| Tomato Bacterial wilt (TBW) caused by Ralstonia solanacearum is the most serious and worldwide bacterial disease. Due to its wide host range and ability to survive freely in the soil for extended period, no single strategy can provide effective control against this disease in regions where the pathogen is endemic. Biological control of TBW combined with other control approaches has proven to be more durable, eco-friendly and maintain stability and productivity of agro-ecosystems.In this study, about200rhizobacterial isolates were isolated from tomato and potato rhizosphere and examined for their antagonistic activities against R. solanacearum T-91, the causal agent of TBW. Among these isolates,10isolates including Am1, Am2, Am5, Am50, Am47, D11, D12, D16, D29and H8showed high potential of antagonistic activity in in vitro assay and further screened for their biocontrol efficacy against R. solanacearum under greenhouse conditions. In greenhouse,81.1-89.0%reduction of disease incidence of TBW was recorded in the tomato plants treated with the isolates Am1, D16, D29and H8. The application of these biocontrol agents significantly (p>0.05) increased plant height by22.7-43.7%and dry weight by47.93-91.55%compared with non-treated control. The four Bacillus isolates showed ability to inhibit growth of the three soil-borne fungi(Fusarium graminearum, Pythium aphanidermatum and Rhizoctonia solani). Identification of four selected isolates using16SrRNA gene sequence, the biochemical and physiological tests and fatty acid methyl esters analysis assigned strains Am1and D29as Bacillus amyloliquefaciens, D16and H8as B. subtilis and B. methylotrophicus, respectively.Another study was carried out to assess the biocontrol efficacy of B. subtilis4812and B. methylotrophicus H8individually or in combination with two plant defense inducers viz. Acetyl salicylic acid (ASA) and DL-Beta-aminobutyric acid (BABA) against R. solanacearum in vitro and in vivo. The results showed that the pathogen was significantly inhibited by all applied treatments in the greenhouse and in vitro tests. Under in vitro test the treatments H8+ASA and4812+H8were found to be the most effective treatments. Applied on tomato seeds and as soil drenching, the disease was most inhibited by H8whereas4812+H8+ASA was the least effective treatment. High activity of phenylalanine ammonia-lyase was observed in the plants treated with4812+H8, H8+ASA and4812+H8+ASA. The highest expression of peroxidase and polyphenoloxidase enzymes was found in the plants treated with H8,4812+H8and H8+ASA. The result of real time PCR confirmed that concentration of the pathogen in stem tissues was significantly reduced in all treated plants and H8+ASA was the most effective treatment.The study of bioactivity mechanisms revealed that all four strains viz. Bacillus amyloliquefaciens D29, Am1, B. subtilis D16and B. methylotrophicus H8were able to produce Indole acetic acid and siderophores and can not produce Hydrogen cyanide (HCN). Moreover three of them D29, Am1and H8had capability to solubilize phosphor. Bacillus strains were able to form biofilm beside their ability to both membrane lysis and the destruction of Ralstonia biofilm. Results of conventional PCR analysis revealed that all the four strains are harboring the lipopeptides biosynthetic genes bmyB,fenD, ituC, srfAA and bacA which were responsible for biosynthesis of lipopeptides (Bacillomycin, Fengycin, Iturin, Surfactin and Bacilysin), respectively. Subsequent reverse transcription-polymerase chain reaction analysis revealed that ituC and srfAA biosynthesis genes in Bacillus strains Am1and D16gave the highest expression levels during in vitro interaction with R solanacearum which could suggest the potential antibacterial related mechanism is associated to their ability to secret the corresponding lipopeptide in surrounding niche. A positive correlation value (0.777and0.686) was observed between IAA amount, fresh weight and dry weight, respectively in treated tomato plants in a greenhouse trial. |
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