半夏软腐病拮抗细菌的筛选与鉴定

为解决半夏软腐病带来的种植弊端及经济损失,采集了湖北钟祥地区半夏种植合作社的健康半夏根际土,并从中筛选到2株具有较强拮抗半夏软腐病的病原菌胡萝卜软腐果胶杆菌性能的菌株JK-4和JK-28。根据其形态特征、生理生化特性并结合16S rRNA基因序列分析,将这2株菌鉴定为枯草芽孢杆菌(Bacillus subtilis),并将其分别命名为B.subtilis JK-4(JK-4)和B.subtilis JK-28(JK-28)。研究发现：JK-4和JK-28均具备较强的产脂肽类抗生素surfactin的能力,在高糖Landy培养基中,经30℃、200 r/min培养48 h后,surfactin的产量分别为1.28和1.12 g/L,且同时具备产NH₃、分泌铁载体、产ACC脱氨酶及吲哚乙酸的能力。本研究筛选得到的菌株JK-4和JK-28具备较好的拮抗性能及植物促生潜力,为半夏绿色种植以及开发高效的生防菌剂提供了基础。     

黄瓜枯萎病病原菌拮抗菌的筛选及其生物防治效应

分离筛选到一株对黄瓜枯萎病病原菌——尖孢镰刀菌具有显著拮抗作用的菌株YHJ15,并根据其生理生化特征和16SrRNA基因序列将其初步鉴定为短小芽孢杆菌;研究了其最适的生长条件。为黄瓜枯萎病的生物防治提供了种质资源。     

柞蚕空胴病病原拮抗菌的分离、鉴定及防治效果研究

【目的】本研究从辽宁省蚕业科学研究所柞园土壤中分离筛选对柞蚕空胴病病原菌有显著拮抗作用的细菌,为该病的生物防治奠定研究基础。【方法】采用稀释涂布平板法分离柞园土壤中细菌,利用抑菌圈法筛选拮抗效果显著的菌株;根据形态学、生理生化及分子生物学对拮抗菌进行鉴定;利用自然转化法对拮抗菌进行荧光蛋白标记,测定其在柞树叶片和柞蚕肠道内的定殖规律,并对其室内和野外防效进行测定。【结果】从柞园土壤中分离获得87株细菌,其中BF-49对柞蚕肠球菌的拮抗效果显著(P<0.001);鉴定结果显示该BF-49与贝莱斯芽孢杆菌(Bacillus velezensis)的亲缘关系最近,故将该菌株鉴定为Bacillus velezensis,命名为B. velezensis BF-49。荧光蛋白标记菌株BF-49-GFP在柞蚕肠道中能定殖5d,在柞树叶片上接种20d后浓度仍达1.25×104CFU/g。BF-49发酵液的10倍稀释液对柞蚕空胴病的室内防效为78.25%,野外防效为74.42%,均显著高于对照药剂。【结论】筛选获得的B. velezensis BF-49对柞蚕空胴病防效显著,可作为开发柞蚕空胴病...     

三株芸豆叶际拮抗细菌的筛选与鉴定

以芸豆锈病病原菌[Uromyces appendiculatus(Pers.)Ung.]为指示菌,从芸豆叶际中筛选到3株具有明显拮抗效果的细菌,编号为SS2,L14b,NEW2。经过形态学观察,生理生化测定,16S rDNA序列及系统发育分析,初步鉴定SS2为短短芽孢杆菌(Brevibacillus brevis),L14b与NEW2为枯草芽孢杆菌(Bacillus subtilis)。SS2,L14b,NEW2的16S rDNA序列的Gen-Bank登录号分别为EU771078、EU771076和EU771079。     

黑麦草根际产铁载体细菌HMGY6B的筛选鉴定及对病原菌的拮抗作用

【目的】从石灰性土壤中分离筛选出铁载体合成能力强、抗病效果好的菌株。【方法】采用刃天青(Chrome azural S,CAS)平板检测法,定性、定量筛选产铁载体能力较强的菌株,通过菌株形态、生理生化特征、16S r RNA基因序列相似性和系统发育分析,鉴定细菌类型,然后采用平板对峙法研究菌株与病原菌的拮抗作用。【结果】从多年生黑麦草根际土壤中分离得到一株产铁载体能力很强的菌株HMGY6B,经鉴定,该菌属假单胞菌属Pseudomonas菌株,其产生的儿茶酚型铁载体对黄瓜灰霉病(Botrytis cinerea)有显著的拮抗作用,在低铁条件下(0.16、2、5、10μmol/L FeCl_3)对黄瓜灰霉病的生长抑制率高达91.2%,但在富铁条件下(50μmol/L FeCl_3)降为30.2%,100μmol/L FeCl_3抑制率仅为5.5%。【结论】菌株HMGY6B可用于今后复合型抗病生物菌肥的开发研制。     

两株生防芽孢细菌筛选、鉴定及拮抗研究

【目的】筛选出广谱、高效的生防芽孢细菌,并对其拮抗作用进行研究。【方法】以8种植物病原真菌为靶标菌,通过皿内拮抗和发酵液拮抗能力的测定筛选出2株广谱性和高效性的芽孢细菌B06和B07。【结果】B06对8种植物病原真菌的R2/R1为0.4-1.8,无菌滤液对8种植物病原真菌的抑制率为66.7%-87.5%。B07对8种植物病原真菌的R2/R1为0.23-1.21,无菌滤液对8种植物病原真菌的抑制率为55.56%-81.25%。经16S rRNA序列鉴定,菌株B06和B07都被鉴定为解淀粉芽孢杆菌。【结论】芽孢细菌能够抑制多种植物病原真菌,具有较好的抑病作用。广谱和高效芽孢细菌的筛选在农业生物防治方面具有很大的开发和应用价值。     

大豆内生细菌的分离及根腐病拮抗菌的筛选鉴定

内生细菌存在于健康植物体内,一些内生细菌具有促生长、抗病和固氮等生物学功能.本项研究采用化学药剂表面灭菌方法从黑龙江省大豆品种合丰25的根、茎、叶和种子中分离到大量内生细菌,其种群数量在根部最多,为3.4×103CFU/g,在叶部次之,为2.8×103CFU/g,在茎部和种子中最少,为2.9×102 CFU/g和1.4×102CFU/g.从121株内生细菌中筛选到31株对大豆根腐病菌Fusarium oxysporum f. sp.soybean具有较强抑制作用的拮抗内生细菌,其中菌株TF28抑菌谱广,抑菌率高,对不同植物的病原菌F. oxysporum的抑菌率为80.2%～96.7%.经形态、生理生化和16S rRNA鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens).     

黄瓜苗根围拮抗细菌X3的分子鉴定

采用生理生化、Biolog和16S rDNA分子鉴定3种不同方法,对抑制黄瓜苗期猝倒病病原真菌的细菌菌株X3进行了鉴定．生理生化鉴定显示该菌株为Pseudomonas aeruginosa;而Biolog鉴定显示其为P．spinosa;进一步对该菌株作16S rDNA基因的测定与分析,表明其与已报道的P．aeruginosa 16S rDNA具有93．7％的同源性,二者在所建系统发育树中处于同一分枝,据此确定该菌株为P．aeruginosa。     

魔芋软腐病拮抗菌GZA12的分离鉴定及其防病促生作用

【目的】软腐病是影响魔芋产量和品质的重要病害之一。本研究旨在从魔芋根际土壤中筛选出一株对软腐果胶杆菌(Pectobacteriumaroidearum)具有拮抗作用的菌株,从而为花魔芋软腐病害的生物防治提供种质资源。【方法】通过平板对峙法筛选出一株拮抗菌,并测定该菌株对多种病原真菌的拮抗效果。通过魔芋球茎组织接种、生防和灌根试验验证GZA12对魔芋软腐病的防治效果。同时对该菌株的促生能力进行室内测定,并通过番茄盆栽试验初步验证该菌株促生效果。【结果】筛选出一株具有拮抗能力的菌株GZA12并鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis),该菌株对软腐果胶杆菌的抑菌圈直径达21.33 mm,对葡萄座腔菌(Botryosphaeria dothidea)、尖孢镰刀菌(Fusarium oxysporum)和茄病镰刀菌(F.solani)的抑菌率分别为58.16%、47.30%和54.53%。在魔芋球茎组织接种试验中,魔芋组织病情指数比单独接种软腐果胶杆菌菌液分别降低了26.67%、33.33%和40.00%;在生防盆栽试验中,与对照组相比,GZA12菌悬液处理组病情指数降低了22.85%,防效达53.31%。在灌根试验中,与清水灌根相比,GZA12发酵液灌根处理后,病情指数降低了4.89%,防效达21.57%。室内促生试验表明菌株GZA12具有固氮、解磷、产铁载体和产吲哚乙酸(indole-3-acetic acid,IAA)的能力,接种GZA12菌悬液能促进番茄幼苗生长,其中高浓度的菌悬液效果更佳。【结论】菌株GZA12对魔芋软腐病病原菌有较好的拮抗效果,并具有促生特性,有进一步开发利用的潜力。     

广西岩溶区烟草黑胫病拮抗细菌的筛选鉴定及其抗病机理

从广西岩溶区靖西县优质烟叶生产区分离烟草黑胫病病原及土壤细菌,通过拮抗试验、离体及田间抗病能力测试等方法筛选拮抗细菌;利用形态观察、生理生化测试和16SrDNA序列分析三种方法相结合,对抗病效果良好的菌株进行分类鉴定;并对拮抗菌抗黑胫病机理进行初步研究。结果表明:从8份土壤样品中,共分离出土壤细菌340株,获得抗黑胫病效果良好的拮抗细菌3株,编号为8-23、6-70和13,它们分别属芽孢杆菌属、溶菌杆属和假单胞杆菌属细菌;三个拮抗细菌的抗黑胫病机理是通过胞外分泌一些可溶解病原菌细胞壁的酶或其它化学物质,破坏菌丝的细胞壁和细胞膜等生理结构,使细胞质渗漏、凝集,从而导致病原死亡;其中,菌株8-23和13的抗病活性物质主要为蛋白质类化合物;而菌株6-70除蛋白质外,还有其它一些非蛋白因子起作用。     

大白菜软腐菌种群组成及优势菌致病型的研究

对黑龙江省成熟大白菜[Brassica pekinensis（Lour．）Rupr．]生产田采集的软腐病菌进行分离、纯化,并根据形态学特征分析了大白菜软腐病菌的种群组成。结果表明,引起黑龙江省秋季大白菜软腐病的主要致病菌是胡萝卜软腐欧文氏菌胡萝卜软腐亚种[Erwinia carotovora（Jones）Bergey et al．subsp、carotovora]（Ecc）;利用20个Ecc菌株的混合菌对来源于不同生态型及不同地区的大白菜品种进行接种,筛选出5个鉴别寄主,以此将20个Ecc菌株划分为5个致病力类型,其中V型为优势致病菌,其分布广且致病力强。     

高效钾长石分解菌株的筛选、鉴定及解钾活性研究

目的 筛选高效的钾长石分解细菌,并进行初步鉴定和解钾活性测定.方法 采集湖南省桂东县钾长石开采区土壤,利用钾长石为唯一钾源的选择性培养基筛选分离解钾细菌,通过摇瓶释钾试验复筛高效解钾菌株;同时,利用ICP-OES测定解钾菌的释钾效率.采用形态特征观察、生理生化特性检测和基于16S rRNA基因序列的系统发育分析初步鉴定高效解钾菌株.结果 分离获得11株生长良好的钾长石分解细菌,其中菌株JKC1、JKC2、JKC5和JKC7的解钾能力较强,解钾率分别为11.00％、11.50％、12.70％和11.70％.经初步鉴定JKC1为Bacillus megaterium,JKC2为Bacillus aryabhattai,JKC5为Azotobacter chroococcum,JKC7为Microbacterium trichothecenolyticum.结论 菌株JKC1、JKC2、JKC5和JKC7是高效的钾长石分解菌,可作为微生物浸矿(钾长石)机制研究的候选菌株.     

Affinity purification of a siderophore that exhibits an antagonistic effect against soft rot bacterium

Bacterial colonies were isolated from different Egyptian soil samples. From these isolates, one bacterial species was found to produce siderophore. Using classical and biochemical identification methods, the siderophore producing isolate was identified as Pseudomonas fluorescens. Based on the affinity of siderophores for metal ions, an affinity chromatography system was designed for the purification of the siderophore in one step. It was possible to isolate 25 mg siderophore per liter of culture media. The purified siderophore was found to exist in two forms of approximately 30 and 90 kD. They are believed to be polymers of several siderophore molecules. Both forms were found to be active against the pathogen Erwinia carotovora var. carotovora, the causal bacteria of soft rot disease on potato tubers. The advantage of this method over other purification methods is that it uses metal ion so it can be applied for the purification of the known types of siderophores. Moreover, the purification is based on affinity chromatography, so the siderophore purity state permits several biotechnological applications without further treatments.     

白色念珠菌拮抗菌株的筛选

目的筛选对白色念珠菌具有明显拮抗作用的菌株.方法通过纸片琼脂扩散法(K-B法)观察各菌株对白色念珠菌的拮抗作用;再利用试管法观察有拮抗作用菌株对白色念珠菌的抑菌效果.结果 K-B法表明大肠埃希菌、甲型链球菌和卡他球菌对白色念珠菌无抑菌作用,表皮葡萄球菌、微球菌有抑菌作用,但抑菌环小;枯草杆菌有明显抑菌作用.试管法表明表皮葡萄球菌和枯草杆菌对白色念珠菌均有生物拮抗作用,其抑菌率分别为97%和89.7%.结论枯草杆菌是白色念珠菌的理想拮抗菌株.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

1株产细菌素乳酸菌的筛选和鉴定

目的 从植物性材料中筛选产细菌素的乳酸菌。方法 琼脂扩散法。结果 所筛选的产细菌素R260菌株经鉴定为植物乳杆菌。排除有机酸、过氧化氢等干扰因素后,发酵液仍有很强的抑菌作用;用胰蛋白酶和胃蛋白酶处理后,发酵液抑菌活性急剧下降,因而确定产生的抑菌物质具有蛋白质性质,是一种细菌素。抑菌谱试验测定表明,此菌株的发酵液不仅抑制革兰阳性菌,而且对部分革兰阴性菌也有抑制作用,因此产生的是一类广谱细菌素。结论筛选到了1株产广谱细菌素的乳酸菌。     

Diversity of antagonistic bacteria isolated from medicinal plant Peganum harmala L.

The antimicrobial activity of plant extract of Peganum harmala, a medicinal plant has been studied already. However, knowledge about bacterial diversity associated with different parts of host plant antagonistic to different human pathogenic bacteria is limited. In this study, bacteria were isolated from root, leaf and fruit of plant. Among 188 bacterial isolates isolated from different parts of the plant only 24 were found to be active against different pathogenic bacteria i.e. Escherichia coli, Methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium, Enterococcus faecalis and Pseudomonas aeruginosa. These active bacterial isolates were identified on the basis of 16S rRNA gene analysis. Total population of bacteria isolated from plant was high in root, following leaf and fruit. Antagonistic bacteria were also more abundant in root as compared to leaf and fruit. Two isolates (EA5 and EA18) exhibited antagonistic activity against most of the targeted pathogenic bacteria mentioned above. Some isolates showed strong inhibition for one targeted pathogenic bacterium while weak or no inhibition for others. Most of the antagonistic isolates were active against MRSA, following E. faecium, P. aeruginosa, E. coli and E. faecalis. Taken together, our results show that medicinal plants are good source of antagonistic bacteria having inhibitory effect against clinical bacterial pathogens.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

小碎斑鱼蛉幼虫肠道细菌分离及鉴定研究

目的从微生态学角度研究小碎斑鱼蛉幼虫的营养生理活动。方法从小碎斑鱼蛉幼虫肠道环境中进行分离、纯化和培养,获得3个细菌菌株,对其菌体形态、染色反应、培养性状和生理生化反应进行系统研究。结果上述3个菌株均属于芽胞杆菌属(Bacillus),1号菌株为巨大芽胞杆菌(Bacillus megaterium),2号菌株为枯草芽胞杆菌(Bacillus subtilis),3号菌株为地衣芽胞杆菌(Bacillus licheniformis)。结论小碎斑鱼蛉幼虫肠道环境中的不同细菌,其数量存在明显差异。