一株扩展青霉拮抗菌的分离、鉴定及抑菌活性物质

【目的】筛选有效抑制扩展青霉(Penicillium expansum)的拮抗菌,并鉴定其所产抑菌物质的主要种类及相对含量。【方法】从苹果表面分离到拮抗扩展青霉的菌株BA-16,经形态学、生理生化及16S r RNA基因序列分析对该菌进行鉴定;根据已知脂肽类抗生素合成相关基因序列设计3对特异性引物对菌株BA-16进行检测,对PCR产物克隆、测序和BLAST分析,采用酸沉淀法从菌株发酵液中制备出抑菌物质粗提液,对活性粗提物进行HPLC和MALDI-TOF-MS分析。【结果】经鉴定,BA-16被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),所得PCR产物经测序和BLAST分析,证实BA-16带有sfp和fen B基因。HPLC和MS结果显示菌株发酵液中含有Fengycin和Surfactin两种脂肽类产物,Fengycin是拮抗扩展青霉的主要因素。【结论】本研究对于苹果采后青霉病的生物防治具有良好的应用开发前景。     

Screening the Fusarium graminearum inhibitory mutant strain from Bacillus subtilis by atmospheric-pressure plasma jet

Aims:  The aim of this study was to improve the antagonistic activity of Bacillus subtilis JA towards Fusarium graminearum by screening high-yielding mutant using the atmospheric-pressure plasma jet (APPJ).
Methods and Results:  Atmospheric-pressure plasma jet was applied as mutagenic source for breeding high-yielding mutant strain. Helium was used as APPJ operating gas. The mutation effects of different treatment times of APPJ were studied. The mutant strain designated as B. subtilis B06 was successfully screened out, which showed higher antagonistic activity against F. graminearum in vitro . Its inhibition zone against the indicator fungus increased by 23% compared to the original one. HPLC and ESI (electrospray ionization) mass spectrometry analysis indicated that antifungal compounds produced by the mutant and original strain belonged to the lipopeptide, surfactin and iturin families. The mutant strain showed favourable properties of faster growth in the fermentation process and higher production of antibiotics. The lipopeptide production of the mutant was 2·3-fold as that of the original strain.
Conclusions:  A mutant strain with strong antagonistic activity and high yielding of antibiotics was obtained by APPJ in this study. The mutant could be used as a promising biocontrol agent in agriculture.
Significance and Impact of Study:  This study provides a novel mutagenic source for breeding high-yielding microbial mutant, which would be very useful in the application of some valuable metabolites from micro-organism.     

Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans, the causative agent of blackleg disease of canola

A bacterial isolate capable of inhibiting the growth of Leptosphaeria maculans (Desmaz.) Ces. & De Not., the causative agent of blackleg disease of canola (Brassica napus L. and Brassica rapa L.), was identified as a potential biological control agent. This environmental isolate was determined to be Paenibacillus polymyxa based on its (i) biochemical and growth characteristics and (ii) 16S rRNA sequence similarity, and was given the strain designation PKB1. Antifungal peptides were produced by P. polymyxa PKB1 around the onset of sporulation, with optimal production on potato dextrose broth. The antifungal peptides were extracted from P. polymyxa PKB1 cells and (or) spores using methanol and were purified using size exclusion and reverse-phase chromatography. Characterization of the antifungal peptides was done using amino acid compositional analysis, Edman degradation sequencing from partially hydrolyzed material, and a variety of mass spectrometric methods. The purified antifungal material was found to be a mixture of related peptides of molecular masses 883, 897, 948, and 961 Da, with the most likely structure of the 897 Da component determined to be a cyclic depsipeptide with an unusual 15-guanidino-3-hydroxypentadecanoic acid moiety bound to a free amino group. These compounds are therefore members of the fusaricidin group of cyclic depsipeptides.     

Isolation and analysis of bacteria associated with spores of Gigaspora margarita

Aims:  The aim of this work was to observe bacteria associated with the spores of Gigaspora margarita , an arbuscular mycorrhizal fungus (AMF).
Methods and Results:  First, a direct analysis of DNA from sterilized spores indicated the bacteria belonging to the genus Janthinobacterium . In the second assay, two bacterial strains were isolated by osmosis from protoplasts, which were derived from spores by using two particular enzymes: lysing enzymes and yatalase. After isolation, cultivation and identification by their DNA as performed in the first experiment, the species with the closest relation were Janthinobacterium lividum (KCIGM01) and Paenibacillus polymyxa (KCIGM04) isolated with lysing enzymes and yatalase respectively. Morphologically, J. lividum was Gram negative and oval, while P. polymyxa was also oval, but Gram positive. Both strains had antagonistic effects to the pathogenic fungi Rosellimia necatrix, Pythium ultimum , Fusarium oxysporum and Rhizoctonia solani . In particular, J. lividum was much stronger in this role. However, in phosphorus (P) solubilization P. polymyxa functioned better than J. lividum.
Conclusions:  This experiment had revealed two new bacteria species ( P. polymyxa and J. lividum ), associated with AMF spores, which functioned to suppress diseases and to solubilize P.
Significance and Impact of the Study:  AMF spores could be a useful source for bacterial antagonists to soil-borne diseases and P solubilization.     

Paenibacillus polymyxa antagonizes oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum

Aim:  To find sustainable alternatives to the application of synthetic chemicals for oomycete pathogen suppression.
Methods and Results:  Here, we present experiments on an Arabidopsis thaliana model system in which we studied the antagonistic properties of rhizobacterium Paenibacillus polymyxa strains towards the oomycete plant pathogens Phytophthora palmivora and Pythium aphanidermatum . We carried out studies on agar plates, in liquid media and in soil. Our results indicate that P. polymyxa strains significantly reduced P. aphanidermatum and P. palmivora colonization in liquid assays. Most plants that had been treated with P. polymyxa survived the P. aphanidermatum inoculations in soil assays.
Conclusions:  The antagonistic abilities of both systems correlated well with mycoidal substance production and not with the production of antagonistic substances from the biocontrol bacteria.
Significance and Impact of the Study:  Our experiments highlight the need to take biofilm formation and niche exclusion mechanisms into consideration for biocontrol assays performed under natural conditions.     

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25

Aims:  Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages.
Methods and Results:  A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16.
Conclusions:  The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F.
Significance and Impact of the Study:  The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100°C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.     

Antifungal and phytostimulating characteristics of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Ch-13 rhizospheric strain,producer of bioprepations

Bacillus subtilis Ch-13 industrial strain was shown to have a wide spectrum of antagonistic activities against different species of phytopathogenic fungi and bacteria. The B. subtilis Ch-13 strain produces lytic enzymes; cyanide and other antifungal metabolites; stimulates plant growth, producing phytohormones—auxin derivatives. This strain by 2.5 times reduced the quantity of tomato plants infected with phytopathogenic fungus Fusarium oxysporum during inoculation. Fungi abundance on roots with bacterial inoculation was 6.9 times less than in the absence of inoculation. The application of detected antifungal metabolites as biochemical markers for the strain enables to control the stability of physiologic and biochemical characteristics of the producer, and ensures a rapid quality assay of biopreparations with high performance liquid chromatography (HPLC).     

Mycofumigation with Oxyporus latemarginatus EF069 for control of postharvest apple decay and Rhizoctonia root rot on moth orchid

Aims:  To characterize the volatile antifungal compound produced by Oxyporus latemarginatus EF069 and to examine in vitro and in vivo fumigation activity of the fungus.
Methods and Results:  An antifungal volatile-producing strain, O. latemarginatus EF069 inhibited the mycelial growth of Alternaria alternata , Botrytis cinerea , Colletotrichum gloeosporioides , Fusarium oxysporum f. sp. lycopersici , and Rhizoctonia solani by mycofumigation. An antifungal volatile compound was isolated from the hexane extract of wheat bran–rice hull cultures of O. latemarginatus EF069 by repeated silica gel column chromatography and identified as 5-pentyl-2-furaldehyde (PTF). The purified PTF inhibited mycelial growth of R . solani in a dose-dependent manner. The mycofumigation with solid cultures of EF069 also reduced effectively the development of postharvest apple decay caused by B. cinerea and Rhizoctonia root rot of moth orchid caused by R. solani .
Conclusions:  Oxyporus latemarginatus EF069 showed in vitro and in vivo fumigation activity against plant pathogenic fungi by producing 5-pentyl-2-furaldehyde.
Significance and Impact of the Study:  Oxyporus latemarginatus EF069 producing an antifungal volatile compound may be used as a biofumigant for the control of fungal plant diseases.     

Detection and quantification of Paenibacillus polymyxa in the rhizosphere of wild barley (Hordeum spontaneum) with real-time PCR

Aim:  To detect and quantify the plant drought tolerance enhancing bacterium Paenibacillus polymyxa in a collection of 160 Hordeum spontaneum rhizosphere samples at the 'Evolution Canyon' ('EC'), Israel.
Methods and Results:  PCR primers and a FAM-TAMRA probe (6-carboxyfluorescein, 6-carboxy-tetramethyl-rhodamine) targeting 16S rRNA genes were designed and used to detect and quantify the target strain. Two commercial kits, Bio101 Fast Spin and Mo Bio Ultra Clean Soil DNA, were tested for DNA isolation from the rhizosphere and surrounding soil. Population densities of P. polymyxa were studied in the rhizosphere of wild barley and surrounding soil from the contrasting climatic slopes at the 'EC' using the real-time PCR and culture based methods.
Conclusion:  Paenibacillus polymyxa is one of the best established species in wild barley rhizosphere at the 'EC' slopes. With the real-time PCR assay we are able to detect 1 pg of DNA per PCR corresponding to 100 cells per ml. The results at the 'EC' correlate well to bacterial estimations by culture based methods.
Significance and Impact of the Study:  Significantly higher P. polymyxa cell number was detected in the rhizosphere of arid 'African' microclimate indicating possible role of adaptive co-evolution with plants.     

Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits

AIM: To study the antifungal activity and plant beneficial traits of a broad-spectrum antagonistic fluorescent pseudomonad strain, PUPa3. METHODS AND RESULTS: Strain PUPa3 was isolated from the rhizosphere soil of rice and identified as Pseudomonas aeruginosa on the basis of biochemical tests and by comparison of 16S rDNA sequences. This bacterium exhibits a broad-spectrum antifungal activity towards phytopathogenic fungi. The antifungal metabolite by PUPa3 was extracted, purified and characterized using nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Production of indole-3-acetic acid (IAA), siderophores, phosphatase and protease in PUPa3 was determined. Strain PUPa3 did not produce hydrogen cyanide, cellulase and pectinase. CONCLUSION: The antifungal metabolite produced by PUPa3 has been identified as phenazine-1-carboxamide (PCN) on the basis of NMR and MS data. Strain PUPa3 showed a broad-spectrum antifungal activity towards a range of phytopathogenic fungi. This bacterium also showed several plant growth-promoting traits but did not show the traits attributed to deleterious rhizobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the production of PCN as well as IAA for the first time by a saprophytic P. aeruginosa strain PUPa3. Because of the production of siderophore, growth hormone, protease and phosphatase and its innate fungicidal potential, this strain can be used as biofertilizer and antagonist against a range of phytopathogenic fungi that infect rice, groundnut, tobacco, chili, mango, sugarcane, tea, cotton and banana.     

Identification of a Passiflora alata Curtis dimeric peptide showing identity with 2S albumins

Antifungal proteins and peptides, essential compounds for plant defense, have been isolated from several tissues of various plants. These proteins could be used as a natural alternative to control phytopathogenic fungi. In this report a heterodimeric antifungal protein named Pa-AFP1, showing higher identity with the 2S albumin family, was purified by using 70-100% ammonium sulfate saturation and further purification steps such as anionic exchange Q-Sepharose chromatography associated with HPLC reversed-phase C4 chromatography. Analysis by Tricine-SDS-PAGE revealed two peptidic molecular masses of approximately 4500 Da and 7000 Da, in the presence of β-mercaptoethanol, while by removing the reducing agent a single protein with molecular mass of about 11,500 Da was obtained. Moreover, dimer mass was confirmed by MALDI-TOF analyses (11,569.76 Da). The antifungal protein, named Pa-AFP1, efficiently inhibited the growth of filamentous fungi Colletotrichum gloeosporioides, and was added to a short list of 2S albumins with antimicrobial properties. Otherwise, this same peptide showed no activity toward bacteria and yeasts. In summary, this compound could be used in the future to develop biotechnological products for the control of phytopathogenic fungi.     

Cyclosporine A from a nonpathogenic Fusarium oxysporum suppressing Sclerotinia sclerotiorum

AIMS: To evaluate the antagonistic activity of Fusarium oxysporum nonpathogenic fungal strain S6 against the phytopathogenic fungus Sclerotinia sclerotiorum and to identify the antifungal compounds involved. METHODS AND RESULTS: The antagonistic activity of Fusarium oxysporum strain S6 was determined in vitro by dual cultures. The metabolite responsible for the activity was isolated by chromatographic techniques, purified and identified by spectroscopic methods as cyclosporine A. The antifungal activity against the pathogen was correlated with the presence of this metabolite by a dilution assay and then quantified. Cyclosporine A caused both growth inhibition and suppression of sclerotia formation. In a greenhouse assay, a significant increase in the number of surviving soybean (Glycine max) plants was observed when S. sclerotiorum and F. oxysporum (S6) were inoculated together when compared with plants inoculated with S. sclerotiorum alone. CONCLUSION: Fusarium oxysporum (S6) may be a good fungal biological control agent for S. sclerotiorum and cyclosporine A is the responsible metabolite involved in its antagonistic activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Cyclosporine A has not been previously described as an inhibitor of S. sclerotiorum. Its minimum inhibitory concentration (MIC) of 0.1 microg disc(-1) makes it suitable to use as a biofungicide. In vivo experiments showed that F. oxysporum (S6) is a good candidate for the biocontrol of S. sclerotiorum in soybean.     

一株抗真菌内生多粘芽孢杆菌的分离鉴定及对水稻恶苗病菌的抑制作用

目的：从番茄叶片中筛选具广谱抑真菌活性的拮抗内生细菌,研究其对水稻恶苗病菌的抑制作用。方法：采用对峙培养法筛选拮抗内生细菌,根据菌株形态、生理生化特性结合16S rRNA基因序列分析鉴定菌株;采用硫酸铵沉淀法提取抗菌粗蛋白,研究其对水稻恶苗病菌菌丝生长和孢子萌发的影响。结果：从番茄叶片中筛选到一株抗真菌内生多粘芽孢杆菌（Paenibacillus polymyxa）SD-6,该菌株具有广谱抑菌活性,对供试的13种植物病原真菌均具较强的抑制作用;该菌株产生的抗菌粗蛋白能够显著抑制水稻恶苗病菌菌丝生长和孢子萌发,并能导致萌发孢子畸形和破裂。结论：从番茄叶片中分离到一株能产生抗真菌蛋白并具有广谱高效抑真菌作用的内生多粘芽孢杆菌,该菌株及其抗菌蛋白具有防治水稻恶苗病的潜力。     

Comparision between Bacillus subtilis RP24 and its antibiotic-defective mutants

Bacillus subtilis RP24, a promising plant growth-promoting rhizobacterium and a potent biocontrol agent isolated from pigeonpea rhizosphere was mutagenized with ethyl methanesulphonate to study the possible mechanism/s involved in the potential antagonistic properties of the strain. Over 10,000 mutants were screened against the phytopathogenic fungus Macrophomina phaseolina on potato dextrose agar plates to select ten mutants showing partial antagonism as compared to the parent strain and one negative mutant showing no antagonism. The parent strain RP24 was compared with its mutants for the presence of different possible mechanisms behind antagonism. Production of hydrogen cyanide, ammonia, siderophores, and hydrolytic enzymes like lipase, amylase, and protease were detected in all the mutants as well as the parent strain, whereas fungal cell-wall-degrading enzymes, β-1, 3-glucanase and chitosanase were not detected in any of the mutants and the parent strain, indicating that none of these mechanisms was involved in the antagonistic trait of the strain. Two possible mechanisms detected behind the antifungal trait of the strain RP24 were production of antifungal volatiles and extra-cellular diffusible antibiotics. An attempt was made for extraction, partial characterization of the extra-cellular diffusible antifungal metabolite/s by thin layer chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The extracellular, methanol soluble, hydrophobic, ninhydrin-negative, thermostable and pH-stable antifungal metabolites were characterized as cyclic lipopeptides belonging to the iturin group of peptide antibiotics.     

Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus

Aims:  Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence.
Methods and Results:  A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production.
Conclusions:  The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis.
Significance and Impact of the Study:  The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism.     

Chitinolytic activity of Bacillus Cohn.--phytopathogenic fungus antagonist

Among the 70 tested Bacillus spp. strains antagonistic to phytopathogenic fungi, 19 were found to possess chitinolytic activity when grown on solid media with 0.5% colloidal chitin. The chitinolytic activity of almost all of these 19 strains grown in liquid cultures ranged from 0.1 to 0.3 U/ml. One of the 19 strains exhibited exochitinase activity. In addition to chitinase, two strains also produced chitosanase and one strain, beta-1,3-glucanase. No correlation was found between the antifungal activity of the bacillar strains studied and their ability to synthesize extracellular chitinase. Among the 19 chitinolytic strains, the correlation between these parameters was also low (r x,y = 0.45), although the enzymatic preparations of most of these strains inhibited the growth of the phytopathogenic fungus Helminthosporium sativum.     

多粘类芽孢杆菌HY96-2产脂肽类抗真菌物质的研究

对一株已经商业化的可防治植物枯萎病的生防菌-多粘类芽孢杆菌HY96-2发酵液中抗真菌活性物质进行了分离纯化,采用酸化、正丁醇萃取、乙酸乙酯沉淀、硅胶柱层析、Sephadex LH-20柱层析、高效液相色谱(HPLC)等方法分离得到了一个抗真菌活性化合物6B,经NMR、MS、MS/MS等光谱学方法鉴定其为Fusaricidin A。琼脂扩散法抑菌试验结果表明,6B对西瓜枯萎病菌、水稻纹枯病菌、灰霉病菌等15种植物病原真菌的最小抑菌浓度为12.5～50μg/mL。盆栽实验结果表明,6B对黄瓜灰霉病具有明显的防治效果,当6B的浓度达到250μg/mL时,防治效果高达95%。     

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 μM added iron (Fe⁺²). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.