Degradation of the fungal cell wall by clostridial strains isolated from soil subjected to biological soil disinfestation and biocontrol of Fusarium wilt disease of spinach

Abstract

Biological soil disinfestation (BSD) involves elimination of soil-borne plant pathogens in an environmentally friendly manner. Two anaerobic bacterial strains (H110 and TB8) isolated from BSD-treated soil samples were analyzed for their roles in elimination of pathogenic fungi. The two strains were identified as Clostridium beijerinckii based on 16S rRNA gene sequences and phenotypic properties. The strains fermented various carbohydrates and produced acetate, butyrate, and n-butanol as major products as well as abundant gases (H and CO). For evaluation of the antifungal potential of these strains, cells of a pathogen (spinach wilt disease, Fusarium oxysporum f. sp. spinaciae) were co-inoculated into anaerobic media with each anaerobic strain. After incubation for ~3 weeks at 30 °C, 10-30% of the cells of the pathogen survived when incubated without the anaerobic isolates, whereas the pathogen was eliminated when co-incubated with each anaerobe because of the growth of the anaerobic bacterium. It was found by microscopic examination that mycelial cells of the fungal pathogen were severely degraded during the first 3-7 days of the co-incubation. The two strains utilized major cell wall polysaccharides of ascomycetous fungi-chitosan and ß-1,3-glucan (curdlan and laminarin)-as fermentative substrates added to the medium. Furthermore, both isolates degraded a cell wall preparation isolated from the mycelium of the Fusarium pathogen of spinach wilt disease. We concluded that the two anaerobic strains kill the pathogen of spinach wilt disease by degrading major fungal cell wall components as antifungal activities.

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.…”