MSc

Microorganisms associated with aerial parts of plants such as stems
(cauloshere), phylloplane (leaves), anthosphere (flowers) and carposphere (fruits)
which are collectively known as phyllosphere. Phyllosphere microbes includes
bacteria, filamentous fungi and yeasts. They offer natural biological control of
diseases affecting aerial plant parts. Being better adapted to the phyllosphere niche,
they are potential biocontrol agents which are under exploited in agriculture which
allow them to adapt to the phyllosphere environment and inhibit the growth of
microbial pathogens, thus sustaining plant health. Hence, microbial
biocontrolbyphyllosphere microorganisms is a fast growing field of research.
Considering the importance of the potential of phyllosphere antagonists, the research
project entitled “Phyllosphere microorganisms for the management of anthracnose
disease of cowpea (Vigna unguiculata (L.) Walp.).” was carried out. Phyllosphere
microbiome enhance plant tolerance to withstand biotic and abiotic stress conditions
in the current facet of climate change.
A purposive sampling survey was conducted in 21 locations of Kerala where
cowpea and/or yard long bean are grown as a vegetable crop. The survey covered six
districts namely, Thrissur, Palakkad, Malappuram, Kozhikode, Kannur and Kasaragod
and five agro ecological units. Fungi associated with anthracnose symptoms and
phylloshere microrganisms associated with healthy and symptomless plants from the
fields were isolated. On pathogenicity test, six fungi were confirmed as pathogens and
they were identified as Colletotrichum Siamense, three isolates of Colletotrichum sp.,
Ectophoma multirostrata and Curvularia verruculosa based on cultural, morphological
and molecular characterization.C. siamense and Ectophoma multirostrata infecting
cowpea is the first report in the world. Similarly, first report of Curvularia verruculosa
infecting cowpea and producing anthracnose symptoms from India.
Enumeration of phyllosphere microorganisms on cowpea grown at different
locations showed that population of fungi distributed on all plant parts and more
abundant on flowers. Likewise, bacteria are more abundant on cowpea flowers, and
fluorescent pseudomonads and yeasts are more abundantly seen on cowpea pods.
Based on colony characters, a total of 183 different isolates of phyllosphere microbes
were made into pure cultures and were screened for antagonism towards C. siamense.
Forty one isolates expressed antagonism in varying degrees which were again
subjected to dual culture screening in vitro. Five promising anatgonists were selected
based on per cent inhibition of mycelium of the pathogen on PDA. The five were
identified as Trichoderma longibrachiatum, Trichoderma asperellem, Bacillus
amyloliquefaciens, Bacillus velezensis and Candida tropicalis based on cultural,
morphological and molecular characterization. Cell free culture filtrates of promising
phyllosphere antagonists showed maximum growth inhitbion of C. siamense ranging
from 86 per cent for Candida tropicalis to 100 per cent for Trichoderma
longibrachiatum and Trichoderma asperellem
In order to study the induction of systemic resistance in cowpea by
phyllosphere antagonists, a pot culture experiment was conducted and the results
showed that all the antagonists accelerate the production of defence related enzymes
such as peroxidase, polyphenol oxidase and phenylalanine ammonia lyase when
challenge inoculated with the pathogen. Bacillus amyloliquefaciens induced higher
activity of peroxidase (1.2 min-1g-1 fresh tissue over control), similarly polyphenol
oxidase by Candida tropicalis (0.5min-1g-1 fresh tissue over control) and
phenylalanine alanine by Trichoderma asperellem (0.8 μmol of transcinnamic acid
formed g-1 fresh tissue over the control).
To test the biocontrol efficacy and plant growth promotion of phyllosphere
antagonists against C. siamense causing cowpea anthracnose, a field experiment was
conducted. The semi trailing cowpea variety ‘Anashwara’ was used in the experiment.
The treatments included conventional biocontrol agents such as Trichoderma sp.
(KAU) and Pseudomonas fluorescens (KAU) and chemical check with hexaconazole
(systemic), mancozeb (contact) and carbendazim + mancozeb (systemic +contact) and
a control without treatments were also included along with five phyllsophere
antagonists such as T. longibrachiatum, T. asperellem, B. amyloliquefaciens, B.
velezensis and C. tropicalis. All the treatments were given as seed treatment, and foliar
spray. Higher vigour index of seedlings was recorded in the treatment with Bacillus
amyloliquefaciens (1408). Biometric observations such as plant height, number of main
branches, number of pods per plant, number of seeds per pod, fresh weight of pods,
pod length, days to first harvest, days to last harvest and total yieldand per cent disease
incidence and severity were recorded at regular interval. Results of the field experiment
indicated significant reduction in disease incidence and severity effected by
phyllosphere antagonists such as T. asperellem, B. amyloliquefaciens, B. velezensis, C.
tropicalis, Trcihoderma sp. (KAU), Pseudomonas fluorescens, hexaconazole,
mancozeb and carbendazim + Mancozeb. In terms of yield all the five antagonists
along with Trichoderma sp. (KAU) recorded significant yield increase in cowpea
compared to other treatments.
The bacteria, B. amyloliquefaciens has gained more attention and an emerging
evidence has identified its health beneficial effects as probiotics, hence farmers can
safely use this as a biocontrol agent. Focusing on efficient and safer (non pathogenic
nature), the phyllosphere antagonists such as T. asperellem and B. amyloliquefaciens
are selected as the best among all the treatments. So, it can be concluded that out of the
eleven treatments, T. asperellem and B. amyloliquefaciens could promote the growth of
the plants and reduce the disease incidence and severity than the other treatments.
However, multilocational field studies should be conducted to validate the findings of
the current study, so that these bioagents can be formulated and made available to the
farming community.