生姜根际茎腐病拮抗菌的筛选及鉴定

从生姜根际土样分离获得的86个细菌分离物中,通过离体拮抗试验,筛选出2株对生姜茎腐病有良好拮抗效果的细菌,编号分别为JF24和JF3。通过对它们进行形态学观察、生理生化测定以及16S rRNA序列分析,初步确定,JF24为荧光假单胞菌(Pseudomonas fluorescens),JF3为粪产碱菌(Alcaligenes faecalis)。JF24和JF3的16S rRNA序列的GenBank登录号分别为FJ999730和FJ999731。     

烟草根黑腐病拮抗内生细菌的筛选及其抑菌作用

【目的】从烟草根、茎中分离获得对烟草根黑腐病菌(Thielaviopsis basicola)有较好控病作用的内生细菌。【方法】采用平板对峙培养,测定分离获得的306个内生细菌对烟草根黑腐病菌的抑制作用;通过菌株形态特征、生理生化特性及16S rDNA序列对菌株进行分类鉴定。【结果】筛选获得6个菌株对烟草根黑腐病菌有较强拮抗作用,室内测定其对病原菌的抑菌带宽达6.5 mm-11.0 mm,温室控病效果达11.9%-77.1%,其中来自茎部的内生细菌T295菌株对烟草根黑腐病的防效达77.1%;无菌滤液实验表明,拮抗内生细菌T295无菌滤液在实验浓度范围内对病菌菌丝生长、孢子萌发有较好地抑制作用。【结论】菌株T295对烟草根黑腐病具有较好的防治作用,经鉴定为枯草芽孢杆菌(Bacillussubtilis)。     

云南芒果采后炭疽病病原菌的鉴定及室内生防菌的筛选

随着对云南芒果需求量的增加,其种植面积日趋扩大,并不断引入新品种,这也导致云南省芒果炭疽病害发生日趋加重。为了有针对性地开展芒果炭疽病的生物防治,采用组织块分离法分离芒果采后炭疽病病原菌,通过形态学观察初步鉴定,并遵循柯赫氏法则对病原菌进行验证。随后,利用rDNA-ITS序列和系统发育树分析明确病原菌的分类学地位。最后,采用5种生防细菌对病原菌进行拮抗试验。通过对芒果采后炭疽病病原菌进行分离鉴定,确定胶孢炭疽菌(Colletotrichum gloeosporioides)是引起云南芒果采后炭疽病的病原菌,该菌株内转录间隔区(internal transcribed spacer,ITS)序列长度为536 bp,登录号为MH744668;5株生防细菌对芒果炭疽病病原菌都有一定的抑菌作用,具有较好的生防开发潜能,其中抗生素溶杆菌L-44的抑菌效果最好,抑制率达53.7%。研究结果为云南省芒果采后病害的生物防治提供了新的思路。     

桃褐腐病菌拮抗性内生细菌的筛选及其抑病效果

【目的】从桃树根部组织中分离、筛选能够防治桃褐腐病害的内生拮抗菌,从细胞学水平探究其抑制机理。【方法】采用平板对峙法筛选对桃褐腐病菌(Monilinia fructicola)有拮抗作用的内生细菌,调查内生拮抗细菌的形态学及生理生化特性,通过细菌16S r RNA基因测序分析菌株的系统发育学特征。通过果实离体实验检测内生拮抗细菌的抗菌效果,使用电子显微镜观察受抑制的桃褐腐病菌菌丝、孢子的形态以及细胞内部结构变化。【结果】从桃树根部组织中复筛分离得到的3株内生细菌对桃褐腐病防效高且稳定,经鉴定3株细菌均为枯草芽孢杆菌。果实离体试验表明,3株内生细菌均明显抑制了桃褐腐病的发生。通过电子显微镜观察,发现受拮抗细菌抑制的桃褐腐病菌的菌丝明显变细、杂乱且打结缠绕成团,孢子普遍干瘪变大、破裂,细胞质外渗。【结论】从桃树根部组织中得到的3株内生细菌对桃褐腐病菌的生长均有明显的抑制作用,可以作为防治桃褐腐病的新选择,具有进一步研究价值。     

玉米茎基腐病生防菌的筛选及应用

【目的】拟针对禾谷镰孢进行生防菌的筛选和应用研究,以期为玉米茎基腐病生防菌剂的研制奠定基础。【方法】通过对峙培养法对玉米茎基腐病的主要致病菌禾谷镰孢进行玉米内生生防细菌的筛选,从玉米主栽品种九单48幼苗内部获得一株具有较强抑菌效果的内生生防细菌(简称48SJ7-1);基于传统鉴定、16S rRNA基因序列测定及系统发育分析,对48SJ7-1进行鉴定;并通过盆栽试验测定该生防菌株的防效。【结果】菌株48SJ7-1经鉴定为甲基营养型芽孢杆菌,GenBank登录号为KU377993,48SJ7-1盆栽防效为68.47%,与对照药剂2%戊唑醇悬浮种衣剂差异不显著。【结论】48SJ7-1对玉米茎基腐病的主要致病菌禾谷镰孢有较好防效,对玉米生长还有明显的促进作用,而且表观上无药害发生。     

玉米种质和新品种对腐霉茎腐病和镰孢穗腐病的抗性分析

玉米是我国最重要的农作物之一,腐霉茎腐病和镰孢穗腐病是玉米生产上的重要病害。2006-2012年期间,对1647份玉米种质进行了抗肿囊腐霉茎腐病和拟轮枝镰孢穗腐病鉴定,筛选出高抗茎腐病和穗腐病的种质分别为564份和27份,占鉴定总材料的34.2%和1.6%,抗性材料分别为209份和352份,占比为12.7%和21.4%,表明高抗肿囊腐霉茎腐病的资源较为丰富,高抗镰孢穗腐病的种质相对匮乏。其中,13份种质对2种病害均表现高抗,207份种质对2种病害均表现抗性或对其中一种表现高抗而另一种表现抗性。自交系中对肿囊腐霉茎腐病和拟轮枝镰孢穗腐病表现抗性以上(含HR和R)的种质分别占总鉴定种质的56.5%和23.6%,在农家种中分别为21.2%和21.4%,表明玉米自交系中的抗性资源较农家种丰富。2009-2013年期间参加国家玉米区试的品种中,对腐霉茎腐病表现高抗、抗性、中抗、感病和高感的品种分别占11.5%、11.9%、40.1%、17.6%和18.9%。2009-2011年间,中抗以上的育成品种所占比例呈现明显上升趋势,但2012-2013年间,中抗以上的品种所占比例呈下降趋势。     

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).     

花生侵脉新赤壳菌果腐病生防芽孢杆菌的分离鉴定及防病效果

【目的】为了分离鉴定对花生侵脉新赤壳菌果腐病病原菌Neocosmospora vasinfecta具有抑制作用的根际芽孢杆菌。【方法】利用平板稀释法从花生的根际土壤分离芽孢杆菌,再采用平板对峙法筛选出对N.vasinfecta具有抑制作用的根际芽孢杆菌,通过形态观察、生理生化特性和分子生物学相结合的多相分类方法对生防根际芽孢杆菌进行分类鉴定,检测脂肽类抗生素合成基因类型,并进行花生侵脉新赤壳菌果腐病的田间防治试验。【结果】从花生根际土壤中分离到28株芽孢杆菌,其中对花生果腐病病原菌具有明显抑制作用有8株。多相分类法结果显示2株为枯草芽孢杆菌(Bacillus subtilis),6株为解淀粉芽孢杆菌(B.amyloliquefaciens)。脂肽类抗生素合成基因检测显示,8株生防芽孢杆菌含有至少1种脂肽类抗生素,其中所有生防菌均含有丰原素B合成基因,推测这些芽孢杆菌对N.vasinfecta的抑制机制可能与脂肽类抗生素的合成相关。田间防病实验结果显示,B.amyloliquefaciens GF-3和GF-22制备的生物有机肥均能有效降低NPRP的发病指数,其防治效率分别为32.35%和79.41%,增产率分别为19.12%和25.85%。【结论】分离鉴定了2株对花生侵脉新赤壳菌果腐病具有明显防治效果的根际芽孢杆菌,这不仅为花生侵脉新赤壳菌果腐病的生防制剂研制提供了菌株,还为研究防治机理奠定了基础。     

红树林内生细菌的分离及拮抗菌筛选

红树林内生细菌及拮抗菌分离筛选结果表明:各品种红树体内均有大量的内生细菌,不同红树品种及部位内生细菌的数量不同,其中以红海榄体内的含量最高,达4．225×104cfu／g(fw),其它依次为木榄、桐花树、秋茄和白骨壤等;不同部位以茎组织体内内生细菌的含量最多,达1．649×104cfu/g(fw),其次为根和叶。获得的内生细菌中约有43．53％的内生细菌菌株对枯萎病菌(Fusarium oxysporum)、炭疽病菌(Colletotrichum sp．)等病原真菌及番茄青枯病菌(Ralstonia solanaceance)具有较强拮抗作用。对番茄生长测定发现,13株菌株中有9株(69．23％)表现为促进生长效果,4株(30．77％)表现为抑制生长作用。经初步鉴定,上述拮抗细菌均为芽孢杆菌(Bacillus sp．)。     

Mycolytic effect of extracellular enzymes of antagonistic microbes to Colletotrichum falcatum,red rot pathogen of sugarcane

Strains of selected bacteria and Trichoderma harzianum isolated from sugarcane rhizosphere and endosphere regions were tested for the production of chitinolytic enzymes and their involvement in the suppression of Colletotrichum falcatum, red rot pathogen of sugarcane. Among several strains tested for chitinolytic activity, 12 strains showed a clearing zone on chitin-amended agar medium. Among these, bacterial strains AFG2, AFG 4, AFG 10, FP7 and VPT4 and all the tested T. harzianum strains produced clearing zones of a size larger than 10 mm. The antifungal activity of these strains increased when chitin was incorporated into the medium. Trichoderma harzianum strain T5 showed increased levels of activity of N-acetylglucosaminidase and -1,3-glucanase when grown on minimal medium containing chitin or cell wall of the pathogen. Lytic enzymes of bacterial strains AFG2, AFG4, VPT4 and FP7 and T. harzianum T5 inhibited conidial germination and mycelial growth of the pathogen. Enzymes from T. harzianum T5 were found to be the most effective in inhibiting the fungus. When mycelial discs of the pathogen were treated with the enzymes, electrolytes were released from fungal mycelia. The results indicated that antagonistic T. harzianum T5 caused a higher level of lysis of the pathogen mycelium, and the inhibitory effect was more pronounced when the lytic enzymes were produced using chitin or cell wall of the pathogen as carbon source.     

Detection of Colletotrichum falcatum causing red rot of sugarcane by enzyme-linked immunosorbent assay

Red rot disease of sugarcane caused by Colletotrichum falcatum Went is one of the most destructive diseases of sugarcane (Saccharum officinarum L.) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease-free setts is essential to prevent the disease. In order to develop immunological method for detection of C. falcatum, two proteins with molecular weights of 27 kDa and 45 kDa were purified from the mycelium of C. falcatum race Cf 05 and used as antigen source to raise polyclonal antibodies in NewZealand white rabbit. The developed polyclonal antibodies were tested for detection of C. falcatum by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. The polyclonal antibodies specifically detected C. falcatum in extracts from infected plants, both in immunoblot and ELISA. The ELISA results showed that the developed polyclonal antibodies were highly specific to C.falcatum. The developed antibodies were very sensitive and could detect C.falcatum proteins even at a dilution of 1:50,000. Higher ELISA absorbance values were recorded even at an antigen dilution of 1:500. In western blot analysis, protein bands with molecular weights of 27 kDa and 45 kDa reacting to antisera raised against 27 kDa and 45 kDa mycelial proteins of C. falcatum, respectively, were detected in protein samples from red rot infected canes. The high specific reactivity and sensitivity of the antisera indicate its potential suitability for ELISA-based detection of C. falcatum.     

向日葵菌核病拮抗菌的筛选、鉴定及防效测定

【背景】由核盘菌(Sclerotinia sclerotiorum)引起的菌核病是影响向日葵产量的重要病害,近年来在我国内蒙古和甘肃等地频繁发生。【目的】挖掘能够对向日葵菌核病进行生物防治的拮抗菌株和有效方法。【方法】用4种不同的培养基通过稀释涂布法对向日葵健康植株的根际土壤菌群进行分离,利用平板对峙实验筛选出对核盘菌有抑制作用的菌株。选取拮抗作用较强的菌株进行向日葵离体叶片防效测定,采用形态学特征、生理生化特性结合16SrRNA基因序列分析进行菌种鉴定,并配制成不同单菌剂和复合菌剂进行盆栽实验,测定活体防效。【结果】从土壤中共分离出142株菌,从中筛选到12株抑菌圈明显的拮抗菌株。其中拮抗菌Bacillussp.NM63、JQ134、J7、J33和Streptomyces sp. Z9、ZX6抑菌圈直径大于25 mm,这6株菌在向日葵离体叶片防效测定实验中效果显著。菌株NM63、JQ134、J7、Z9、J33和ZX6单菌剂盆栽实验的防治效果依次为79.06%、74.10%、70.72%、67.83%、65.11%和57.11%。菌株配比为Z9:NM63:JQ134:J7=1:1:1:...     

关防风根腐病拮抗细菌筛选与鉴定

【背景】由木贼镰刀菌[Fusarium equiseti(Corda)Sacc.]引起的关防风根腐病,是近年来导致关防风产量及质量下降的主要土传病害之一。生物防治因其对环境安全及人畜无害等优势,成为目前植物病害防治的一种有效手段。【目的】挖掘关防风根际土壤中对木贼镰刀菌具有良好拮抗作用的生防菌株。【方法】采用稀释平板法分离根际土壤细菌;用滤纸片法和牛津杯法对拮抗细菌进行筛选和抑菌谱检测;用抗生素标记法标记拮抗细菌并测定其定殖能力;通过盆栽实验研究其对关防风根腐病的防效;通过形态学、生理生化特征及16S rRNA基因序列确定分类学地位。【结果】从健康关防风根际土壤中分离纯化了157株细菌,筛选获得对关防风根腐病菌具有显著抑菌作用的拮抗细菌SC-119,无菌滤液的抑菌率可达68.53%,而且兼具广谱抑菌能力和良好的定殖能力;盆栽实验表明,菌株SC-119对关防风根腐病的防治效果达到67.39%,其相对防效较接种哈茨木霉菌剂、枯草芽胞杆菌菌剂和代森锰锌分别提高了29.03%、32.26%和16.13%;对菌株SC-119进行分类学鉴定,确定其为萎缩芽胞杆菌(Bacillus atrophae...     

广西甘蔗根际高效联合固氮菌的筛选及鉴定

对广西主要甘蔗产区的根际联合固氮细菌进行了收集和评价,拟筛选获得对甘蔗具有潜在促生性能的联合固氮菌,为甘蔗生产节肥减耗提供依据。结合nifH基因扩增和固氮酶活性分析方法筛选获得36个固氮细菌菌株;进一步对所获得固氮菌株的固氮能力、溶磷性、分泌植物生长素IAA的特性等促进植物生长潜能进行评价,获得了5个同时具有较强固氮能力、降解无机磷和分泌植物生长激素IAA的功能菌株;通过Biolog鉴定系统和16S rRNA序列分析对5个具有较好应用潜力的固氮菌进行分类鉴定。结果表明这5个菌株分别属于Klebsiella sp.、Bacillus megaterium、Pseudomonas sp.、Pantoea sp.和Burkholderia sp.。本研究结果表明广西甘蔗根际联合固氮菌具有较大的开发利用潜力。     

Molecular detection of Colletotrichum falcatum causing red rot disease of sugarcane (Saccharum officinarum) using a SCAR marker

Red rot disease of sugarcane caused by Colletotrichum falcatum is one of the most destructive diseases of sugarcane (Saccharum officinarum) worldwide. The pathogen spreads primarily through infected sugarcane setts and hence the use of disease‐free planting materials is essential for preventing disease development in the field. In the present study a polymerase chain reaction (PCR) assay was developed for accurate and sensitive detection of C. falcatum in planting materials. Randomly amplified polymorphic DNA (RAPD) analysis identified a 566 bp PCR fragment that was specific to C. falcatum. The DNA sequence of this fragment was determined and used to design oligonucleotides amplifying a 442 bp sequence characterised amplified region (SCAR). The specificity of the SCAR primers was evaluated using purified DNA from C. falcatum and other Colletotrichum spp. as templates in PCR. The results indicated that the SCAR primers were highly specific to C. falcatum since the 442 bp fragment was amplified only from DNA of isolates and races of C. falcatum but not from any other Colletotrichum spp. tested. The detection sensitivity of C. falcatum was 0.1 ng for genomic DNA of C. falcatum and 5 ng for DNA extracted from infected sugarcane tissue. This new PCR‐based assay is a convenient tool for detection of this important pathogen in seed canes to ensure production of sugarcane.     

贵州半夏块茎腐烂病拮抗细菌的筛选与鉴定

【目的】从半夏根际土壤筛选对半夏块茎腐烂病致病菌有拮抗活性的细菌并进行鉴定。【方法】采用稀释分离法和平板对峙法分离拮抗菌,然后根据菌落形态、生理生化特性,结合分子生物学方法进行鉴定。【结果】从半夏根际土壤共分离到228株细菌,其中菌株GZDF2、GZDF3、GZDF4对病原细菌及病原真菌均有拮抗活性,抑菌圈大小达23 mm,并且抑菌谱广。经形态学观察、生理生化特性分析和分子生物学方法,将菌株GZDF2、GZDF3、GZDF4鉴定为短短芽胞杆菌(Brevibacillus brevis)。进一步采用gyr B和rpo B基因对3株短短芽胞杆菌进行聚类分析,结果显示GZDF3菌株与其他2株短短芽胞杆菌存在遗传差异。【结论】半夏根际土壤中分离到的3株短短芽胞杆菌抑菌活性强、抑菌谱广,具有开发成生防剂的潜力。     

Molecular Assessment of Diversity among Pathotypes of Colletotrichum falcatum Prevalent in Sub-Tropical Indian Sugarcane

Summary Six pathotypes of Colletotrichum falcatum, responsible for Red-rot in sugarcane, prevalent in subtropical India were examined for genetic relationships using RAPD markers. A high degree of polymorphism (78.6%) was observed using 40 RAPD markers. More than 50% genetic divergence was found among the pathotypes and UPGMA cluster analysis of genetic similarity indices grouped the six pathotypes into two clusters. Cluster I comprised pathotypes Cf01 and Cf09, while cluster II comprised the remaining four pathotypes. Cf02 and Cf08 were the most closely related among all the pathotypes. Pathotype-specific unique bands generated in RAPD profiling are being used for developing markers for pathotype identification in diseased cane samples.     

药用野生稻拮抗稻瘟病内生细菌的筛选、鉴定及发酵条件优化

【背景】稻瘟病是水稻的三大重要病害之一,每年对水稻生产都会造成较大的损失,生物防治稻瘟病已成防治该病害的重要手段,而目前鲜少有报道从药用野生稻中分离内生菌用以防治稻瘟病。【目的】从药用野生稻内生菌中挖掘对稻瘟病具有拮抗作用的微生物资源。【方法】采用分离法从勐遮药用野生稻中先分离出内生菌,再通过平板对峙法筛选对稻瘟病菌具有拮抗作用的菌株,通过形态学和16S rRNA基因序列分析对菌株进行鉴定,同时筛选该菌株的最适培养基、pH、培养温度和摇床转速。【结果】筛选得到拮抗菌株Z5,在LB培养基上对稻瘟病菌菌丝生长的抑制率达到96.43%,经形态学和16S rRNA基因序列分析,初步鉴定为短小芽孢杆菌(Bacillus pumilus)。在NYBD、CM、LB、NA和YSP这5种培养基中,菌株Z5的最适培养基为NYBD培养基、最适pH值为8.0-9.0、最适培养温度为35℃、最适摇床转速为250 r/min。【结论】短小芽孢杆菌Z5对稻瘟病菌的生长具有较好的抑制作用,且该菌株耐高温、较耐酸碱,作为稻瘟病生防制剂研发具有一定的价值。     

甘蔗内生解淀粉芽孢杆菌CGB15的分离、鉴定及生防活性

真菌病害占作物病害种类的一半以上,病原真菌是目前已知种类最多的作物病原菌。从作物根际与/或体内分离筛选具有生防活性的微生物,并应用于病害的防控,是除作物品种改良与化学防治外的另一种高效的病害防控策略。【目的】本研究拟筛选并分离鉴定对重要作物病原真菌具有拮抗作用的甘蔗内生细菌,为开发生物防治作物真菌病害新策略提供理论依据。【方法】采用平板对峙法初步筛选对病原真菌具有拮抗能力的甘蔗叶片内生细菌,通过16SrRNA基因测序鉴定其种属;进一步检测候选拮抗内生细菌对甘蔗鞭孢堆黑粉菌(Sporisorium scitamineum)致病发育过程关键步骤：有性配合/菌丝生长、冬孢子萌发的抑制率,田间试验检测其对甘蔗鞭黑穗病的防治效果;检测候选拮抗内生细菌对稻梨孢菌(Pyricularia oryzae)附着胞形成、离体叶片及盆栽条件下叶片病斑形成的抑制作用。【结果】分离自甘蔗叶片的细菌菌株,编号为CGB15,经分子鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。CGB15菌株能有效抑制甘蔗鞭孢堆黑粉菌有性配合/菌丝生长,对峙培养条件下使真菌菌落呈现光滑;抑制冬孢子萌发,...