苦瓜枯萎病菌的鉴定及其遗传多样性分析

 对分离获得的32株苦瓜枯萎病菌菌株进行形态学特征和寄主专化型测定, 结果表明, 测试的苦瓜枯萎病菌株均为尖孢镰刀菌苦瓜专化型 (Fusarium oxysporum f. sp. momordicae), 这些菌株可以侵染苦瓜和瓠瓜幼苗, 但不侵染其他葫芦科瓜类作物。对苦瓜枯萎病菌菌株的rDNA-ITS区 (ITS1、5.8S和ITS2)序列进行扩增测序, 结果显示其序列长度均为456 bp;聚类分析表明测序菌株与镰刀菌属中尖孢镰刀菌不同专化型的菌株聚为一群。利用RAPD标记技术分析苦瓜枯萎病菌的遗传多样性, 结果显示苦瓜枯萎病菌株与其他葫芦科瓜类作物枯萎病菌株间的遗传相似系数范围为0.59~0.99, 当遗传相似系数为0.85时, 供试的48个菌株分成10个类群 (G_1~10)。在RAPD聚类树中所有苦瓜枯萎病菌株聚在一个分支上 (G₁群), 菌株间的遗传相似系数范围为0.92~1.00, 具有较高的遗传相似性, 且菌株的聚群与地理来源存在一定的相关性。     

大豆枯萎病菌尖孢镰孢遗传多样性及大豆品种抗性

 了解大豆枯萎病菌的群体遗传特征及明确大豆种质对大豆枯萎病的抗性,对抗病育种、抗性品种的合理布局以及制定更有效的病害防治策略具有重要的参考价值。本研究利用随机扩增多态性DNA(random amplified polymorphic DNA,RAPD),对采自我国不同地区的大豆枯萎病菌—尖孢镰孢菌(Fusarium oxysporum)进行遗传多样性分析,筛选到10个多态性随机引物,共扩增出75条RAPD条带,其中55条为多态性条带,占73.3%。利用UPGMA法对DNA扩增图谱进行聚类分析,以相似系数0.68为阈值,55个分离物可分为9个遗传聚类组,表明我国大豆枯萎病菌具有丰富的种内遗传多样性,所划分的群体与分离物来源地不相关。同时,对上述分离物进行致病性分析,发现我国的大豆枯萎病菌具有明显的致病力分化现象。进一步利用3个代表性分离物对来自我国不同大豆产区的180个大豆品种(资源)进行抗大豆枯萎病鉴定,发现皖豆28、中黄13、中黄51、中作X08076和5D034等5个品种对大豆枯萎病具有良好抗性,占供试材料的2.8%,表明不同大豆品种对枯萎病的抗性存在一定的差异。     

香蕉枯萎病菌RAPD分析及4号生理小种的快速检测

 用随机扩增多态性DNA(RAPD)技术,对采自广东、广西的香蕉和粉蕉上的30个香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense)菌株和3个其它尖孢镰刀菌专化型的菌株进行比较及聚类分析。在遗传相似系数0.67时,可将供试菌株划分为3个RAPD群(RGs),其中香蕉枯萎病菌4号生理小种(FOC4)共15个菌株属于RGⅠ,1号生理小种(FOC1)共15个菌株属于RGⅡ,供试的其它尖孢镰刀菌专化型的3个菌株则属于RGⅢ。这说明香蕉枯萎病菌和供试3个其它专化型菌株与致病性间存在明显的相关性。1号生理小种内菌株间的遗传分化大于4号生理小种内菌株间的遗传分化。从90条RAPD随机引物中筛选出2条引物可产生4号生理小种的RAPD标记2个。将这2个RAPD标记电泳切胶回收、克隆及测序,并根据这2个特异片段序列设计SCAR上下游特异引物,通过对30个菌株的PCR扩增检验,其中一个RAPD标记成功地转化为SCAR标记,初步建立了以此为基础的4号生理小种快速检测技术,其检测灵敏度为2 ng新鲜菌丝。对采自不同地区的显症样品、吸芽、室内接种未显症的香蕉苗以及发病的香蕉植株不同部位进行检测,能够准确灵敏地鉴定出4号生理小种,从而为香蕉枯萎病菌的快速检测及防治奠定了基础。同时,快速检测结果发现,田间发病植株果柄的各部位及果实内并没有枯萎病菌的存在。     

香蕉枯萎病菌致病力分化与ISSR遗传多样性分析

香蕉枯萎病是一种破坏香蕉维管束的全株性土传病害。本研究旨在探讨香蕉枯萎病菌致病力分化和遗传多样性。以30株采自我国广西的香蕉枯萎病菌,16株分别来自澳大利亚和我国广东、海南、福建、云南等地的香蕉枯萎病菌为对象,采用伤根灌淋法测定香蕉枯萎病菌的致病力,然后用筛选到的ISSR引物对46个香蕉枯萎病菌菌株和4个对照菌株(3个非致病性尖孢镰刀菌和1个茄腐镰刀菌)进行ISSR遗传多样性分析。结果显示,分离到广西香蕉枯萎病菌1号生理小种(FOC1)8株,致病力强、中、弱类型比例分别为62.5%、12.5%和25%;广西香蕉枯萎病菌4号生理小种(FOC4)22株,致病力强、中、弱类型比例分别为18.18%、63.64%和18.18%。14条ISSR引物扩增出237个条带,多态性条带161个,多态性比例为67.93%,遗传相似系数0.76～0.96。聚类分析显示,以遗传距离0.80为阈值时菌株被分为8个类群,所占比例分别为4%、10%、60%、16%、4%、2%、2%、2%。第三类群全部为香蕉枯萎病菌4号生理小种。第一、二、四和五类群总量的70.59%为香蕉枯萎病菌1号生理小种。第八类群为香蕉枯萎病菌3号生理小种。结果表明,在香蕉枯萎病菌与寄主协同进化中,广西的FOC1和FOC4出现明显致病力分化。1号生理小种的遗传多样性比4号生理小种丰富。广西香蕉枯萎病菌4号生理小种与海南、广东的FOC4遗传相似性较高。香蕉枯萎病菌生理小种类型与遗传多样性相关。致病力变异与遗传多样性无相关性。研究结果对香蕉枯萎病菌种群扩张机制探讨、遗传动态分析以及有效防控措施的制定具有一定的指导意义。     

新疆棉花枯萎病菌群体结构的研究

 采自新疆24个不同植棉县(市或团场)的37株棉花枯萎病菌代表菌株,经人工接种于国际通用鉴别寄主,致病性反应均表现为典型的7号生理小种特征。RAPD分析结果也显示出这37个供试菌株与7号小种各对照菌株间基因组DNA的指纹图谱高度相似,属同一遗传相似组,而与3号和8号小种的对照菌株间遗传差异较大,亲缘关系较远,即7号生理小种是组成目前新疆棉花枯萎病菌群体的优势小种,而原分布于新疆吐鲁番等地的3号小种在本研究中未被发现。结合部分自选辅助鉴别寄主对其中18个菌株进行的致病力分化研究表明,在7号小种内部还存在着侵染力的分化,显示出棉花枯萎病菌较强的变异性和适应性。     

杂草和水稻梨孢菌亲缘关系研究

利用12个引物的随机扩增多态性DNA标记(RAPD)对来自18种寄主的49个梨孢菌株进行了亲缘关系分析,从DNA电泳谱带中可以直观地看出,来自同一种或同一属或亲缘关系相近寄主上的梨孢菌带型较相似,相反,亲缘关系较远的寄主上的梨孢菌带型差异较大,茭白、碎米莎草和马唐寄主上的梨孢菌的谱带均非常特异,与牛筋草和狗尾草等其他15种寄主的梨孢菌有较大差异,牛筋草和狗尾草等15种寄主的梨孢菌存在比较多的共有谱带.对引物S380扩增的RAPD谱带进行聚类分析表明,18种寄主上的49个梨孢菌株可分为5个类群.种与种之间的DNA(RAPD谱带)电泳谱带差异非常明显,而种内品种间的带型差异比较小.     

稻曲病菌遗传多样性与群体结构的初步分析

 利用随机扩增多态性DNA (random amplified polymorphic DNA,RAPD)初步分析了稻曲病菌(Ustilaginoidea virens)的群体遗传结构。从1 60个随机引物中筛选32个扩增带型清晰、重复性好的引物,对不同年份采自辽宁、云南、湖北和浙江等水稻种植区的5 6个菌株进行扩增。32个引物扩增出2 2 3条带,绝大多数引物对不同年度采自不同稻区的菌株扩增的DNA谱型相同,大多数菌株间相似性系数达0.80以上。根据扩增DNA片段的多态性,从空间分布来看,来源于北方、长江流域和南方的菌株难以划分出明显的地理宗谱;不同年度的菌株DNA多态性也无明显的差异。上述结果初步表明稻曲病菌遗传稳定,寄主选择作用(寄主的基因型及其时空分布)对稻曲病菌变异的影响较小。但是尚需采用其它的分子技术测试更多的菌系,才能较系统地分析我国稻曲病菌系的遗传变异及群体结构特点。     

玉米弯孢叶斑病菌生理分化和分子生物学研究

 本论文采用同工酶电泳技术、鉴别寄主鉴定技术、基因组随机多态性扩增技术(RAPD)及人工诱变,从寄主一病原互作角度研究了玉米弯孢叶斑病菌的遗传、变异与致病性分化,明确了该病菌存在致病性分化和生理分化,并从遗传物质DNA水平证实病菌致病性分化并非是表型差异,而是一种基因的分化,具有较稳定的遗传基础。     

运用随机扩增多态性DNA标记检测中国东北大豆灰斑病菌株遗传变异

 运用致病力和DNA多态性检测中国东北地区的35个大豆灰斑病菌分离物的遗传变异、根据菌株在9个品种(系)上的致病力反应可将其分为7个组。利用13个随机引物扩增供试菌株共计产生105个RAPD标记,其中78.1%具有多态性。通过聚类分析计算了各菌株间的遗传距离,并产生树状图,发现同一地区内及不同地区间的病菌表现遗传变异、致病性和DNA多态性间具有一定相关性。     

贵州省烟草黑胫病菌遗传多样性分析

运用RAPD分子标记技术分析了采自贵州各个地区的38个烟草黑胫病菌株的遗传多样性,探索了贵州省主要烟草种植区间的烟草黑胫病病菌的遗传分化关系.结果表明,受试38个菌株共产生65条谱带.其中多态性为59条,占90.7％.说明贵州省主要烟草种植区的烟草黑胫病病菌具有丰富的遗传多样性,不同地点烟草黑胫病病菌的遗传结构之间都有一定的差异.根据引物扩增的指纹图谱,运用UPGMA分析法,以遗传相似系数0.74,将供试38个菌株划分为9个基因型,发现除少数地区的菌株被划分为同一基因型外,其他不同地理区域的菌株基因型的划分与地理来源没有直接的相关性.     

Genetic Variability and Host Specialization in the Latin American Clade of Ceratocystis fimbriata

ABSTRACT The Ceratocystis fimbriata complex includes many undescribed species that cause wilt and canker diseases of many economically important plants. Phylogenetic analyses of DNA sequences have delineated three geographic clades within Ceratocystis fimbriata. This study examined host specialization in the Latin American clade, in which a number of lineages were identified using sequences of the internal transcribed spacer (ITS) region of the rDNA. Three host-associated lineages were identified from cacao (Theobroma cacao), sweet potato (Ipomoea batatas), and sycamore (Platanus spp.), respectively. Isolates from these three lineages showed strong host specialization in reciprocal inoculation experiments on these three hosts. Six cacao isolates from Ecuador, Trinidad, and Columbia differed genetically from other cacao isolates and were not pathogenic to cacao in inoculation tests. Further evidence of host specialization within the Latin American clade of Ceratocystis fimbriata was demonstrated in inoculation experiments in growth chambers using sweet potato, sycamore, Colocasia esculenta, coffee (Coffea arabica), and mango (Mangifera indica) plants; inoculation experiments in Brazil using Brazilian isolates from cacao, Eucalyptus spp., mango, and Gmelina arborea; and inoculation experiments in Costa Rica using Costa Rican isolates from cacao, coffee, and Xantho-soma sp. Hosts native to the Americas appeared to be colonized by only select pathogen genotypes, whereas nonnative hosts were colonized by several genotypes. We hypothesize that local populations of Ceratocystis fimbriata have specialized to different hosts; some of these populations are nascent species, and some host-specialized genotypes have been moved to new areas by humans.     

Ceratocystis species infecting stem wounds on Eucalyptus grandis in South Africa

Reports of Ceratocystis spp. causing disease of exotic plantation hardwood species have increased in recent years. Ceratocystis fimbriata causes wilt and canker on Eucalyptus spp. in Africa and South America, and C. albofundus results in wilt and death of Acacia mearnsii in Africa. Ceratocystis spp. generally infect wounds on trees, and artificial stem wounding can thus be used to determine the presence of these fungi. The aim of this study was to identify Ceratocystis spp. infecting wounds on Eucalyptus grandis in South Africa. Isolated Ceratocystis spp. were identified using morphological characteristics and comparisons of DNA sequence data for the ITS and 5·8S regions of the rRNA operon. Pathogenicity trials were conducted in the greenhouse to determine the possible role that these Ceratocystis spp. could have in disease development. These trials were also conducted under field conditions. Three Ceratocystis spp. were collected: C. fimbriata , C. moniliformis and C. pirilliformis . This is the first report of C. fimbriata and C. pirilliformis from Eucalyptus spp. in South Africa, and the first report of the latter fungus outside Australia. Both C. fimbriata and C. pirilliformis caused significant lesions on inoculated E. grandis trees. This is the first evidence that C. pirilliformis is a pathogen of Eucalyptus spp. From the results of both greenhouse and field trials, it has the potential to cause serious disease problems in Eucalyptus plantations.     

Pathogenicity, Internal Transcribed Spacer-rDNA Variation, and Human Dispersal of Ceratocystis fimbriata on the Family Araceae

ABSTRACT Ceratocystis fimbriata is a complex of many cryptic, host-specialized species that causes wilt and canker of woody species and rot diseases of storage roots and corms of many economically important plants worldwide. With the exception of the family Araceae, all confirmed hosts of C. fimbriata are dicotyledonous plants. We hypothesized that the isolates from members of the family Araceae would form a monophyletic lineage specialized to infect these unique hosts. Analyses of sequences of the internal transcribed spacer (ITS) region of nuclear rDNA indicate that isolates and herbarium specimens of C. fimbriata from the family Araceae represent three different groups: an Xanthosoma/Syngonium group on corms of Xanthosoma spp. from the Caribbean region and on ornamental S. podophyllum from greenhouses in Florida, Hawaii, Australia, and Brazil; an inhame group on corms of Colocasia esculenta in Brazil; and a distantly related taro group on Colocasia esculenta in Hawaii and China and on X. sagittifolium in Fiji. Inoculations of three species of Araceae (Caladium bicolor, S. podophyllum, and Colocasia esculenta) showed that isolates from all three groups are pathogenic to these three hosts. Brazilian isolates from Mangifera indica and Ficus carica were only weakly pathogenic to Caladium and Syngonium sp. and were not pathogenic to Colocasia sp. Syngonium plants appeared to be most susceptible to isolates of the Xanthosoma/Syngonium group, and Colocasia plants were least susceptible to isolates from Syngonium spp. Thus, it appears that adaptations to the family Araceae have evolved more than once in the C. fimbriata complex. It is hypothesized that the three groups of C. fimbriata on the family Araceae are native to the Caribbean, Brazil, and Asia, respectively, but they have been spread elsewhere by humans.     

Genetic variation in populations of the cacao wilt pathogen, Ceratocystis cacaofunesta

Ceratocystis cacaofunesta (=  Ceratocystis fimbriata ) causes a lethal wilt disease of cacao ( Theobroma cacao ) in Latin America. Polymorphic microsatellite markers, (CAT)₅ nuclear DNA fingerprints and Hae III mitochondrial DNA fingerprints were used to compare genetic diversity among isolates of C. cacaofunesta collected from populations in western Ecuador, Costa Rica, Colombia, and Rondônia and Bahia in Brazil. Microsatellite markers and nuclear DNA fingerprints separated Ecuadorian isolates from isolates of the other four populations, and these two major groups correspond to genetic lineages already identified from ITS-rDNA sequences and intersterility groupings. Mitochondrial DNA fingerprints also demonstrated substantial diversity and split the Ecuadorian isolates into two groups. All marker types showed limited variation in the Colombian, Costa Rican and Bahian populations, as might be expected for introduced populations that have gone through recent genetic bottlenecks. In contrast, the Rondonian and western Ecuadorian populations showed gene diversity values similar to natural populations of other Ceratocystis species. The Rondonian population was the only sampled population in the native range of T. cacao (the Upper Amazon), and the putatively introduced populations were more closely related to the Rondonian population than to the western Ecuadorian population. The Ecuadorian population is in an area with other native Theobroma species, which may serve as natural hosts.     

Movement of genotypes of Ceratocystis fimbriata within and among Eucalyptus plantations in Brazil

Ceratocystis wilt on eucalyptus, caused by Ceratocystis fimbriata, was first recognized in 1997 in the state of Bahia, Brazil, but is now known in five other states and in four other countries. C. fimbriata is a native, soilborne pathogen in some parts of Brazil but we hypothesized that genotypes of the pathogen have been moved among plantations in rooted cuttings collected from diseased trees and within plantations on cutting tools. We used six microsatellite markers to identify 78 genotypes of C. fimbriata among 177 isolates from individual trees in 20 eucalyptus plantations. The highest gene and genotypic diversity values were found in plantations on formerly wild Cerrado forest in Minas Gerais, suggesting that the fungus was in the soil prior to planting eucalyptus. In contrast, one or only a few genotypes were found in plantations on previous pastureland (with no woody hosts) in Bahia and S?o Paulo, and most of these genotypes were found in a Bahian nursery or in one of two Bahian plantations that were sources for rooted cuttings. Sources of cuttings tended to be dominated by one or a few genotypes that may have been spread within the plantation on cutting tools.     

国内一种果树新病害--石榴枯萎病

2001年在云南省蒙自县相继发生石榴枯萎病，经作者分离鉴定以及致病性测定，证明病害是由甘薯长喙壳Ceratocystis fimbriata Ellis and Halsted引起的，为国内果树新病害。对病害的症状、鉴定方法、病原菌形态及发生危害情况进行了描述。     

Genetic variation and variation in aggressiveness to native and exotic hosts among Brazilian populations of Ceratocystis fimbriata

Ceratocystis fimbriata is a complex of many species that cause wilt and cankers on woody plants and rot of storage roots or corms of many economically important crops worldwide. In Brazil, C. fimbriata infects different cultivated crop plants that are not native to Brazil, including Gmelina arborea, Eucalyptus spp., Mangifera indica (mango), Ficus carica (fig), and Colocasia esculenta (inhame). Phylogenetic analyses and inoculation studies were performed to test the hypothesis that there are host-specialized lineages of C. fimbriata in Brazil. The internal transcribed spacer region ribosomal DNA sequences varied greatly but there was little resolution of lineages based on these sequences. A portion of the MAT1-2 mating type gene showed less variation, and this variation corresponded more closely with host of origin. However, mango isolates were found scattered throughout the tree. Inoculation experiments on the five exotic hosts showed substantial variation in aggressiveness within and among pathogen populations. Native hosts from the same families as the exotic hosts tended to be less susceptible than the cultivated hosts, but there was little correlation between aggressiveness to the cultivated and native hosts of the same family. Cultivation and vegetative propagation of exotic crops may select for strains that are particularly aggressive on those crops.     

Ceratocystis wilt of cacao-a disease of increasing importance

ABSTRACT Ceratocystis cacaofunesta (formerly C. fimbriata) causes a lethal wilt disease of cacao (Theobroma cacao) in the Caribbean and Central and South America. Recent studies employing phylogenetics, intersterility, and host range separate the cacao pathogen from other strains of the C. fimbriata complex. Ceratocystis wilt has been managed through genetic resistance, but the disease is an emerging problem in Bahia, Brazil, where it was recently introduced. Genetic studies indicate that populations of the fungus in Costa Rica, Colombia, and Bahia may have been introduced on cacao cuttings; whereas populations in Rond?nia, Brazil, and western Ecuador appear to be native. The fungal genotype present in Bahia is similar to those found in Rond?nia and may have been introduced on propagative material with witches' broom resistance.     

甘薯品种对黑斑病菌的抗性与抗氰呼吸

选用对黑斑病菌具有不同抗感程度的甘薯品种(R:85-2458,S:85-180)块根组织为材料。研究了不同甘薯品种对黑斑病菌的抗性与抗氰呼吸、乙烯释放量、过氧化物酶活性的相互关系。结果表明,不同抗感材料接种后的抗氰呼吸活性明显高于伤害组织(对照)。抗病品种在接种和伤害情况下,其抗氰呼吸活性明显高于感病品种,而且持续时间较长。乙烯释放量、过氧化物酶活性与抗氰呼吸的变化趋势相一致。甘薯品种块根组织对黑斑病菌的抗性与抗氰呼吸活性及对总呼吸的贡献大小有关。     

Comparison of populations of the wilt pathogen Ceratocystis albifundus in South Africa and Uganda

Ceratocystis albifundus is an important fungal pathogen of Acacia mearnsii trees in South Africa. In a previous study, a high level of gene diversity was demonstrated in a South African population of C. albifundus . This, together with the occurrence of the pathogen on native Protea species and its exclusive occurrence in South Africa, led to the hypothesis that C. albifundus is probably native to that country. More recently, C. albifundus has been reported from A. mearnsii in south-western Uganda. The aim of this study was to compare the populations of C. albifundus from Uganda and South Africa based on genetic diversity, population structure and possible gene flow. This was achieved using codominant microsatellite markers developed for the closely related species Ceratocystis fimbriata . Available isolates for comparison were from six different areas of South Africa and six jungle stands in Uganda. Eight of the 11 available markers amplified loci in C. albifundus . Gene diversity was higher in the Ugandan population, but genotypic diversity was greater for the South African isolates. There were no common genotypes between the two populations and they shared only 22% of the total alleles. The populations were genetically isolated from each other and highly substructured within. There was no association between isolates collected from the same geographic locations, and gene flow between the two populations was low. Results suggest that C. albifundus was probably not introduced into Uganda from South Africa but rather that an ancestral population, yet to be discovered, is the source of both populations.