白菜黑腐病拮抗菌Lysobacter enzymogenes CX03的分离鉴定及生防效果研究

从白菜根际土壤中分离到一株对白菜黑腐病菌具有显著拮抗效果的生防细菌CX03。根据生物学特征、生理生化分析、Biolog测定及多基因系统发育分析，鉴定菌株CX03为产酶溶杆菌Lysobacter enzymogenes。酶学试验结果表明，菌株CX03在代谢过程中产生纤维素酶、蛋白酶和几丁质酶。通过抑菌谱分析，证明菌株CX03具有广谱拮抗作用，对野油菜黄单胞野油菜致病变种、密执安棒杆菌密执安致病变种等5种病原细菌和尖孢镰孢菌、辣椒疫霉菌等4种病原真菌具有显著的拮抗效果。通过盆栽试验结果显示，菌株CX03处理组的白菜黑腐病症状明显减轻，防治效果达到90.4%，显著高于春雷霉素的防治效果84.1%。综上所述，本文首次报道了一株对植物病原细菌具有抗菌活性的产酶溶杆菌生防菌株CX03，其生防性状优良，在植物病害生物防治中具有一定的应用潜力，为国内生物防治研究提供了新的微生物资源。     

草莓根围拮抗细菌的多样性分析及其田间应用

为研究草莓相关拮抗细菌的多样性,以期筛选对草莓土传病害有良好防效的生防细菌。本研究从土传病害重发田块的草莓健株根围分离得504株草莓根围细菌。用平板对峙培养法,以草莓黄萎病菌、草莓疫霉和草莓枯萎病菌作指示病原真菌,筛选得24株有显著拮抗活性的细菌。通过测定拮抗细菌的胞外酶活性(纤维素酶,蛋白酶,几丁质酶)、次生代谢物(噬铁素)活性,初步确定拮抗细菌的拮抗机理。通过细菌16S rDNA限制性酶切图谱分析(ARDRA)和16S rRNA基因序列测定比对结果,阐明拮抗细菌种群结构。同时把分离得的拮抗细菌用于防治草莓黄萎病试验。研究结果表明,24个拮抗细菌的ARDRA图谱共有12种;结合菌株16SrDNA序列测定结果得出,12个ARDRA类群分属于6个细菌属(芽孢杆菌、假单胞菌、伯克氏菌、土壤杆菌、不动杆菌和食酸菌属)。其中芽孢杆菌、假单胞菌和伯克氏菌为优势种群。田间防治结果表明,菌株SB13对草莓黄萎病的田间防效达93.5%,菌株SB336和SB479的防效达100%。     

芽孢杆菌类生物杀菌剂的研发与应用

本文概述了芽孢杆菌防治植物病害的机理,包括营养和空间位点竞争、分泌抗菌物质、溶菌作用、诱导植物抗病性。简述了国内外芽孢杆菌类生物杀菌剂的研发现状,分析了生防芽孢杆菌在农业生产应用中存在的问题,并提出了相应的对策。最后展望生防芽孢杆菌类杀菌剂由于能够有效控制植物病害,同时具有对人畜安全、环境相容性好、植物病原菌不易产生抗性、生产成本相对比较低廉等优点,必将在现代农业中有广阔的应用前景,并产生巨大的经济、社会和生态效益。     

地衣芽孢杆菌W10对灰葡萄孢的抑制作用及其抗菌物质

地衣芽孢杆菌（Bacillus licheniformis）W10是一株对多种植物病原菌有抑制作用的拮抗细菌，对番茄灰霉病的田间防效与化学药剂腐霉利相当，可达60％以上。该菌株除了对病菌有较强的抑制作用外，还具有在番茄植株体表定殖竞争能力、诱导植株产生系统抗性能力。但地衣芽孢杆菌W10对灰葡萄孢的作用方式和产生的抗菌物质种类尚不清楚，为此作者进行了该项研究。现将研究结果报道如下。     

拮抗细菌B11的鉴定及其分泌的拮抗蛋白抗菌谱

芽孢杆菌 (Bacillusspp .)是一群好氧兼性厌氧的产芽孢革兰氏阳性杆菌的总称 ,它不仅具有显著的生防潜力 ,还能产生耐热抗逆的芽孢 ,有利于生防菌剂的生产、剂型加工及在环境中存活、定殖与繁殖 ,是植物病害生防微生物的重要组成部分。广西地处亚热带 ,菌物资源丰富 ,生防资源 (基因库 )也多种多样 ,因此 ,在广西植物病害生物防治的研究具有较好的前景。黎起秦等[1] 从作物根围分离得到对西瓜枯萎病菌具有强烈抑制作用的拮抗细菌B11,本文对其进行了鉴定 ,并对其分泌的拮抗蛋白的抗菌谱进行了测定。现将结果报道如下。1　材料与方法1 .1　供…     

枯草芽孢杆菌对稻瘟病的防治效果评价及机制初探

稻瘟病（Magnaprothe grisea）严重危害水稻,是重点防治对象.目前,化学防治仍是必要手段,但会污染环境,严重危害人畜健康.因此,生物防治被认为是非常有发展潜力的防治植物病害的方法之一.枯草芽孢杆菌（Bacillus subtilis）因其广谱抗菌活性及极强的抗逆能力等优势,而极具生防应用价值,目前已有多个产品在推广应用.将拮抗细菌用于稻瘟病的防治曾有一些报道.本文测定了枯草芽孢杆菌B-332菌株对稻瘟病菌及其孢子萌发的抑制作用,对其致畸作用进行了观察和测定.现将研究结果报道如下.     

致病杆菌和发光杆菌抗菌代谢产物研究进展

致病杆菌Xenorhabdus和发光杆菌Photorhabdus是昆虫病原线虫肠道共生细菌,具有对宿主昆虫致病和对寄主线虫共生的双重特性,是一类特殊的生防细菌资源,其代谢产物具有广泛的杀虫和抑菌功能。本文综述了近年来昆虫病原线虫共生细菌产生的抗菌物质、编码抗菌代谢产物的基因及抗菌机制的研究进展。     

生防细菌BRF-1和BRF-2鉴定及生物学特征

BRF-1和BRF-2是从大豆根际土壤中筛选得到的两株革兰氏阳性生防细菌,平板对峙培养表明它们对7种植物病原真菌具有拮抗作用。BRF-1芽孢球型中生,无鞭毛;而BRF-2芽孢球型端生,周生鞭毛。部分生物学特征研究表明,两菌株之间存在较大的差异。采用Biolog微生物自动鉴定仪鉴定和对两菌株的16S rDNA基因序列分析表明,菌株BRF-1和BRF-2分别与多粘类芽孢杆菌和枯草芽孢杆菌亲缘关系最近,故把菌株BRF-1和BRF-2分别定为多粘类芽孢杆菌和枯草芽孢杆菌。     

三种生防芽孢杆菌对5种烟田常用化合物的敏感性

芽孢杆菌是植物病害生物防治中重要的生防细菌。采用浊度测定法研究了生防细菌——枯草芽孢杆菌、解淀粉芽孢杆菌及多粘芽孢杆菌对烤烟专用复合肥[m(N)∶m(P2O5)∶m(K2O)=10∶10∶25]、生石灰、甲霜灵、代森锰锌及硫酸链霉素5种烟草生产上常用化学品的敏感性。结果表明:甲霜灵对3种芽孢杆菌的生长无影响,而烤烟专用复合肥和生石灰对3种生防细菌均有一定的促生长作用;而代森锰锌和硫酸链霉素能显著抑制枯草芽孢杆菌、解淀粉芽孢杆菌和多粘芽孢杆菌的生长,其EC50值分别为14.15、11.88、11.30μg/mL和5.20、5.69和2.64μg/mL。     

防治烟草根黑腐病拮抗芽孢菌株的筛选

由基生根串珠霉菌(Thielaviopsis basicola)引起的烟草根黑腐病是烟草生产上重要根部真菌病害,近年在我国烟草种植区有加重危害的趋势,目前防治措施主要依赖化学药剂,给卷烟卫生带来一系列问题[1].探索该病生物防治途径,克服农药残留和病原菌抗药性十分重要.芽孢杆菌是土壤和植物微生态区系的优势生物种群,具有优良生防特性[2],采用芽孢杆菌作为烟草根黑腐病生防因子尚未见报道.本研究从烟草根际土壤中分离筛选对烟草根黑腐病病菌有较强拮抗作用的芽孢菌株,研究其抑菌活性、控病作用及其分类学地位.     

The degradation fragments of gamma-glutamyl transpeptidase from Bacillus subtilis BU108 have antimicrobial activity against Streptomyces scabiei

Journal of Plant Diseases and Protection - Bacillus species are often regarded as ideal biocontrol agents against plant pathogens, which can produce various antimicrobial peptides (AMPs). In this...     

石榴干腐病生防菌株Z2的鉴定及其培养条件的优化

 从石榴根际土壤中分离获得一株生防细菌菌株Z2。根据16S rDNA、gyrA 基因序列分析和特定基因的选择性扩增,结合形态学、生理与生化特性鉴定和Biolog 检测,将生防菌株Z2鉴定为解淀粉芽胞杆菌(Bacillus amyloliquefaciens)。通过单因素试验研究菌株的培养条件,并经过正交实验设计对培养条件进行优化,探究生防菌对Zythia versoniana引起的石榴干腐病的防治效果。生防菌株Z2的优化培养条件为:温度27℃,pH =6或8,LB培养基,培养时间3 d。采用新鲜离体石榴果实针刺接种方法进行菌株生防效力检测,与其他供试植物病原真菌相比,菌株Z2对石榴果实干腐病菌Z. versoniana具有较强的抑菌活性,对石榴果实干腐病的病斑抑制率达31.27%~81.89%。本研究获得的生防菌株Z2对石榴干腐病生物防治具有开发应用潜力,优化的培养条件可为该生防菌的产业化和大田应用奠定基础。另外,菌株Z2对植物病原真菌有较宽的抑菌谱,表明该菌株对其他作物病害的防治也具有潜在的应用价值。     

木霉菌生物防治作用机理与应用研究进展

木霉菌Trichoderma是国际上应用非常普遍的生防真菌.随着植物免疫的MAMPs(微生物互作分子模式)理论的发展,利用木霉菌防治病害和提高作物抗逆性机理研究进入了一个新阶段,尤其是系统生物学方法的应用,使得人们在"组学"尺度上认识木霉菌与植物和病原菌互作的本质成为可能,这将极大丰富木霉菌生物防治植物病害的理论基础.在我国建立木霉菌多样化应用技术途径是发挥木霉菌在农业可持续发展中作用的重要发展方向.本文重点介绍木霉菌生物防治分子机理研究的新进展.     

一株具有防治水稻条斑病潜力的高地芽胞杆菌181-7

 水稻条斑病(Bacterial leaf streak, BLS)由稻黄单胞菌种下的致病变种条斑病菌(Xanthomonas oryzae pv. oryzicola, Xoc)侵染引起,已成为我国南方水稻种植区的一个重要病害。为了筛选防治BLS的生防细菌,本研究以Xoc的模式菌株RS105为靶标菌,采用平板稀释和抑菌圈法,从辣椒根际土壤中分离筛选到具有拮抗活性的菌株181-7。通过形态学、生理生化特征以及16S rRNA序列分析鉴定该菌株为高地芽胞杆菌,命名为Bacillus altitudinis 181-7。针对14种植物病原细菌和3种病原真菌的拮抗谱试验发现,181-7对Xoc和水稻白叶枯病菌(X. oryzae pv. oryzae, Xoo)表现特异性的拮抗作用,对禾谷镰刀菌也具有较明显的抑制作用。基因组信息显示,181-7携带一个环状的质粒,含有3 826个开放阅读框,含有涉及抗真菌、抗逆、诱导植物抗性和促进植物生长等特性的相关基因。AntiSMASH的分析显示,181-7含有地衣杆菌素(lichenysin)、丰原素(fengycin)、菌溶素(bacilysin)、细菌素(bacteriocin)以及噬铁素等抑菌活性代谢产物。温室的初步试验结果显示,在感病水稻品种‘原丰早'上, 181-7对Xoc在水稻叶片上引起的水渍症状具有明显的抑制作用。这表明, B. altitudinis 181-7具有防治水稻条斑病的潜力。这些研究为水稻条斑病的生物防治提供了新的微生物资源,也为后续生防机理的探究奠定了理论基础。     

Studies on Endophytic Actinomycetes ( I ) <Emphasis Type="Italic">Streptomyces </Emphasis>sp. Isolated from Rhododendron and Its Antifungal Activity

To survey endophytic actinomycetes as potential biocontrol agents against fungal diseases of rhododendron, young plants of rhododendron were surface-sterilized for use as an isolation source. Nine, six and two isolates, with distinguishing characteristics based on the macroscopic appearance of colonies, were obtained from roots, stems and leaves, respectively, suggesting that various species of actinomycetes grow in the respective organs of this plant as symbionts or parasites. On an agar medium, only isolate R-5 commonly formed a clear growth-inhibition zone against two major fungal pathogens of rhododendron, Phytophthora cinnamomi and Pestalotiopsis sydowiana, indicating that this isolate can produce antifungal material(s). Acetone extracts of a liquid culture of R-5 had a broad antimicrobial spectrum against Gram-positive bacteria, yeast and filamentous fungi. Isolate R-5 was identified as a Streptomyces sp. based on morphological, physiological and chemotaxonomical characteristics. The present results indicate that isolate R-5 is a suitable candidate for the biocontrol of diseases of rhododendron. Received 25 March 2000/ Accepted in revised form 18 May 2000     

防治药用植物土传病害的芽胞杆菌制剂开发的制约因素分析

芽胞杆菌是土传病害生防制剂开发中最具应用潜力的微生物之一,但目前针对药用植物土传病害,市场上可供选择和使用的,以其为生防因子的产品和数量较少,远远不能满足生产实际需要。本文从菌株的筛选获得、定殖与生态适应性、安全性评价等方面概述了影响其开发的制约因素,并提出了相应的建议和展望,以期为促进芽胞杆菌制剂在防控药用植物土传病害中的应用提供参考。     

Biocontrol of soil-borne fungal plant diseases by 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA

Fluorescent pseudomonads producing the antimicrobial compound 2,4-diacetylphloroglucinol (Phl) are being studied extensively for use as biocontrol agents of soil-borne fungal diseases. Some of them can produce pyoluteorin (Plt) in addition to Phl, whereas others synthesise only Phl. Here, a collection of seven Phl⁺ Plt^- pseudomonads, seven Phl⁺ Plt⁺ pseudomonads and seven Phl^- biocontrol pseudomonads were compared for protection of plant roots against fungal pathogens. The seven Phl⁺ Plt⁺ pseudomonads were identical by restriction analysis of amplified spacer ribosomal DNA (spacer ARDRA), whereas the Phl⁺ Plt^- pseudomonads and especially the Phl^- biocontrol pseudomonads were quite diverse by spacer ARDRA. Collectively, the Phl⁺ Plt^- pseudomonads proved superior to the Phl⁺ Plt⁺ pseudomonads and the Phl^- biocontrol pseudomonads for protection of tomato against Fusarium crown and root rot (in rockwool microcosms) or cucumber against Pythium damping-off (in non-sterile soil microcosms). There was no correlation between protection in vivo and inhibition of the corresponding fungal pathogen on plates. However, there was a significant correlation between the amount of Phl produced on plates and protection of tomato against Fusarium crown and root rot, but not with protection of cucumber against Pythium damping-off. Interestingly, the minority of strains unable to produce HCN, an extracellular protease, or both, were among those unable to protect plants in both pathosystems. A seedling assay was developed to compare pseudomonads for suppression of Fusarium crown and root rot in vitro, and a significant correlation was found between disease severity in vitro and in vivo. Overall, results suggest that promising biocontrol pseudomonads may be identified based on the ability to produce Phl and/or specific ARDRA-based fingerprints.     

洋葱伯克霍尔德氏菌JX-1防治番茄青枯病机理的初步分析

本研究分离得到一株对茄科雷尔氏菌Ralstonia solanacearum具有明显拮抗作用的根围细菌JX-1，其温室防治番茄青枯病的防效达80.89%。形态学、生理生化、16S rDNA序列和gyrB序列分析表明，该菌株属于洋葱伯克霍尔德氏菌Burkholderia cepacia。生防相关性状分析表明，菌株JX-1可产生嗜铁素、蛋白酶等抗菌物质。抗生素合成相关基因分析发现，菌株JX-1还可能产生硝吡咯菌素和藤黄绿脓菌素。转座子随机突变获得2株明显影响菌株JX-1拮抗能力的突变菌株，其中突变体M1710对茄科雷尔氏菌拮抗作用完全消失，序列分析表明Tn5破坏了与非核糖体寡肽产生相关基因tlpks/nrps；而突变体M645则对茄科雷尔氏菌的拮抗作用显著增强，序列分析表明Tn5破坏了gntR基因。上述结果表明，菌株JX-1可产生多种次生代谢产物，并且tlpks/nrps基因和gntR基因在防治番茄青枯病过程中起到重要调整作用。     

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.     

Identification and Characterization of Novel Genetic Markers Associated with Biological Control Activities in Bacillus subtilis

ABSTRACT Suppressive subtractive hybridization (SSH) was used to identify genetic markers associated with biological control of plant pathogens by Bacillus subtilis. The genomes of two commercialized strains, GB03 and QST713, were compared with that of strain 168, which has no defined biocontrol capacities, to obtain a pool of DNA fragments unique to the two biocontrol strains. The sequences of 149 subtracted fragments were determined and compared with those present in GenBank, but only 80 were found to correspond to known Bacillus genes. Of these, 65 were similar to genes with a wide range of metabolic functions, including the biosynthesis of cell wall components, sporulation, and antibiotic biosynthesis. Sixteen subtracted fragments shared a high degree of similarity to sequences found in multiple B. subtilis strains with proven biocontrol capacities. Oligonucleotide primers specific to nine of these genes were developed. The targeted genes included five genes involved in antibiotic synthesis (bmyB, fenD, ituC,srfAA, and srfAB) and four additional genes (yndJ, yngG, bioA, and a hypothetical open reading frame) not previously associated with biological control. All nine markers were amplified from the commercialized B. subtilis strains GB03, QST713, and MBI600, with the exception of ituC, which was not detected in GB03. The markers also were amplified from four other B. subtilis isolates, but they were not amplified from other related Bacillus strains, including the plant growth-promoting rhizobacteria IN937a and IN937b. Sequencing of the amplified markers revealed that all seven of the isolates that scored positive for multiple markers were genotypically distinct strains. Interestingly, strains scored positive for the amplifiable markers generally were more effective at inhibiting the growth of Rhizoctonia solani and Pythium ultimum than other Bacillus isolates that lacked the markers. The potential utility of the defined genetic markers to further define the diversity, ecology, and biocontrol activities of B. subtilis are discussed.