| Abstract | Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious and widespread
disease in tomatoes and capsicums. The control of this soil-borne pathogen presents a
challenge to growers. The persistence and its wide host range often restrict the
effectiveness of the cultural and chemical control measures. Therefore this study was
conducted to explore antagonistic microorganisms for biological control of BW disease
(R. solanacearum) in tomato and capsicum at the Agricultural System Laboratories of the
Asian Institute of Technology, Thailand during March 2008 to July 2009. There were four
steps: 1) Isolation of potential antagonists and pathogen, 2) Multiplication, screening of
effective antagonists against R. solanacearum in in vitro conditions, production
optimization and identification of isolated antagonists 3) In vivo evaluation of antagonistic
strains on capsicum and tomato wilt in sterilized soils, and 4) In vivo evaluation of
selected antagonists under unsterilized soils against R. solanacearum.
Bacterial wilt diseased plant parts of capsicum annuum, tomato and infested soil were
collected from various fields (Duc Trong district, Lam Dong Province, Vietnam, and
Banglen district, Nakhon Pathom Province, Thailand) where BW disease was prevalent.
The BW pathogen, R. solanacearum was also isolated from diseased plant parts using
TZC medium. The suspected antagonistic microbes were isolated from roots and soils by
the triple layer agar technique. There were 73 different colonies of microorganisms, of
which twenty colonies were potentially high as antagonists for inhibiting R. solanacearum.
Twenty antagonists were screened using in vitro: cross- culture method, the filter-paper
disk method and the perforated agar plate method. Finally six antagonists from all isolates
coded as TR2, TR6, TR7, TR10, TR12 and TR15, and two antagonists available from
previous studies [viz. LR10 - isolated from soils, and Candida ethanolica - isolated from
commercially available Effective Microorganism solution (EM)] were selected for further
production optimization. Biochemical test and physical properties indicated that TR6, TR7
were Bacillus sp.; TR2, TR10, TR12, TR15 were in family Enterobacteriaceae and LR10
was Yeast.
The follow up, in vivo evaluations were conducted in greenhouse to assess the eight
screened antagonists against BW pathogen, using tomato and capsicum annuum seedlings
in sterilized and non-sterilized spoils by introducing both antagonist and pathogen under
three scenarios: 1) both of antagonists and pathogen were inoculated to tomato and chillie
seedlings at the same time; 2) antagonists were introduced one week prior to pathogen
inoculation; and 3) antagonists were introduced after pathogen inoculation and heavy
infestation.
All bio-control agents tested significantly reduced disease symptoms. Among eight
isolated antagonists, Bacillus sp. TR6, Enterobacteriaceae TR12, Yeast LR10 and C.
ethanolica showed a high potential for disease suppression and also promoted plant height,
biomass and fruit weight. The disease severity varied with the antagonists, time of
applications and host plants, which reduced when antagonists were applied one week prior
to transplanting or seedlings. In in vivo studies, the disease severity was reduced to 8.3%,
29.3%, 29.2% and 22.9% in capsicum annuum and 18.8%, 12.5%, 29.2% and 18.8% in
tomato by Bacillus sp. TR6, Enterobacteriaceae TR12, Yeast LR10 and C. ethanolica,
respectively, and in non-sterilized conditions the lowest disease severity was 18.8%,
iv
29.2%, 31.3% and 18.8% in capsicum annuum, and 25%, 12.5%, 26% and 18.8% in
tomato, respectively.
Using partial 16S rDNA sequencing, the strain TR6 was determined as Bacillus
megaterium and the strain TR12 as Enterobacter cloacae. LR10 was determined as Pichia
guillermondii (type strain: U45709) by D1/D2 domain of 26S ribosomal RNA sequence
method.
These results provide encouragement for continuing research on biological control of
bacterial wilt by antagonistic isolates on chillie pepper and tomato and other Solanaceae
crops. |