Biological control of postharvest disease in the perishable fruit industry by bacillus lipopeptides

[摘要] ENGLISH SUMMARY: The work presented in this study aimed to investigate the suitability of Bacillus spp. lipopeptides to be used as effective biocontrol agents that have the potential to replace chemically synthesised pesticides that are currently in use in post-harvest disease control. The screening criteria were based on the ability of differentBacillus species to produce antifungal lipopeptides which in turn could inhibit phytopathogens isolated from post-harvest grapes. The study further aimed to identify the lipopeptides responsible for any noted antifungal activity and investigate possible downstream isolation methods that could be included into process optimisation. With these goals in mind, the workpresented here hopes to inform on the way forward for Future studies that are focused towards the controlled in vitro production of a customised cell-free biocontrol agent for post-harvest disease control. A phytopathogen culture bank was established from 79 crude isolates of fungi frompost-harvest grapes supplied by the South African Table Grape industry. These isolates were purified into 59 pure cultures using 5 sequential rounds of subculturing on PDA, MEA and NA agar plates. Of these, 16 isolates weresuccessfully sequenced using Sanger sequencing with ITS 4 and ITS 5 primers. Of the identified phytopathogens, 50% belonged to Penicillium spp., 33% to Botrytis spp. and the remaining 17% grouped as Aspergillus spp., Alternaria spp.and Lewia spp. These phytopathogens are responsible for noble rot, blue-green mould, bunch rot, black mould and leaf blight in the grape industry respectively. Four different strains of lipopeptide producing bacteria, Bacillus amyloliquefaciens DSM 23117, B. licheniformis DSM 13, B. subtilis ATCC 21332 and B. subtilis spizizini DSM 347 were obtained from culture collections and screened in triplicatein shake flasks over a seven day period using media from Kim et al (1997) with modifications. Bacillus amyloliquefaciens DSM 23117 was identified as the superior producer of lipopeptides. In addition to the significant amounts of lipopeptides produced by this strain, crude lipopeptides from B. amyloliquefaciens were shown to exhibit efficacy (either by causing death or static inhibition) towards all 9 isolated fungal phytopathogen strains tested in radial diffusion assays (RDA). The strains incorporated in RDA assays belonged to Botrytis, Penicillium, Alternaria, Aspergillus and Lewia genera.TLC separation in combination with antifungal efficacy assays further narrowed down the Iturin family, peaks 3, 4, 5, 8 and 9, as compounds that exerted significant antifungal activity. Thus indicating these compounds are important targets for isolation and incorporation into antifungal studies towards the development of cell-free biocontrol products for use in post-harvest applications and crop protection. Using different downstream methods for lipopeptide isolation and purification, salt precipitation was identified as a bulk isolation method while solvent extraction from isobutanol and n-hexane proved superior in separating antifungal lipopeptides from surfactin. Through the use of various analytical and experimental methods all the aims set outin this study were met by large. B. amyloliquefaciens was identified as a target strain for culture and process optimisation, targeting iturins, and for incorporationtowards the development of a cell-free biocontrol agent targeting phytopathogens found in, but not limited to, the South African Table Grape Industry.

[发布机构] Stellenbosch University