Biological soil disinfestation (BSD) using plant biomass incorporation is an effective method and a good alternative to chemical fumigants for controlling soil-borne plant pathogens. In this study the bacterial communities in pot soil treated with three different BSD conditions (without plant biomass and with Brassica juncea L. plants or bran of wheat, Triticum aestivum L.) were analyzed using mainly molecular techniques. Earlier dropping of redox potential of both biomass-treated soils indicated rapid development of anaerobic conditions in the soil. The population of Fusarium oxysporum F. pathogen incorporated in the soil at the start was decreased considerably during the treatment, and the number of culturable anaerobic bacteria increased in both biomass-treated soils. Rather high concentrations of acetate and butyrate were detected from the biomass-treated soils. The polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis for the V3 region of 16S rRNA gene sequences revealed that the profiles of both biomass-treated soils were initially represented by similar and dominant groups, many of which were closely related to the species in the classes Clostridia and Bacilli of the phylum Firmicutes. Based on the clone library analysis, the control soil samples showed diverse bacterial groups with a few number of anaerobic clones. In contrast, for both biomass-treated libraries, clones belonging to the class Clostridia, a strictly anaerobic spore-forming bacterial group, appeared exceedingly dominant. The clostridial group detected was composed of phylogenetically diverse members, and it seemed likely that the diverse clostridial species were responsible for suppression of pathogens by making various compounds including volatile fatty acids and other compounds during anaerobic decomposition of plant biomass.

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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.

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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.