【作者】 朱荷琴；

【导师】 杨家荣；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2013， 博士

【摘要】 采集并分离到我国三大棉区308个棉花黄萎病大丽轮枝菌菌株，采用无底纸钵定量蘸菌液接种法测定了167个代表菌株的致病力，采用特异引物检测了167个供试菌株的落叶型与非落叶型种类，并研究了致病力分化与SSR和ISSR指纹图谱的关系。同时，采用农杆菌介导的遗传转化方法，构建了棉花黄萎病菌强致病力菌株Vd080的T-DNA插入突变体库，成功克隆3个与致病力相关的基因，并对其基因的功能进行初步分析和预测。取得以下主要结果：1.采集分离到的308株大丽轮枝菌菌株，在PDA培养基上产生5种不同的培养类型：A型（0.6%）和B型（72.7%）菌株均产生大量的微菌核，A型有黑色菌丝，B型菌落边缘有白色圆环；C型菌株产生的微菌核较少，大多分布在菌落周围，形成黑色圆环（23.7%）；D型（1.6%）和E型（0.1%）菌株在室温下不产生微菌核，E型菌株的菌落有明显凸起。其中，B型菌株为优势类群。长江流域棉区来源菌株的培养类型多样性丰富，其次是黄河流域棉区，新疆内陆棉区的最小。2.对棉花黄萎病菌167个代表菌株的致病力测定结果表明，参试菌株的致病力分化明显，可划分为强（平均病指为53.4~71.5）、中（平均病指为20.9~51.7）、弱（平均病指为6.4~27.4）3个致病力类型，中等致病力类型的菌株占测试菌株的57.5%。黄河流域棉区和长江流域棉区的菌株致病力分化趋势一致，致病力水平也比较接近，均以中等致病力类型的菌株为优势类群，新疆棉区的菌株以弱致病力类型为优势类群，致病力水平较低。3.大丽轮枝菌落叶型菌株特异引物D-1/D-2和非落叶型菌株特异引物ND-1/ND-2对167个菌株的检测结果表明，参试菌株中落叶型菌株占91.0%，分布于我国的各产棉地区。落叶型菌株多数为中等和强致病力类型，非落叶型菌株多数致病力较弱。温室生物测定结果表明，非落叶型菌株和落叶型菌株均能导致棉苗落叶，只是落叶型菌株出现落叶的时间早3~6天，且落叶株率高。在接种浓度为1×10~7孢子/ml时，落叶型菌株的落叶株率显著高于非落叶型菌株，但增加接种浓度能显著提高非落叶型菌株的落叶株率，当接菌量达到1×10~8孢子/ml时，落叶型菌株和非落叶型菌株之间差异不明显。表明非落叶型菌株导致棉花落叶需要的时间较长、接种剂量较大。4.依据莴苣黄萎病菌（V. dahliae）全基因组开发SSR标记，并根据侧翼序列设计高特异SSR引物，筛选出13对多态性较高的引物。SSR图谱的聚类分析结果将供试167个菌株划分为2个群体，群体Ⅰ以中等和强致病力类型为主，占84.4%；群体Ⅱ以弱致病力类型为主。9对ISSR引物对棉花黄萎病菌扩增图谱将供试菌株划为强致病力、中等致病力和弱致病力3个群体，中等致病力群体为优势类群。供试菌株的SSR和ISSR指纹图谱均与黄萎病菌的致病力存在明显相关性，致病力水平相近的菌株亲缘关系比较近。5.以强致病力落叶型菌株Vd080为初始菌株，利用农杆菌介导遗传转化（ATMT）的方法，成功构建了容量为2000个转化子的棉花黄萎病菌T-DNA插入突变体库。最佳转化体系是采用新鲜的农杆菌菌液和棉花黄萎病菌孢子悬浮液，农杆菌OD600为0.4左右，诱导剂乙酰丁香酮（AS）浓度为200μM，25℃共培养48h，转化效率可达150～540个转化子/106个孢子。对随机挑取的突变体检测表明，T-DNA成功插入到棉花黄萎病菌Vd080中，且多为单拷贝，突变体菌株能够稳定遗传。6.从突变体库中筛选到3株生物学性状或致病力发生显著变异的突变体。VdT286的变异主要表现为丧失微菌核产生的能力、分生孢子产量骤减、菌落生长速率显著降低、致病力略有下降。VdT1023和VdT1053均表现致病力显著降低，Vd1053丧失微菌核产生的能力，VdT1023还表现粗毒素产量显著增加。结合TAIL-PCR技术，对上述3个突变体中T-DNA侧翼序列扩增，成功克隆与棉花黄萎病菌生物学性状或致病力等重要性状相关的基因3个。VdT286中T-DNA插入到了CYC8（葡萄糖阻遏蛋白）上， CYC8是一个与分生孢子产生、菌丝生长及微菌核形成相关的基因，全长为3201bp，位于第五条染色体上，包括7个外显子和6个内含子，cDNA的长度为2679bp，编码892aa，CYC8在大丽轮枝菌中的相关功能目前还未见报道。VdT1023中T-DNA插入到了VDAG00467的上游启动子区，该基因位于第二条染色体上，基因长度为1152bp，无内含子，编码383aa，是一个尚未进行注释功能的未知新基因。VdT1053中T-DNA破坏了VDAG00607，分析得知该基因位于第二条染色体上，基因长度为2053bp，cDNA长度为1947bp，有一个内含子，编码648aa。编码产物是磷酸甘油变位酶（phosophoglyceratemutase），目前关于磷酸甘油变位酶的研究报道较少。更多还原

【Abstract】 308Verticillium dahliae isolates were recovered from three cotton-planting regions inChina and of which, pathogenicity of167isolates was identified using a method namedvermiculate and sand in bottomless paper pot dipping in quantitative spores. The number ofdefoliating and non-defoliating isolates was also tested based on specific deofliating andnon-defoliating primers. Correlation between fingerprint of ISSR and ISSR and thepathogenicity was analysised. And a mutant library, which initiated from a high virulent V.dahliae strain Vd080, was constructed by agrobacterium tumefaciens-mediatedtransformation (ATMT). Three genes related to biological characteristics and pathogenicitywere cloned and analyzed. The main conclusions showed as fllowing:1. According to cultural characteristics on potato dextrose agar (PDA),308V. dahliaeisolates were divided into five culture types (A-E). Both type A (0.6%) and type B (72.7%)produced massive microsclerotia, moverover, type A produced black mycelia while there wasa white circle at the colony edge of Type B. Type C (23.7%) produced a few microsclerotiadistributed at the edge of colony which formed a black circle. Both type D (1.6%) and type E(0.1%) did not produce microsclerotia under room temperature, in addition, there were raisedaerial mycelium at the colony surface of type E Among them, Type B was the dominant groupaccounted for72.9%. The cultural characteristics of isolates from Yangtze River regionshowed highest variation, followed by those from Yellow River and Xinjiang.2. Based on the results of the pathogenicity tests,167V. dahliae isolates were clusteredinto three groups, which showed strong (average disease index varied53.4~71.5), moderate(average DI varied20.9~51.7%) and weak (average DI varied6.4~27.4) pathogenicity oncotton, respectively. Moderate virulent isolates distributed dominantly in China (57.5%).Differentiation tendency of isolates recovered from Huanghe Basin cotton-planting region andYellow River Basin showed consistently. Isolates exhibited similarly pathogenicity. Amongthem, moderate pathogenic isolates belonged to the dominant group. However, in Xinjiang,the number of weak pathogenic isolates was largest. 3. Among167tested isolates,91%of them were identified as defoliating V. dahliae withthe specific deofliating and non-defoliating primers (D-1/D-2, ND-1/ND-2), which widelydistributed main cotton producing regions. Most of the defoliating isolates belonged to strongand moderate pathogenicity groups. Howerver, The majority of non-defoliating isolatesexhibited weak pathogenicity. The results of greenhouse tests showed that bothnon-defoliating and defoliating isolates could lead to the leaf-defoliation of cotton seedlings.Compared with non-defoliating isolates, the cotton seedlings inoculated with defoliatingisolates showed defoliation3-6days earlier and the defoliating percentage significantly higher.Under the inoculating concentration of1×10~7spores per milliliter, the defoliating percentageof cotton seedlings inoculated with defoliating isolates was significantly higher than thoseinoculated with non-defoliating isolates. With the increasing of inoculating concentration, thedefoliating percentage of cotton seedlings inoculated non-defoliating isolates obviouslyincreased. When the inoculating concentration reached to1×10~8spores per milliliter, there wasnot distinct difference between cotton seedlings inoculated with non-defoliating anddefoliating isolates. The above results showed that non-defoliating isolates needed longerperiod and higher inoculating concentration to cause the defoliation of cotton leaves.4. SSR markers were explored from the genome database of V.dahliae on lettuce. Highspecific primers were designed based on the flanking sequence of SSR.13pairs of SSR withhigher polymorphism were selected for molecular fingerprint analysis. The results showedthat167tested isolates of V. dahliae from China were distinctly classified into two groups.GroupⅠcontained141isolates, of which，strong and moderate pathogenicity isolatesaccounted for89.4%；GroupⅡcontained26isolates, of which，84.6%of the isolates wereweak pathogenicity. Nine pairs of ISSR primer were used to analyze the genetic diversity of167tested isolates of V. dahliae from China. Clustering analysis results showed that167tested isolates were classified into strong, medium and weak three different pathogenicitygroups (strong, moderate and weak). There were17(10.2%),123(73.7%) and27(16.2%)isolates in strong, moderate and weak pathogenicity groups respectively. The study indicatedthat there was a significant correlation between fingerprint of ISSR and ISSR and thepathogenicity. Genetic relationship among isolates with similar pathogenicity was relativelyclose.5. A mutant library containing2000mutants of a high virulent and deofliating V. dahliaestrain Vd080was constructed by the ATMT. Fresh A.tumefaciens cells with OD600≈0.4, freshV. dahliae spore suspension,200μM Acetosyringone and48hours co-cultivation period at25℃were optimizing conditions. Mutant ratio could reach150~540transformants per106spores under the optimizing conditions.The results of PCR and Southern blot indicated thatT-DNA inserted into the genome of Vd080successfully, and T-DNA in mutants selected randomly were mostly single copy. All of the tested transformants maintained their resistanceto hygromycin B.6. Three mutants, with significant variation in biological characteristics andpathogenicity compared with wild strain Vd080, were screened from the mutantlibrary.VdT286showed significantly lower spore yield, growth rate and non-microsclerotia.In addition, its pathogenicity decreased slightly. Both VdT1023and VdT1053exhibitedsignificantly lower pathogenicity. Vd1053did not produce microsclerotia and the secretioncrude toxin of VdT1023increased significantly. Using TAIL-PCR, three genes related tobiological characteristics and pathogenicity of V. dahliae were cloned by amplying flankingsequence of inserted T-DNA. In VdT286, T-DNA was inserted into CYC8(glucose repressionmediator protein)，which was a key gene in the control of spore yield, growth rate of myceliaand microsclerotia producing of V. dahliae. It was located in the fifth chromosome with3201bp in full length and contained7exons and6introns. The length of its cDNA was2679bpcoding892aa. The function of CYC8was still not reported in V. dahliae yet. In VdT1023,T-DNA was inserted upside promoter region of VDAG00467, which was a new unknown genewithout annotation. It was located in the second chromosome with1152bp in full lengthwithout intron, Coding protein contained383aa. In VdT1053, T-DNA disrupted VDAG00607,which was located in the second chromosome with1947bp in full length and contained1intron. Its coding product was phosophoglycerate mutase containing648aa.更多还原