Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp. Strain M10 antagonistic to Botrytis cinerea

A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.     

番茄灰霉病生防链霉菌筛选及鉴定

【背景】由灰葡萄孢侵染所致的番茄灰霉病是一类重要的真菌病害,生物防治具有环境友好、病原菌不易产生抗药性等特点,是果蔬灰霉病绿色防控的有效措施。【目的】筛选对番茄灰霉病具有防病作用且能促进番茄种子发芽的广谱拮抗性链霉菌,并明确该菌株种级分类地位。【方法】采用琼脂块法筛选拮抗番茄灰霉病菌的链霉菌菌株,采用对峙培养法和生长速率法检测菌株T22抑菌谱,通过产胞外酶活性检测、离体叶片防效和种子发芽试验明确该菌株的防病促生相关特性,根据形态学特征、生理生化特性和分子生物学方法对该菌株进行种类鉴定。【结果】从分离的56株放线菌中筛选到14株对番茄灰霉病菌具有拮抗效果的放线菌菌株,其中链霉菌T22对番茄灰霉病菌抑制作用最强,且具有较广抑菌谱,同时菌株T22具有产生纤维素酶和几丁质酶的能力。菌株T22无菌发酵滤液对番茄灰霉病菌、桃褐腐病菌、黄瓜枯萎病菌抑菌率分别为84.6%、81.5%和79.1%;其无菌发酵滤液原液对番茄灰霉病离体防效为55.1%;100倍稀释液处理番茄种子,胚轴、胚根和种子活力指数分别增加15.1%、29.7%和43.9%。根据形态学特征、生理生化特性和多基因聚类分析将链霉菌T22鉴定为白黑链霉菌(Streptomycesalboniger)。【结论】白黑链霉菌T22具有较强的抗真菌、产胞外酶、防病和促生活性,在番茄灰霉病生物防治中具有较好的开发应用潜力。     

Biological activity of phenylpropionic acid isolated from a terrestrial Streptomycetes.

The strain ANU 6277 was isolated from laterite soil and identified as Streptomyces sp. closely related to Streptomyces albidoflavus cluster by 16S rRNA analysis. The cultural, morphological and physiological characters of the strain were recorded. The strain exhibited resistance to chloramphenicol, penicillin and streptomycin. It had the ability to produce enzymes such as amylase and chitinase. A bioactive compound was isolated from the strain at stationary phase of culture and identified as 3-phenylpropionic acid (3-PPA) by FT-IR, EI-MS, 1H NMR and 13C NMR spectral studies. It exhibited antimicrobial activity against different bacteria like Bacillus cereus, B. subtilis, Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, P. flourescens, Staphylococcus aureus and some fungi including Aspergillus flavus, A. niger, Candida albicans, Fusarium oxysporum, F. udum and Penicillium citrinum. The antifungal activity of 3-PPA of the strain was evaluated in in vivo and in vitro conditions against Fusarium udum causing wilt disease in pigeon pea. The compound 3-PPA is an effective antifungal agent when compared to tricyclozole (fungicide) to control wilt caused by F. udum, but it exhibited less antifungal activity than carbendazim.     

水霉拮抗菌的筛选及其拮抗活性物质稳定性初步研究

【目的】从海底沉积物中分离、筛选水霉拮抗放线菌菌株,鉴定目标菌株及其无菌发酵液对水霉生长的抑制效果,并初步分析拮抗活性物质的稳定性。【方法】用稀释涂布法从采集的海底沉积物中分离得到海洋放线菌,以水霉为靶菌,通过平板对峙法在PDA平板上筛选出对水霉有拮抗作用的菌株;利用其发酵液对水霉菌丝和孢子进行初步拮抗效果研究;通过16S rRNA基因序列分析对目标菌株的种属进行初步鉴定。【结果】从分离到的数十株海洋放线菌中筛选到5株水霉拮抗菌,其中拮抗效果最强的为S26菌株,16S rRNA基因序列分析结果显示其为链霉菌,并与紫色链霉菌具有较近的亲缘关系;S26马铃薯葡萄糖液体培养基发酵液在平板抑菌圈实验中,对水霉孢子萌发的抑菌圈直径达32.00 mm±0.81 mm,其5倍浓缩无菌发酵液对水霉菌丝的抑菌圈直径达39.75 mm±0.50 mm;5倍浓缩无菌发酵液抑菌活性的3.125%即能完全抑制水霉孢子的萌发;5倍浓缩液对温度具有较强耐受性,经100 °C高温30 min处理后平板抑菌圈直径为25.50 mm±0.58 mm;经不同pH值处理12 h后,pH 5.0–9.0之间仍保持较好的拮抗活性;在37 °C下蛋白酶处理2 h后实验组与对照组存在显著性差异,但平板抑菌圈直径仍可达33.25 mm以上,推测拮抗物质活性成分由多肽和非多肽类代谢物共同组成。【结论】海洋链霉菌株S26产生的活性物质对病原水霉真菌有较强的抑制作用,并对外界环境变化有较强的适应能力,因而在水霉病的生物防治中具有潜在的应用价值。研究结果同时也显示海洋链霉菌在水产病害生物防治应用领域有较好的发展前景和更广阔的挖掘空间。     

Comparison of beta-Glucosidase Activities in Different Streptomyces Strains

Cellobiase (beta-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of beta-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the beta-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose.     

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.     

β-Xylosidase and xylanase characterization and production by Streptomyces sp. CH-M-1035

Extracellular xylanase activity and cell-bound β-xylosidase production by a selected strain of Streptomyces sp. CH-M-1035 was characterized during growth on three xylans, sugar cane bagasse pith and lemon peel as sole carbon source. The cell-bound β-xylosidase and extracellular endoxylanase had pH optima of 6·0 and 5·0, and temperature optima of 50°C and 60°C, respectively. The highest level of β-xylosidase activity was obtained when Streptomyces sp. CH-M-1035 was grown on larchwood xylan, whereas the maximal endoxylanase production was found on lemon peel. Reducing sugars accumulated in the culture media when Streptomyces sp. CH-M-1035 was grown on xylans, but not on agroindustrial residues.     

链霉菌LA5高产菌株诱变育种研究初报

抗生素LA5具有开发成抗真菌农用抗生素的广阔前景,但由于该菌株的野生型菌株发酵效价低,不能满足工业化开发的要求。通过以链霉菌LA5为出发菌株,采用紫外线(UV)、微波、亚硝基胍(NTG)、紫外线 亚硝基胍、亚硝基胍 紫外线等方法进行诱变筛选,结果表明,以紫外线照射80 s 亚硝基胍处理80 min的诱变的正突变率最高,为30%,其他依次为亚硝基胍处理80 min 紫外线照射40 s诱变和亚硝基胍处理60min 紫外线照射120 s诱变,正突变率均为26%。然后依次使用亚硝基胍处理80 min 紫外线照射80 s、亚硝基胍处理80 min 紫外线照射40 s、亚硝基胍处理60 min 紫外线照射120 s进行第2、第3、第4轮复合诱变筛选,最后选育出突变菌株U8-N8A-196,其产抗生素能力比链霉菌LA5出发菌株提高了34.5%,极显著高于LA5菌株。     

高温链霉菌24＃的初步研究

从土壤中筛选得到一株产广谱、高活性抗真菌物质的链霉菌 2 4 # ,经测定对 2 5种植物病原真菌有显著拮抗作用 ,其次生代谢产物对病菌菌丝有断裂、扭曲、缢缩等致畸效应。 16SrDNA序列分析显示本菌株与模式菌株链霉菌DSM4 4 2 93T的 16SrDNA同源性为 98 93% ,但菌株形态特征、培养特征、细胞壁化学组分、生理生化特性等均不同于模式菌株 ,且DNA杂交率只有 2 2 5 4 % ,建议为链霉菌属的一个新种 ,命名为山东链霉菌 (Streptomycesshandon gensissp .nov .)。     

Activity of some aminoglycoside antibiotics against true fungi, Phytophthora and Pythium species

AIMS: To investigate the in vitro antifungal and antioomycete activities of some aminoglycosides against true fungi and Phytophthora and Pythium species and to evaluate the potential of the antibiotics against Phytophthora late blight on plants. METHODS AND RESULTS: Antifungal and antioomycete activities of aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin and streptomycin) and a paromomycin-producing strain (Streptomyces sp. AMG-P1) against Phytophthora and Pythium species and 10 common fungi were measured in potato dextrose broth (PDB) and on seedlings in pots. Paromomycin was the most active against Phytophthora and Pythium species with a minimal inhibitory concentration of 1-10 microg ml(-1) in PDB, but displayed low to moderate activities towards other common fungi at the same concentration. Paromomycin also showed potent in vivo activity against red pepper and tomato late blight diseases with 80 and 99% control value, respectively, at 100 microg ml(-1). In addition, culture broth of Streptomyces sp. AMG-P1 as a paromomycin producer exhibited high in vivo activity against late blight at 500 microg freeze-dried weight per millilitre. CONCLUSIONS: Among tested aminoglycoside antibiotics, paromomycin was the most active against oomycetes both in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study show that aminoglycoside antibiotics have in vitro and in vivo activities against oomycetes, suggesting that Streptomyces sp. AMG-P1 may be used as a biocontrol agent against oomycete diseases.     

An antifungal protein from the marine bacterium streptomyces sp. Strain AP77 is specific for Pythium porphyrae,a causative agent of red rot disease in Porphyra spp

A novel antifungal protein (SAP) was found in the culture supernatant of a marine bacterium, Streptomyces sp. strain AP77, and was purified. This protein was characterized by chemical, biochemical, and biological analyses. By using gel filtration, the molecular mass of SAP was estimated to be 160 kDa. Structural analysis of SAP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry suggested that SAP is composed of three heterologous protein subunits of 41.7 kDa (SAP1), 21.7 kDa (SAP2), and 18.7 kDa (SAP3) at a molar ratio of 1:1:5 (or 1:1:6). N-terminal amino acid sequence analysis and a homology search revealed that SAP1, SAP2, and SAP3 exhibit 64.3, 68.4, and 86.7% similarity to three Streptomyces coelicolor polypeptides, puromycin resistance protein (Pur8), a conserved hypothetical protein, and bacterioferritin, respectively. The MIC of purified SAP against Pythium porphyrae was determined to be 1.6 microg/disk, whereas no inhibitory effect was observed at concentrations up to 100 microg/disk against most of the fungal and bacterial strains tested; the only exception was relatively strong antifungal activity against Pythium ultimum (MIC, 6.3 microg/disk). In vitro and in vivo toxicity tests demonstrated that SAP showed no toxicity against Porphyra yezoensis cells, human normal dermal fibroblasts, and mice at doses up to 700 microg/ml (for 24 h), 250 microg/ml (for 12 h), and 75 mg/kg (for 35 days), respectively. SAP was labile when it was subjected to a heated-air drying treatment, which is a great advantage in food production procedures. These results indicated that Streptomyces sp. strain AP77 might be useful as a gene source for safe transgenic Porphyra breeding for tolerance to Pythium infection.     

Isolation and identification of Streptomyces sp. Act4Zk,a good producer of Staurosporine and some derivatives

In this study, strain Streptomyces sp. Act4Zk was isolated based on a method developed for the isolation of myxobacteria. Due to the low efficiency of the majority of conventional DNA extraction techniques, for molecular identification of the strain Streptomyces sp. Act4Zk, a new technique for DNA extraction of Actinobacteria was developed. In order to explore potential bioactivities of the strain, extracts of the fermented broth culture were prepared by an organic solvent (i.e. ethyl acetate) extraction method using. These ethyl acetate extracts were subjected to HPLC fractionation against standard micro-organisms, followed by LC/MS analysis. Based on morphological, physiological, biochemical and 16S rRNA gene sequence data, strain Streptomyces sp. Act4Zk is likely to be a new species of Streptomyces, close to Streptomyces genecies and Streptomyces roseolilacinus. Antimicrobial assay indicated high antifungal activity as well as antibacterial activity against Mycobacterium smegmatis and Gram-positive bacteria for the new strain. HPLC and LC/MS analyses of the extracts led to the identification of three different compounds and confirmed our hypothesis that the interesting species of the genus Streptomyces being a good producer of staurosporine and some derivatives.     

The variation of antioxidant defense system of Streptomyces sp. M4018 with respect to carbon sources

The effect of glycerol, glucose, and starch as carbon sources on the antioxidant defense system such as superoxide dismutase (SOD) and catalase (CAT) activities, pyruvate levels, and membrane lipid peroxidation (LPO) levels of Streptomyces sp. M4018, after isolation from the rhizosphere samples of Colutea arborescens and identification as a strain of S. hiroshimensis based on phenotypic and genotypic characteristics, were investigated. As an antioxidant defense enzyme, SOD activities increased up to 20 g/L of glycerol and 15 g/L of starch, while they showed negative correlation with glucose concentration. CAT activity variations of glycerol- and glucose-supplemented mediums showed significant positive correlations with the trend of SOD activities. However, CAT activity, in contrast to SOD, in Streptomyces sp. M4018 tended to decrease as the starch concentration increased. The production of pyruvate increased with respect to glycerol and starch up to 15 g/L, while it was positively correlated with glucose concentration. The highest pyruvate production was seen at 20 g/L glucose. Membrane LPO levels were negatively correlated with the activities of SOD and CAT enzymes, and the minimum LPO level was determined at 5 g/L of glucose, where SOD and CAT activities reached their maximum levels. Nevertheless, the higher SOD and CAT activities in a wider range of incubation period compared to the beginning by resulting in insignificant increases in membrane LPO levels showed the unusual antioxidant response capacities of the in Streptomyces sp. M4018 against the potentially deleterious effects of reactive oxygen species (ROS) for glycerol, glucose, and starch as carbon sources.     

抗真菌多肽捷安肽素发酵中试研究

对筛选出的一株芽孢杆菌ZK在1000L发酵罐中进行了代谢产物抗真菌多肽捷安肽素的放大中间试验。研究菌体的生长、还原糖的利用及产素的变化规律,为进行捷安肽素的工业化生产提供了依据。     

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.     

ste7与ste15双基因敲除对依博素生物合成的影响

摘要：【目的】研究ste7和ste15基因双敲除对依博素生物合成的影响。【方法】通过基因同源重组双交换,对ste15基因缺失突变株Streptomyces sp. 139 (ste15 -)再进行ste7基因的敲除,经Southern杂交验证,获得了ste7和ste15双基因缺失变株Streptomyces sp. 139 (ste7 - ste15 -)。对该突变株进行了基因互补。气相色谱分析ste7和ste15双基因缺失突变株及互补株产生的胞外多糖单糖组分,排阻色谱测定衍生物的重均分子量,ELISA法     

康宁木霉对龟裂链霉菌MY02诱导活性及其诱导组分的初步分析

利用真菌作为诱导菌株评价其对龟裂链霉菌(Streptomyces rimosus) MY02的活性代谢产物的影响,并对其诱导条件进行优化。当以康宁木霉(Trichoderma koningii)和香菇真菌(Lentinus edodes)作为诱导菌株,黄瓜枯萎病菌(Fusarium oxysporum f sp.cucumarinum)作为指示菌,以菌株MY02作为被诱导菌株时,康宁木霉和香菇真菌对菌株MY02的抗真菌活性均具有明显的正向诱导作用,菌株MY02的抗真菌活性均比对照明显增强。以康宁木霉作为诱导菌株时,其最佳诱导条件为接种量2%,添加时间为发酵36 h。通过初步分析康宁木霉的诱导组分为其发酵产生的多糖类物质。     

微白黄链霉菌G-1发酵液抗真菌特性研究和发酵条件优化

为明确微白黄链霉菌(Streptomyces albidoflavus)G-1发酵液抗真菌特性和较优发酵条件,本研究采用平板对峙法、生长速率法、单因素试验、RDA分析和响应面CCD设计分别对其抗真菌特性、产抗菌物质关键限制因子和发酵条件较优组合进行了探究.结果 表明:S.albidoflavus G-1发酵液对马铃薯早...     

Identification of extracellular N-acylhomoserine lactone acylase from a Streptomyces sp. and its application to quorum quenching

N-acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.