应用聚合酶链式反应鉴定新疆棉花落叶型黄萎病菌

用一对棉花落叶型黄萎病菌的特异性引物D1和D2进行PCR扩增,对于落叶型黄萎病菌,该对引物可特异性地扩增产生一段550bp的产物,而非落叶型黄萎病菌则不能被扩增.供试的35个新疆黄萎菌系中,有3个菌系扩增出550bp大小的落叶型黄萎病菌特异性片段,表明目前新疆已存在落叶型黄萎病菌,用此技术可快速、准确地检疫和鉴定落叶型黄萎病菌.     

落叶型棉花黄萎病菌的致病力分化

为明确我国长江流域和黄河流域主要植棉省落叶型菌系的致病力差异,选用4个抗性不同的陆地棉品种银瑞361、豫棉21、冀棉228和冀棉11作为鉴别品种,在温室可控条件下,采用苗期移苗蘸根接种法,对不同植棉省32个落叶型黄萎病菌的致病性进行了测定,并根据供试菌系在鉴别品种上病情指数进行聚类分析。苗期接种14 d后,抗、感品种间的病情指数差异较大,是落叶型菌系病情指数调查的合适时间。32个落叶型菌系致病力差异显著,可分为3个类型。第Ⅰ类型只有1个菌系,致病力弱;第Ⅱ类型菌系占供试菌系的62.5%,平均病情指数为36.8,致病力中等;第Ⅲ类型菌系占供试菌系34.4%,平均病情指数52.7,致病力较强,4个鉴别品种均表现感病。研究表明来源于不同植棉省菌系的平均致病力有一定差异,且来源于同一植棉省的落叶型菌系可归属于不同的致病类型。     

棉花黄萎病菌致病型的AFLP分析

 选用41个棉花黄萎病菌(Verticillium dahliae)代表菌系,在温室条件下,对4个棉花品种鄂荆1号(感)、中棉所12(耐)、文-5(抗)和唐棉2号(抗)进行致病性测定,结果可将供试菌系分为落叶型与非落叶型2类。选取8对AFLP引物PCR扩增的结果中,统计带型稳定、清晰且有多态性的条带,共169条作系统聚类分析,将上述菌系分为2大类,第一类为非落叶型菌系,包括10个非落叶型菌系和1个过渡菌系;第二类为30个落叶型菌系。根据聚类分析建立树状图,发现菌系与地理来源存在一定的相关性,而依据菌系致病力强弱分类则相关关系不大。选用25对EcoRⅠ和MseⅠ引物组合,对供试的41个V.dahliae进行AFLP扩增,筛选到2对引物E64(GACTGCGTACCAATTCGAC)、M53(GATGAGTCCTGAGTAACCG)和E49(GACTGCGTACCAATTCCAG)、M65(GAT-GAGTCCTGAGTAAGAG),能分别扩增出433bp和110bp2条仅为V.dahliae非落叶型菌系独有的特异片段,可将落叶型与非落叶型菌系分开,这2条特异片段被命名为EM₄₃₃和EM₁₁₀。     

江苏省大丽轮枝菌的培养、遗传及致病特性分析

由大丽轮枝菌引起的黄萎病是我国棉花和茄子的主要病害之一,对棉花和茄子的生产造成巨大损失。为了研究大丽轮枝菌的群体遗传变异以及对棉花和茄子的交互致病性,本文对分离自江苏省的63个棉花黄萎菌和10个茄子黄萎菌进行了培养、遗传和致病特性分析。根据菌株在PDA培养基上生长时形成微菌核的多少来划分培养类型,结果菌核型占83.6%,成为主要的培养类型。用PCR技术检测菌株的致病类型、交配型以及是否具有无毒基因Ave1,结果落叶型菌株占86.3%,为优势种群,但是10株茄子黄萎菌100%都是非落叶型菌株;供试的所有江苏菌株交配型都是MAT1-2型,并且都没有无毒基因Ave1。选择江苏省的6个棉花黄萎菌和4个茄子黄萎菌在室内苗期接种棉花和茄子,进行交互致病性测定,结果这10个菌株都可以侵染棉花和茄子,而且来源于不同寄主作物的菌系之间致病力分化明显,表现在不同菌株对同一寄主的致病力不同,同一菌株对不同寄主的致病力也不同。研究结果为深入研究大丽轮枝菌群体遗传结构和制定黄萎病防治措施提供了理论支持。     

棉田土壤中棉花黄萎病菌的致病力分化

为了明确同一棉田生态系中棉花黄萎病菌的群体组成和致病力分化特点,利用病害反应型及病情指数测定法,对来自同一棉田的43个棉花黄萎病分离菌株在5个鉴别寄主上进行了致病力测定。结果表明,43个菌株可划分为落叶型致病力强的Ⅰ型、混合型致病力中等的Ⅱ型和非落叶型致病力弱的Ⅲ型。其中Ⅰ型菌株19个,占44.2%,平均病情指数大于50;Ⅱ型菌株16个,占37.2%,平均病情指数在30～50之间;Ⅲ型菌株8个,占18.6%,平均病情指数小于30。由此证明,同一棉田棉花黄萎病存在落叶型和非落叶型两类病害表现类型,其病原菌存在强、中、弱三种不同致病类型的生理型,揭示出棉花黄萎病菌本身是一个易变异的混合基因型群体,群体中的不同个体组成的亚群体具有不同的致病性。     

新疆棉花黄萎病的发生现状及其病原菌的分子鉴定与ISSR分析

为掌握新疆主要植棉区棉花黄萎病的发生现状及其病原菌大丽轮枝菌Verticillium dahliae的落叶型菌系分布以及遗传变异情况,于2015年对26个新疆主要植棉区棉花黄萎病的发生情况进行了随机调查,统计新疆大丽轮枝菌的培养性状,利用大丽轮枝菌落叶型特异引物D1/D2、INTD2F/INTD2R与非落叶型特异性引物ND1/ND2、INTNDF/INTNDR对新疆大丽轮枝菌菌系进行互补鉴定,并对部分菌系的遗传变异进行简单序列重复区间(inter simple sequence repeat,ISSR)分析。结果表明:2015年新疆棉花黄萎病发病田比例为54.0%,其中病情指数在10.0以上的发病田与2013年持平,而病情指数在20.0以上的严重发病田比例为10.8%,比2013年增加3.8个百分点;新疆大丽轮枝菌的培养性状以菌核型为主,比例为70.1%,菌丝型与中间型比例分别为13.4%和16.5%;新疆大丽轮枝菌落叶型菌系比例为53.2%,26株菌株的来源地全部检出落叶型菌系;聚类分析结果显示,当遗传相似系数为0.66时,新疆大丽轮枝菌落叶型与非落叶型菌系聚为2个谱系,菌系地理来源、培养性状与大丽轮枝菌的遗传分化无明显相关性。     

不同植棉省区落叶型黄萎病菌的培养特性及致病力比较

近年来落叶型症状的棉花黄萎病对棉花生产的危害越来越大,为明确落叶型棉花黄萎病菌的分化特点,采用特异性分子标记方法,对我国10个主要植棉省区30个落叶型棉花黄萎病菌的培养特性和致病力进行了比较。落叶型黄萎病菌系在我国主要植棉省区已普遍存在,该菌系以菌核型为主,其微菌核呈放射状或环状。63.3%菌系的菌丝发达致密,其余菌系的菌丝较为疏松。落叶型黄萎病菌系之间的产孢量和致病力差异较大,产孢量变幅为3.7×107～18.8×107个孢子/mL;71.9%为强致病力菌系,25.0%为中等致病力。研究表明,不同植棉省区间落叶型黄萎病菌系的产孢量和致病力具有一定差异。     

北方棉区棉花黄萎病菌落叶型菌系鉴定

 采用RAPD扩增与温室致病性测定2种方法,以黄萎病菌Verticillium dahliae的5个落叶型、7个非落叶型菌系和V.albo-atrum的2个菌系为对照,对采自北方棉区6省(自治区)的34个棉花黄萎病菌菌系进行致病型鉴定。94.1%的菌系在2种鉴定结果中表现一致,与对照菌系相比较,确定其中26个菌系为落叶型菌系,6个菌系为非落叶型菌系。从而证实了黄萎病菌落叶型菌系在北方棉区河北、河南、山东3省的存在,并发现所确定的26个北方落叶型菌系中的22个与来自美国的对照落叶型菌系T₉、V₄₄的关系比与来自江苏的对照落叶型菌系V_B、V₉₉₁的关系更近。本实验还初步筛选到2条用于鉴别V.dahliae落叶型与非落叶型菌系的RAPD特异条带OPB-19₉₆₆和OPM-20₁₆₉₁,将它们用于对34个北方菌系的RAPD扩增鉴定,则与温室致病性测定结果的一致性分别为88.2%和94.1%,证明这2条特异条带在鉴定棉花黄萎病菌落叶型菌系上具有一定的实用价值,并为进一步制作特异探针以形成一套简便、准确、规范化的鉴定技术奠定了基础。     

北京地区草莓灰霉病菌的转座子及其分布频率

为了解北京地区草莓灰霉病菌的转座子类型及其分布,本研究用转座子Boty和Flipper的特异性引物对北京地区2012-2013年从12个草莓园采集和分离的60株草莓灰霉病菌进行PCR扩增。结果表明,北京地区草莓灰霉病菌群体中共存在3种转座子类型:transposa型、Boty型和Flipper型。其中,以transposa型菌株最多,占供试菌株的63.3%,Boty型菌株占供试菌株的28.3%,Flipper型菌株最少,仅占8.4%,未检测到vacuma型菌株。选取属于不同转座子类型的18株菌株测定其对草莓叶片的致病力,结果显示Boty型菌株所致病斑的平均直径显著大于Flipper型。草莓灰霉病菌转座子类型与致病力的关系有待进一步研究。转座子类型的检测为进一步研究灰葡萄孢的遗传多样性及遗传变异提供了基础。     

新疆棉花黄萎病菌营养体亲和群的研究

 由大丽轮枝菌(Verticillium dahliae)引起的棉花黄萎病是一种世界性分布的毁灭性病害。依据其对不同寄主品种致病力的强弱,可被划分为强致病力的落叶型、中度致病力非落叶型(或混合型)和弱致病力非落叶型3种致病类群。而依据不能利用硝酸盐的营养突变体(nit mutants)间亲和性,又可将其划分为多个不同的营养体亲和群(VCGs),其中我国的棉花黄萎病菌可归为2个VCGs,分别对应于落叶型和非落叶型(包括中度致病力和弱致病力)类群,即VCGs群与生物学测定得致病力类群间存在着相关性,测定VCGs可作为鉴定强致病力落叶型棉花黄萎病菌的一种稳定可靠的辅助手段。     

河南商丘地区棉花黄萎病菌分离鉴定和致病力分析

为探讨河南商丘地区棉花黄萎病菌的致病型群体变异,对该地区棉花上分离的8株单孢菌株的菌落形态、显微结构、致病力、ITS序列、系统进化及菌体蛋白等方面进行了研究。结果表明:这些菌株均属于棉花黄萎病菌Verticilliumdahliae;系统进化树显示8株菌株并没有聚在同一进化枝上;8株黄萎菌菌株存在致病力差异,SQ4菌株致病力最强,属于落叶型,而其它致病力较弱的7个菌株属于非落叶型;不同致病力的菌株间蛋白谱带存在差异。     

Comparative Study of Genetic Diversity and Pathogenicity Among Populations of Verticillium Dahliae from Cotton in Spain and Israel

Genetic diversity and phenotypic diversity in Verticillium dahliae populations on cotton were studied among 62 isolates from Spain and 49 isolates from Israel, using vegetative compatibility grouping (VCG), virulence and molecular assays. In Spain, defoliating V. dahliae isolates (D pathotype) belong to VCG1, and non-defoliating isolates (ND) belong to VCG2A (often associated with tomato) and VCG4B (often associated with potato). The D pathotype was not identified in Israel. The ND pathotype in Israel is comprised of VCG2B and VCG4B. Isolates in VCG2B and VCG4B ranged in virulence from weakly virulent to highly virulent. The highly virulent isolates induced either partial defoliation or no defoliation. Virulence characteristics varied with inoculation method and cotton cultivar. Highly virulent isolates from Israel were as virulent as D isolates from Spain under conditions conducive to severe disease. The D pathotype is pathologically and genetically homogeneous, whereas the ND pathotype is heterogeneous with respect to virulence, VCG, and molecular markers based on single-primer RAPD and on PCR primer pairs.     

棉花落叶型黄萎病菌的检测技术

采用Nit硝酸还原酶缺陷型突变体技术，共得到棉花黄萎病菌Nit突变体301个，其中A型155个，B型97个，C型29个。共检测到落叶型棉花黄萎病菌11株，除以前报道的6株外，发现了5个新菌株。针刺接种后，在棉株上均引起典型的落叶型症状。通过聚丙烯酰受凝胶电泳比较落叶型和非落叶型的8个代表菌株的蛋白质电泳图谱的差异，发现在浓度为7.5%的凝胶中，落叶型菌株有两条特异性的蛋白，其迁移率分别为Rf5=0.135,Rf9=0.405。用非落叶型菌株DF4、落叶型菌株SY12菌体的可溶性蛋白制备获得两个抗血清。用间接ELISA法检测，即能区分棉花黄萎病菌的立枯、枯萎、炭疽病菌，落叶型和非落叶型黄萎病菌均呈阳性反应，但落叶型黄萎病菌的OD值高于非落叶型黄萎病菌。     

Vegetative Compatibility Groupings of Verticillium dahliae from Cotton in Mainland China

One hundred and fourteen isolates of Verticillium dahliae obtained from cotton and eggplant in mainland China were successfully assigned to two vegetative compatibility groups (VCGs) except for one self-incompatible isolate. Eleven isolates were strongly compatible with T9, the tester strain of the cotton defoliating pathotype, forming a linear growth of wild type with abundant microsclerotia and dense mycelia between compatible nitrate-nonutilizing mutants. The remaining 102 isolates were grouped into the non-defoliating VCG2, although the strength of the reaction varied; some isolates were strongly compatible with the tester strain while others were only slightly compatible. All VCG1 isolates including T9 showed the same defoliating symptom in greenhouse inoculation tests. This study confirmed the presence of the defoliating pathotype (VCG1) of V. dahliae in mainland China.     

土壤中棉花黄萎病菌快速检测技术研究

 Verticillium wilt,caused by Verticillium dahliae,is the most important disease of cotton.In this work,the previously developed defoliating(D) and nondefoliating(ND) V.dahliae-specific primers were adopted for detection of pathotypes of V.dahliae in soil by using nested PCR.The results showed that the detection assay was efficient when used in infested soil and was an useful technique for rapid and accurate assessment of soil contamination by V.dahliae.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

不同来源番茄溃疡病菌致病力差异研究

采用打顶法接种、半选择性培养基再分离发病植株中的病原菌,以及特异性PCR验证方法,对来自3个国家9个不同地区的46株番茄溃疡病菌进行了致病性测定,以病情指数评价不同菌株的致病力。结果显示,分离自我国河北滦平县、内蒙古包头市等地的24株菌株的病情指数达到75以上,属于强致病力水平;11株菌株的病情指数为50~75,属于中等致病力;而9株菌株的病情指数为50以下,属于弱致病力;检测同时证实,有2株属于无致病力菌株。强致病力、中等致病力、弱致病力和无致病力菌株占供试菌株总数的比例分别为52.2%、23.9%、19.6%和4.3%,表明供试的46株番茄溃疡病菌存在不同程度的致病力差异。