Asian Program

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Dissertation Abstracts

Thailand

  Ralstonia solanacearum Yabuuchi et al. (1995), the former name Pseudomonassolanacearum E.F.Smith (1896), is one of the most important diseases in the world. Since it is a severe and devastating, this disease causes tremendous damage on many crops in tropical, subtropical and warm temperate regions. It has been documented that this disease is soil-borne and the pathogen survives for long time in agricultural soils. The pathogen distributed around the world by seeds such as potato. Also it dispatches easily by irrigation water, planting material or equipment, animal, insect, human etc. Since chemical control is not always useful because of its cost and efficacy, the introduction of the resistant variety has been highly stressed. However, the resistance to R. solanacearum is not vertical and rather horizontal. Such horizontal resistance varied with the circumstances as soil condition and climate, especially temperature. Thus, the uses of resistant varieties and chemicals, the common control measures to plant diseases, are unreliable. This pathogen has been called Ralstonia solanacearum complex and involves so many varieties of strains. Furthermore, it has been documented the existence of VNC (viable but non-cultural) state in the whole life cycle. Therefore, the measurement of the pathogen density in soils with the general procedure, counting colonies appeared on the culture media, will not be reliable.
  For efficient control of this complicated disease, the survey of the distribution of the strains and estimation of the pathogen density in soils with novel procedures have been required. The author collected totally 120 isolates from the wilt-diseased 17 economic plants and a weed of 18 provinces in Thailand. Hypersensitivity and pathogenicity of these 120 isolates against original and collateral hosts were tested. Furthermore, the virulence of these bacteria was verified by the behavior on TTC medium. Biovars were decided with biochemical characteristics and races were decided by the inoculation to seven collateral economic hosts. Results obtained were as follows: Anthurium 4 strains are race 1 biovar 3, holy basil 1 strain is race 1 biovar 3, chili 5 strains are race 1 biovar 3 and race 3 biovar 2a, curcuma 6 strains are race 1 biovar 3 and 4, eggplant 7 strains are race 1 biovar 4, eucalyptus 1 strain is race 1 biovar 3, ginger 10 strains are race 1 biovar 3 and 4, groundnut 8 strains are race 1 biovar 3 and 4, marigold 4 strains are race 1 biovar 3, pepper (yellow) 2 strains are race 1 biovar 3, pepper 10 strains are race 1 biovar 3 and 4, race 3 biovar 2a, potato 35 strains are race 1 biovar 3,4 and race 3 biovar 2a,3 and 4, sesame 2 strains are race 1 biovar 3,tobacco 6 strains are race 1 biovar 3,tomato 6 strains are race 1 biovar 3, tomato(proa) 8 strains are race 1 biovar 3,4, water convolvulus 2 strains are race 1 biovar 4,weed 3 strains are race 1 biovar 3.
  In general, the serological tests with polyclonal and monoclonal antibodies are useful for efficient and precise identification of the strains of the microorganisms. It was assumed that the serological techniques will be useful for the detection of R. solanacearum strains in the latent state of VNC. The author obtained a polyclonal antibody (Pab) and 22 monoclonal antibodies (Mab) with R. solanacearum potato strains. Seventy-seven isolates which were isolated from 17 various plants were tested for the serological reaction to the Pab and Mabs by the indirect ELISA assay. Most reactions with the monoclonal antibodies (Mabs) were more sensitive than polyclonal antibody, but the correlation among races, biovars and serotypes was ambiguous.
  The phylogenetics with 16SrRNA gene analysis has been highly focused and also used for identification of microorganisms. The author performed the sequencing for 1545 nucleotides of 16S rRNA of R. solanacearum, 20 isolates of 3 biovars from 15 host plants in Thailand and compared with the database for R. solanacearum type culture No.U27986 from DDBJ (DNA Database Gene Bank of Japan). The results of sequencing showed 97.5% similarity and intraspecific differences less than 2.5%. The differences were recognized at 39 positions on whole sequences. The differences were found at 405,459-460,511,566,612,1025 in potato strain biovar 2;405,433,435,1113 in ginger strain biovar 3 and 612,813,1437 in biovar 4;435,502,597,612,642,650,824,1409,1439 in ground nut strain biovar 3; 566,809 in pepper strain biovar 3 and 809 in biovar 4; 405,458-460,474,532, 649,1419,1423,1451 in chili strain biovar 3; 459,460, 502,535-537, 1240 in curcuma strain biovar 4; 459,460,612,809,1096,1315,1423 in tobacco strain biovar 3; 411,459,460,474,502,549,566,913,914,1096 in sesame strain biovar 3; 1185,1314 in tomato strain biovar 3. Whereas in the cases of eggplant strain biovar 3, potato strain biovar 3 and 4, anthurium strain biovar 3, eucalyptus strain biovar 3, water convolvurous strain biovar 4 and weed (Mexican fire plant) strain biovar 3, no differences were observed. No significant correlation was found among the results of sequencing, races and biovars.
  Since the use of the chemicals and the resistant variety are not reliable control measures to the bacterial wilt caused by R. solanacearum, the biological control has long been stressed. The author isolated 319 bacteria from the rhizoplanes of symptomless host plants in moderately to heavily infested fields of 18 provinces in Thailand. The antagonistic bacteria were screened by the confronting bioassay method on King B medium. Fifteen antagonistic bacteria were selected as a antagonist to R. solanacearum and 5 isolates screened after the tests in green house. The efficacy of the antagonists were obvious in the case of pre-treatment of seedling before infection and not obvious at post-treatment. These 5 antagonists were effective in field condition and the striking outcomes were obtained at 2 locations in the northern Thailand at which the fields had been heavily infected during 2003-2005. In this biological control, the potato were dressed with the suspension of the antagonists at the concentration of 109 cfu/ml before planting and drenched every 7 days for four times. The results showed that these antagonists controlled significantly the bacterial wilt disease at 24-70%. The same trials were also conducted at bigger areas (farm trial) in 2 locations at Chaing mai and Kanchanaburi during 2004-2005. The results showed that these antagonists are strikingly effective and controlled 62-83% of this notorious wilt disease. Furthermore, the treatment with the antagonists promoted well the potato yields. A novel method which was invented for rapid identification of phytopathogenic bacteria by using TLC with aminolipids was successfully applied for the identification of these antagonistic bacteria. The 5 antagonists were identified as Bacillus subtilis which the common spot at Rf. 0.36 existed on their TLC chromatogram.

Typical symptom is green wilt symptom and clearly bacterial exudates (bacterial ooze).

Population of Ralstonia solanacearum in soil under biocontrol by antagonist bacteria in pre disease infected application