Biological control of postharvest disease in the perishable fruit industry by bacillus lipopeptides
Date
2018-03
Authors
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Publisher
Stellenbosch : Stellenbosch University
Abstract
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 different
Bacillus 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 work
presented 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 from
post-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 were
successfully 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 triplicate
in 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 out
in this study were met by large. B. amyloliquefaciens was identified as a target strain for culture and process optimisation, targeting iturins, and for incorporation
towards the development of a cell-free biocontrol agent targeting phytopathogens found in, but not limited to, the South African Table Grape Industry.
AFRKIKAANSE OPSOMMING: In die studie is die bakterium Bacillus spp bestudeer as moontlike produseerder van anti-fungiese middels wat as plaasvervanger kan dien vir die huidige chemiese middels wat as plaagdoders gebruik word teen fungi. Die kriteria waaraan die siftingstoets onderworpe was, is gebasseer op die vermoeë van die bakterium om anti-fungiese middels te kon produseer en/ of hierdie middels anti-fungiese aktiwiteit kon toon teen die fungi wat ge-isoleer is uit die natuur, meer spesifiek, tafeldruiwe. Met die studie is verder gepoog om die anti-fungiese middels, lipopeptiedes, wat verantwoordelik is vir anti-fungiese aktiwiteite, te identifiseer asook moontlike prosesse te identifiseer vir isolasie. Met die doelwitte word beoog om toekomstige navorsing te lei wat fokus op die in vitro produksie van ‘n selvrye produk wat kan dien as ‘n biologiese plaagdoder. ‘n Kultuurbank vir anti- fungiese eksperimente is geskep vanuit 79 fungiese monsters wat vanaf die Suid-Afrikaanse Tafeldruifbedryf verkry is. Dit is verder omskep in 59 suiwerkulture. DNS identifikasie is gedoen op 18 kulture en 16 is suksesvol geidentifiseer met behulp van DNS herhalings en morfologie studies. Die fungi is gegroep as 50% Penicillium spp., 33% to Botrytis spp. en 17% Aspergillus spp., Alternaria spp. en Lewia spp. saamgegroepeer. Vier spesies van Bacillus naamlik Bacillus amyloliquefaciens DSM 23117, B. licheniformis DSM 13, B. subtilis ATCC 21332 en B. subtilis spizizini DSM 347 is bekom vir die anti-fungiese middels wat hulle produseer, naamlik lipopeptiedes. Bacilluskulture is gekultiveer op selmedia soos gebubliseer deur Kim et al. (1997) met modifikasies en Bacillus amyloliquefaciens is geidentifiseer as die top spesie vir lipopeptiedproduksie. Die resultate van B. amyloliquefaciens gedurende kultivering dui daarop dat die spesie van Bacillus ‘n oorproduseerder is van anti-fungiese middels. Die ongesuiwerde anti-fungiese middels het ook sterk anti-fungiese eienskappe teen al 9 van die fungi-isolate waarteen dit getoets is, getoon. Die fungiese kulture getoets, het behoort aan Botrytis, Penicillium, Alternaria, Aspergillus en Lewia genera. Dunlaag chromatografie in kombinasie met anti-fungiese toetse het Iturins, pieke 3, 4, 5, 8 en 9 geidentifiseer as die middels verantwoordelik vir anti-fungiese aktiwiteit wat die groep ‘n belangrike onderwerp vir verdere studie maak met die doel op die ontwikkeling van ‘n selvrye produk. Deur gebruik te maak van verskillende isolasie- en suiweringsmetodes is soutpresipitasie geidentifiseer as ‘n metode vir algemene isolasie van alle lipopeptiedes terwyl isobutanol en n-hexaan spesifiek anti-fungiese middels geteiken het vir isolasie. Deur die gebruik van verskeie analitiese- en eksperimentele metodes is al die doelwitte wat in hierdie studie uiteengesit is, behaal. B. amyloliquefaciens is geïdentifiseer as 'n teikenspesie vir verdere prosesoptimalisering Iturins en vir inkorporasie in studies vir die ontwikkeling van 'n selvrye, biologiese plaagdoder wat op fytopatogene in die Suid-Afrikaanse Tafeldruifbedryf fokus.
AFRKIKAANSE OPSOMMING: In die studie is die bakterium Bacillus spp bestudeer as moontlike produseerder van anti-fungiese middels wat as plaasvervanger kan dien vir die huidige chemiese middels wat as plaagdoders gebruik word teen fungi. Die kriteria waaraan die siftingstoets onderworpe was, is gebasseer op die vermoeë van die bakterium om anti-fungiese middels te kon produseer en/ of hierdie middels anti-fungiese aktiwiteit kon toon teen die fungi wat ge-isoleer is uit die natuur, meer spesifiek, tafeldruiwe. Met die studie is verder gepoog om die anti-fungiese middels, lipopeptiedes, wat verantwoordelik is vir anti-fungiese aktiwiteite, te identifiseer asook moontlike prosesse te identifiseer vir isolasie. Met die doelwitte word beoog om toekomstige navorsing te lei wat fokus op die in vitro produksie van ‘n selvrye produk wat kan dien as ‘n biologiese plaagdoder. ‘n Kultuurbank vir anti- fungiese eksperimente is geskep vanuit 79 fungiese monsters wat vanaf die Suid-Afrikaanse Tafeldruifbedryf verkry is. Dit is verder omskep in 59 suiwerkulture. DNS identifikasie is gedoen op 18 kulture en 16 is suksesvol geidentifiseer met behulp van DNS herhalings en morfologie studies. Die fungi is gegroep as 50% Penicillium spp., 33% to Botrytis spp. en 17% Aspergillus spp., Alternaria spp. en Lewia spp. saamgegroepeer. Vier spesies van Bacillus naamlik Bacillus amyloliquefaciens DSM 23117, B. licheniformis DSM 13, B. subtilis ATCC 21332 en B. subtilis spizizini DSM 347 is bekom vir die anti-fungiese middels wat hulle produseer, naamlik lipopeptiedes. Bacilluskulture is gekultiveer op selmedia soos gebubliseer deur Kim et al. (1997) met modifikasies en Bacillus amyloliquefaciens is geidentifiseer as die top spesie vir lipopeptiedproduksie. Die resultate van B. amyloliquefaciens gedurende kultivering dui daarop dat die spesie van Bacillus ‘n oorproduseerder is van anti-fungiese middels. Die ongesuiwerde anti-fungiese middels het ook sterk anti-fungiese eienskappe teen al 9 van die fungi-isolate waarteen dit getoets is, getoon. Die fungiese kulture getoets, het behoort aan Botrytis, Penicillium, Alternaria, Aspergillus en Lewia genera. Dunlaag chromatografie in kombinasie met anti-fungiese toetse het Iturins, pieke 3, 4, 5, 8 en 9 geidentifiseer as die middels verantwoordelik vir anti-fungiese aktiwiteit wat die groep ‘n belangrike onderwerp vir verdere studie maak met die doel op die ontwikkeling van ‘n selvrye produk. Deur gebruik te maak van verskillende isolasie- en suiweringsmetodes is soutpresipitasie geidentifiseer as ‘n metode vir algemene isolasie van alle lipopeptiedes terwyl isobutanol en n-hexaan spesifiek anti-fungiese middels geteiken het vir isolasie. Deur die gebruik van verskeie analitiese- en eksperimentele metodes is al die doelwitte wat in hierdie studie uiteengesit is, behaal. B. amyloliquefaciens is geïdentifiseer as 'n teikenspesie vir verdere prosesoptimalisering Iturins en vir inkorporasie in studies vir die ontwikkeling van 'n selvrye, biologiese plaagdoder wat op fytopatogene in die Suid-Afrikaanse Tafeldruifbedryf fokus.
Description
Thesis (MEng)--Stellenbosch University, 2018.
Keywords
Bacillus amyloliquefaciens -- Biological control, UCTD, Iturin, Postharvest diseases and pests -- Biological control, Grapes -- Diseases and pests -- Biological control, Lipopeptides as pest control agents, Biological pest control agents, Phytopathogenic fungi -- Biological control