of
highest chitinase activity in these bacterial isolates. Among various carbon and metal
ions, Luria broth supplemented with fructose and with Fe2+ ions supported maximum
chitinase activity in both the selected bacteria (Pseudomonas spp. strain NS-1 and
Bacillus spp. strain NS-22).
Under in vitro studies, extracellular, diffusible and volatile metabolites produced
by both these bacterial cultures exhibited antagonism against F. udum. Growth inhibition
by means of reduction in mycelial dry weight of F. udum was also observed when both
these bacteria were grown simultaneously/at different growth time of pathogen in PD
broth. Hydrolytic enzymes (endochitinase, exochitinase, chitobiase, protease and lipase)
related to biological disease suppression were also detected in both the selected cultures.
Additionally, other biocontrol traits like production of siderophore, and ammonia were
also detected in both antagonistic bacteria, while HCN was observed only in
Pseudomonas spp. strain NS-1. Light and scanning microscopy study also revealed lysis,
distortation, deformation and irregularity in hyphae morphology during interaction of the
pathogen with the selected cultures.
In vivo pot experimental study also supported the results obtained under in vitro
study. Seed bacterization with the bacterial cultures of the pathogen challenged pigeon
pea plants led to a higher induction of the different defense enzymes such as
phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (PO),
increase in total phenolics and chlorophyll content and reduced root mortality (%) over
control. There was also increase in plant height, biomass and number of pods in treated
plants. Application of selected cultures enhanced soil FDA, dehydrogenase activity and
soil microbial biomass. It also reduced fusarium population in the soil.
Thus, both these cultures, Pseudomonas spp. strain NS-1 and Bacillus spp. strain
NS-22, exhibited good antagonistic potential against F. udum and can prove as promising
candidates for biocontrol of fusarium wilt in pigeon pea.
Description
t9595
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