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

Abstract

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.

β-1,3-Glucanase production as an anti-fungal enzyme by phylogenetically different strains of the genus Clostridium isolated from anoxic soil that underwent biological disinfestation

“…The purity of the strains was confirmed through repetition of the anaerobic roll tube method for each isolate (Holdeman et al 1977) as well as observation of cell morphology by microscopy. The strains were cultivated under anaerobic conditions at 30°C using a peptone-yeast extract (PY) medium as a basal medium with oxygen-free mixed gas (95% N 2 and 5% CO 2 ) in the headspace as described earlier (Ueki et al 2017(Ueki et al , 2019. Each test tube containing the medium was closed tightly with an inner butyl rubber stopper equipped with an outer screw cap.…”

“…Motility of the cells was evaluated by phase contrast microscopy. Physiological examination of the isolates was performed as reported previously (Ueki et al 2016(Ueki et al , 2017(Ueki et al , 2019. All the cultivation experiments were performed at least in duplicate, and reproducibility of the results was confirmed.…”

“…Especially, the abilities to degrade insoluble substrates were determined by measuring fermentation products in the culture supernatants after the cultivation. Fermentation products, such as volatile fatty acids (VFAs), alcohols, and gases (H 2 and CO 2 ), were analyzed by gas chromatography (GC) (Hitachi G-3000, G-5000 or G-163 Tokyo, Japan) as described previously (Ueki et al 2017(Ueki et al , 2019. All the experiments for measurement of the products were performed in duplicates.…”

“…The three strains were similar in utilizing β-1,3glucans (curdlan and laminarin) as growth substrates and producing β-1,3-glucanase in the culture supernatants, whereas none of them decomposed chitosan. Thus, the three strains had different physiological properties compared to strains H110 and TB8 (Ueki et al 2017(Ueki et al , 2019 for the decomposition of polysaccharides that comprise the cell wall of the ascomycetes. It was also shown that although the three clostridial strains are similar in producing β-1,3-glucanase as an antifungal enzyme, the strains attack the cells of the Fusarium pathogen of spinach wilt disease in different manners.…”

“…We isolated anaerobic bacteria from the soil samples subjected to the treatments in the model experiments shown above (Mowlick et al 2012(Mowlick et al , 2013a to investigate their role in suppressing soil-borne plant pathogens. In the previous studies (Ueki et al 2017(Ueki et al , 2019, we explored the degradation of the fungal cell wall of ascomycetes using the two anaerobic bacterial strains (H110 and TB8 identified as Clostridium beijerinckii) out of these isolates. The two anaerobic bacterial strains that produced β-1,3glucanase and chitosanase were shown to almost completely decompose cells of the ascomycete fungal pathogen, F. oxysporum f. sp.…”