Bacterial communities associated with the surface of sweet pepper and their selection for biocontrol

Abstract

Biocontrol agents (especially, microbial antagonists) can sustainably and effectively protect yield losses in crops. In sweet peppers (Capsicum annum), one of the most nutritionally rich fruit crop, widely grown worldwide, productivity is threatened by microbial pathogens, particularly those that cause post-harvest spoilage. The identities as well as the roles that could be played by microbial antagonistic microorganisms in protecting yield loses for this important crop are poorly established. The aim of this project was: i) to investigate how the effect of growing conditions (hydroponic system versus direct sowing), inorganic pesticides treatment (i.e., application of a fungicide) and maturity status (green versus red), could influence the structure and composition of bacterial communities on the surfaces of fresh pepper fruits; ii) to predict the phenotypic changes in the microbiota of pepper samples; iii) to identify bacterial taxa with potential to minimize postharvest losses of peppers; and also, iii) to identify bacterial antagonists of R. solanacearum, residing on the surfaces of red and green sweet pepper fruits. To achieve this, amplicon sequencing, targeting the 16S rRNA marker gene, and microbial functions assays to depict the identities and the potential antagonistic functions of bacteria were employed. Amplicon sequencing showed bacteria belonging to the phylum Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, to be enriched in the fungicide-treated compared to fungicide-untreated samples, in open field compared to the hydroponic system samples, and in the green compared to the red samples. Phenotypic predictions (at phylum level) detected high abundance of potentially pathogenic, biofilm forming and stress tolerant bacteria on samples grown on open soils than those from hydroponic systems. Furthermore, bacterial species of genera mostly classified as fungal antagonists including; Acinetobacter, Agrobacterium and Burkholderia were the most

abundant on the surfaces. Microbial isolations and functional analysis successfully identified four potential antagonists from the surface of the sweet pepper fruit surfaces viz. Bacillus cereus strain HRT7.7, Paenibacillus polymyxa strain SGT5.3, Serratia marcescens strain SRT9.1 and Enterobacter hormaechei strain SRU4.4, and these indicated antagonism against R. solanacearum (the most devastating pathogen of peppers). Optimisation studies under different carbon and nitrogen sources revealed that these potentially antagonistic isolates can effectively suppress R. solanacearum at 3% (w/v) starch and 2,5% (w/v) tryptone at pH of 7 and temperature of 30oC. The mode of action exhibited by the strains against the pathogen was secretion of lytic enzymes (i.e., cellulase and protease). Furthermore, the antagonists also displayed plant growth-promoting (PGP) capabilities through phosphate solubilisation and siderophores production. Overall, results demonstrated the ability of sweet peppers to accommodate different microbial taxa on its fruit surfaces, and that some of the microbial constituents can potentially protect the plant under disease pressures from potential pathogenic microbial strain. Results also unequivocally indicated the potential of agronomic choices (especially, site selection/medium of growth) in reducing risk of disease pressures on plants. These results presents a starting point in development of effective biological control as well as integrated pest management (IPM) measures in peppers, for optimization of yield and its protection, globally.