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Evaluation of Bacillus velezensis Kos for the control of Lecanicillium fungicola

ABSTRACT: Lecanicillium fungicola, the causative agent of dry bubble disease on Agaricus bisporus results in significant crop losses for mushroom growers worldwide. Dry bubble disease is treated through strict hygiene control methods and the application of chemical fungicides but an increase in fungicide resistant L. fungicola strains has resulted in a need to develop alternative biocontrol treatment methods. The aim of the work presented here was to evaluate the response of L. fungicola to a Bacillus velezensis isolate to assess its potential as a novel biocontrol agent. The bacterial species in Serenade, a commercially available biocontrol treatment was also included in this analysis. Exposure of 48 hr L. fungicola cultures to 25% v/v 96h B. velezensis culture filtrate resulted in a 45% reduction in biomass (P < 0.0002) and a disruption in hyphal structure and morphology. Characterisation of the proteomic response of L. fungicola following exposure to B. velezensis culture filtrate revealed an increase in the abundance of a variety of proteins associated with stress response (Norsolorinic acid reductase (+8 fold), isocitrate lyase (+7 fold) and MMS19 nucleotide excision repair protein (+4 fold). There was also a decrease in the abundance of proteins associated with transcription (40S ribosomal protein S30 (-33 fold), 60S ribosomal protein L5 (-45 foldThe results presented here indicate that B. velezensis culture filtrate is capable of inhibiting the growth of L. fungicola and inducing a stress response, thus indicating its potential to control this important pathogen of mushrooms.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Lecanicillium Fungicola Var. Fungicola

SUBMITTER: Joy Clarke

LAB HEAD: Kevin Kavanagh

PROVIDER: PXD028506 | Pride | 2022-06-01

REPOSITORIES: Pride

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Project description:Aspergillus fumigatus is an opportunistic fungal pathogen of the lungs resulting in both chronic and acute infection. The fungus interacts with numerous bacteria that compose the microflora of the lung including Pseudomonas aeruginosa and Klebsiella pneumoniae, both common isolates from Cystic fibrosis sputum. It is well documented that bacterial and fungal interactions both physically and through production of secreted products which can alter the growth and virulence of other species. The interaction with P. aeruginosa has been characterised previously indicating decreased growth and increased gliotoxin production in the presence of cells and the opposite when only exposed to culture filtrate. In our analysis exposure of cell free culture filtrate inhibited fungal growth and increased gliotoxin production. This was supported with proteomic analysis indicating reduction in 1,3-beta-glucanosyltransferase (-3.97 fold), methyl sterol monooxygenase erg25B (-2.9 fold) and Calcium/calmodulin dependent protein kinase (-4.2 fold) involved in fungal development. Coupled with increased expression of Glutathione S-transferase gliG (6.17 fold), non-ribosomal peptide synthase gliP (3.67 fold), O-methyltransferase gliM (3.5 fold), Gamma-glutamyl acyltransferase gliK (2.89 fold) and thioredoxin reductase gliT (2.33 fold) involved in Gliotoxin production indicate a possible shift towards secondary metabolism. Qualitative proteomic analysis of the bacterial supernatant also elucidates possible mechanisms for these alterations including physical interaction by Outer membrane protein A, High-affinity zinc uptake system protein ZnuA and Iron uptake system component EfeO reducing essential nutrient availability, enzymatic activity including Periplasmic serine endoprotease DegP-like and redox active proteins including Alkyl hydroperoxide reductase C. These proteomic alterations to the fungus could have implications for the severity of virulence in the context of coinfection and may impact disease progression.

Project description:Lipopeptide biosurfactant producing Bacillus strains have many useful applications in biotechnology and agriculture, based on their emulsifying, surface activity and antimicrobial properties. In the current study, lipopeptide production kinetics, and biocontrol potentials of two new B. velezensis strains, ES1-02 and EFSO2-04 were analyzed and compared with those of commercial strains QST713 and FZB42. ES1-02 and EFSO2-04 showed higher specific growth rates than FZB42, but lower growth rates than QST713. All strains produced surfactin lipopetides, while fengycin production was not observed in ES1-02 and EFSO2-04. Production of fengycin A, B, X and Y were however confirmed in strains QST713 and FZB42. Significant differences were observed in the production of lipopeptides of the iturin family. While ES1-02 and EFSO2-04 produced bacillomycin L, QST713 produced iturin A, and FZB42 produced bacillomycin D. This was in line with the PCR analysis of corresponding genes encoding the identified lipopeptides. Highest surfactin titer of 97.4 mg/L was observed in ES1-02, while QST713 produced highest amount of iturin/bacillomycin (8.5 mg/L). Surfactin isoforms C12 to C17, and iturin/bacillomycin isoforms C11 to C17 were identified by mass spectrometry. ES1-02 and EFSO2-04 showed biocontrol potentials comparable with that of QST713 against Diaporthe spp., while FZB42 showed superior antifungal potentials. Up to 41%, 43%, 47 % and 68.9 % inhibition of D. caulivora were achieved by ES1-02, EFSO2-04, QST713 and FZB42 respectively. Upon exposure to B. velezensis strains, morphological changes to Diaporthe hyphae in form of swellings, distortion, and complete disruption occurred. Interaction of D. longicolla DPC_HOH20 with ES1-02 and EFSO2-04 induced 10-fold and 5-fold increase in surfactin synthesis, respectively. Antagonist interaction with D. longicolla induced significant changes in the proteome of ES1-02 including an increased abundance of several proteins associated with biosynthesis of antimicrobial compounds and fatty acids, while proteins associated with phosphate uptake were decreased in abundance.

Project description:Bacillus thuringiensis, a well-known and effective bio-insecticide, has attracted considerable attention as a potential biological control agent for the suppression of plant diseases. Treatment of tomato roots with a filter-sterilized cell-free filtrate (CF) of B. thuringiensis systemically suppresses bacterial wilt caused by Ralstonia solanacearum through systemic activation of the plant defense system. Comparative analysis of the expression of the Pathogenesis-Related 1(P6) [PR-1(P6)] gene, a marker for induced resistance to pathogens, in various tissues of tomato plants treated with CF on their roots suggested that the B. thuringiensis-induced defense system was activated in the leaf, stem, and main root tissues, but not in the lateral root tissue. At the same time, the growth of R. solanacearum was significantly suppressed in the CF-treated main root tissue but not in the CF-treated lateral root tissue. This distinct activation of the defense reaction and suppression of R. solanacearum were reflected by the differences in the transcriptional profiles of the main and lateral tissues in response to the CF. In the CF-treated main root tissue, but not CF-treated lateral root tissue, the expression of several salicylic acid (SA)-responsive defense-related genes was specifically induced, whereas jasmonic acid (JA)-related gene expression was either down-regulated or not induced in response to the CF. On the other hand, genes encoding ethylene (ET)-related proteins were induced equally in both the main and lateral root tissues. Taken together, the co-activation of SA-dependent signaling pathway with ET-dependent signaling pathway and suppression of JA-dependent signaling pathway may play key roles in B. thuringiensis-induced resistance to R. solanacearum in tomato plants. Gene expression was measured in main and lateral root tissues of tomato treated with Bacillus thuringiensis or distilled water-treated control at 48 hours after treatment. Two independent experiments were performed at each tissue (main root or lateral root tissue) for each treatment (Bacillus thuringiensis or distilled water control).

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Evaluation of Bacillus velezensis Kos for the control of Lecanicillium fungicola

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