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Genetic Marker Assisted Detection Of Bacillus Spp. Antimicrobial Metabolites And Induced Systemic Resistance By Lipopeptides Against Sclerotinia Sclerotiorum

Posted on:2020-06-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:Ayaz FarzandFull Text:PDF
GTID:1483306605491624Subject:Plant pathology
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Sclerotinia sclerotiorum is a devastating necrotrophic pathogen that infects multiple crops and causes significant yield loss.Antimicrobial compounds included lipopeptides and polyketides produced by Bacillus species play an important role in antifungal activity against fungal pathogens and induced systemic resistance without any hazardous effect on the environment.Bacillus species produce multiple antifungal lipopeptides that can control plant disease by suppressing the level of inoculum of pathogens or induction of systemic resistance in plants.The aim of this study was to detect antimicrobial compound biosynthetic genes through genetic markers in different Bacillus species and their validation through LC-MS,Expression profiling of antimicrobial genes in EZ1509 and virulence genes in S.sclerotiorum during co-culturing.Moreover,to understand the mechanism of action of fengycin and its ability to elicit systemic resistance in tomato against S.sclerotiorum.In the first part of this study genetic markers and LC-MS were used simultaneously to detect eight antimicrobial compounds viz.,surfactin,bacillomycin,iturin,fengycin/plipastatin,bacilysin,bacillaene,bacillibactin and plantazolicin in 47 Bacillus strains.Among them 19 strains possessed marker sequences encoding secondary metabolites under study.Bacillus strains FZB42,EZ1509,EZ1507,VM10,GBAC46,VM49 and LLCG43 indicated the presence of maximum number of antimicrobial biosynthetic genes.LC-MS confirmed corresponding results obtained from genetic marker-based detection but was unable to detect fengycin and surfactin in 168 and fengycin in OKB105 by LC-MS due to absence of regulatory sequences responsible for the production of these compounds.Nineteen Bacillus strains and their methanolic extracts were tested in vitro for their antifungal activity against S.sclerotiorum.Among nineteen strains EZ1509,VM10,GBAC46,VM49 and FZB42 showed highest inhibitory activity against the pathogen.A bioassay on detached rapeseed leaves revealed that strains VM10,EZ1509,FZB42 and GBAC46 showed highest reduction in lesion diameter,while,the strains OKB105 and 168 were completely ineffective to control S.sclerotiorum.The antifungal activity of Bacillus strains was found to be positively correlated to the number of antimicrobial genes possessed by them,indicating their vital role in antifungal activity.To explore the mechanisms underlying the suppression of S.sclerotiorum by Bacillus species transcriptomic analysis of diffusible lipopeptides and fungal virulence genes was carried out.Among five Bacillus strains,B.amyloliquefaciens EZ1509 possessing four LPs biosynthetic genes viz.,surfactin,iturin,fengycin,and bacilysin exhibited promising antifungal activity against S.sclerotiorum in a dual culture assay.The pivotal role of lipopeptides in the promising antifungal activity of Bacillus amyloliquefaciens EZ1509 was evaluated.A remarkable upregulation in LPs biosynthetic gene expression was observed during the in vitro interaction assay with S.sclerotiorum.The highest upsurge in LPs biosynthetic genes was observed on the 2nd and 3rd day of in vitro interaction.Iturin and fengycin were the highly expressed genes on the 2nd and 3rd day.MALDI-TOF-MS analysis was used to confirm the presence of LPs in the inhibition zone.Furthermore,disintegration,shrinkage,plasmolysis,and breakdown of fungal hyphae were observed by scanning electron microscopy analysis.S.sclerotiorum previously challenged with EZ1509 showed significantly reduced pathogenicity on detached leaves of tobacco and rapeseed during In Planta evaluation.S.sclerotiorum previously confronted with EZ1509 also showed the reduced synthesis of oxalic acid.The expression of major virulence genes of S.sclerotiorum including endopolygalacturonase-3,oxalic acid hydrolase,and endopolygalacturonase-6 was significantly downregulated during in vitro confrontation with EZ1509.The suppression of S.sclerotiorum by induction of systemic resistance and regulation of antioxidant pathways in tomato plants was studied.Fengycin extracted from Bacillus amyloliquefaciens FZB42 mutant AK1S(which is a double mutant producing fengycin only and unable to produce surfactin and bacillomycin D)induced systemic resistance in tomato against S.sclerotiorum.In a dual culture assay B.amyloliquefaciens FZB42 wild type,its mutant AK1S,and their corresponding metabolites showed inhibition of S.sclerotiorum.Fengycin derived from AK1S mutant was detected and identified through HPLC and MALDI-TOF-MS analysis respectively.Structural deformities and alterations in fungal mycelium were observed by scanning electron microscopy and transmission electron microscopy.Fengycin induced the accumulation of Reactive Oxygen Species(ROS)in S.sclerotiorum mycelium by downregulating ROS-scavenging genes viz.,superoxide dismutase(SsSOD1),peroxidase(SsPO)and catalase(SsCAT1)in fengycin treated mycelium compared to untreated control.The effect of fengycin on disease development was evaluated in a greenhouse experiment indicating that the lesion size was dramatically reduced in fengycin treated tomato plants compared to infected control plants.Transcriptomic analysis of plant defense-related genes GST,SOD,PAL,HMGR,and MPK3 showed the highest upregulation at 48 hpi and their expression was subsequently decreased at 96 hpi in fengycin+S.sclerotiorum treatment compared to the plants treated with fengycin only.In contrast,the expression of PPO increased in a linear manner up to 96 hpi.The upsurge in the expression of defense-related genes as a response to fengycin treatment indicates the possible role of fengycin in the elicitation of plant defense against S.sclerotiorum.Fengycin played an important role in defense elicitation to protect the plantsagainst the pathogen.Collectively,our study provides new insights regarding the potential of antimicrobial compounds or lipopeptides in biocontrol activity against S.sclerotiorum.
Keywords/Search Tags:Bacillus amyloliquefaciens, Sclerotinia sclerotiorum, Lipopeptides, Transcriptional regulation, Non-volatile compounds of Bacillus spp., antifungal activity, Induced systemic resistance, pathogenic traits
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