一种评价甘蓝枯萎病菌转化子致病力的方法

为建立甘蓝枯萎病菌(Fusarium oxysporumf.sp.conglutinans)转化子致病力评价方法,从接种方法、甘蓝品种、苗龄、病原菌接种体浓度等几方面探索,建立了一种评价甘蓝枯萎病菌转化子致病力差异的体系。结果表明:蘸根法和伤根法均适于甘蓝枯萎病菌转化子致病力的评价,其中伤根法更优;其他适合甘蓝枯萎病菌转化子致病力评价的因素有:甘蓝品种为‘中甘21’,苗龄为三叶期,甘蓝枯萎病菌孢子悬浮液浓度为孢子含量1×106个/mL。该致病力评价方法的建立为甘蓝枯萎病菌转化子致病力衰弱或增强突变体的筛选提供了方法支持,也为下一步尖孢镰刀菌致病机理的解析奠定了基础。     

不同抗性黄瓜品种连作对枯萎病菌群体遗传分化的影响

尖孢镰刀菌黄瓜专化型(Fusarium oxysporum f. sp.cucumerinum,Foc)是引起黄瓜枯萎病的病原真菌,为了明确不同抗性黄瓜品种连作对枯萎病菌遗传分化的影响,本研究采用TEF1-α、H3基因片段对不同抗性黄瓜品种连作及对轮作后分离的枯萎病菌群体进行遗传分化分析。结果表明,感病品种连作及轮作后菌群分化程度较低(0.007ST<0.045);与种植抗病品种一茬后的菌群相比,连作三、五茬后的菌群均发生显著分化(F_ST> 0.050),其中连作三茬后的菌群遗传分化程度最高(F_ST> 0.300)。单倍型分析发现,抗、感病品种连作后部分菌株形成新的单倍型,且分离自抗病品种的病原菌群体分化产生的单倍型数量多于感病品种。主成分分析结果进一步证明,与感病品种连作及轮作相比,抗病品种连作后病原菌菌群呈现出更显著的菌群分化。综上所述,抗病品种连作能够加剧黄瓜枯萎病菌群体的遗传分化。     

甘蓝枯萎病菌1号和2号生理小种的快速检测与鉴定

 甘蓝枯萎病菌生理小种传统鉴定方法费时费力,不能满足生产的要求,因此需要建立一种快速、可靠的分子检测技术。本研究在甘蓝枯萎病菌1号和2号生理小种基因组测序的基础上,通过比较基因组学方法筛选1、2号生理小种各自的特异基因片段并设计引物,并分别以10个甘蓝枯萎病菌1号生理小种菌株、2个2号生理小种菌株、7个尖孢镰刀菌其他专化型菌株及4个外围菌株DNA为模板进行常规PCR扩增,筛选出甘蓝枯萎病菌1号和2号生理小种特异性引物,同时引入尖孢镰刀菌通用引物W106R/W106S,建立起一步三重PCR检测甘蓝枯萎病菌1、2号生理小种的分子检测技术。结果表明,该分子检测技术实现了在一次PCR反应中快速、准确地同步检测出甘蓝枯萎病菌DNA、罹病甘蓝组织和土壤中的甘蓝枯萎病菌1号和2号生理小种,对检测甘蓝植株是否感染枯萎菌及甘蓝种植区土壤是否受到枯萎菌的污染有实用价值。     

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。     

利用香蕉水培系统测定枯萎病病原菌致病力

 为建立一种评价香蕉枯萎病病原菌(Fusarium oxysporum f.sp. cubense)菌株间致病力差异的体系,在改进的香蕉水培系统基础上,对影响香蕉枯萎病菌致病力的接种菌液浓度、类型及处理方式等因素开展分析,同时与不同菌株盆栽测定结果进行比较以验证该方法的可靠性。结果表明:在香蕉水培系统下,将摇培5 d的菌液稀释10倍以上,使孢子初始浓度为1×10⁶ cfu·mL^-1,直接加入该菌液50 mL即可用于致病力评价,且不同菌株用该测定方法与盆栽测定的致病力结果基本一致。该方法的建立为香蕉枯萎病菌致病力的评价提供了快速简便的方法,也为下一步解析尖孢镰刀菌致病相关基因功能和致病机理及抗病品种的选育奠定了基础。     

常熟地区蚕豆枯萎病病原菌鉴定及其致病力初探

2000、2001年在江苏省常熟地区采集了有典型枯萎症状的蚕豆标样各50个,获94个镰刀菌单孢菌株。经鉴定分别属于尖孢镰孢(Fusarium axysporum)、燕麦镰孢(F.avenaceum)、串珠镰孢(F.moniliforme )、木贼镰孢(F.equiseti)、三线镰孢(F.tricinctum)、禾谷镰孢(F.graminearum)和茄镰孢(F.solani),其中尖孢镰孢、燕麦镰孢、串珠镰孢、木贼镰孢为该地区蚕豆镰刀菌枯萎病的主要病原菌。测定了48个镰刀菌菌株对蚕豆的致病力,尖孢镰孢、木贼镰孢、串珠镰孢和燕麦镰孢对蚕豆的致病力都较强。用蚕豆枯萎病菌和棉花枯萎病菌交叉接种棉花和蚕豆,结果表明两者存在着较强的交互侵染能力。     

芝麻枯萎病病原菌致病力室内鉴定方法

 芝麻枯萎病(Sesame Fusarium wilt, SFW)是由尖孢镰刀菌芝麻专化型(Fusarium oxysporum Schl. f. sp. sesami (Zap.), FOS)引起的一种土传真菌病害, 是世界芝麻生产上的主要病害之一。为测定FOS致病力, 本文选用郑芝98N09等4个芝麻品种, 在苗期对15个FOS菌株的致病力强弱进行了室内鉴定和评价。结果表明, 采用1×10⁶个/mL分生孢子悬浮液与无菌蛭石和无菌土壤按V1∶V2∶V6比例混合接菌(即最终接菌浓度为1.4×10⁵孢子/g土壤), 在接菌后第7 d幼苗开始出现枯萎病症状, 调查菌株致病性的最佳时间为接菌后第25 d~28 d;在供试15个FOS菌株中, 对4个品种均表现为强致病力的菌株有8个(DI>50), 均表现为弱致病力的菌株有5个(DI<20);不同芝麻品种对不同菌株的抗性有一定差异。该方法可应用于芝麻枯萎病病原菌致病性测定和芝麻种质抗枯萎病特性评价, 并为后续的机理研究提供了技术支持。     

苦瓜枯萎病菌Ⅱ型卤酸脱卤酶基因FoHAD-type Ⅱ功能分析

苦瓜枯萎病是由尖孢镰刀菌苦瓜专化型(Fusarium oxysporum f. sp.momordicae Sun&Huang)侵染引起的一种严重制约苦瓜安全生产的土传病害。明确苦瓜枯萎病菌的致病机理对苦瓜枯萎病的防治具有重要的指导意义,但目前苦瓜枯萎病菌的致病机理尚不明确。前期分析苦瓜枯萎病菌强致病力菌株SD-1和弱致病力菌株SD-V(携带真菌病毒)转录组差异表达基因时,发现Ⅱ型卤酸脱卤酶(FoHAD-typeⅡ)基因在SD-V菌株中表达量显著下调,推测该基因与苦瓜枯萎病菌的致病性相关。本研究根据同源重组的原理,利用分割标记法(Split-Marker PCR)获得了FoHAD-typeⅡ基因的融合片段,分别通过PEG介导的原生质体转化和农杆菌介导的遗传转化获得该基因的敲除突变体和回补突变体,并对敲除和回补突变体的生物学特性和致病力进行了分析。结果表明,敲除突变体的生长速率和产孢量与野生型菌株相比没有显著差异,菌丝尖端形态和孢子形态也无明显差异,但气生菌丝减少,对渗透胁迫耐受性降低,致病力显著下降;回补突变体的生物学性状和致病力与野生型菌株一致。表明FoHAD-typeⅡ基因...     

棉花枯萎病菌镰刀菌酸的产生和致病力的关系

 镰刀菌酸是棉花枯萎病菌的次生代谢产物,对一种非特异性的毒素。为了探索棉花枯萎病原菌的镰刀菌酸产量与致病力的关系及不同棉花品种对镰刀菌酸的反应,本实验测定了10个新疆吐鲁番地区的菌株和20个内地的菌株,发现其产镰刀菌酸的数量多少与生理型无关.培养初期各菌株的镰刀菌酸产量与菌株的致病力有关.陆地棉的10个品种对镰刀菌酸的抗性与它们在生产中表现出对枯萎病的抗性一致.     

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。     

Comparison of Secreted in Xylem (SIX) genes in two fusarium wilt pathogens of ornamental palms

Fusarium oxysporum ff. sp. canariensis (Foc) and palmarum (Fop) cause lethal fusarium wilt on ornamental palms. Foc infects Phoenix canariensis and has a worldwide presence, whereas Fop primarily infects Washingtonia robusta and Syagrus romanzoffiana and is currently primarily restricted to Florida (USA). A Secreted in Xylem (SIX) gene profile was obtained for Fop and Foc isolates from the USA. SIX1, SIX7, SIX10 and SIX12 were detected in most Foc isolates and, with the exception of SIX12, with minimal sequence polymorphism. SIX8 and SIX9 were detected in all Fop isolates, with no sequence polymorphism. SIX10 was also present in seven of the Fop isolates. Phylogenetic trees were constructed using EF-1α-, SIX1, SIX7 and SIX10 gene sequences from this study and previously derived sequences from Foc isolates obtained in Australia, the Canary Islands and Japan. The topology of the housekeeping EF-1α gene indicated Foc is polyphyletic, with the Australian isolates separating into a distinct group from all other Foc isolates. However, the topologies of SIX1, SIX7 and SIX10 phylogenetic trees for Foc indicate monophyly. As has been proposed previously, this suggests the core genome of Foc evolved into a separate lineage after the SIX genes were conserved within the whole genome. This study is the first to generate SIX12 sequences for Foc. Interestingly, the topology of the SIX12 phylogenetic tree suggests polyphyly. For Fop, the topologies of the EF-1α, SIX8 and SIX9 phylogenetic trees support monophyly. The distinct SIX gene profile and EF-1α sequence of Fop demonstrates an independent evolutionary origin from Foc.     

Influence of chickpea genotype and Bacillus sp. on protection from Fusarium wilt by seed treatment with nonpathogenic Fusarium oxysporum

Seeds of kabuli chickpea cultivars ICCV 4 and PV 61 were treated with conidia of nonpathogenic Fusarium oxysporum isolate Fo 90105 suspended in methylcellulose (3 × 10⁶ conidia.seed^-1), or with methylcellulose alone, and sown in soil artificially infested with 500 or 1,000 chlamydospores.g^-1 of F. oxysporum f. sp. ciceris race 5. At an inoculum concentration of 500 chlamydospores.g^-1, seed treatment with Fo 90105 significantly increased the incubation period of the disease by 11 (ICCV 4) or 25 (PV 61) days, and reduced the final disease incidence, disease intensity and the standardized area under the curve of disease intensity over time. This protection from disease was higher and more consistent in PV 61 than in ICCV 4. However, it was annulled with an inoculum concentration of 1,000 chlamydospores.g^-1, except for the incubation period in PV 61 which was increased by 10 days. When ICCV 4 seeds were treated with Fo 90105 (3 × 10⁶ conidia.seed^-1) and/or Bacillus sp. isolate RGAF 51 (1 × 10⁷ cfu.seed^-1), then sown in infested soil, there was no influence by the Bacillus isolate on protection conferred by Fo 90105. However, the degree of protection by the nonpathogenic F. oxysporum was higher and more consistent when plants from treated seeds were grown in sterile sand for 6 days, then transplanted into infested soil.     

Sequence variation in the putative effector gene SIX8 facilitates molecular differentiation of Fusarium oxysporum f. sp. cubense

Fusarium oxysporum f. sp. cubense (Foc), causal agent of fusarium wilt of banana, is among the most destructive pathogens of banana and plantain. The development of a molecular diagnostic capable of reliably distinguishing between the various races of the pathogen is of key importance to disease management. However, attempts to distinguish isolates using the standard molecular loci typically used for fungal phylogenetics have been complicated by a poor correlation between phylogeny and pathogenicity. Among the available alternative loci are several putative effector genes, known as SIX genes, which have been successfully used to differentiate the three races of F. oxysporum f. sp. lycopersici. In this study, an international collection of Foc isolates was screened for the presence of the putative effector SIX8. Using a PCR and sequencing approach, variation in Foc‐SIX8 was identified which allowed race 4 to be differentiated from race 1 and 2 isolates, and tropical and subtropical race 4 isolates to be distinguished from one another.     

大蕉枯萎病病原菌鉴定及TEF-1α序列分析

 近年来,大蕉枯萎病在广东省东莞市发生严重,为了有效控制病害发生蔓延,生产上急需明确大蕉枯萎病的病原。本研究收集了我国华南地区的12株大蕉枯萎病病原菌及19株包括1号及4号生理小种的单孢菌株,以来源于澳大利亚的1号、2号、3号和亚热带4号生理小种以及4株非病原尖孢镰孢菌作对照,通过病原菌形态鉴定、致病性测定、4号小种(Foc 4)及热带4号小种(TR4)的分子特异检测、以及基于翻译延伸因子(TEF-1α)序列的系统发育分析,对大蕉枯萎病病原菌进行鉴定。同时,对我国华南地区不同来源的香蕉枯萎病病原菌的遗传发育关系及致病性分化情况进行了研究。结果表明:(1)引起大蕉枯萎病的病原菌主要是1号生理小种或者是与1号生理小种亲缘关系较近的一个新的系统发育谱系,该谱系可能为 1 号生理小种变异演化而来;(2)大蕉枯萎病病原菌对大蕉和粉蕉都有较强的致病力,但不能侵染香蕉;我国的1号小种存在一定的分化,其中有一个类群只能感染粉蕉,另一个类群既能感染粉蕉也能感染大蕉;(3)大蕉与粉蕉枯萎病的病原菌在致病性及遗传发育关系上都存在一定的交叉和分化。     

Isolation of Pathogenicity Mutants of Fusarium oxysporum f. sp. melonis by Insertional Mutagenesis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to isolate mutants of Fusarium oxysporum f. sp. melonis impaired in pathogenicity. The race 2 strain Mel02010 was transformed with linearized pSH75, conferring resistance to hygromycin B, with or without the enzyme used to linearize the plasmid. Addition of restriction enzymes did not affect the transformation frequency. A total of 2929 REMI transformants were tested for pathogenicity to three melon cultivars, Amus, Ogon 9 and Ohi. The race 2 strains are pathogenic to Amus and Ogon 9, but not to Ohi. Of 43 transformants with reduced pathogenicity on susceptible melon cultivars, 12 mutants were examined in detail for pathogenicity, vegetative growth and integrative mode of pSH75. The levels of pathogenicity varied among these mutants. Two mutants (B48 and B137) almost completely lost pathogenicity to both susceptible cultivars, and the others had reduced pathogenicity. Mutants B48, B241, B886 and X36 were also impaired in vegetative growth. Mutant B809 was a biotin auxotroph. By DNA gel blot analysis, nine mutants were found to contain a single copy of the transformation vector. These mutants may thus be useful in isolating genes involved in pathogenicity. Received 22 December 2000/ Accepted in revised form 16 April 2001     

Identification of differential resistance to six Fusarium oxysporum f. sp. cepae isolates in commercial onion cultivars through the development of a rapid seedling assay

Fusarium oxysporum isolates collected from onions in the UK and other countries were characterized using sequences of the transfer elongation factor 1‐α (TEF) gene and compared with published sequence data for 10 other isolates. Isolates associated with diseased onion bulbs in the UK formed two clades. Isolates from both clades were selected for pathogenicity testing and to develop a rapid seedling assay to screen commercial onion cultivars for resistance to F. oxysporum f. sp. cepae (FOC), the cause of basal rot. Differences in the levels of aggressiveness between isolates were observed and isolates from both clades were pathogenic. Differences in resistance/susceptibility were also observed amongst 10 commercial onion cultivars, with cvs Ailsa Craig Prizewinner and White Lisbon showing the highest levels of resistance. The results from the seedling assay were supported by those from a subsequent onion bulb rot assay. Thus, this study reports the development of a rapid, simple and repeatable seedling assay that can be used to screen large numbers of onion cultivars for resistance to FOC and which is indicative of resistance at the bulb stage.     

香蕉枯萎病菌myosin-1基因的功能分析及外施dsRNA介导的抗性评估

 香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)引起的香蕉毁灭性土传病害,其中 4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。SMART在线软件分析myosin-1基因具有肌球蛋白马达蛋白(myosin motor domain, MMD),肌动蛋白尾结构TH1(myosin tail)和 Src家族同源结构域SH3(src homology domain 3),与禾谷镰刀菌中氰烯菌酯靶标基因myosin-5具高度的蛋白同源性,相似性高达83%。利用Split-marker基因重组技术获得Foc4的myosin-1基因敲除突变体,Δmyosin-1突变株丧失了对氰烯菌酯的敏感性,菌丝生长缓慢,产孢量减少且孢子畸形,对香蕉致病力严重下降,证实myosin-1是氰烯菌酯在Foc4中的作用靶标基因。外施靶向myosin-1体外转录的dsRNA,能抑制菌丝的生长,降低菌丝活性;菌丝膨胀扭曲分枝增多,出现典型的球状结构,与氰烯菌酯处理后的表型一致。在盆栽活体人工接种实验中,体外施用dsRNA可以明显抑制枯萎病外部症状的发展,推迟发病时间,赋予寄主抗性,结果说明体外施用dsRNA可以作为新型杀菌剂防治香蕉枯萎病。综上,myosin-1基因作为氰烯菌酯在Foc4中的靶标基因具有高度的序列保守,在调控菌丝生长发育,产孢以及致病力等方面发挥重要作用,而外施dsRNA具有防治香蕉枯萎菌的巨大潜力。     

Back to the roots: Understanding banana below-ground interactions is crucial for effective management of Fusarium wilt

Global banana production is affected by Fusarium wilt, a devastating disease caused by the soilborne root-infecting fungus, Fusarium oxysporum f. sp. cubense (Foc). Fusarium wilt is notoriously difficult to manage because infection arises through complex below-ground interactions between Foc, the plant, and the soil microbiome in the root–soil interface, defined as the rhizosphere. Interactions in the rhizosphere play a pivotal role in processes associated with pathogen development and plant health. Modulation of these processes through manipulation and management of the banana rhizosphere provides an auspicious prospect for management of Fusarium wilt. Yet, a fundamental understanding of interactions in the banana rhizosphere is still lacking. The objective of this review is to discuss the state-of-the-art of the relatively scant data available on banana below-ground interactions in relation to Fusarium wilt and, as a result, to highlight key research gaps. Specifically, we seek to understand (a) the biology of Foc and its interaction with banana; (b) the ecology of Foc, including the role of root-exuded metabolites in rhizosphere interactions; and (c) soil management practices and how they modulate Fusarium wilt. A better understanding of molecular and ecological factors influencing banana below-ground interactions has implications for the development of targeted interventions in the management of Fusarium wilt through manipulation of the banana rhizosphere.     

Phylogenetic position and nucleotide diversity of Fusarium oxysporum f. sp. vanillae worldwide based on translation elongation factor 1α sequences

Fusarium oxysporum f. sp. vanillae is considered the most important fungus affecting vanilla crops around the world, causing rot on vanilla roots and stems. Previous studies showed that the ability to infect vanilla plants is a polyphyletic trait among strains of the Fusarium oxysporum species complex (FOSC). The same studies proposed a single origin for F. oxysporum f. sp. vanillae isolates sampled from Mexico, the centre of origin and distribution of vanilla. The aim of this work was to test the hypothesis of the monophyletic origin of a wider sample of isolates of F. oxysporum f. sp. vanillae infecting Mexican vanilla and estimate nucleotide diversity of pathogen isolates from the main vanilla‐producing countries. Sequence data for the TEF1α gene from 106 isolates was assembled. The phylogenetic analyses suggest that some Mexican isolates of F. oxysporum f. sp. vanillae belong in two well‐supported clades, mixed with isolates from Madagascar, Indonesia, Réunion and Comoros. The phylogenetic position of other Indonesian and Mexican isolates is unresolved. Estimations of nucleotide diversity showed that the population from Mexico is genetically more diverse than the other three populations from Madagascar, Indonesia and Réunion. The results support a polyphyletic origin of vanilla‐infecting isolates of F. oxysporum worldwide, and also reject the proposition that Mexican isolates have a single origin. The phylogenetic optimizations over the strict consensus tree of the ability to infect vanilla plants suggest that pathogenic strains around the world are the product of multiple shifts of pathogenesis and dispersion events.     

甘蓝枯萎病菌寄主范围研究

<正>甘蓝枯萎病于2001年最先在北京延庆发现~([1]),目前已扩散到山西、河北、甘肃及陕西等北方甘蓝生产基地,重病区发病率高达80%以上,对甘蓝生产造成了严重的威胁。国内外对甘蓝枯萎病寄主范围的研究仅限于十字花科作物~([2-4]),对该病原菌是否侵入其他类蔬菜及1、2号生理小种侵染十字花科寄主的差异尚未见报道。本文采用人工接种的方法,综合分析甘蓝枯萎病菌对主要大田蔬菜作物的侵染寄生能力,为制定合理的轮作防病