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The purpose of this study is to isolate the beneficial microorganisms whose growth is promoted in the presence of charcoal materials. We successfully isolated strain IA, whose growth is promoted on an agar plate with charcoal materials, and identified it as a novel strain of the Bacillus sp. The growth of strain IA in the liquid medium was promoted by the addition of both activated charcoal (AC) and rice husk biochar (RHB). Moreover, the sporulation of strain IA in the RHB medium and the antifungal activity of the culture supernatant of the RHB medium were much higher than those with AC. HPLC and MS analyses revealed that strain IA produced an antifungal lipopeptide iturin A, and the yield of iturin A in the RHB medium was 8 times higher than that in the medium without RHB. This is the first paper to describe the positive effect of RHB on microbial metabolisms.

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In a previously study, the Bacillus sp. strain IA was successfully isolated with high sensitivity to rice husk biochar (RHB). Moreover, RHB promoted an antibiotic iturin A production by strain IA. In order to develop the biocontrol agent, we attempted to reveal the functions of the RHB in promoting the production of iturin A by strain IA. The promotion effects of growth, sporulation and iturin A production of strain IA by the RHB were explained as follows. First, the manganese ion, released from RHB, promoted the sporulation and iturin A production of strain IA. Second, the silicon dioxide contained in RHB adsorbed the metabolic inhibitor(s) and promoted the iturin A production of strain IA. Finally, the combination of manganese ion and silicon dioxide promoted the growth, sporulation and iturin A production of the Bacillus sp. strain IA. To culture strain IA in the medium combining manganese ion and silicon dioxide, the total cells, spore cells and iturin A production increased 15 times, 10,000 times and 18 times higher than the control medium, respectively.

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Plant disease resistance gene (R gene)-like sequences were screened from the Physcomitrella patens genome. We found 603 kinase-like, 475 Nucleotide Binding Site (NBS)-like and 8594 Leucine Rich Repeat (LRR)-like sequences by homology searching using the respective domains of PpC24 (Accession No. BAD38895), which is a candidate kinase-NBS-LRR (kinase-NL) type R-like gene, as a reference. The positions of these domains in the genome were compared and 17 kinase-NLs were predicted. We also found four TIR-NBS-LRR (TIR-NL) sequences with homology to Arabidopsis TIR-NL (NM_001125847), but three out of the four TIR-NLs had tetratricopeptide repeats or a zinc finger domain in their predicted C-terminus. We also searched for kinase-LRR (KLR) type sequences by homology with rice OsXa21 and Arabidopsis thaliana FLS2. As a result, 16 KLRs with similarity to OsXa21 were found. In phylogenetic analysis of these 16 KLRs, PpKLR36, PpKLR39, PpKLR40, and PpKLR43 formed a cluster with OsXa21. These four PpKLRs had deduced transmembrane domain sequences and expression of all four was confirmed. We also found 14 homologs of rice OsXB3, which is known to interact with OsXa21 and is involved in signal transduction. Protein-protein interaction was observed between the four PpKLRs and at least two of the XB3 homologs in Y2H analysis.

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Bacterial endophytes were found from 6 plant leaves among 35 plant leaves screened. Two of the isolated bacteria showed antagonistic activity against fungal plant pathogens. An isolate named KL1 showed the clear inihibition against plant pathogens, Fusarium oxysporum and Rhizoctonia solani, on PDA as well as TSA plate. Supernatant of the bacterial culture also showed the clear inhibition against the fungal growth on the plate and the antibiotic substance was identified as iturin A by HPLC analysis. KL1 was identified as Bacillus sp. from the 16S rRNA gene analysis. Very thin hyphae of R. solani was miccroscopically observed when the fungus was co-cultivated with KL1.

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An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.

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To enhance the production of lipopeptide antibiotic iturin A, nutrient contents of the culture mediums were investigated in both submerged and biofilm fermentations. As a carbon source maltose and as nitrogen source, fish protein was used. In submerged fermentation maltose uptake was found lower (12%) compared to biofilm fermentation (15%) that was associated with higher cellular growth in biofilm. However, requirement of nitrogen (fish protein) concentration was found similar in both submerged and biofilm fermentations. Production of iturin A in submerged fermentation with 12% maltose and 5% fish protein was 4450 mg/L, and in biofilm fermentation it was 5050 mg/L when 15% maltose and 5% fish protein was used.

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Interest in microbial surfactants has been steadily increasing in recent years due to their diversity, mass production possibility, selectivity, performance under extreme conditions and potential applications in environmental protection. In this study two pentose sugars (xylose and arabinose) were investigated for the submerged fermentation (SmF) of Bacillus subtilis in surfactant production medium for bio-surfactant surfactin production. An excellent vegetative growth of B. subtilis (× 10(10) CFU/mL) was observed for xylose and arabinose containing medium which were comparable to glucose supplemented medium. Low growth (× 10(8) CFU/mL) was found when medium was not supplemented with any of the sugars. Surfactin production in xylose, arabinose and glucose containing medium was 2700, 2600 and 2000 mg/L, respectively, whereas, medium without any sugar showed low surfactin (700 mg/L) production. These results clearly indicate the effect of pentose sugars on production of surfactin. Gradual depletion of the xylose and arabinose were confirmed by HPLC analysis during the growth phase of the strain that ultimately produced the surfactin.

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