Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.     

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae)

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm‐isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)‐like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria‐colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high‐affinity sucrose‐specific proton‐dependent importer. Plasma‐membrane/tonoplast localization of GeSUT4‐GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant‐microbe interactions.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Efficient secretory production of large-size heterologous enzymes in Bacillus subtilis: A secretory partner and directed evolution

Secretory production of recombinant proteins provides a simple approach to the production and purification of target proteins in the enzyme industry. We developed a combined strategy for the secretory production of three large-size heterologous enzymes with a special focus on 83-kDa isoamylase (IA) from an archaeon Sulfolobus tokodaii in a bacterium Bacillus subtilis. First, a secretory protein of the B. subtilis family 5 glycoside hydrolase endoglucanase (Cel5) was used as a fusion partner, along with the NprB signal peptide, to facilitate secretory production of IA. This secretory partner strategy was effective for the secretion of two other large enzymes: family 9 glycoside hydrolase from Clostridium phytofermentas and cellodextrin phosphorylase from Clostridium thermocellum. Second, the secretion of Cel5-IA was improved by directed evolution with two novel double-layer Petri-dish-based high-throughput screening (HTS) methods. The high-sensitivity HTS relied on the detection of high-activity Cel5 on the carboxymethylcellulose/Congo-red assay. The second modest-sensitivity HTS focused on the detection of low-activity IA on the amylodextrin-I₂ assay. After six rounds of HTS, a secretory Cel5-IA level was increased to 234 mg/L, 155 times the wild-type IA with the NprB signal peptide only. This combinatory strategy could be useful to enhance the secretory production of large-size heterologous proteins in B. subtilis.     

Potency of Bacillus thuringiensis and Bacillus subtilis against the cotton leafworm,Spodoptera littoralis (Bosid.) Larvae

The biological activities of two species of bacteria isolated from soil of cotton fields identified as Bacillus subtilis strain NRC313 (BS NRC313) and Bacillus thuringiensis strain NRC335 (BT NRC335) were evaluated against the third larval instar of the cotton leafworm, Spodoptera littoralis (Boisd.). The different entomopathogenic bacteria of BS NRC313and BT NRC335 contained 10 × 10⁸ cell/ml, and caused mortality of 100 and 97.3% for the above mentioned strains, respectively. Concentrations of 2.5 × 10⁸ to 10 × 10⁸ cell/ml of strains BS NRC313 and BT NRC335 were applied to the larvae: LC₅₀ were 3.3 × 10⁸ and 3.9 × 10⁸ cell/ml respectively. The influence of exposure to toxin concentrations manifested in terms of decreasing the adult emergence and prolongation of the generation period. The percentage of larvae that survived and succeeded to pupate increased by decreasing the concentration. The longevity of adult emergence that resulted from larvae treated with Bacillus subtilis were 6.0 ± 0.51 and 9.0 ± 0.63 days at 5 × 10⁸ and 2.5 × 10⁸ cell/ml, respectively compared with 9.8 ± 0.47 in control. The results indicated that Bacillus subtilis was more potent than Bacillus thuringiensis. Field applications of B. thuringiensis, B. subtilis and Reldan achieved 55.6, 67.4 and 89.4% reduction of the cotton leafworm larvae Spodoptera littoralis in clover plants under field conditions.     

PGPR mediated management of stem blight of Phyllanthus amarus (Schum and Thonn) caused by Corynespora cassiicola (Berk and Curt) Wei

Bacillus subtilis (BSCBE4), Pseudomonas chlororaphis (PA23), endophytic P. fluorescens (ENPF1) inhibited the mycelial growth of stem blight pathogen Corynespora casiicola (Berk and Curt)Wei under in vitro. All these bacterial isolates produced both hydroxamate and carboxylate type of siderophores. But the siderophore production was maximum with the isolate ENPF1. Delivering of talc based formulation of BSCBE4 through seedling dip and foliar application effectively reduced stem blight disease incidence and increased the dry matter production under pot culture and field conditions. Application of BSCBE4, PA23 and ENPF1 increased the defense related enzymes such as peroxidase, polyphenol oxidase, chitinase and β-1,3 glucanase in P. amarus up to ten days after challenge inoculation with C. cassicola. Native gel electrophoretic analysis revealed that challenge inoculation of pathogen with BSCBE4 and PA23 induced both peroxidase and polyphnol oxidase isoforms.     

Direct effect of biocontrol agents on wilt and root-rot diseases of sesame

In Egypt, sesame cultivation is subject to attack by wilt and root-rot diseases caused by Fusarium oxysporum f.sp. sesami (Zap) Cast. and Macrophomina phaseolina (Maubl) Ashby causing losses in quality and quantity of sesame seed yield. Bacillus subtilis and Trichoderma viride isolates which were isolated from sesame rhizosphere were the most effective to antagonise fungal pathogens, causing high reduction of hyphal fungal growth. Trichoderma viride was found to be mycoparasitic on Fusarium oxysporum f.sp. sesami and M. phaseolina causing morphological atternation of fungal cells and sclerotial formation. In general, Bacillus subtilis, T. viride, avirulent Fusarium oxysporum isolate and Glomus spp. (Amycorrhizae) significantly reduced wilt and root-rot incidence of sesame plants at artificially infested potted soil by each one or two pathogens. Data obtained indicate that Glomus spp significantly reduced wilt and disease severity development on sesame plants followed by T. viride. Meanwhile, avirulent Fusarium oxysporum isolate followed by Glomus spp. were effective against root-rot disease incidence caused by M. phaseolina. Glomus spp. followed by B. subtilis significantly reduced wilt and root-rot disease of sesame plants. All biotic agents significantly reduced F. oxysporum f.sp. sesami and M. phaseolina counts in sesame rhizosphere at the lowest level. Glomus spp. and the avirulent isolate of F. oxysporum eliminated M. phaseolina in sesame rhizosphere. Meanwhile T. viride was the best agent at reducing F. oxysporum at a lower level than other treatments. Application of VA mycorrhizae (Glomus spp.) in fields naturally infested by pathogens significantly reduced wilt and root-rot incidence and it significantly colonised sesame root systems and rhizospheres compared to untreated sesame transplantings.     

Biological control of onion leaf blight disease by bulb and foliar application of powder formulation of antagonist mixture

Abstract

Three antagonists: Pseudomonas fluorescens (Pf1), Bacillus subtilis and Trichoderma viride, were tested alone and in combination for suppression of onion leaf blight (Alternaria palandui) disease under glasshouse and field conditions. The average mean of disease reduction was 24.81% for single strains and 42.44% for mixtures. In addition to disease suppression, treatment with a mixture of antagonists promoted plant growth in terms of increased plant height and ultimately bulb yield. Though seed treatment of either single strain or strain mixtures alone could reduce the disease, subsequent application to root, leaves or soil further reduced the disease and enhanced the plant growth. The mixture consisting of Pseudomonas fluorescens Pf1 plus Bacillus subtilis plus Trichoderma viride was the most effective in reducing the disease and in promoting plant growth and bulb yield in greenhouse and field tests.     

Knockout of pgdS and ggt genes improves γ‐PGA yield in B. subtilis

One of the emerging biopolymers that are currently under active investigation is bacterial poly(γ‐glutamic acid) (γ‐PGA). However, before its full industrial exploitation, a substantial increase in microbial productivity is required. γ‐PGA obtained from the Bacillus subtilis laboratory strain 168 offers the advantage of a producer characterized by a well defined genetic framework and simple manipulation techniques. In this strain, the knockout of genes for the major γ‐PGA degrading enzymes, pgdS and ggt, leads to a considerable improvement in polymer yield, which attains levels analogous to the top wild γ‐PGA producer strains. This study highlights the convenience of using the laboratory strain of B. subtilis over wild isolates in designing strain improvement strategies aimed at increasing γ‐PGA productivity. Biotechnol. Bioeng. 2013; 110: 2006–2012. © 2013 Wiley Periodicals, Inc.     

Mycelial β-N-Acetylglucosaminidase of Streptomyces sp. Having β-N-Acetylgalactosaminidase Activity

Formation of transfer products from soybean arabinogalactan and glycerol by endo-1,4-β-d-galactanase from Penicillium citrinum was described. The amount of transfer products depended on the glycerol concentration. About 50% of the galactose residues which could be liberated from the polysaccharide by the enzyme were transferred to glycerol at an acceptor concentration of 2.5% (w/v). Transfer products with various polymerization degrees were accumulated at the beginning of the reaction and then those with higher polymerization degrees were degraded gradually. At a final stage of the reaction, two transfer products in addition to two hydrolysis products (galactose and galactobiose) were mainly accumulated. The two transfer products were isolated and their structures were examined. They were 2-O-β-d-galactosyl glycerol and O-β-d-galactosyl-(1 → 4)-O-β-d-galactosyl-(1 → 2)glycerol.