M.Sc

The study entitled “Management of bacterial wilt disease of tomato by the root
endophytic fungus Piriformospora indica, rhizobacteria and bacterial endophytes”
was conducted during 2016-2018 at Department of Agricultural Microbiology,
College of Agriculture, Vellayani with the objective of assessing the potential of root
endophytic fungus Piriformospora indica, plant growth-promoting rhizobacteria and
bacterial endophytes in suppressing bacterial wilt incidence in tomato.
The bacterial wilt pathogen, Ralstonia solanacearum was isolated from infected
tomato plants on Semi selective medium from South Africa (SMSA). Koch’s
postulates were proved by artificial inoculation of tomato seedlings. A functional
relationship between population and optical density of the broth culture was worked
out and was used for determining the inoculum density for challenge inoculation in
the biocontrol experiment.
In vitro antagonistic interaction between the bioagents and the pathogen was
worked out both by direct and indirect assays. In all direct assays which involved
cross streak plating, agar plug diffusion technique, disc diffusion and spot on lawn
method, it was found that Bacillus amyloliquefaciens VLY24, Bacillus velezensis
PCSE10 and Streptomyces leeuwenhoekii KBT004 exhibited antagonism against
Ralstonia solanacearum.
Indirect antagonism was checked by agar well diffusion and disc diffusion
methods using culture filtrate. Out of the eight bacterial bioagents tested, only two
i.e., Bacillus velezensis PCSE10 and Bacillus amyloliquefaciensVLY24 had
inhibitory effect on the pathogen.
Dual culture plate assay on PDA has shown that three bacterial bioagents,
Rhizobium radiobacter PCRE10, Bacillus megaterium NAT001 and Streptomyces
leeuwenhoekii KBT004 were compatible with Piriformospora indica. However, when
the compatible bacteria were co-cultured in a single fermentor system along with
Piriformospora indica it was observed that Bacillus megaterium NAT001 failed to
grow along with the fungal endophyte.
Suppression of bacterial wilt incidence by the individual and combined
application of bacterial bioagents and fungal endophytes were tested with the wilt
susceptible tomato variety Naveen (Indo-American hybrid seeds Pvt. Ltd, Bengaluru)
and the moderately tolerant KAU variety Vellayani Vijay. Bioagents were applied
during the nursery production of seedlings and the 21 days old seedlings were
transplanted to pots filled with unsterile garden soil. Challenge inoculation with the
pathogen was done five days after transplanting by drenching the pots with 10 ml
each of the bacterial suspension (107cfu/ml). The disease incidence was scored at
weekly intervals for 21 days.
When the bioagents were tested individually for the suppression of bacterial
wilt incidence in the hybrid variety Naveen, maximum disease suppression was
observed in plants treated with Rhizobium radiobacter PCRE10 (15 percent) after 21
days. The disease suppressive ability of Bacillus velezensis PCSE10 (25 percent) was
also significantly superior to all other treatments.
Selection of bacterial bioagents for combined application with Piriformospora
indica was done based on compatibility with the fungal endophyte and inhibitory
action against Ralstonia solanacearum. Combined application of the fungal
endophyte and Bacillus amyloliquefaciens VLY24 suppressed the wilt incidence to
the highest extent (40 percent).
When bacterial wilt suppression in Vellayani Vijay was tested by the individual
application of bacterial bioagents, maximum disease suppression was recorded in
plants treated with Streptomyces leeuwenhoekii KBT004 (30 percent), when observed
21 days after challenge inoculation. The combination of Piriformospora indica with
Rhizobium radiobacter PCRE10 (50 percent) and Bacillus velezensis PCSE10 (50
percent) showed significantly lower disease incidence compared to the rest of the
treatments.
In the plant growth promotion experiment done with the variety Vellayani Vijay
in the nursery stage, maximum plant height was observed in plants treated with
Rhizobium radiobacter PCRE10 (14.49cm). However, those plants treated with
Bacillus pumilus VLY17 had the highest number of leaves per plant (4.58).
Combined inoculation of Piriformospora indica and Bacillus amyloliquefaciens
VLY24 resulted in improved shoot fresh weight (2.08 g per plant) which was on a par
with the plants treated with Bacillus velezensis PCSE10 (1.95 g plant-1). However,
shoot weight on dry weight basis was the maximum for plants treated with Bacillus
velezensis PCSE10 (122.55 mg plant-1) which was at par with those treated with
combination of Piriformospora indica and Bacillus amyloliquefaciens VLY24
(104.78 mg plant-1).
The present study revealed that biological management of bacterial wilt in
tomato could be a feasible strategy under controlled conditions. The same has to be
validated under field conditions before making any recommendations.