Ph.D

Tomato (Solanum lycopersicum L.) is a widely grown vegetable and the most
remunerative solanaceous crop in both tropics and sub tropics of the world. It is
vulnerable to various biotic diseases among which, early blight caused by Alternaria
solani is a destructive one. Management of diseases using biocontrol agents is a safe
alternative in the integrated disease management system. However, success of a
bioagent is dependent on its comparative survivability in the new niche. Therefore,
the present investigation was undertaken to identify strains of beneficial
microorganisms adept on the phylloplane of tomato for use against early blight as it is
a disease affecting mainly the foliage.
As part of the investigation, purposive sampling survey was conducted in
Thrissur and Palakkad districts of Kerala. It was found that early blight is prevalent in
all the areas surveyed and the disease severity on foliage varied from 23 to 78 per
cent at various locations. Though several fungi were found to be associated with the
disease, only six isolates collected from Pudukkad, Valapad, Mundathikode,
Vadakarapathy, Nenmara and Parrisakal were identified as Alternaria sp.(PDK), A.
alternata (VAL), Fusarium oxysporum (MUN), A. solani (VAD and NEM) and
Corynespora cassiicola (PAR) which could be proved as pathogens on tomato.
Phylloplane microorganisms were isolated from both the healthy and diseased leaves
collected from plants at various locations and a total of 132 isolates were then
subjected to preliminary in vitro screening for their ability to suppress A. solani and
59 were selected as they could antagonize the pathogen in various degrees. The
antagonists thus selected were further screened by dual culture technique and 36
showed more than 60 per cent inhibition. The antagonism exerted by phylloplane
isolates was clearly discerned by limited growth, or the complete absence of fungal
mycelium in the inhibition zone between the pathogen and the test isolate. Out of the
36, five were selected as promising potential antagonists as they could give more than
80 per cent inhibition of the pathogen. The selected five are identified as
Trichoderma spp. (PF2 and PRF1), Bacillus subtilis (MNB2), Bacillus mojavensis
ii
(TB1) and Ochrobactrum sp. (EB1) which showed per cent inhibition of 91.2, 90,
89.4, 88 and 85 respectively. Furthermore, when these isolates were tested for growth
promotion on tomato, the vigour index of tomato seedlings varied from 2110
(Trichoderma sp., PF2) to 1676 (Bacillus mojavensis, TB1).
As next step, selected antagonists were tested for induction of systemic
resistance (ISR) on tomato by conducting a pot culture experiment. Increased
accumulation of peroxidase (PO), polyphenol oxidase (PPO) and, phenylalanine
ammonia-lyase (PAL) was observed in plants treated with phylloplane antagonists.
Higher activity of PO and PPO was induced by Trichoderma sp. (PF2) and B. subtilis
(MNB2) at fifth day after inoculation (DAI) of pathogen. The phylloplane antagonists
Trichoderma sp. (PF2) and B. subtilis (MNB2) recorded 169.54 min-1
g
-1 and 152.56
min-1
g
-1 respectively for PO and 199.19 min-1
g
-1 and 172.24 min-1
g
-1 respectively for
PPO. Higher activity of PAL at fifth DAI was obtained by Pseudomonas fluorescens
(KAU) followed by Trichoderma sp. (PF2) and B. subtilis (MNB2) recording 209.86
min-1
g
-1
, 198.59 min-1
g
-1 and 180.85 min-1
g
-1 respectively. However, the highest per
cent reduction of early blight after the first spray (68.58) was effected by
Trichoderma sp. (PF2) followed by (64.92 and 60.22) B. subtilis (MNB2) and
Ochrobactrum sp. (EB1), respectively. The disease reduction at early stage was
reflected on yield of tomato and the same treatments; Trichoderma sp. (PF2), B.
subtilis (MNB2) and Ochrobactrum sp. (EB1) gave 410 g, 389.33 g and 340 g fresh
tomatoes per plot respectively. Hence these three phylloplane antagonists were
selected for further evaluation.
An experiment was carried out under rain shelter to evaluate the efficacy of
the selected antagonists, conventional biocontrol agents such as Trichoderma sp.
(KAU) and P. fluorescens (KAU) and a chemical check, propineb (0.2 %). The
results showed that the phylloplane antagonists Trichoderma sp. (PF2) and B. subtilis
(MNB2) offered significant effect against the disease recording 40.74 and 37.04
reduction in per cent disease severity (PDS) which in turn resulted in per cent yield
increase of 32.63 and 28.10 respectively. Enumeration of phyllopane microflora
proved that, there is drastic reduction in microbial population on leaf surface after
iii
spraying with propineb whereas the population increased after application of
bioagents. Further, it was found that Trichoderma sp. (PF2) and B. subtilis (MNB2)
are compatible with P. fluorescens (KAU) but B. subtilis (MNB2) is not compatible
with Trichoderma sp. (KAU). Trichoderma sp. (PF2) is weakly inhibited by propineb
(0.2 %), but azoxystrobin (0.1 %) could reduce its growth by 58 per cent. However,
B. subtilis (MNB2) could not be affected by any of the two fungicides used against
leaf blight. Hence, based on the results of various experiments in this study,
phylloplane antagonists, especially, B. subtilis (MNB2) seems to be suitable for
inclusion in IDM for early blight of tomato.
Enumeration of culturable microflora on tomato leaves revealed that, bacteria
are more on healthy leaves while fungi are more on the infected leaves. However,
metagenomic analysis of the phylloplane revealed great difference in density and
diversity of microbial taxa between healthy and infected leaves. This indicates that,
specific functions of the phylloplane microbes and their antagonistic potential are
also important in determining their efficacy as biocontrol agents. The study also
revealed the presence of plant pathogenic fungal genera like Cladosporium,
Corynespora, Pseudocercospora, Conidiosporomyces and Ustilago on tomato leaves.
Both healthy and diseased tomato leaves harbour great microbial diversity and the
phylloplane dwellers include well-known antagonists like species of Bacillus,
Pseudomonas, Trichoderma and Penicillium. Presence of members of genera like
Klebsiella and Enterobacter which are human pathogens was also revealed by
metagenomic analysis. However, they are beneficial to plants as N fixers, P and K
solubilizers and siderophore and IAA producers. Phylloplane dwellers also include
species of Methylobacterium, Sphingomonas and Massilia and they are reported to
produce different pigments and antibiotics besides being plant growth promoters, and
antagonists to plant pathogens. Furthermore, species of Staphylococcus,
Cladosporium, Coprinellus and Moesziomyces are also detected from tomato
phylloplane and are reported to have antifungal properties with good plant growth
attributes, while species of Meira are reported as potential biological control agents
against phytophagous mites