棉花黄萎病菌的侵染过程

深入揭示黄萎病菌的致病机理,为培育高抗新品种提供理论依据和技术途径,是有效控制黄萎病的根本.本文系统探讨了土壤中微菌核或孢子受寄主根系分泌物的刺激,开始萌发、产生的菌丝在寄主根表面定殖、穿过表皮、在皮层中发生及在寄主体内扩展和症状形成等黄萎病菌侵染棉花的过程,阐述了寄主植物形成多级防御反应阻击病原菌的入侵.对黄萎病菌侵染寄主植物过程及其机理下一步研究的问题和内容进行了讨论.     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花花萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条谱带,其中５５．４％的谱带为多态速,经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

继代对棉花黄萎病落叶型菌系致病力的影响

在棉花黄萎病菌致病力分化的研究中,我们发现落叶型菌系T9的致病力有日渐减弱的倾向,不少单位也遇到此类问题,为明确落叶型菌系是否会在继代过程中致病力发生变化,如何变化等问题,我们采用国内外典型的3个落叶型菌系T9和V991、V76,以及2个非落叶型菌系V146和V250为研究对象,采用连续转接的方法,利用3个不同抗病性品种对它们的致病力进行测定,以明确经过连续转接后这些菌系的致病力是如何变化的.结果表明,经过14代的转接,落叶型菌系T9的致病力发生了显著\降,甚至已失去其致使棉花落叶的能力,即连续转接后,落叶型菌系T9已变成非落叶型菌系,且其致病力有成为弱致病力菌系倾向;但我国落叶型菌系代表V991则仍然保持其强致病力的落叶菌系特性.     

棉花黄萎病拮抗菌KF-43-1的鉴定与3种处理田间防治效果对比

【背景】棉花黄萎病具有棉花“癌症”之称,对棉花的产量和纤维质量造成较重危害,生物防治以绿色安全等优点成为防治棉花黄萎病应用领域的重点研究内容,因此开发新的生物菌剂防治棉花黄萎病对棉花生产工作具有重要意义。【目的】通过对具有拮抗棉花黄萎病菌作用的放线菌KF-43-1进行初步鉴定及生理生化测定,明确最佳使用方式,为开发新的防治棉花黄萎病生物菌剂提供支持。【方法】对菌株KF-43-1进行分子鉴定,观察其形态特征及生长特性,测定菌株KF-43-1对病原真菌的抑菌效果,明确菌株KF-43-1的生理生化特性,验证放线菌KF-43-1不同使用方式对棉花黄萎病的田间防治效果。【结果】培养特征和显微特征及分子鉴定结果将菌株KF-43-1鉴定为白浅灰链霉菌(Streptomyces albogriseolus);菌株KF-43-1对病原菌V991抑制效果达到82.05%,防效高于放线菌5406,对尖孢镰刀菌萎蔫专化型ST89抑制效果达到25.81%,低于放线菌5406的防效;菌株KF-43-1无荧光反应,能够使明胶液化、淀粉水解、甲基红试验阳性、可以产生黑色素,不具备分解纤维素的能力,菌株KF-43-1在pH 7.0-8.0时生长情况良好,在盐浓度为2%的高氏一号培养基上生长受到抑制并表现出一定的耐盐性,使用拮抗菌KF-43-1菌液制作种衣剂对棉花黄萎病的防效高于离心液包衣和离心液叶面喷施,说明拮抗菌KF-43-1在土壤中定殖后对棉花黄萎病暴发具有更好的抑制效果。【结论】棉花黄萎病拮抗放线菌KF-43-1作为一种新型生防菌,可为开发防治棉花黄萎病的生物菌剂提供生防菌资源,具有良好的开发价值和应用价值。     

棉花黄萎病及其抗病育种的研究

就棉花黄萎病菌的致病机理、棉花的抗黄萎病机制、抗黄萎病的遗传方式以及抗黄萎病棉花育种等方面的研究进行了综述。     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

澳大利亚棉花黄萎病菌DNA多态性及其地理分布相关性研究(英文)

应用ＲＡＰＤ技术对澳大利亚东南部八个主要棉花种植区的９９个棉花黄萎病菌菌株进行了ＤＮＡ多态性分析。结果表明用１０个筛选的随机引物对供试菌株的全基因组ＤＮＡ扩增,共获得９２条ＲＡＰＤ谱带,其中５５．４％的谱带为多态带。经类聚分析,供试菌株类聚为１５个ＲＡＰＤ遗传指纹相似组,其中１０个指纹相似组的菌株与其采集区域有明显相关性,其余５个指纹相似组的菌株为普通分布的指纹类型。     

拮抗菌BJB01抗黄萎病的抗病效果评价

棉花是重要的战略与民生物资,在种植过程中面临多种不利因素的影响,其中棉花黄萎病作为一种危害性极大的植物性疾病使棉花大量减产,因此需要一种安全高效的方法应对棉花黄萎病的发生。从新疆棉花重病田中筛选到菌株BJB01并进行提取纯化,通过生理生化鉴定和革兰氏染色试验,对获得的菌株DNA进行16S rDNA测序,将测序结果在NCBI上进行比对;然后利用平板对峙试验初步测试防效;最后通过温室防效试验,筛选到最佳的防治方法及浓度。试验结果显示,该菌与贝莱斯芽孢杆菌相似度极高,为99.71%,由此可确定该菌为贝莱斯芽孢杆菌(Bacillus belleiensis);在平板对峙中,该菌显现出对大丽轮枝菌(Verticillium dahlia Kleb.)的抑制作用;通过温室防效测试发现该菌在浓度为OD₆₀₀=0.6时的防效结果达到89.35%,优于其他方法及处理浓度,并且再次单独测试后防效为85.91%,效果依旧最佳。综合分析,从新疆棉花重病田中获得的贝莱斯芽孢杆菌BJB01,可以对大丽轮枝菌起抑制作用,有望用于制备生物菌剂用以棉花生产防治。     

木霉菌合成银纳米粒子条件的优化及其对甜瓜尖孢镰刀菌抑制作用

随着绿色环保观念的普及,生物合成金属纳米粒子的方法备受青睐。纳米银(Silver nanoparticles,AgNPs)由于其抗菌活性强且不易产生抗药性等特点在农业病害防治中越来越受到关注。文中利用橘绿木霉Trichoderma citrinoviride和毛簇木霉Trichoderma velutinous研究了AgNPs的最适合成条件和AgNPs对尖孢镰刀菌抑菌活性。结果表明,所有合成的AgNPs均在400–500 nm处有吸收峰,两种木霉生物合成AgNPs的最适合成条件为CL法(菌丝滤液)静置光照培养,底物AgNO₃浓度为2.0mmol/L,pH值为7,反应温度为45℃。橘绿木霉和毛簇木霉合成的AgNPs均对尖孢镰刀菌有抑制作用,抑菌效果随浓度的增加而增大,AgNPs在浓度为200 mg/L时,抑菌率分别达到33.745%和36.083%。     

棉花黄萎病高效拮抗菌XJUL-6的筛选鉴定及其特性研究

为探讨棉花黄萎病高效拮抗菌的拮抗机制,从新疆有毒植物焮麻(Urtica cannabina L.)中筛选出一株对棉花黄萎病具有较强抗性的内生菌XJUL-6,对其生物学特性进行初步研究,结果表明,36℃~38℃为最适生长温度,pH6~8为最适生长pH值。根据其形态特征、生理生化检测、16S rDNA、(G C)mo1%,将其鉴定为蜡状芽孢杆菌,XJUL-6的获得为进一步的研究棉花黄萎病高效拮抗菌的拮抗机制提供了实验材料。     

抑制茄子黄萎菌的植物提取物的筛选

从蔬菜、花卉、树木、作物和杂草等植物中选取29科73种植物材料,通过室内生物测定和田间试验,筛选出的葡萄叶、大葱叶提取物对黄萎菌具有较强的抑制作用,菌丝生长抑制率达68.44%和61.31%,孢子萌发抑制率达到95%以上,田间防病效果与茄子"病菌净"药剂达到同一显著水平,防效分别达到了75.76%和71.72%。丁香枝叶、薄荷叶、辣椒叶、番茄叶、醉蝶根、南瓜叶、韭菜叶、核桃叶、银杏叶、大蓟叶、藿香叶、苣卖菜叶、万寿菊叶、柳枝叶、花椒果和橘皮等也具有较好的抑菌活性,可作为其它病原菌的天然抗菌剂筛选研究试材。     

茄子黄萎病菌抗性根际芽孢杆菌的筛选与鉴定

利用离体抑菌圈法从茄子根际土壤筛选出8株对黄萎病菌大丽轮枝菌(Verticillium dahliae)抑菌效果明显的芽孢杆菌,其中F53菌株分泌几丁质酶和-β1,3-葡聚糖酶,对所检测的12种植物病原菌具明显的抑制作用,其发酵液热、酸碱稳定性强,用F53菌株制成的菌剂对茄子黄萎病盆栽试验防效为52.4%~75.8%,初步鉴定F53菌株为环状芽孢杆菌(Bacillus circulans)。     

甘肃省甜瓜黄萎病的病原鉴定

【背景】2013年11月在甘肃省兰州市皋兰县的日光温室秋冬茬甜瓜种植棚发现黄萎症状的甜瓜植株,病株率约为1%。【目的】明确甜瓜黄萎病的病原。【方法】采用组织分离法进行病原菌分离;通过科赫氏法则(Koch’s法则)明确分出病菌的致病性;采用形态学和分子生物学方法对病原菌进行种类鉴定。【结果】分离得到轮枝菌属真菌8株,轮枝菌属真菌的病株分出率达100%;2个代表性菌株GLTG-2和GLTG-5(显微特征相似但菌落形态和生长速率不同),在温度18-24℃及昼/夜光周期为11.5 h/12.5 h的试验条件下,人工接种可引起甜瓜苗矮化、枯萎;接种后40 d,枯死株率分别为70%和40%;BLASTn分析结果显示,菌株GLTG-2的rDNA-ITS序列与Verticillium dahliae菌株MRHf7的序列相似性达99.78%,菌株GLTG-5的rDNA-ITS序列与V.dahliae菌株MRHf7和Vd414的序列相似性达100.00%。【结论】引起甜瓜黄萎病的病原菌被鉴定为大丽轮枝菌(V. dahliae),这是大丽轮枝菌引起甜瓜黄萎病在我国和亚洲地区的首次报道。     

Isolation of Glycoproteins from Verticillium dahliae and Their Phytotoxicity

Verticillium dahliae Kleb. is a phytopathogenic fungus that causes cotton wilt-disease. Glycoproteins secreted by V. dahliae have been found to play an important role in wilting syndrome. In this study the glycoproteins were purified consecutively by ConA-Sepharose 4B affinity chromatography, Sephadex G-150 gel filtration, two-dimensional gel electrophoresis and SDS gradient gel electrophoresis. The N-terminal residual sequence of a 26 kD glycoprotein was analyzed. Plant-wilting tests were carried out by injection of glycoproteins, and those treated by heat, ConA and zeatin, into cotton leaves, respectively. Results showed that heat and ConA treatment abolished the wilt-causing activity of the glycoproteins, and zeatin alleviated the wilt syndrome of cotton. Furthermore, the glycoproteins were found to be effective elicitors in inducing the biosynthesis of sesquiterpene aldehyde phytoalexins in suspension cell cultures of Gossypium barbadense L., and heat-treatment lowered, but not abolished the elicitor activity. However, application of native glycoproteins at the concentration higher than 5 mg/L resulted in cell death.     

GhADF6-mediated actin reorganization is associated with defence against Verticillium dahliae infection in cotton

Several studies have revealed that actin depolymerizing factors (ADFs) participate in plant defence responses; however, the functional mechanisms appear intricate and need further exploration. In this study, we identified an ADF6 gene in upland cotton (designated as GhADF6) that is evidently involved in cotton's response to the fungal pathogen Verticillium dahliae. GhADF6 binds to actin filaments and possesses actin severing and depolymerizing activities in vitro and in vivo. When cotton root (the site of the fungus invasion) was inoculated with the pathogen, the expression of GhADF6 was markedly down-regulated in the epidermal cells. By virus-induced gene silencing analysis, the down-regulation of GhADF6 expression rendered the cotton plants tolerant to V. dahliae infection. Accordingly, the abundance of actin filaments and bundles in the root cells was significantly higher than that in the control plant, which phenocopied that of the V. dahliae-challenged wild-type cotton plant. Altogether, our results provide evidence that an increase in filament actin (F-actin) abundance as well as dynamic actin remodelling are required for plant defence against the invading pathogen, which are likely to be fulfilled by the coordinated expressional regulation of the actin-binding proteins, including ADF.     

大丽轮枝菌寡糖素诱导的棉花组培细胞的过氧化物酶表达分析

将已在察氏液体培养基中培养3d的强毒性（V44)或弱毒性（V64）大丽轮枝菌接种到新鲜的相同培养基中,加入棉花黄萎病感病品种（S）或抗病品种（R）的悬浮培养细胞（或其细胞匀浆）。培养4d后收获真菌孢子,用于致萎力测定,收获菌丝体细胞壁用于制备真菌寡糖素（H,来自V44或L,来自V64）,然后用该寡糖素诱导悬浮培养的棉花细胞（S或R）,棉花细胞的过氧化物酶（POD）同工酶结果表明;（1）所有寡糖素均诱导棉花细胞产生新的过氧化物酶;（2）感病品种豫棉6号的活组培细胞S（或其细胞匀浆）显著影响真菌,用生长过程中接触该活细胞（或其细胞匀浆）的真菌所制备的寡糖素再诱导该活细胞（即S→L（H）→S）时,棉花细胞在36h的POD同工酶谱与其对照,即L（H）→S比较,酶带增减变化显著,致萎力测定结果表明感病品种的活细胞提高了病原毒性和致病力;（3）耐病（豫棉8号）或抗病品种（中棉12号）的活组培细胞R（或其细胞匀浆）对真菌的影响不同于感病品种,R→L（H）→R在36h的POD同工酶谱与L（H）→R比。酶带没有增减变化。但是在H→R或H→S中于9h和24h出现的酶带在R→H→R或R→H→S中分别提前到6h和9h,致萎力测定结果表明抗病品种细胞对病原毒性影响很小。     

大丽轮枝菌微菌核的萌发条件及致死温度

微菌核是大丽轮枝菌在土壤中的主要存活结构和初侵染源,在土壤中可存活14年之久,其数量及存活状况直接影响着大丽轮枝菌型黄萎病的发生为害程度。以棉花黄萎病菌Verticillium dahliae XJ2008菌株为试材,研究了微菌核萌发的最佳条件、致死温度及土壤温度对微菌核存活的影响。结果表明,20℃、pH8.0是微菌核萌发的最佳条件。大丽轮枝菌微菌核具有很强的耐高温特性,随着处理时间的延长,微菌核的萌发率呈下降趋势,在55℃及以上处理时其萌发率下降迅速,而在50℃及以下处理时其萌发率下降相对较慢,在55℃处理360min可使微菌核完全致死,而在40℃、45℃和50℃处理1,440min,仍然有少量的微菌核存活,但其萌发率随着时间的推移呈下降趋势。土壤微菌核模拟试验结果表明,土壤温度对微菌核有很强的致死作用,40℃条件下处理4d后土壤中的微菌核已全部死亡。该研究结果为通过覆膜增温防治作物黄萎病提供了理论依据。     

iTRAQ-based proteomics analysis of autophagy-mediated immune responses against the vascular fungal pathogen Verticillium dahliae in Arabidopsis

The mechanisms underlying the functional link between autophagy and plant innate immunity remain largely unknown. In this study, we investigated the autophagy-mediated plant defense responses against Verticillium dahliae (V. dahliae) infection by comparative proteomics and cellular analyses. An assessment of the autophagy activity and disease development showed that autophagic processes were tightly related to the tolerance of Arabidopsis plant to Verticillium wilt. An isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics analysis was performed, and we identified a total of 780 differentially accumulated proteins (DAPs) between wild-type and mutant atg10-1 Arabidopsis plants upon V. dahliae infection, of which, 193 ATG8-family-interacting proteins were identified in silico and their associations with autophagy were verified for several selected proteins. Three important aspects of autophagy-mediated defense against V. dahliae infection were revealed: 1) autophagy is required for the activation of upstream defense responses; 2) autophagy-mediated mitochondrial degradation (mitophagy) occurs and is an important player in the defense process; and 3) autophagy promotes the transdifferentiation of perivascular cells and the formation of xylem hyperplasia, which are crucial for protection against this vascular disease. Together, our results provide several novel insights for understanding the functional association between autophagy and plant immune responses.     

大丽轮枝菌分泌蛋白激发子的分离纯化及生物功能研究

利用硫酸铵沉淀、(A)KTA explorer 10蛋白纯化仪、非变性电泳、割胶电洗脱等方法,从大丽轮枝菌(Verticillium dahliae)发酵液中分离纯化出一种蛋白激发子,经SDS-PAGE电泳检测单一条带,相对分子量为20 kD.该蛋白激发子能够诱导烟草的过敏反应,处理6h后,处理部位出现水溃状,24h后出现坏死斑.该激发子可以诱导烟草细胞在较短时间内产生防卫反应信号分子H2O2和NO,并引起活性氧爆发.     

Characteristics of Bacteria from Oilseed Rape in Relation to their Biocontrol Activity against Verticillium dahliae

The potential of bacteria that are adapted to the oilseed rape root environment for use in the biological control of Verticillium dahliae, Kleb was investigated in both controlled and non‐sterile growth conditions. Bacterial strains dominated by the red‐pigmented members of enterobacteriaceae were isolated from thoroughly washed and air‐dried root segments of symptomless young rape plants. Other associated strains found either belonged to Alcaligenes sp., Stenotrophomonas spp. and Pseudomonas spp. (Pseudomonas acidovorans and Pseudomonas putida) or were unidentified according to fatty acid methyl ester profile analysis. A total of 19 strains isolated in this study together with two previously studied strains, Serratia proteamaculans and Pseudomonas chlororaphis, were characterized on the basis of their interactions with V. dahliae and a number of functional characteristics. In line with earlier observations with root‐colonizing fungi also from oilseed rape, all bacterial strains suppressed the pathogen not only directly and but also indirectly in in vitro assays. Mechanisms of suppression were apparently multifold among the strains, but production of hydrogen cyanide does not seem to be involved in indirect inhibition. The majority of the strains possessed the ability to produce cellulases, proteases and phosphatases and some even produced chitinases and induced hypersensitive responses, indicating their potential for nutrient acquisition as well as colonization capacity and active recognition by the plant cells. Investigations in non‐sterile field soil revealed that some strains protected rape plants from V. dahliae partly by delaying symptom development. None of the strains, however, was strongly deleterious to rape growth either in the presence or absence of the pathogen. Light microscopic observations of roots and results based on agar printing techniques revealed the potential of the studied strains to colonize or interfere with the pathogen colonization. This study provides some insight into the evolved relationship of bacterial residents with their host in terms of their potential importance in its fitness.