The influence of Bacillus subtilis RB14-C on the development of Rhizoctonia solani and indigenous microorganisms in the soil

The effect of soil inoculation with an antagonistic strain Bacillus subtilis RB14-C on the development of Rhizoctonia solani and changes occurring in soil and rhizosphere microbial communities were studied. RB14-C was added to the soil as a water suspension of the cells or as a broth culture. Application of cell suspensions to non-planted soil reduced the number of culturable bacteria. The density of R. solani and the number of filamentous fungi were not significantly affected by RB14-C. A similar effect was observed in the rhizosphere of tomato plants growns in bacterized soil. Broth cultures of RB14-C suppressed R. solani 1 d after inoculation, but after 3 d there was no difference in the pathogen density between soil amended with broth culture and control soil. In microcosm studies, cell suspensions of RB14-C also did not inhibit growth of R. solani on filters buried in soil. However, an inhibitory effect was obtained when a broth culture of the bacterium was used. The effect of RB14-C on fungal biomass was also estimated by measurement of ergosterol concentration in soil. It was found that ergosterol was mostly derived from R. solani and that there were no significant differences in its content between untreated soil and soil treated with RB14-C. The results suggest that suppression of Rhizoctonia damping-off by B. subtilis RB14-C probably is not related to the reduction of the pathogen population in the soil.     

Production of a lipopeptide antibiotic, surfactin, by recombinant Bacillus subtilis in solid state fermentation

Production of a lipopeptide antibiotic, surfactin, in solid state fermentation (SSF) on soybean curd residue, Okara, as a solid substrate was carried out using Bacillus subtilis MI113 with a recombinant plasmid pC112, which contains lpa-14, a gene related to surfactin production cloned at our laboratory from a wild-type surfactin producer, B. subtilis RB14. The optimal moisture content and temperature for the production of surfactin were 82% and 37 degrees C, respectively. The amount of surfactin produced by MI113 (pC112) was as high as 2.0 g/kg wet weight, which was eight times as high as that of the original B. subtilis RB14 at the optimal temperature for surfactin production, 30 degrees C. Although the stability of the plasmid showed a similar pattern in both SSF and submerged fermentation (SMF), production of surfactin in SSF was 4-5 times more efficient than in SMF. (c) 1995 John Wiley & Sons, Inc.     

Cloning, sequencing, and characterization of the genetic region relevant to biosynthesis of the lipopeptides iturin A and surfactin in Bacillus subtilis

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.     

Isolation of new variants of surfactin by a recombinant Bacillus subtilis

A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized. Received: 18 December 1996 / Received revision: 20 February 1997 / Accepted: 28 February 1997     

产表面活性素和伊枯草菌素A菌株的筛选及其脂肽类产物的特性

采用血琼脂平板法, 从菜园土壤中分离到8株代谢表面活性剂的菌株, 比较各菌株的排油性、抑菌性, 根据合成脂肽类物质表面活性素(Surfactin)和伊枯草菌素A (Iturin A)必需的sfp、ituD和lpa-14基因设计引物, 结合PCR的方法筛选到一株具广谱抗菌性且含有sfp、ituD和lpa-14 3个关键基因的细菌XZ-173。经过生理生化试验测定和16S rDNA序列系统发育学分析, 将其鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过红外光谱(FT-IR)分析该菌株代谢产物, 初步鉴定为脂肽类物质, 并对照高效液相色谱(HPLC)与标准品比对结果, 确定含有Surfactin和Iturin A组分。该菌株产生的脂肽粗品能使纯水的表面张力降低至26.6 mN/m, 临界胶束浓度(CMC)为500 mg/L, 具有很好的乳化性能, 对立枯丝核菌和青枯菌表现出很好的拮抗活性。因此, 产脂肽细菌XZ-173是一株应用前景广阔的功能菌。     

Second stage production of iturin A by induced germination of Bacillus subtilis RB14

Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995     

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.     

Isolation and characterization of a halotolerant <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">subtilis</Emphasis> BBK-1 which produces three kinds of lipopeptides: bacillomycin L,plipastatin, and surfactin

Twenty-three halotolerant and biosurfactant producing strains were collected from salty conditions in central Thailand. One of the strains designated BBK-1 produced the biosurfactants with the highest activity. BBK-1 was isolated from fermented foods and was identified as B. subtilis based on its physiological characteristics and 16S rRNA gene sequence. We show that the strain grows in media containing NaCl up to 16% (w/v) and produces biosurfactants in NaCl up to 8%. We found that B. subtilis BBK-1 produces three kinds of surface-active lipopeptides simultaneously. By their respective molecular weights and amino acid compositions, it is indicated that these lipopeptides are bacillomycin L, plipastatin, and surfactin. In order to analyze the production mechanism of lipopeptides further in the strain, a generally important biosynthetic gene encoding 4'-phosphopantetheinyl transferase was cloned and sequenced. The gene existed in a single copy in the genome and the deduced amino acid sequence was almost identical to that of Lpa-14 from B. subtilis strain RB14, which co-produces iturin A and surfactin.     

Cloning,Sequencing, and Characterization of the Genetic Region Relevant to Biosynthesis of the Lipopeptides Iturin A and Surfactin in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis B3 was found to produce lipopeptides iturins and fengycin that have activity against several plant pathogens such as Fusarium graminearum, Rhizoctonia solani, Rhizoctonia cerealis, and Pyricularia grisea. A 3642-bp genomic region of B. subtilis B3 comprising srfDB3, aspB3, lpaB3, and yczEB3 genes that resulted in biosynthesis of surfactin in B. subtilis 168 was cloned, sequenced, and characterized. Among them, the srfDB3 gene encodes thioesterase, which is required for biosynthesis of surfactin in B. subtilis; the aspB3 gene encodes a putative aspartate aminotransferase-like protein; the lpaB3 encodes phosphopantetheinyl transferase, which shows high identity to the product of lpa-14 gene regulating the biosynthesis of iturin A and surfactin in B. subtilis RB14; the yczEB3 encodes a YczE-like protein with significant similarities in signal peptide and part of the ABC transport system. The genetic regions between the srfD gene and lpa gene from B. subtilis B3 and B. subtilis A13, which produces iturin A, contain an approximate 1-kb nucleotide fragment encoding an aspartate aminotransferase-like protein; however, the relevant regions from B. subtilis 168 and B. subtilis ATCC21332 producing surfactin comprise an approximately 4-kb nucleotide fragment encoding four unknown proteins. There is 73% identity between the Lpa family and the Sfp family, although both are highly conserved.Received: 29 October 2002 / Accepted: 6 December 2002     

Studies on lipopeptide biosynthesis by Bacillus subtilis: Isolation and characterization of iturin−, surfactin+ mutants

Abstract Two mutant strains, M35 and M89, were obtained by UV irradiation from a wild-type Bacillus subtilis producing iturin and surfactin. Sporulation and surfactin production were similar in both mutants and in the parent strain, while the iturin production of M35 was 300-fold less than that of the wild-type strain; M89 did not produce any iturin. The analysis of the incorporation of sodium [1-¹⁴C]acetate into cellular lipids and lipopeptides showed that M89 still synthesized β-amino fatty acids, the lipid moiety of iturin.     

Isolation and characterization of lipopeptide antibiotics produced by Bacillus subtilis

Aims:  Antibiotics from Bacillus subtilis JA show strong pathogen inhibition ability, which has potential market application; yet, the composition of these antibiotics has not been elucidated. The aim of this paper is to isolate and identify these antibiotics.
Methods and Results:  The antagonistic activity of JA was tested in vitro ; it exhibited strong inhibition against some important phytopathogens and postharvest pathogens. Crude antibiotic production was extracted with methanol from the precipitate by adding 6 mol l⁻¹ HCl to the bacillus-free culture broth. The crude extract was run on Diamonsil C₁₈ column (5  μ m, 250 × 4·6 mm) in HPLC system to separate the antibiotics. Major antibiotics were classified into three lipopeptide families according to electrospray ionization–mass spectrometry analysis. Subsequently, the classification of antibiotics was confirmed with typical collision-induced dissociation fragments.
Conclusions:  Three kinds of antibiotics were isolated from B. subtilis JA and were identified to the lipopeptide families, surfactin, iturin and fengycin. These compounds could function as biocontrol agents against a large spectrum of pathogens.
Significance and Impact of the Study:  This study provided a reliable and rapid method for isolation and structural characterization of lipopeptide antibiotics from B. subtilis .     

Coproduction of surfactin and iturin A, lipopeptides with surfactant and antifungal properties, by Bacillus subtilis

Of the 13 strains of Bacillus subtilis tested for the coproduction of the lipopeptide surfactin and the antifungal lipopeptides of the iturin family, only 1 produced both lipopeptides with a high yield. The cultures were made in a synthetic medium. Several L-amino acids and various carbon sources were good substrates for the lipopeptide production. The maximum yield of surfactin was about 110 mg/liter and that of iturin A about 39 mg/liter/absorbance unit for the best strain, B. subtilis S 499.     

黑水虻肠道细菌抗菌筛选及其活性物质分子鉴定

【目的】从昆虫黑水虻分离的肠道细菌进行抗植物病原菌的拮抗菌筛选,对获得有拮抗活性的肠道细菌进行活性物质的分子鉴定。【方法】用稀释涂布法从水虻肠道中分离菌株,采用平板对峙法进行抗菌筛选,对有抗菌活性的菌株通过生理生化实验、16S rRNA鉴定和进化树分析确定其种属。参考已知脂肽合成关键基因设计引物,以拮抗菌总DNA为模板进行PCR扩增,对目的片段进行测序。【结果】通过抗菌筛选获得一株对水稻黄单胞菌以及小麦纹枯病病原菌等有很强抑制效果的水虻肠道细菌BSF-CL,经鉴定为枯草芽胞杆菌。脂肽合成关键基因PCR结果显示BSF-CL菌株具有脂肽Iturin和Surfactin合成的关键基因。推测BSF-CL很可能合成脂肽Iturin和Surfactin。【结论】从水虻肠道中分离出对水稻黄单胞菌有很强抑菌活性的菌株,分离菌被鉴定为一种枯草芽胞杆菌,通过活性物质的分子克隆鉴定初步推测其活性物质可能为脂肽Iturin和Surfactin。     

The Effect of Mode of Application of Bacillus subtilis RB14-C on its Efficacy as a Biocontrol Agent Against Rhizoctonia solani

Bacillus subtilis RB14‐C, which produces the antibiotic iturin A, was investigated for its effectiveness as a biocontrol agent against Rhizoctonia solani infecting tomato using seed coating and/or direct introduction of the bacteria to the soil. The ability of RB14‐C to colonize plant roots and produce iturin A in soil, depending on the method of bacterial application, was also determined. Seed coating and the combined treatment (soil and seed bacterization) did not protect seedlings against damping‐off caused by R. solani. By contrast, RB14 introduced only to the soil controlled the disease. The total number of RB14‐C bacteria on the roots of plants grown from coated seeds was significantly lower than on the roots of plants grown in soil mixed with the bacteria. In the combined treatment, application of B. subtilis with seeds to soil preinoculated with this bacterium, at first suppressed the population of RB14‐C in the soil. Then the colonization was generally uniform. The concentration of iturin A in non‐planted soil was highest at the beginning of the experiment (i.e. after application of the bacterial suspension) but then decreased, and was undetectable 3 days after incubation. However, after seed planting the antibiotic was produced again around young roots. Bacteria introduced to the soil as a seed coating also released the antibiotic around the seeds.     

Effect of Herbicides on the Toxicity of Fungicides Against Rhizoctonia solani Causing Damping-off of Cotton

The effect of two herbicides (paraquat and simazine on the antifungal activity of two fungicides (captan and mounsrin) against Rhizoctonia solani was studied. when the herbicides paraquat and simazine were applied to soil they altered the effectiveness of both fungicides in controlling R. solani , thus causing damping-off of cotton. Both herbicides increased the toxicity of both fungicides against mycelial growth of the fungus. In pot tests, seeds or soil treated with captan or mounsrin, gave better control of R. solani damping-off disease when the soil was treated with paraquat or simazine compared to untreated soil. Captan was, however, found to be more effective in controlling the disease than mounsrin.     

Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A.

Iturin A and surfactin are two lipopeptides extracted from a same strain of Bacillus subtilis. Iturin A possesses antibiotic and antifungal activities and surfactin is a strong surfactant. The presence of surfactin, at a concentration at which, alone, it is inactive, increases to a very large extent the haemolysis percent induced by iturin A. This synergistic effect seems to be in relation with interactions between iturin A and surfactin. Iturin A adsorbs to and penetrates into surfactin monolayers. Iturin A and surfactin are miscible and interact specifically in mixed monolayers.     

Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is facilitated by biofilm formation and surfactin production

Relatively little is known about the exact mechanisms used by Bacillus subtilis in its behavior as a biocontrol agent on plants. Here, we report the development of a sensitive plant infection model demonstrating that the bacterial pathogen Pseudomonas syringae pv tomato DC3000 is capable of infecting Arabidopsis roots both in vitro and in soil. Using this infection model, we demonstrated the biocontrol ability of a wild-type B. subtilis strain 6051 against P. syringae. Arabidopsis root surfaces treated with B. subtilis were analyzed with confocal scanning laser microscopy to reveal a three-dimensional B. subtilis biofilm. It is known that formation of biofilms by B. subtilis is a complex process that includes secretion of surfactin, a lipopeptide antimicrobial agent. To determine the role of surfactin in biocontrol by B. subtilis, we tested a mutant strain, M1, with a deletion in a surfactin synthase gene and, thus, deficient in surfactin production. B. subtilis M1 was ineffective as a biocontrol agent against P. syringae infectivity in Arabidopsis and also failed to form robust biofilms on either roots or inert surfaces. The antibacterial activity of surfactin against P. syringae was determined in both broth and agar cultures and also by live-dead staining methods. Although the minimum inhibitory concentrations determined were relatively high (25 microg mL(-1)), the levels of the lipopeptide in roots colonized by B. subtilis are likely to be sufficient to kill P. syringae. Our results collectively indicate that upon root colonization, B. subtilis 6051 forms a stable, extensive biofilm and secretes surfactin, which act together to protect plants against attack by pathogenic bacteria.     

Survival of bacillus subtilis NB22, an antifungal-antibiotic iturin producer,and its transformant in soil-systems

Bacillus subtilis NB22 is an antifungal-antibiotic iturin producer that expresses broad suppressibility against phytopathogenic microorganisms. The survival of B. subtilis NB22-1, which is a spontaneous streptomycin-resistance mutant of NB22 was investigated in four different soils. After a gradual decline, the bacterial viable cell number stabilized at a level of 10⁴–10⁵ colony forming units/g-dry soil irrespective of soil differences. A similar decline and stabilization pattern was observed in the case of the transformant of B. subtilis NB22-1 with the plasmid pC194 in nonsterile soil. The transformant reached a much higher stabilized level in sterile soil than in nonsterile soil. However, significant loss of the plasmid was observed in both the soil systems after 10 to 20 d incubation. Plasmid pC194 was stable over a hundred generations in the strain when cultivated in a liquid complex medium, but unstable in minimal medium, indicating that the plasmid stability in soil does not necessarily reflect that in liquid culture.     

Medium optimization of antifungal lipopeptide,iturin A,production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in solid-state fermentation by response surface methodology

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 μg/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.