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1.
香蕉枯萎病菌RAPD分析及4号生理小种的快速检测 总被引：3，自引：0，他引：3
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。 相似文献
2.
香蕉枯萎病菌生理小种鉴定及其SCAR标记 总被引：8，自引：0，他引：8
通过室内人工接种蕉类鉴别寄主,对采集于广东蕉区的18个蕉类枯萎病菌菌株进行鉴定,KP021、KP022、GZ981和JL021 4个菌株属Racel,其余14个菌株属Race4,说明广东蕉区同时存在尖孢镰刀菌古巴专化型Race1和Race4。用RAPD技术对上述18个菌株进行分析,从200条随机引物中筛选出8条引物可产生生理小种RAPD标记12个,其中标记Racel的8个,标记Race4的4个。对这些RAPD标记带分别进行回收、克隆、测序,根据这些特异片段序列分别设计相应的SCAR引物,通过对18个菌株的PCR扩增检验,有4个RAPD标记成功地转化为SCAR标记,其中Race1-SCAR标记1个、Race4-SCAR标记2个、同时能鉴定出2个小种的SCAR标记1个。应用这4个SCAR标记同时对采自田间的9个病菌分离物进行检测,能够准确地鉴定出广东蕉区的尖孢镰刀菌古巴专化型Racel和Race4,这为下一步开展香蕉枯萎病菌生理小种的分子鉴定及各生理小种田间流行动态监测奠定了基础。 相似文献
3.
香蕉枯萎病拮抗细菌的分离筛选与鉴定 总被引：8，自引：0，他引：8
从采集的香蕉根际土壤中经分离、纯化获得细菌菌株74个,采用对峙培养法,获得对香蕉枯萎病菌4号小种具有强抑制作用的菌株S-1,相对抑制率达76%。抑菌谱测定表明,菌株S-1对尖孢镰刀菌古巴专化型1号小种、番茄枯萎病菌、玉米弯孢叶斑病菌、胶孢炭疽病菌、棉花枯萎病菌、棉花黄萎病菌、小麦赤霉病菌、西瓜枯萎病菌等病原菌具有抑制作用。经Biolog系统鉴定及16S rDNA序列同源性分析,菌株S-1为枯草芽孢杆菌。该菌株理想的培养条件为：PYTG或PDA培养基,26-30℃,pH值7.0-8.0,振荡培养40-48h。 相似文献
4.
香蕉枯萎病菌致病力分化与ISSR遗传多样性分析 总被引：1，自引：0，他引：1
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。 相似文献
5.
尖孢镰刀菌的遗传多态性 总被引：13，自引：0，他引：13
尖孢镰刀菌是重要的植物维管束病原真菌。近年来,有关该菌的遗传多态性研究报道很多,本文着重综述了利用营养体亲和群、DNA多态性技术研究尖孢镰刀菌专化型、小种及其相互关系等方面的进展,同时介绍了对棉花枯萎病菌、瓜类枯萎病菌及尖孢镰刀菌小种起源等的研究概况。 相似文献
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以ITS测序与SCAR分子标记相结合的香蕉病原菌FOC4分子检测方法,利用通用引物ITS1和ITS4对病原菌SF001的rDNA中ITS序列进行PCR扩增,获得560bp左右的特异条带。经Blast分析比对,确定该菌为FOC。再利用FOC4的特异引物PCL/PDL对基因组上的特征序列扩增区域(SCAR)进行PCR扩增,获得677bp的特有序列,鉴定菌株SF001是尖孢镰刀菌古巴专化型4号生理小种。经过致病性测试,菌株SF001表现出典型4号生理小种的病理特征。 相似文献
7.
对30个西瓜枯萎病菌Fusarium oxysporum f.sp.niveum菌株基因组DNA进行相关序列扩增多态性(SRAP)分子标记分析,以探究其遗传多样性与地理来源的关系。采用尖孢镰刀菌西瓜专化型Fusarium oxysporumf.sp.niveum0、1、2号生理小种的基因组DNA为模板,对225对SRAP引物进行筛选,筛选出20对多态性、重复性较好且条带清晰的引物,对30个菌株进行PCR扩增,共扩增出386条带,其中多态性条带有371条,多态性比率为96.11%,平均每对引物扩增出19.3个位点和18.55个多态性位点。UPGMA法聚类分析结果显示,供试菌株两两之间的遗传相似系数范围为0.69~0.90,平均为0.79,说明尖孢镰刀菌西瓜专化型的遗传多样性较为丰富。基于SRAP标记聚类分析表明,30个菌株在遗传相似系数为0.70处被划分为3个类群,I类群包含24个菌株,其中18个来自湖南省,Ⅱ类群只包含1个来自黑龙江省哈尔滨市的菌株,它和另一个来自黑龙江地区的菌株被划分到不同的类群,且遗传距离相对较远;Ⅲ类群包含了5个菌株,其中3个来自海南三亚,其余两个来自湖南省。根据菌株的分布情况来看,菌株的聚集与地理来源没有明显的相关性。 相似文献
8.
香蕉枯萎病菌Fusarium oxysporum f. sp. cubense和细菌性软腐病菌Dickeya zeae的复合侵染为害给香蕉产业发展带来严重挑战, 有必要建立相关病害的多重聚合酶链式反应(multiplex polymerase chain reaction, multiplex PCR)检测技术。本文基于尖孢镰刀菌古巴专化型1号生理小种(F. oxysporum f. sp. cubense race 1, FOC1)基因组contig 438区间(35 631-37 693 bp)(GenBank: AMGP01000438.1)和4号生理小种(F. oxysporum f. sp. cubense race 4, FOC4)基因组contig 195区间(4 028-6 126 bp)(GenBank: AMGQ01000195.1)存在160 bp插入序列差异设计特异扩增引物FOC-F/-R, 同时以香蕉细菌性软腐病菌D. zeae的促旋酶B 亚单位基因(the subunit B of gyrase gene)(GenBank: JQ284039)序列设计特异扩增引物gyrB-F/-R。多重PCR检测结果显示:本技术可在一次PCR扩增反应内同时检测香蕉枯萎病菌1号、4号生理小种和细菌性软腐病菌; 多重PCR的灵敏度结果表明:检测香蕉枯萎病菌的DNA浓度最低限为0.1 ng/μL, 细菌性软腐病菌的灵敏度为10 3cfu/mL;检测结果稳定可靠。因此, 本研究建立的多重PCR检测方法可有效应用于检测香蕉发病组织中的香蕉枯萎病菌和细菌性软腐病菌, 也可用于香蕉种苗和田间土壤带病菌的监测, 为香蕉种植保驾护航。 相似文献
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通过URP(Universal Rice Primers)-PCR分析尖孢镰孢菌苦瓜专化型基因组DNA扩增片段多态性,筛选检测尖孢镰孢菌苦瓜专化型的特异性引物,并建立了基于该引物的PCR检测方法。结果表明,特异性引物为FOMM-SPF/FOMM-SPR, PCR检测体系为25 SymbolmAL,包括2SymboltB Green Taq Master Mix 12.5 SymbolmAL,10 mmol·L-1 的上下游引物各1 SymbolmAL,模板DNA 1 SymbolmAL,灭菌去离子水补足至25 SymbolmAL;PCR程序为95℃预变性3 min,94℃变性15 s,57℃退火30 s,72℃延伸20 s,共30个循环,循环结束后72℃延伸5 min;特异性扩增片段大小294 bp,检测灵敏度为2 ng·μL-1 DNA或50个孢子·500 mg-1土壤。该引物及其检测方法对尖孢镰孢菌苦瓜专化型的检测特异性好、灵敏度高,可以从土壤和植物样品中快速准确地检测出苦瓜枯萎病菌,无需病原菌的分离培养和致病性检测,对苦瓜枯萎病的早期诊断和预警及有效防控具有重要的指导意义。 相似文献
10.
GA1-2菌株的分离鉴定及其对香蕉尖孢镰刀菌的抑菌效果 总被引：2，自引：0，他引：2
为了从土壤中分离筛选对香蕉尖孢镰刀菌具有良好拮抗作用的放线菌,采用平板稀释涂布法从四川省会理县干热河谷小麦根际土壤中进行放线菌分离,并采用平板对峙法和孢子萌发法进行筛选,通过形态特征、培养特征、生理生化特征以及16S r DNA序列分析对筛选菌株进行鉴定。结果表明,从四川省会理县干热河谷小麦根际土壤筛选获得一株对香蕉尖孢镰刀菌4号生理小种Fusarium oxysporum f.sp.cubense race 4(FOC4)菌丝和孢子萌发都有很强抑制作用的菌株GA1-2,对FOC4菌丝和孢子萌发抑制率分别为36.34%和94.81%。菌株GA1-2与薰衣草灰链轮丝菌Streptoverticillium lavenduligriseum的亲缘关系最近,相似率达99.85%,且其形态特征、培养特征、生理生化特征也与薰衣草灰链轮丝菌基本相符,因此将菌株GA1-2初步鉴定为薰衣草灰链轮丝菌。 相似文献
11.
瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测 总被引：2，自引：0，他引：2
通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。 相似文献
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Fusarium oxysporum is one of the most important phytopathogens and cause Fusarium wilt disease in cucumber, watermelon and melon, etc.In this study, a pair of species-specific primers Fc-1 and Fc-2 was synthesized based on differences in internal transcribed spacer sequences of Fusarium genus.With the primers, a specific 315 bp PCR product was amplified from five F.oxysporum isolates isolated from cucumber, watermelon and melon, infected cucumber and watermelon tissues, while no product was obtained from other fourteen fungi, healthy cucumber and watermelon tissues.The detection sensitivity is 100 fg for genomic DNA of F.oxysporum and 1 000 spores/g soil for the soil pathogens.In contrast, the nested PCR with two pairs of primers(ITS1/ITS4 and Fc-1/Fc-2) increased the sensitivity by 100-fold.In addition, one-step PCR could also detect F.oxysporum in symptomless cucumber root of 7 dpi(days post inoculation) and in infected cucumber and watermelon tissues at the early stage of disease development.Therefore, the developed PCR-based method enabled rapid, sensitive and reliable detection of F.oxysporum.It also provides the detection method for early monitoring and diagnosis of the pathogen as well as the plant disease management guidance. 相似文献
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Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large- and small-flowered Gladiolus cultivars. Race 2 isolates infect only small-flowered cultivars but can be present as epiphytes on large-flowered plants. When 160 arbitrary 10-mer oligonucleotide primers were tested on FOG by PCR to find RAPD markers specific for race 1, the RAPD primer G12 amplified two discriminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates only. Both fragments were cloned and sequenced. Two pairs of race 1-specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolates by PCR showed that, in almost all cases, race 1 isolates of vegetative compatibility group 0340 could be distinguished with these primers. Seven putative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latently infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28°C in a semiselective medium to induce growth of Fusarium . Cultivated mycelium was isolated and subjected to the developed multiplex PCR after standard DNA isolation or disruption by microwave treatment. 相似文献
15.
香蕉枯萎病菌1号和4号生理小种细胞壁降解酶的比较 总被引：8，自引：1，他引：7
对香蕉枯萎病菌1号和4号生理小种的细胞壁降解酶进行比较。通过测定4号生理小种在寄主体内细胞壁降解酶的活性发现,能检测到多聚半乳糖醛酸酶(PG)、果胶甲基半乳糖醛酸酶(PMG)、多聚半乳糖醛酸反式消除酶(PGTE)、果胶甲基反式消除酶(PMTE)和纤维素酶(Cx)的活性。在不同碳源培养条件下,2个生理小种均有以上5种酶的活性,以1%柑桔果胶为碳源时产生的PMG和PG活性明显高于其他几种酶的活性,而以1%CMC为碳源时,所产生的Cx都比其他几种酶的活性高。细胞壁降解酶同工酶电泳后发现,4号生理小种在寄主体内和体外培养时都比1号生理小种多分泌一种PG。2个生理小种在体外培养时分泌的PMG、PGTE和PMTE没有差异。4号生理小种在寄主体内比1号生理小种多分泌一种PMG,却少分泌一种PGTE,2个生理小种在寄主体内的PMTE则没有差异。 相似文献
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M. A. Dita C. Waalwijk I. W. Buddenhagen M. T. Souza Jr G. H. J. Kema 《Plant pathology》2010,59(2):348-357
This study analysed genomic variation of the translation elongation factor 1α (TEF‐1α) and the intergenic spacer region (IGS) of the nuclear ribosomal operon of Fusarium oxysporum f. sp. cubense (Foc) isolates, from different banana production areas, representing strains within the known races, comprising 20 vegetative compatibility groups (VCG). Based on two single nucleotide polymorphisms present in the IGS region, a PCR‐based diagnostic tool was developed to specifically detect isolates from VCG 01213, also called tropical race 4 (TR4), which is currently a major concern in global banana production. Validation involved TR4 isolates, as well as Foc isolates from 19 other VCGs, other fungal plant pathogens and DNA samples from infected tissues of the Cavendish banana cultivar Grand Naine (AAA). Subsequently, a multiplex PCR was developed for fungal or plant samples that also discriminated Musa acuminata and M. balbisiana genotypes. It was concluded that this diagnostic procedure is currently the best option for the rapid and reliable detection and monitoring of TR4 to support eradication and quarantine strategies. 相似文献
18.
Chiocchetti A Bernardo I Daboussi MJ Garibaldi A Gullino ML Langin T Migheli Q 《Phytopathology》1999,89(12):1169-1175
ABSTRACT Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transposable elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi strains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolates of race 4. Genomic DNAs flanking race-associated insertion sites of Fot1 (from races 1, 2, and 8) or impala (from race 4) were amplified by the inverse polymerase chain reaction (PCR) technique. These regions were cloned and sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DNA as template in PCR experiments, primer pairs generated amplification products of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with genomic DNA belonging to races 1 and 8, 2, or 4, single amplimers were generated, allowing clear race determination of the isolate tested. PCR was successfully performed on DNA extracted from susceptible carnation cv. Indios infected with isolates representative of races 1, 2, 4, and 8. 相似文献
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A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata. 相似文献