Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP-1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH-20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP-1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1-10 μg ml(-1) and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 μg ml(-1) under greenhouse conditions. The efficacy of FP-1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp -tagged strain of KPE62302H confirmed its ability to colonize tomato plants.     

Isolation and structure elucidation of a new antifungal and antibacterial antibiotic produced by Streptomyces sp. 201

An antibacterial and antifungal antibiotic was isolated from the culture filtrate of Streptomyces sp. 201, and its structure was determined as 2-methyl-heptyl isonicotinate by extensive use of NMR spectroscopy. The compound exhibited marked antimicrobial activity against Bacillus subtilis, Shigella sp., Klebsiella sp., E. coli, Proteus mirabilis, and the pathogenic fungi, Fusarium moniliforme, F. semitectum, F. oxysporum, F. solani and Rhizoctonia solani.     

Isolation and Structure Elucidation of a New Antifungal and Antibacterial Antibiotic Produced by Streptomyces sp. 201

An antibacterial and antifungal antibiotic was isolated from the culture filtrate of Streptomyces sp. 201, and its structure was determined as 2-methyl-heptyl isonicotinate by extensive use of NMR spectroscopy. The compound exhibited marked antimicrobial activity against Bacillus subtilis, Shigella sp., Klebsiella sp., E. coli, Proteus mirabilis, and the pathogenic fungi, Fusarium moniliforme, F. semitectum, F. oxysporum, F. solani and Rhizoctonia solani.     

Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic

An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus.     

Biological control of fusarial wilt of pigeon pea by Bacillus brevis

A virulent strain of pigeon pea wilt pathogen was isolated from wilted pigeon pea plants and was identified as Fusarium oxysporum f. sp. udum. Many bacterial cultures showing antagonism to the pathogen were isolated from various ecological niches. When tested under pot and field conditions, development of fusarial wilt symptoms was prevented in pigeon pea seeds treated with one such antagonist, Bacillus brevis. A formulation of B. brevis with vermiculite as a carrier had a shelf life of at least 6 months. Bacillus brevis produced an extracellular antagonistic substance which induced swelling of the pathogen's hyphal tips, and cells were bulbous and swollen with shrunken and granulated cytoplasm. The antagonistic substance also inhibited germination of conidia, and was fungicidal to the vegetative mycelia of the pathogen. Comparison of the properties of our antagonistic substance with that of known antibiotics produced by B. brevis suggests that our antagonistic substance is a novel compound. The observations reported here indicate that this strain of B. brevis may have potential as a biocontrol agent against fusarial wilt in pigeon pea.     

对黄瓜枯萎病具防效的海洋源芽孢杆菌Y3F的鉴定

【目的】对一株从连云港海域海藻样品中获得的拮抗细菌Y3F进行鉴定并且研究菌株Y3F对黄瓜土传枯萎病害的控制效果。【方法】对Y3F进行形态、生理生化特征和16S r RNA基因序列分析,采用平板对峙法测定菌株Y3F的活菌液和无菌滤液的抑菌活性,利用盆栽试验测定Y3F对黄瓜枯萎病害的防治效果。【结果】初步鉴定该菌属于蜡样芽孢杆菌(Bacillus cereus),用2216E培养基振荡培养24 h的Y3F的无菌滤液对黄瓜尖孢镰刀菌有较强的抑菌活性,表明菌株能分泌抑制病原菌生长的活性物质。盆栽试验表明,种植30 d后,浸种和灌根同时处理(JG)的防治效果达到50.46%,对黄瓜枯萎病害有明显的控制效果,显著提高黄瓜植株生物量,显著降低黄瓜根际土的真菌和尖孢镰刀菌数量,增加根际的细菌和放线菌数量。【结论】菌株Y3F能有效防治黄瓜枯萎病害,改善根际微生物结构,具有进一步开发应用的前景。     

Isolation, characterization and antifungal activity of Streptomyces sampsonii GS 1322

For new antifungal antibiotics from actinomycetes, a strain of Streptomyces GS 1322 was isolated from a sample of garden soil. The strain was found to possess antagonistic activity against four fungi i.e., Candida albicans, Aspergillus niger, Microsporum gypseum and Trichophyton sp. The strain was identified as Streptomyces sampsonii and the antifungal compound produced by it was found to be the heptaene group of polyene antibiotics.     

STUDIES ON THE EFFECT OF ASSOCIATED SOIL MICROFLORA ON FUSARIUM UDUM BUTL., THE FUNGUS CAUSING WILT OF PIGEON-PEA (CAJANUS CAJAN (L.) MILLSP.), WITH SPECIAL REFERENCE TO ITS PATHOGENICITY

The interaction of certain soil saprophytes and Fusarium udum , the wilt organism of pigeon-pea, with special reference to their effect on pathogenicity, has been studied. The filtrates of Aspergillus niger, Rhizopus nigricans and mixed filtrates of all the saprophytes inhibited the growth of Fusarium udum on solid medium. This inhibition of F. udum has been shown to be due to unfavourable reaction of the medium rather than to food exhaustion or the presence of toxic substances. The culture filtrates after passage through soil beds failed to affect adversely the growth of F. udum because of the change in pH. Inoculation experiments have indicated that only Rhizopus nigricans is effective in reducing the incidence of wilt because of its faster rate of growth. The mixed inocula of the organisms and mixed filtrates of all the saprophytes have also been observed to be effective in reducing wilt incidence. Aspergillus terreus appears to enhance the virulence of Fusarium udum.     

Seed Bacterization with Bacillus subtilis AF 1 Increases Phenylalanine Ammonia-lyase and Reduces the Incidence of Fusarial Wilt in Pigeonpea

Cell free culture filtrate of a biocontrol PGPR strain of Bacillus subtilis showed a concentration-dependent growth and conidiation inhibition of Fusarium udum . the incitant of pigeonpea wilt. In vitro interaction of cell free filtrate of AF 1 induced bulb-like structures in the hyphae of F. udum . Seed treatment with B. subtilis significantly reduced the incidence of pigeonpea wilt, but not with the cell free culture filtrate of AF 1. Seeds bacterized with B. subtilis AF 1 showed an increase in phenylalanine ammonia-lyase (PAL) from day 1, while the peroxidase activity increased from day 6. The results are discussed in terms of induction of host plant resistance in biological control of plant pathogens.     

HPLC analysis of fusaric acid, 9,10-dehydrofusaric acid and their methyl esters,toxic metabolites from weed pathogenic Fusarium species

A simple and rapid HPLC method, using a high-density C18 column, has been developed for the quantitative analysis of fusaric and dehydrofusaric acids and their methyl esters in the methanol extract of lyophilised culture filtrates of species of Fusarium. The method has been used to determine the content of these metabolites in two strains of Fusarium oxysporum and in strains of F. nygamai and F. udum. Fusaric acid has been isolated and identified from a strain of F. udum for the first time.     

Antifungal activity of venenatine, an indole alkaloid isolated fromAlstonia venenata

The indole alkaloid venenatine exhibited antifungal activity against some plant pathogenic and saprophytic fungi. Venenatine in an aqueous acetic acid solution inhibited spore germination of all the 10 tested fungi, Fusarium udum, Alternaria brassicicola, Ustilago cynodontis and Aspergillus flavus showed an especially high sensitivity towards this compound, exhibiting germination levels below 10%. The spore germination and colony development of the parasitic fungus Erysiphe pisi, which causes powdery mildew in pea (Pisum sativum), on excised leaves of pea was also significantly affected. Pre-inoculation rather than post inoculation treatment of the leaves was more inhibitory against spore germination and colony development.     

向日葵枯萎病拮抗芽胞杆菌的筛选与生防评价

目的筛选向日葵枯萎病拮抗芽胞杆菌菌株并研究其抗菌谱,探讨环境条件对菌株抑菌活性的影响并通过植物栽培完成生防评价。方法从向日葵根际土壤中选择性分离芽胞杆菌,通过5点对峙法确定1株高效拮抗菌株,进行鉴定后,测定其抑菌谱;单因素实验探讨环境条件对抑菌活性的影响;向日葵发芽实验完成生防评价。结果确定1株高效的枯萎病拮抗菌株WBFL-1,经鉴定为枯草芽胞杆菌,该菌对镰刀菌属具有广谱抗性,其抑菌活性的最佳条件是温度40℃,pH值7.0,接种量150μL,发酵时间48 h。生防评价表明该菌对枯萎病的拮抗效果显著。结论该菌可为农作物枯萎病的生物防治提供有效菌种储备。     

高产铁载体棉田土壤细菌SS05的筛选与鉴定

【目的】研究从棉田土壤中筛选得到的高产铁载体细菌产铁载体能力、分类地位和抑菌活性。【方法】通过改良蔗糖-天冬氨酸培养基选择性筛选产铁载体细菌,通过分光光度计法测定铁载体活性,通过混菌法测定产铁载体细菌上清液对棉花枯萎病致病菌尖孢镰刀菌(Fusarium oxysporum)的抑菌效果,采用形态学、生理生化鉴定及16S rDNA序列系统发育分析对高产铁载体菌株进行鉴定。【结果】从棉田土壤中筛选到162株产铁载体细菌,30株产铁载体能力较强的细菌中21株具有较高产铁载体能力,菌株SS05的铁载体活性单位达到98.3%;在低铁条件下,SS05上清液对F.oxysporum具有显著的抑制作用;SS05与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近。【结论】SS05是高产铁载体菌株,与莫哈韦芽孢杆菌(Bacillus mojavensis)最为接近,在低铁培养条件下其上清液对F.oxysporum具有显著的抑制作用。     

链霉菌IMS002的分类鉴定及其抗真菌活性物质解析

【目的】对一株分离自植物根际土壤的具有抗真菌活性的链霉菌IMS002进行菌株分类鉴定,通过活性追踪分离纯化并鉴定有机相中的活性物质。【方法】通过16S rDNA和5个不同基因(atpD,gyrB,recA,rpoB,trpB)串联聚类分析以及生理生化实验分析,对链霉菌IMS002进行菌株分类鉴定,用扫描电子显微镜观察该株链霉菌的菌丝及孢子形态,以尖孢镰刀菌(Fusarium oxysporum)为指示菌进行生物活性追踪,通过硅胶柱层析、凝胶柱层析及高压液相色谱(HPLC)对活性物质进行分离和纯化,使用液质联用高分辨质谱仪、500 MHz核磁共振波谱仪以及圆二色光谱仪确定该物质的化学结构。【结果】IMS002经初步鉴定与产二素链霉菌(Streptomycesambofaciens)具有较近的亲缘关系,其发酵液对尖孢镰刀菌具有良好的抑菌效果,经分离和纯化以及现代波谱技术分析,确定有机相中的抑菌活性组分为Borrelidin。【结论】链霉菌IMS002能够产生化合物Borrelidin,该化合物对尖孢镰刀菌具有抑制活性。     

Plant-growth-promoting traits and antifungal potential of the Bacillus amyloliquefaciens YL-25

Biological control of plant soil-borne diseases has been shown as an attractive and an environment friendly alternative to chemical fungicides. Different microbial strains have been reported effective in controlling plant pathogens. Among those, Bacillus strains have their own importance. Bacillus amyloliquefaciens strain YL-25, isolated from the rhizosphere of healthy banana plant, was evaluated as bio-organic fertiliser (BIO) for its ability to promote plant growth and suppress Fusarium wilt of banana in pot experiment. The results showed that the application of the BIO containing strain YL-25 significantly promoted the growth of banana plants and decreased the incidence of Fusarium wilt compared to the organic fertiliser and chemical fertiliser (CF). In order to explore the beneficial mechanisms of strain YL-25, experiments were conducted in vitro. The phytohormones including indole-3-acetic acid and gibberellin A3 and stable antifungal compounds three homologous of iturin A were identified in the culture broth of strain YL-25. The strain YL-25 also showed the ability to degrade extracellular phytate in plate experiment. Owing to its innate multiple functional traits and biocontrol activity, the strain YL-25 may be used as plant-growth-promoting rhizobacterium and biocontrol agent against Fusarium wilt of banana.     

Novel Production of Isochainin by a Strain of <Emphasis Type="Italic">Streptomyces </Emphasis>sp<Emphasis Type="Italic">.</Emphasis> Isolated from Rhizosphere Soil of the Indigenous Moroccan Plant <Emphasis Type="Italic">Argania Spinosa</Emphasis> L

Summary An actinomycete strain (designated Ap1) isolated from the rhizosphere soil of Argania spinosa L. strongly inhibited the growth of two plant pathogens: Fusarium oxysporum f.sp. albedinis and Verticillium dahliae. The spore morphology suggested that the Ap1 strain belonged to the genus Streptomyces. The antifungal compound produced by Ap1 was purified by HPLC and identified as the polyene macrolide, isochainin, by NMR and mass spectroscopy. Ap1 showed normal biosynthesis of isochainin in comparison with S. cellulosae ATTC 12625, in which precursor-directed biosynthesis by feeding ethyl (Z)-16-phenylhexadec-9-enoate to the culture medium is required. In addition, Streptomyces sp. strain Ap1 produces isochainin with a 6.5-fold higher concentration than Streptomyces cellulosae ATTC 12625.     

Production, purification, and characterization of antifungal metabolite from Pseudomonas aeruginosa SD12, a new strain obtained from tannery waste polluted soil

A new strain, SD12, was isolated from tannery waste polluted soil and identified as Pseudomonas aeruginosa on the basis of phenotypic traits and by comparison of 16S rRNA sequences. This bacterium exhibited broad-spectrum antagonistic activity against phytopathogenic fungi. The strain produced phosphatases, cellulases, proteases, pectinases, and HCN and also retained its ability to produce hydroxamate-type siderophore. A bioactive metabolite was isolated from P. aeruginosa SD12 and was characterized as 1-hydroxyphenazine ((1-OH-PHZ) by nuclear magnetic resonance (NMR) spectral analysis. The strain was used as a biocontrol agent against root rot and wilt disease of pyrethrum caused by Rhizoctonia solani. The stain is also reported to increase the growth and biomass of Plantago ovata. The purified compound, 1-hydroxyphenazine, also showed broad-spectrum antagonistic activity towards a range of phytopathogenic fungi, which is the first report of its kind.     

Pyrrolnitrin Production by Biological Control Agent Pseudomonas cepacia B37w in Culture and in Colonized Wounds of Potatoes

Bacterial strain B37w (= NRRL B-14858), an isolate noteworthy because it inhibits the growth of the bioherbicide fungus Colletotrichum truncatum, was selected for further studies of bacterial antifungal properties. This isolate was identified as a Pseudomonas cepacia strain by performing carbohydrate utilization and fatty acid profile analyses, as well as other biochemical and physiological tests. Petri plate assays revealed that strain B37w exhibited antifungal activity against the potato dry rot fungus Fusarium sambucinum. Using bioautography, we correlated antifungal activity with production of a specific compound. Isolation from strain B37w and identification of the antifungal antibiotic pyrrolnitrin are described. A whole-potato assay revealed B37w's ability to colonize potato wounds. Wounded potatoes were inoculated with B37w, and pyrrolnitrin was detected in these potatoes by thin-layer chromatography-bioautography at a concentration on the order of nanograms per wound. We performed an assay in which we examined efficacy against F. sambucinum-incited potato dry rot and found that B37w inhibited disease development. This is the first report of P. cepacia or pyrrolnitrin activity against the economically important potato pathogen F. sambucinum.     

THE EFFECT OF ASSOCIATED SOIL MICROFLORA ON FUSARIUM UDUM BUTL., THE FUNGUS CAUSING WILT OF PIGEON-PEA (CAJANUS CAJAN (L.) MILLSP.)

Inoculation with Fusarium udum Butl. produced more wilt of pigeon-pea in sterilized than in unsterilized soils at the same pH. From unsterilized soils with low disease incidence, nine fungi, Bacillus subtilis and an Actinomyces were isolated. The number of isolations of a particular organism varied from month to month during the cropping season of pigeon-pea in Delhi. Interaction of Fusarium udum and other organisms isolated was studied. Aspergillus niger and A. terreus secreted inhibitory substances in potato-dextrose broth: Bacillus subtilis inhibited growth on solid medium and also produced a toxic substance in potato-dextrose broth. The nature of the medium employed and period of growth were important factors in the production of the inhibitory principle, which is thermostable. The low incidence of pigeon-pea wilt in unsterilized soils may result from the inhibitory activity of the associated microflora in the soil.     

一株抗香蕉枯萎病内生细菌的分离鉴定及其抗病促生作用

从巴西蕉中分离到一株能够明显抑制尖孢镰刀菌古巴专化型4 号生理小种(FOC4)的内生细菌DB09208, 通过生理生化、形态特征及16S rDNA 序列同源性比较, 初步认为它和类芽孢杆菌(Paenibacillus)比较接近。该菌株能够显著抑制FOC4 菌丝生长, 抑菌率为74.09%。盆栽试验表明,该菌株能够抑制香蕉枯萎病的发生, 防治效果达为65.2%, 该菌同时能显著促进香蕉苗生根, 促生效果与植物生长素IAA 相似, 显示出良好的开发前景。