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.

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.

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.

Although seed priming is well known to be an effective method for enhancing seed vigor and seed performance, the efficacy of priming (soaking in water and re-drying) on the physiological performance of iron-coated rice (Oryza sativa L.) seeds under submerged conditions, and the physiological mechanisms of coleoptile elongation of the primed rice seeds under anoxia, have not been well elucidated. In the present study, primed and unprimed rice seeds with or without iron-coating were produced. We examined the physiological performance of these rice seeds and the physiological efficacy of priming on coleoptile elongation under anoxia. For the primed rice seeds, seed germination, coleoptile elongation, seedling emergence and the establishment of iron-coated rice seeds all improved considerably, which resulted in an increase of plant height and dry weight for the iron-coated primed rice seeds. Increases in -amylase activity and glucose concentration over time were significantly higher in the primed rice seeds than in the unprimed rice seeds. Under anoxia, the -amylase activity and soluble sugar concentration were significantly higher in the primed rice seeds than in the unprimed rice seeds. The glucose and fructose concentrations in the coleoptiles were significantly higher in the primed rice seeds than in the unprimed rice seeds, suggesting that the stimulated coleoptile elongation is partly related to the increased availability of soluble sugar from the seed to the coleoptiles. The increased availability of soluble sugar from the rice seeds to the coleoptiles can be assumed to help maintain glycolytic flux and alcoholic fermentation under the submerged conditions. These results clearly indicate the efficacy of priming on iron-coated rice seeds, and that the coleoptile elongation of the primed rice seeds can be partly ascribed to the sugar availability from the rice seeds to the coleoptiles. Therefore, the use of primed rice seeds for iron coating is highly effective to induce the emergence and establishment of stable seedlings in direct rice sowing systems.

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