ß-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.

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 ß-1,3-glucans (curdlan and laminarin), and ß-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 ß-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 ß-1,3-glucanase.•All strains killed the Fusarium pathogen in the anaerobic co-incubation conditions.•One of the strains produced ß-1,3-glucanase with the fungal cell wall as a substrate.•The strain degraded the cell wall almost completely, except for the mycelial septa.

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

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 ß-1,3-glucan and chitosan, two major polysaccharide components of ascomycetes fungal cell wall, supplemented in the medium. ß-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.

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.

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 (H2 and CO2). 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.

Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

Changes and recovery of soil bacterial communities influenced by biological soil disinfestation as compared with chloropicrin-treatment.

Soil bacterial composition, as influenced by biological soil disinfestation (BSD) associated with biomass incorporation was investigated to observe the effects of the treatment on the changes and recovery of the microbial community in a commercial greenhouse setting. Chloropicrin (CP) was also used for soil disinfestation to compare with the effects of BSD. The fusarium wilt disease incidence of spinach cultivated in the BSD- and CP-treated plots was reduced as compared with that in the untreated control plots, showing effectiveness of both methods to suppress the disease. The clone library analyses based on 16S rRNA gene sequences showed that members of the Firmicutes became dominant in the soil bacterial community after the BSD-treatment. Clone groups related to the species in the class Clostridia, such as Clostridium saccharobutylicum, Clostridium tetanomorphum, Clostridium cylindrosporum, Oxobacter pfennigii, etc., as well as Bacillus niacini in the class Bacilli were recognized as the most dominant members in the community. For the CP-treated soil, clones affiliated with the Bacilli related to acid-tolerant or thermophilic bacteria such as Tuberibacillus calidus, Sporolactobacillus laevolacticus, Pullulanibacillus naganoensis, Alicyclobacillus pomorum, etc. were detected as the major groups. The clone library analysis for the soil samples collected after spinach cultivation revealed that most of bacterial groups present in the original soil belonging to the phyla Proteobacteria, Acidobacteria, Bacteroidetes, Gemmatimonadetes, Planctomycetes, TM7, etc. were recovered in the BSD-treated soil. For the CP-treated soil, the recovery of the bacterial groups belonging to the above phyla was also noted, but some major clone groups recognized in the original soil did not recover fully.

Proliferation of diversified clostridial species during biological soil disinfestation incorporated with plant biomass under various conditions.

Biological soil disinfestation (BSD) involves the anaerobic decomposition of plant biomass by microbial communities leading to control of plant pathogens. We analyzed bacterial communities in soil of a model experiment of BSD, as affected by biomass incorporation under various conditions, to find out the major anaerobic bacterial groups which emerged after BSD treatments. The soil was treated with Brassica juncea plants, wheat bran, or Avena strigosa plants, irrigated at 20 or 30 % moisture content and incubated at 25-30 °C for 17 days. The population of Fusarium oxysporum f. sp. spinaciae incorporated at the start of the experiment declined markedly for some BSD conditions and rather high concentrations of acetate and butyrate were detected from these BSD-treated soils. The polymerase chain reaction-denaturing gradient gel electrophoresis analysis based on the V3 region of 16S rRNA gene sequences from the soil DNA revealed that bacterial profiles greatly changed according to the treatment conditions. Based on the clone library analysis, phylogenetically diverse clostridial species appeared exceedingly dominant in the bacterial community of BSD soil incorporated with Brassica plants or wheat bran, in which the pathogen was suppressed completely. Species in the class Clostridia such as Clostridium saccharobutylicum, Clostridium acetobutylicum, Clostridium xylanovorans, Oxobacter pfennigii, Clostridium pasteurianum, Clostridium sufflavum, Clostridium cylindrosporum, etc. were commonly recognized as closely related species of the dominant clone groups from these soil samples.

Use of a Nitrate-Nonutilizing Mutant and Selective Media to Examine Population Dynamics of Fusarium oxysporum f. sp. spinaciae in Soil.

ABSTRACT Determining the population density of the spinach wilt pathogen Fusarium oxysporum f. sp. spinaciae in soil with conventional Fusarium-selective media is quite difficult because nonpathogenic strains of F. oxysporum also grow on those media and are indistinguishable from the pathogen. Therefore, a nitrate-nonutilizing (nit) mutant of the pathogen and corresponding selective media were tested in an experimental approach to determine the population density of the pathogen. Colony forming units of the pathogen were countable after soil-dilution plating onto nit mutant-selective media MMCPA, CMP, and CGMBP. Colony forming units of wild-type Fusarium spp. were countable using a wildtype Fusarium-selective medium, GMBP. By combining nit mutant- and wild-type-selective media, the population densities of pathogenic and nonpathogenic F. oxysporum in the same soil could be measured selectively. This method was useful in studying population dynamics of the pathogen after different soil treatments. Soil disinfested with hot water or chloropicrin was amended with the nit mutant pathogen, and subsequent changes in population densities of the pathogen were compared with those in nontreated field soil. The pathogen rapidly proliferated in disinfested soil and wilt developed faster than in nontreated soil. When a nonpathogenic isolate of F. oxysporum was added at high density to sterilized soil prior to the pathogen, growth of the pathogen was greatly suppressed. Nonpathogenic F. oxysporum could not, however, reduce the density of preexisting pathogen.