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

Abstract

Biological (or reductive) soil disinfestation (BSD or RSD) is a bioremediation process to control soil-borne plant pathogens using activities of indigenous bacteria in the soil. Three obligate anaerobic bacterial strains (TW1, TW10, and TB10), which were isolated from anoxic soil subjected to BSD treatments, were examined for their abilities to produce anti-fungal enzymes. All strains were affiliated with the different lineages of the genus Clostridium. The three strains decomposed β-1,3-glucans (curdlan and laminarin), and β-1,3-glucanase activities were detected from their culture supernatants with these glucans. The three strains also produced the enzyme with wheat bran as a growth substrate and killed the Fusarium pathogen (Fusarium oxysporum f. sp. spinaciae) in the anaerobic co-incubation conditions. Observation by fluorescence microscopy of the pathogen cells showed that the three strains had degraded the fungal cells in different manners upon co-incubation with wheat bran. When the three strains were cultivated with the dead cells or the cell wall samples prepared from the Fusarium pathogen, strain TW1 utilized these materials as easily decomposable substrates by releasing β-1,3-glucanase. When observed by fluorescence microscopy, it appeared that strain TW1 degraded the mycelial cell wall nearly thoroughly, with the septa remaining as undecomposed luminous rings. In contrast, the other two strains decomposed neither the dead cells nor the cell wall samples directly. The results indicate that the various anaerobic bacteria proliferated in the soil under the BSD treatments should play key roles as an organized bacterial community to eliminate fungal pathogens, namely by release of anti-fungal enzymes with different properties. Key points • Three clostridial strains isolated from BSD-treated soils produced β-1,3-glucanase. • All strains killed the Fusarium pathogen in the anaerobic co-incubation conditions. • One of the strains produced β-1,3-glucanase with the fungal cell wall as a substrate. • The strain degraded the cell wall almost completely, except for the mycelial septa.

Clostridium fungisolvens sp. nov., a new β-1,3-glucan-decomposing bacterium isolated from anoxic soil subjected to biological soil disinfestation

“…Furthermore, another isolate (TW1 T ) produced β-1,3-glucanase, but not chitosanase, and also degraded cell wall of the Fusarium pathogen. Furthermore, it was shown that these strains killed the living Fusarium pathogen under the anaerobic cultivation conditions [16]. Thus, it was shown that anaerobic bacteria grown in soils under the BSD treatments have the potential to eliminate fungal pathogens living in the soil.…”

“…PY medium contained (l −1 ) 10 g Trypticase (BBL), 5 g yeast extract, 0.2 g Na 2 CO 3 (as a buffering agent for CO 2 in the headspace), 0.3 g l -cysteine⋅HCl⋅H 2 O (as a reducing agent) and 1 mg sodium resazurin (as a redox indicator) as well as salt solutions [18, 19]. Isolation and maintenance of strains were conducted as described previously using PY4S agar [16]. The pH value of media was usually adjusted to pH 7.0–7.2 (as measured after autoclaving) with a NaOH solution.…”

“…As described earlier [16], when strain TW1 T was cultivated with freeze-dried dead cell biomass of F. oxysporum f. sp. spinaciae strain M2-1 (the Fusarium pathogen causing spinach wilt disease) as a growth substrate (1 % w/v), the strain decomposed the Fusarium biomass and produced acetate (8.1 mmol l −1 ), butyrate (7.5 mmol l −1 ) and gases.…”

“…Both endo-β-1,3-glucanases (EC 3.2.1.39) and β−1,3 (4)-glucanases (EC 3.2.1.6) are known as laminarinases and act in hydrolysis of β-1,3-glucans [41]. Because strain TW1 T decomposed β−1,3-glucans such as curdlan and laminarin as growth substrates as shown above and had β−1,3-glucanase activity [16], the presence of the genes encoding these enzymes was searched in the genome of strain TW1 T . Two proteins (WP_183279845.1 and WP_183279608.1) annotated as enzymes β-glucanase or laminarinase belonging to glycosylhydrolase family 16 (GH16), to which most of bacterial laminarinases belong [41], were found in the genome (Table S4).…”

“…When observed the pathogen cells after incubation with strain TW1 T by fluorescence microscopy, it appeared that strain TW1 T degraded the mycelial cell wall severely, leaving the septa as undecomposed luminous bars or rings (Fig. S3) [16]. Both endo-β-1,3-glucanases (EC 3.2.1.39) and β−1,3 (4)-glucanases (EC 3.2.1.6) are known as laminarinases and act in hydrolysis of β-1,3-glucans [41].…”