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。     

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.     

一种丹参新病害的病原菌鉴定及致病力测定

 调查发现河北省安国市丹参生产区发生一种丹参新病害。为有效防治该病害,开展了丹参新病害病原菌分离鉴定及致病力测定。采用常规组织分离法,从丹参病株中分离并单孢纯化获得15个真菌分离物。柯赫氏法则证明这15个真菌分离物可造成丹参组培苗表现与田间病株相似症状,并分离获得了与丹参病株初分离物菌落形态相同的真菌菌株。显微观察发现15个病株初分离物的分生孢子梗具有明显的轮状分枝,在培养基中能够产生黑色放射状微菌核,据此将罹病丹参分离物确定为轮枝菌属真菌(Verticillium)。rDNA-ITS序列比对发现分离物与大丽轮枝菌(V. dahliae)亲缘关系最近,进一步通过特异性引物扩增证明该分离物为大丽轮枝菌。利用大丽轮枝菌落叶型和非落叶型引物扩增发现,13株分离物为落叶型菌株,2株为非落叶型菌株。人工切根接种鉴定结果表明从丹参分离的轮枝菌菌株间存在致病力差异,不同丹参品种对黄萎病存在抗病性差异。本研究首次报道了由大丽轮枝菌引起的丹参黄萎病,研究结果将为防治丹参新病害提供重要依据。     

大丽轮枝菌核糖体基因ITS区段的特异扩增

 根据棉花黄萎病菌(Verticillium dahliae)核糖体基因ITS区段的碱基编码序列,设计合成了1对为26 bp的PCR特异扩增引物(引物1:5'CATCAGTCTCTCTGTTTATACCAACG,和引物2:3'CGATGCGAGCTGTAACTACTACGCAA),进行了大丽轮枝病菌PCR特异扩增试验。试验结果表明:本试验设计合成的这对引物,能对大丽轮枝菌全基因组DNA和人工接种棉花黄萎病病株组织特异地扩增到大丽轮枝菌核糖体基因ITS区段的324 bp分子片段,该对引物可用于棉花黄萎病的分子鉴定和分子监测。本试验结果对棉花黄萎病的早期诊断和病原菌的检测和监测有潜在的应用价值。     

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。     

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.     

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。     

北方棉区棉花黄萎病菌RAPD分析

以１４个黄萎病菌代表菌系为对照，对来自我国北方棉区的３４个棉花黄萎病菌菌系进行ＲＡＰＤ分析。选用对所有供试菌系都有扩增条带的１４个引物，取其结果中稳定性和多态性均好的６５条谱带作类平均法系统聚类分析，建立树状图。将上述４８个菌系分为４大类，结果表明北方棉区河北、河南、山东的部分棉田存在黄萎病菌落叶型菌系的危害，而且８５．７％的落叶型菌系与对照的美国落叶型菌系Ｔ９、Ｖ４４的亲缘关系比与对照的江苏落叶型菌系Ｖ_Ｂ、Ｖ_９９１更接近。     

新疆北部棉区黄萎病菌种群致病性分化及变异

采用特异性引物PCR检测技术和鉴别寄主法对新疆北部棉区41个棉花黄萎病菌菌系进行检测,以期明确黄萎病菌种群致病性分化及变异。特异性引物(ND1/ND2和D1/D2)PCR检测结果显示,供试菌系中落叶型菌系16个,占39.0%;非落叶型菌系24个,占58.5%;1个菌系未能检测出致病类型。鉴别寄主法测定结果显示:41个供试菌株中致病性强、中、弱的菌系分别为21、12和8个,分别占51.2%、29.3%和19.5%。落叶型菌系的致病力明显高于非落叶型,平均病情指数分别为39.4和25.8。研究表明,无论落叶型菌系和非落叶型菌系,接种后都可产生落叶症状,但落叶的程度有明显差别,其落叶症状的轻重不仅与菌系的致病类型有关,还与品种的抗病性密切相关。     

棉花黄萎病菌ISSR反应体系优化及其遗传多样性分析

为给棉花黄萎病菌分子变异及遗传多样性研究提供可靠的检测方法,以棉花黄萎病菌基因组DNA为模板,采用正交优化方法对PCR体系中DNA聚合酶、引物、dNTPs、DNA模板、Mg2+及10×Buffer等重要参数进行6因素4水平优化,建立了棉花黄萎病菌ISSR-PCR优化反应体系,并从20条ISSR通用引物中筛选出多态性较好的10条引物。采用该优化反应体系和10条ISSR引物对采自陕西棉花主产区的21个棉花黄萎病菌菌株和3个参照菌株进行ISSR分析。结果显示,10条ISSR引物共扩增出87条谱带,条带分子量均在250~2 000 bp之间,平均每条引物扩增出8.7个条带,其中58条为多态性条带,占65.2%。聚类分析结果显示,在相似系数0.59处,供试菌株分为2个遗传类型。表明棉花黄萎病菌菌株间的亲缘关系与地理来源存在一定的相关性,而与其病害症状类型无相关性。     

Correlation of molecular markers and biological properties in Verticillium dahliae and the possible origins of some isolates

Haploid and amphihaploid Verticillium dahliae isolates were studied using PCR-based molecular markers which: (i) discriminate the defoliating and nondefoliating pathotypes (two primer pairs INTD2f/r and INTND2f/r), and (ii) are species-specific (primer pair 19/22). The results were compared with some known biological and other molecular properties of the isolates. Five discrete sequences of the 19/22 amplicon were found. Sequence 4 was associated with both defoliating isolates from Spain and nondefoliating isolates from Spain and USA; these pathotypes were separated by the primer pairs INTD2f/r and INTND2f/r, but the data showed that the primer espdef01 (derived from the 19/22 amplicon) cannot be used for this purpose. Amplicon sizes and sequences with primers 19/22 divided amphihaploid isolates from crucifers (thought to be interspecific hybrids) into those corresponding to the previously reported α and β groups. The β-group isolates had either sequence 4 or 5 (these two differing by a single base). The distinct amplicon sequence 3 given by the α-group isolates demonstrated that the V. dahliae -like 'parent' of this group was molecularly unlike any haploid isolate yet studied. The overall results are discussed in relation to phytosanitary considerations and the probability of defoliating or crucifer pathotypes arising de novo within Europe, either by selection or by interspecific hybridizations.     

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。     

北疆棉花黄萎病发病率调查及土壤中黄萎病菌微菌核数量与种群类型分析

为了解北疆棉花黄萎病的发生情况及发病与土壤中黄萎病菌微菌核数量的关系和病原菌种群类型, 2021年对北疆石河子?奎屯?博乐等8市(县)棉田棉花黄萎病发病率?土壤中黄萎病菌的微菌核数量?菌株种群类型进行了抽样调查?结果表明, 北疆未发生棉花黄萎病的棉田占49.2%, 0%<发病率<5.0%的棉田占32.7%, 发病率≥5.0%的棉田占18%?与2013年?2015年相比, 2021年无病田率分别增加17.7和12.7百分点, 发病率≥5%的棉田分别减少15.7和21.6百分点?从棉田黄萎病发病率与土壤中微菌核数量的关系来看, 整体上北疆棉田棉花黄萎病发病率与微菌核数量相关性不显著(r=0.119 1); 分区域看, 石河子-沙湾片区?奎屯-乌苏片区?精河-博乐片区棉田黄萎病发病率与微菌核数量的相关系数分别为0.033 2?0.007 6?0.062 3, 均无显著相关性; 而呼图壁-玛纳斯片区棉田黄萎病发病率与微菌核数量的相关系数为0.635 7, 呈中度正相关?土壤中的黄萎病菌菌株以菌核型为主, 占57.9%, 菌丝型占23.2%, 中间型占18.9%?用特异性引物进行PCR检测表明, 土壤中黄萎病菌落叶型菌株占97.6%, 占绝对优势?本研究将为北疆棉区棉花黄萎病的综合防控提供理论依据?     

Hyaline mutants from Verticillium dahliae, an example of selection and characterization of strains for host–parasite interaction studies

The objective of the present work was to select stable well-characterized strains of Verticillium dahliae that could be used as biological tools in genetic and plant–microbe interaction studies. Hyaline mutants, known for their stability in pathogenicity were chosen for the study. Diversity in pathogenicity was found among hyaline subclones obtained from a defoliating wild-type clone, but not within those from nondefoliating ones. Most subclones from the defoliating clone had parental pathotypes, but one (V7-2) exhibited weak pathogenicity. This subclone (V7-2), together with a highly virulent one (V7-7) deriving from the same defoliating parent clone (7), were selected for further characterization, because of their differences in pathogenicity. When studied on the basis of their growth requirements, the two subclones expressed marked differences. V7-7 grew better than V7-2 over a wider range of temperature conditions. Both subclones grew similarly in media supplemented with NH₄ as nitrogen source, but in those with NO₃, V7-7 grew more vigorously than V7-2 and only the former could grow when NO₂ was used. In spite of these differences, the two subclones were found to belong to the same vegetative compatibility group, confirming their genetic proximity. These results highlight the physiological and genetic complexity inherent in V. dahliae . In view of their characteristics, the clones obtained in this study should prove to be valuable tools in furthering the understanding of genetic and host– V. dahliae interactions.     

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。