Production of β-1,3-glucanase and chitosanase from clostridial strains isolated from the soil subjected to biological disinfestation

Biological soil disinfestation (BSD) or anaerobic (reductive) soil disinfestation (ASD/RSD) is a bioremediation method used to eliminate soil-borne plant pathogens by exploiting the activities of anaerobic bacteria in soil. In this study, two obligate anaerobic bacterial strains isolated from BSD-treated soil and identified as Clostridium beijerinckii were examined for their abilities to suppress the spinach wilt disease pathogen (Fusarium oxysporum f. sp. spinaciae) as a representative soil-borne fungal plant pathogen. Both strains degraded β-1,3-glucan and chitosan, two major polysaccharide components of ascomycetes fungal cell wall, supplemented in the medium. β-1,3-Glucanase was detected in the supernatants of cultures supplemented with different types of glucan. Similarly, chitosanase was detected in cultures supplemented with chitosan. Both the enzyme activities were also detected in cultures containing glucose as a substrate. Live cells of F. oxysporum f. sp. spinaciae that were co-incubated with each anaerobic strain under anaerobic conditions using glucose as a substrate died during incubation. Freeze-dried dead fungal biomass of the pathogen, when added to the culture, supported good growth of both anaerobes and production of both enzymes. Severe and nearly complete degradation of both live and dead fungal cells during incubation with anaerobic bacteria was observed by fluorescence microscopy. When individual anaerobic bacterial strain was co-incubated with live pathogenic fungal cells in wheat bran, a popular material for BSD-treatment, both the strains grew well and killed the fungal pathogen promptly by producing both enzymes. These results indicate that both the bacterial strains attack the fungal cells by releasing extracellular fungal cell wall-degrading enzymes, thereby eliminating the pathogen.