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.

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.

An implementation framework for evaluating the biocidal potential of essential oils in controlling Fusarium wilt in spinach: from in vitro to in planta.

Fusarium wilt, caused by Fusarium oxysporum f. sp. spinaciae, causes a significant challenge on vegetative spinach and seed production. Addressing this issue necessitates continuous research focused on innovative treatments and protocols through comprehensive bioassays. Recent studies have highlighted the potential of plant-based compounds in controlling fungal diseases. The present work aims to conduct a series of experiments, encompassing both in vitro and in planta assessments, to investigate the biocontrol capabilities of different essential oils (EOs) at various application rates, with the ultimate goal of reducing the incidence of Fusarium wilt in spinach. The inhibitory effect of four plant EOs (marjoram, thyme, oregano, and tea tree) was initially assessed on the spore germination of five unknown Fusarium strains. The outcomes revealed diverse sensitivities of Fusarium strains to EOs, with thyme exhibiting the broadest inhibition, followed by oregano at the highest concentration (6.66 µL/mL) in most strains. The tested compounds displayed a diverse range of median effective dose (ED50) values (0.69 to 7.53 µL/mL), with thyme and oregano consistently showing lower ED50 values. The direct and indirect inhibitory impact of these compounds on Fusarium mycelial growth ranged from ~14% to ~100%, wherein thyme and oregano consistently exhibiting the highest effectiveness. Following the results of five distinct inoculation approaches and molecular identification, the highly pathogenic strain F-17536 (F. oxysporum f.sp. spinaciae) was chosen for Fusarium wilt assessment in spinach seedlings, employing two promising EO candidates through seed and soil treatments. Our findings indicate that colonized grain (CG) proved to be a convenient and optimal inoculation method for consistent Fusarium wilt assessment under greenhouse conditions. Seed treatments with thyme and oregano EOs consistently resulted in significantly better disease reduction rates, approximately 54% and 36% respectively, compared to soil treatments (P > 0.05). Notably, thyme, applied at 6.66 µL/mL, exhibited a favorable emergence rate (ERI), exceeding seven, in both treatments, emphasizing its potential for effective disease control in spinach seedlings without inducing phytotoxic effects. This study successfully transitions from in vitro to in planta experiments, highlighting the potential incorporation of EOs into integrated disease management for Fusarium wilt in spinach production.

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.