四川省茄子品种资源黄萎病抗性鉴定评价

[目的]开展茄子品种资源黄萎病抗性的田间鉴定研究,为茄子抗病育种提供科学依据。[方法]供试茄子材料为2001年以来从四川省各地收集到的35份茄子资源。从田间采集黄萎病病株,从中分离纯化出茄子黄萎病菌,经鉴定,引起茄子发生黄萎病的病原菌为大丽轮枝孢。选择生长旺盛、产孢量大的茄子黄萎病菌株V6作供试病原菌。采用伤根接菌法对茄子品种资源进行统一标准的抗黄萎病性鉴定。在黄萎病发病盛期,调查各茄子品种资源材料的黄萎病发病植株症状,记载病株显症叶片数、病斑颜色、叶片是否有脱落、卷枯等。当感病对照的病情指数达40左右时,调查各茄子品种资源材料的发病情况,并计算发病株率和病情指数以确定各材料的抗性型。[结果]收集到的四川茄子品种资源对黄萎病具有较好的抗病性。在35份茄子品种资源中,属于抗黄萎病的有242-90P、41—1和35-R5等10份,黄萎病病情指数介于15．71～19．59,占总鉴定材料的28．57％;属于耐黄萎病的有68×47、紫早茄和32—1等23份,病情指数介于21．15-33．18,占总鉴定材料的65．71％;属于感黄萎病类型的有2份,占总数的5．71％。通过鉴定评价,推荐了41—1、35-R5和47-R11等5个抗病材料以及EF5、EF62个耐病高产材料,被育种单位利用后已转育出EF991等抗病品种。[结论]通过分析品种资源的抗病性,明确了四川省茄子资源的抗性水平和今后抗病育种方向,为茄子抗病育种提供了技术指导。     

Cross-pathogenicity of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> between potato and sunflower

This study examined cross-pathogenicity of the soilborne pathogen Verticillium dahliae between potato and sunflower. Four week-old potato and sunflower seedlings were inoculated with ten isolates from each of the two host species. Potato cultivars (Kennebec, susceptible, and Ranger Russet, moderately resistant) and sunflower hybrids (IS8048, susceptible, and 6946, moderately resistant) were assessed for disease severity and percent infection at 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks after inoculation (w.a.i), and for vascular discolouration at 6 w.a.i., using visual scales developed for each host species. The experiments were conducted in 2006 and repeated in 2007. Based on percent infection and disease severity, most V. dahliae isolates were highly aggressive on both host species. The tested isolates caused higher disease levels in the susceptible than in the moderately resistant phenotypes. They also caused more vascular discolouration in their original than in the alternative host. However, the isolates originating from sunflower caused less infection and disease severity on both hosts, compared to their potato counterparts. Cluster analysis based on all of the criteria used to assess pathogenicity led to three groups of isolates: (i) most V. dahliae potato isolates, which ranged with the highly aggressive control isolates, (ii) one V. dahliae sunflower isolate, which showed a similar pathogenicity level to the weakly-aggressive V. albo-atrum sub-group II control isolate, with no more symptoms than in the non-inoculated plants, and (iii) most V. dahliae sunflower isolates with mildly- to weakly-aggressive levels. Based on these results, V. dahliae cross-pathogenicity is very effective between potato and sunflower. Therefore, rotations involving these species should be avoided, especially where sunflower follows potato.     

棉花黄萎病菌拮抗细菌的分离、筛选及鉴定

采用稀释平板法从健康棉花根部土壤中分离得到细菌菌株1520株.通过室内平板多次筛选获得25株对棉花黄萎病菌具有强拮抗抗作用的细菌菌株.25个拮抗细菌温室盆栽防治黄萎病的试验结果表明:2株拮抗细菌在防治棉花黄萎病中表现较好的防病效果,其中868菌株的防治效果为67.3%.根据2个菌株的培养特征和生理生化特性,初步鉴定868菌株为Pseudomonas fluorescens,112菌株为Bacillus megatherium.     

Suppression of clubroot and Verticillium yellows in Chinese cabbage in the field by the root endophytic fungus, Heteroconium chaetospira

Chinese cabbage seedlings inoculated with an isolate of the hyphomycete, Heteroconium chaetospira, were transplanted to the field. After 3 months, they showed a 52–97% reduction in clubroot and a 49–67% reduction in Verticillium yellows compared with noninoculated controls. H. chaetospira colonized the cortical cells, especially in the root tip region. Infected plants showed no disease symptoms. The infection process involves the formation of appressoria on the cell surface and the subsequent growth of hyphae within cells. H. chaetospira colonized 18 plant species, indicating a wide range of hosts. It may have potential as a biocontrol agent for clubroot and Verticillium yellows.     

大丽轮枝菌分泌蛋白提取方法比较

【目的】采用超滤法（UF）、三氯乙酸沉淀法（TCA）、离子交换富集法（IEX）分别从大丽轮枝菌（Verticillium dahliae）高致病性菌株VdG1培养上清液中提取分泌蛋白,对提取样品进行质谱鉴定。寻找适合大丽轮枝菌分泌蛋白的提取方法,为大丽轮枝菌分泌蛋白组深入研究奠定基础。挖掘大丽轮枝菌分泌蛋白中潜在的致病相关蛋白,为其深入功能验证及致病机理研究提供平台。【方法】3种方法提取大丽轮枝菌VdG1分泌蛋白,shot-gun方法进行质谱鉴定。利用生物信息学软件WoLFPSORT、SignalP、TMHMM、PHOBIUS对其中经典分泌蛋白进行预测。通过CAZy和PHI数据库进行经典分泌蛋白中碳水化合物水解酶（CAZy）和病原菌寄主互作因子（PHI）注释,BLASTp软件进行RxLx、LysM、Cys Pattern等模体蛋白比对分析。提取的蛋白样品接种感病棉种（军棉1号）进行样品生物学活性及致病性检测。【结果】TCA法、IEX法和UF法制备大丽轮枝菌分泌蛋白,其提取效率分别为1.18、0.96和0.56 mg?L-1。SDS-PAGE电泳检测显示UF法提取的蛋白样品条带少而模糊。而TCA法和IEX法提取的蛋白样品条带较多,样品的纯度也较高;经生物信息学软件预测分析,提取的蛋白样品中含有经典分泌蛋白分别为124、112和75个;质谱鉴定的分泌蛋白中含有潜在致病因子分别为98、85和57个;另外,IEX法制备大丽轮枝菌分泌蛋白效果较好,具有小分子偏好性,且能够保持样品的生物学活性,该蛋白样品接种棉花叶片能够引起萎蔫、坏死的症状。【结论】3种方法提取大丽轮枝菌分泌蛋白,UF法提取效率最低,纯度最差,蛋白数量最少,蛋白样品中含有潜在的毒素蛋白数量也最少,而且对于大体积处理相当耗时。TCA法和IEX法都适合大丽轮枝菌分泌蛋白的制备,但是TCA法制备过程中不能保持样品的生物学活性,后续适合于2-D电泳检测等试验。IEX法首次应用于病原菌分泌蛋白的制备,操作简单、方便,全部过程均由仪器系统完成。制备的大丽轮枝菌分泌蛋白具有生物学活性,能够引起棉花叶片的萎蔫、坏死病症。另外,数据库及软件比对分析发现大丽轮枝菌分泌蛋白组中含有CAZy酶类61个、PHI相关蛋白38个、RxLx模体蛋白13个、LysM模体蛋白1个、Cys Pattern的模体蛋白15个、VdNEP蛋白家族2个。该结果将为致病相关蛋白的筛选和深入功能验证提供重要依据。     

黄萎病菌诱导下陆地棉抗病品种SSH文库的EST分析

对已构建的黄萎病菌诱导下陆地棉抗病品种冀棉20SSH文库的800个阳性克隆进行了检测和筛选,将插入片段大于400bp的克隆测序,获得非重复克隆的EST序列203条。通过同源比对和序列相似性分析,共有170条ESTs与已知基因同源。将获得的ESTs按比对推测的功能进行分类,主要包括代谢、防御、胁迫、信号转导、核糖体蛋白、细胞结构、细胞发育、能量、渗透调节以及蛋白质合成与分解等10类。功能未知的ESTs也占有不小比例,达16.26%。共发现66条与抗病相关的ESTs,约占全部ESTs的33%。这些与抗病相关的同源序列涉及到与防御、次生代谢、胁迫及信号转导有关的基因,表明当黄萎病菌侵染棉花后,植物体内发生了一系列的反应,抗黄萎病是一个多途径作用的复杂过程。     

大丽轮枝菌致萎毒力指示菌的筛选及拮抗作用初探

为了筛选可指示Verticillium dahliae致萎毒素毒力大小的指示菌,并对致萎毒素抑制指示菌生长的机理进行初步探讨,采用指示菌平板打孔法,对黑曲霉(Aspergillus niger)BF-3758、枯草芽孢杆菌(Bacillus subtilis)、金黄色葡萄球菌(Staphylococcus aureus)、啤酒酵母(Saccharomyces cerevisiae)等12种具有微生物种属代表性的菌株进行筛选;采用拮抗试验、致萎试验结合显微观察的方法,初步探索了Verticillium dahliae致萎毒素对指示菌生长的抑制作用。结果表明,Verticillium dahliae粗毒素对Aspergillus niger BF-3758的生长具有较强的抑制作用,且该抑制作用和粗毒素对离体叶片的致萎作用具有一致性,并存在一定的量效关系,Aspergillusniger BF-3758菌株可以作为Verticillium dahliae致萎毒素的毒力指示菌;Verticillium dahliae粗毒素可抑制Aspergillus niger BF-3758的孢子萌发和菌丝生长,对成熟菌丝也有破坏作用。上述研究可为评价Verticillium dahliae的致病力提供一种操作简便、耗时短、费用低的生物学方法。     

Gene effects for cotton-fiber traits in cotton plant (Gossypium hirsutum L.) under Verticillium conditions

Verticillium wilt (VW), caused by Verticillium dahliae Kleb., has become one of the most serious problems in cotton (Gossypium hirsutum L.). The use of resistant cultivars has long been considered the most practical and effective means of control. The objective of this work was to study the quantitative genetic basis of fiber traits under Verticillium conditions in upland cotton by using five genotypes and their possible crosses without reciprocals, selecting simultaneously for quality fiber, resistance, and agronomic characteristics. Five cotton cultivars and 10 F₁s from half diallel crosses were analyzed for quality fiber under VW conditions. The fiber length, uniformity, strength, elongation, and micronaire were measured during two crop seasons at two different sites each year, consistently in plots with soil naturally infested with Verticillium. Genetic components of variance were analyzed using the Hayman model. Analysis of variance for all traits showed significant differences between genotypes, with the genotype–site interaction in most of the studied traits except for fiber length and micronaire. Both the additive genetic variance component (D) and dominance genetic variance components (H1 and H2) were present in all traits. D was the most important component for uniformity, strength, elongation, and micronaire. Elongation was the trait most correlated with seed-cotton yield. Strength and micronaire were the traits most correlated with VWI. Broad-sense heritability was high for all the traits studied. Narrow-sense heritability was high for uniformity, strength and elongation, and moderate for length and micronaire.     

不同棉花品种(系)的黄萎病抗性及其与经济性状的相关性

研究了若干棉花品种(系)及不同组合棉花杂交群体的经济性状,分析经济性状与病情指数的相关性。结果显示:①海陆杂交群体的抗性比陆陆杂交群体的抗性普遍偏高;②陆地棉品种(系)病情指数与有效果枝、有效铃数呈显著负相关;③陆陆杂交群体M44株系的相对病指与有效果枝、铃数、有效铃数呈显著负相关;④海岛棉品种(系)相对病指和株高呈显著负相关;⑤海陆杂交群体M37株系的相对病指与株高和始节高呈显著负相关;⑥有效果枝和有效铃数可以作为对黄萎病抗性间接鉴定的指标。⑦海陆杂交后代中,与抗病性共同遗传的为株高,因此可将株高作为分子标记辅助抗病育种的参考。     

环介导等温扩增技术检测大丽轮枝菌

 本研究基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),通过比对分析大丽轮枝菌(Verticillium dahliae)与其相近种不同靶标序列间的差异,选取Gpd(glyceraldehyde-3-phosphate dehydrogenase,甘油醛-3-磷酸脱氢酶)基因作为靶标基因,设计并筛选了四条特异性强、灵敏度高的LAMP引物和两条环引物,建立了一种基于颜色判定的简单、快速和灵敏的大丽轮枝菌的检测方法,并进行了特异性、灵敏度实验及田间发病组织的检测。该方法在62 ℃等温条件下进行核酸扩增反应70 min,扩增前加入染料HNB(羟基萘酚蓝),反应后根据染料颜色变化判定扩增结果。特异性试验中,仅含有大丽轮枝菌菌株DNA的反应管扩增后呈天蓝色的阳性反应,而其他供试菌株均呈紫色的阴性反应。该方法的最低检测限为100 pg·μL^-1,在土壤中检测的灵敏度为10个孢子/0.25g土壤。该技术能够检测出棉花发病组织中的目标菌,对采自江苏和山东的24份疑似病害样本进行检测,11份为阳性。该方法的建立为大丽轮枝菌的检测及其所致病害的诊断提供了新的技术。