解淀粉芽胞杆菌胞外抑菌活性物质研究现状

解淀粉芽胞杆菌是芽胞杆菌属的一个种,在生长过程中可以向胞外分泌多种抑菌活性物质。按照胞外抑菌活性物质合成方式的不同分为两大类,一类是通过核糖体途径合成的核糖体类细菌素、几丁质酶和β-1,3-葡聚糖酶等抑菌物质;另一类是通过非核糖体途径合成的细菌素、表面活性素、芬荠素、伊枯草菌素等脂肽类物质。这些代谢产物种类丰富、广谱抑菌、抗逆性强,可应用于农业、食品工业和环境保护等。本文对解淀粉芽胞杆菌抑菌活性物质的研究及应用现状进行综述,为深入研究和全面开发解淀粉芽胞杆菌抑菌活性物质提供充足的理论依据。     

一株抗水稻纹枯病菌的解淀粉芽胞杆菌分离与鉴定

【目的】筛选对水稻纹枯病菌(Rhizoctonia solani)具有强拮抗作用的细菌菌株。【方法】用指示菌法筛选拮抗菌株;通过形态观察、生理生化实验、Biolog及16S rDNA序列分析鉴定目标菌株;利用平板双向培养法和滤纸片扩散法测定抑菌谱及拮抗性质。【结果】分离到一株高活力的水稻纹枯病菌拮抗菌株YB-3,该菌株属于解淀粉芽胞杆菌(Bacillus amyloliquefaciens);菌株YB-3对常见的14株病原真菌和7株细菌具有较强的拮抗作用,并发现其对亲缘关系较近的芽孢菌属有较强的拮抗作用;该菌株的抑制活性具有温度稳定、耐酸、但对蛋白酶敏感的特点。【结论】通过指示菌法筛选到一株对水稻纹枯病菌有强拮抗作用的解淀粉芽胞杆菌(B.amyloliquefaciens)YB-3,它具有广谱、高效的植物病原菌拮抗活性。     

解淀粉芽胞杆菌HR-2的分离鉴定及对水稻稻瘟病菌的拮抗效果

稻瘟病是一种严重威胁全球粮食安全的水稻真菌病害.本研究采用平板对峙法从湖南岳阳地区筛选出1株对水稻稻瘟病菌具有高效拮抗活性的菌株HR-2.通过形态特征验证、16S rRNA、gyrA和tuf基因序列比对分析,鉴定该菌株为解淀粉芽胞杆菌(Bacillus amyloliquefaciens).抑菌广谱性测定结果表明菌株H...     

解淀粉芽胞杆菌分离鉴定及培养优化的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)是芽胞杆菌属的一个种,广泛存在于自然界,具有丰富的生境多样性,其芽胞具有抗逆性,可抵抗不良环境,同时能产生多种抗菌物质,如脂肽、抗菌蛋白和多种酶类,能抗植物病原真菌和有害细菌,该菌能形成生物膜,定植于植物根系,促进植物生长,因此对该菌进行分离鉴定以及培养优化具有重要意义。截至目前,已经从各种生境中分离、鉴定了多种有应用价值的解淀粉芽胞杆菌,通过不同策略对该菌的培养基和培养条件进行优化,使该菌相关功能得以提高。本文综述了近年来对解淀粉芽胞杆菌的生境多样性、分离鉴定及培养基和培养条件优化的策略,简要归纳了国内外关于解淀粉芽胞杆菌重要的工业和商业产品,为更好地研究和应用解淀粉芽胞杆菌提供必要的参考和借鉴。     

解淀粉芽胞杆菌BaX030的分离鉴定及抗菌功能

摘要:【目的】芽胞杆菌是微生物活性物的重要来源,从全国各地采集的72份土壤样品中分离出339株芽胞杆菌,研究各菌株抑菌活性,分离纯化抑菌活性物,为丰富芽胞杆菌菌种资源和微生物次级代谢物的挖掘奠定实际应用基础。【方法】采用水浴加热和稀释平板涂布等方法从河南花生地采集的土壤中筛选得到一株具有很强抑菌活性的芽胞杆菌,结合形态观察、生理生化特征和16S rRNA基因序列同源性比对分析,对该菌株进行鉴定。丙酮沉淀、葡聚糖凝胶柱层析、C18反相柱层析得到Bacillus amyloliquefaciens X030抑菌活性物,LC-MS/MS鉴定其分子量。利用滤纸片扩散法和平板对峙培养法测定抑菌谱及拮抗性质。【结果】筛选分离得到一株解淀粉芽胞杆菌,归类并命名为解淀粉芽胞杆菌Bacillus amyloliquefaciens X030。BaX030对金黄色葡萄球菌(Staphylococcus aureus)、白色念珠菌(Candida albicans)、酵母菌( Saccharomycetes)有较强抑制效果,对水稻稻瘟病菌(Pyriculariaoryzae)、辣椒尖胞炭疽病菌(Chili pointed cell anthrax)、枇杷炭疽病菌(Gloeosporium eriobotryae speg)、烟草黑胫病菌(Phytophthora parasitica)有良好拮抗活性。初步确定BaX030产生的抑菌活性物为多肽类化合物。【结论】分离得到的B． amyloliquefaciens X030产生了一个对病原细菌具有较强抑制作用的多肽,同时该菌株在拮抗植物病原真菌方面也有明显的效果。     

解淀粉芽胞杆菌抗真菌活性研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)具有很强的抑制植物病原真菌的能力。其菌体细胞能产生多种酶类、脂肽类抗生素、生物表面活性素、聚酮类化合物和抑菌蛋白,同时具有诱导植物产生系统抗性(ISR)的能力,因此在工农业、种植业、养殖业、食品加工业、果蔬的采后保鲜和饲料业等行业具有重要价值。本文对解淀粉芽胞杆菌抗真菌作用、抗真菌能力提高策略、抗菌化合物合成调节、抑制真菌机制及其引发的ISR等问题进行了深入探讨和综述。     

生防解淀粉芽胞杆菌的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)因能抑制植物病害和促进植物生长而被广泛研究。随着测序技术的不断发展,解淀粉芽胞杆菌菌株的全基因组序列陆续被测定,综述了其拮抗作用相关的功能基因、生防机制及植物-病原物-生防菌的相互作用,并对后续研究趋势进行了展望,为解淀粉芽胞杆菌的深入研究及其更好的应用提供理论参考     

解淀粉芽胞杆菌SSY1菌株的稳定性研究

目的测定产纤维素酶解淀粉芽胞杆菌SSY1株的稳定性。方法在适宜生长条件下连续传代30代。分别取第2、10、15、20、25和30代细菌,对其进行染色镜检、生化试验、药敏试验、产纤维素酶特性及动物安全性试验观察。结果传至第30代,细菌的形态、生化特性、产纤维素酶特性及药敏特性均未发生明显变化。结论产纤维素酶解淀粉芽胞杆菌SSY1株稳定性良好,有望成为中药发酵菌种。     

1株产细菌纤维素芽胞杆菌的分离及鉴定

从残次水果中分离出1株菌株ZF-7,其产物经纤维素特异性染色反应、红外光谱分析及纤维素酶水解实验后,被确定为细菌纤维素。在对ZF-7菌株常规形态学及生理生化特性鉴定的基础上,对部分长度的16S rDNA同源性进行了分析,发现ZF-7菌株与解淀粉芽胞杆菌相似度可达99.5%,现命名为Bacillus amyloliquefaciens ZF-7。对ZF-7菌株在振荡培养和静置培养条件下的发酵性能进行了初步考察,得到细菌纤维素产率分别为6.6和6.2 g/L。     

鸡肠源芽胞杆菌的分离、鉴定和抗菌活性

从人工饲养的健康三黄肉鸡的盲肠和大肠中分离出10株芽胞杆菌,对其进行菌落形态观察、革兰染色、芽胞染色和生理生化特性鉴定,确定为枯草芽胞杆菌、地衣芽胞杆菌、蜡状芽胞杆菌、巨大芽胞杆菌、球形芽胞杆菌、短小芽胞杆菌、凝结芽胞杆菌。同时测定了它们对动物病原性大肠埃希菌和链球菌的抑菌活性。其中有9株芽胞杆菌对大肠埃希菌有抑制作用,8株芽胞杆菌对链球菌有抑制作用。结果表明芽胞杆菌分离株Y2、Y3、Y4、Y5、Y6、Y7、Y8具有作为益生菌开发的潜力。     

Evaluation of Brassica germplasm for field resistance against clubroot (Plasmodiophora brassicae Woron)

Of the 124 germplasm accession of oil seed Brassicas screened under field condition against clubroot disease (Plasmodiophora brassicae), 80% were susceptible and 17, 3, 1 and 1 of Brassica juncea, Brassica rapa var. toria, B.rapa var. yellow sarson and B. rapa, respectively, were resistant.     

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

This paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins. The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight. Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β-glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH₂₀ in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose-6-phosphate after treatment with β-glucosidase.     

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae‐infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.     

Selection for resistance to clubroot (Plasmodiophora brassicae) in marrowstem kale (Brassica oleracea var. acephala L.)

Ninety-six cultivars of Brassica oleracea were screened for clubroot resistance in a seedling test using two populations of Plasmodiophora brassicae. The most resistant cultivars were kales. Sixteen resistant marrowstem kale cultivars of diverse geographical origin were used to start a selection programme for clubroot resistance. Four generations of selection, involving single plants, half-sib and full-sib families, reduced a disease index averaged over six clubroot populations from 41.2 to 12.5. This was lower than the most resistant cultivar in the original population, cv. Mixti 28.8, and as good as a German landrace of cabbage noted for its resistance, Bohmerwaldkohl 10.5. In comparison, the mean of five kale controls, cvs Bittern, Canson, Condor, Kestrel and Merlin, was 61.1 and the value for the most susceptible control, cabbage cv. Septa, was 89.3. In the final assessment, there were no clubroot population x B. oleracea genotype interactions and in the initial assessment of cultivars there were only small interactions which could be removed by an angular transformation of the data. It was concluded that a high level of non-differential resistance had been achieved and that it may prove durable. It was also concluded from a small field trial that this level of resistance would prevent serious yield losses in practice.     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

Effect of surfactants and fungicides on clubroot (Plasmodiophora brassicae) of brassicas

Two formulations of the surfactant sodium dioctyl sulphosuccinate (Aerosol OT, Monawet MO70), one of alkyl phenyl ethylene oxide (Agral) and three fungicides (PP192, dichlorophen and a thiabendazole/iodophor complex -Byatran) were tested in field trials for control of clubroot (Plasmodiophora brassicae) on cabbage in 1988 and 1989. A standard mercurous chloride treatment (pre-sowing compost incorporation) was included in all experiments. Plants were raised in 64 cm³ blocks (1988) or 15 cm³ free-fill cells (1989). Mercurous chloride reduced disease severity and increased yield in both years. Byatran was ineffective. The surfactants and dichlorophen, applied as pre-planting soaks to the plant-raising compost, restricted disease development and increased yield in 1988 but not in 1989. A similar treatment with PP192 restricted disease severity and enhanced yield in both years. Pouring the surfactants and dichlorophen into the planting hole was more effective than using them to soak the compost. In 1989 pour treatments with Agral and a combined soak/pour treatment with dichlorophen reduced disease severity and increased yields by 250% and 97% respectively; compost treatment with PP192 gave a 150% increase.     

The host range of Plasmodiophora brassicae and its relationship to endogenous glucosinolate content

The host range of the soilborne obligate biotroph, Plasmodiophora brassicae was investigated. Evidence is presented that infection by P. brassicae might occur in non- Brassica species, leading to the potential formation of resting spores. Structures resembling P. brassicae were found in the root cortex of Tropaeolum majus , Carica papaya , Reseda alba and Beta vulgaris as demonstrated by scanning electron microscopy. Inoculation of Brassica rapa roots with spores extracted from either T. majus or B. vulgaris roots which had been previously inoculated with P. brassicae led to development of clubroot in the roots of B. rapa . It was also shown that the development of the symptom might be correlated with glucosinolate content, although other host factors are implicated in the B. vulgaris interaction with P. brassicae . In the glucosinolate-containing non-Brassicas, T. majus and C. papaya , the concentrations of benzylglucosinolate increased markedly in roots inoculated with P. brassicae , compared with the controls. There were also increases in concentrations of benzylglucosinolate in leaves of T. majus after P. brassicae infection. However, in R. alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls. High root concentrations of 2-OH-2-phenylethylglucosinolate (glucobarbarin) compared with low root indole glucosinolates in this species might limit P. brassicae infection and development. The importance of our investigations in relation to cultivation of non- Brassica species on fields infested with P. brassicae is discussed.     

Pathotypes of Plasmodiophora brassicae, the cause of clubroot, in Australia

Variation in pathogenicity of Plasmodiophora brassicae in Australia was studied using the European Clubroot Differential series of brassica hosts. From 41 collections of P. brassicae originating from important vegetable brassica production regions in Victoria, Western Australia, Tasmania, Queensland and New South Wales, 23 triplet codes were generated. These were more similar to populations of P. brassicae reported from the USA than those from Europe. The most common Australian pathotypes had triplet codes of 16/3/12 and 16/3/31 and were each assigned seven times to pathogen collections originating from three states of Australia. Other codes that occurred more than once were 16/2/31, which was assigned to six collections from four states of Australia, and 16/19/31, which was assigned twice to collections originating from Western Australia.     

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims: To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens. Methods and Results: An unknown bacterial species cultured from the Yogu Farm? probiotic dairy beverage was identified through 16S ribosomal RNA analysis as B. amyloliquefaciens, a phylogenetically close relative of Bacillus subtilis. The cell‐free supernatant (CFS) of overnight cultures was active against Listeria monocytogenes and also against clinical isolates of Gardnerella vaginalis and Streptococcus agalactiae. At the same time, several isolates of vaginal probiotic Lactobacilli were resistant to the CFS. The nature of the compound causing inhibitory activity was confirmed as proteinaceous by enzymatic digestion. The protein was isolated using ammonium sulfate precipitation, and further purified via column chromatography. PCR analysis was conducted to determine relatedness to other bacteriocins produced by Bacillus spp. Conclusion: The antimicrobial protein isolated from B. amyloliquefaciens was shown to be subtilosin, a bacteriocin previously reported as produced only by B. subtilis. Significance and Impact of the Study: This is the first report of intra‐species horizontal gene transfer for subtilosin and the first fully characterized bacteriocin isolated from B. amyloliquefaciens. Finally, this is the first report on subtilosin’s activity against bacterial vaginosis‐associated pathogens.     

Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.