宁夏马铃薯镰刀菌根腐病病原菌的ISSR-PCR标记体系建立及其遗传多样性分析

为明确宁夏马铃薯镰刀菌根腐病病原菌种内的遗传差异与亲缘关系,利用ISSR-PCR技术对影响PCR扩增效果的因素和ISSR引物进行优化和筛选,建立适合马铃薯镰刀菌根腐病病原菌的ISSR-PCR反应体系,并通过ISSR分子标记技术对30株地理来源不同的镰刀菌进行遗传多样性分析。结果显示：马铃薯镰刀菌根腐病病原菌的ISSR-PCR扩增最适引物为807,20 μL反应体系中2×Easy Taq PCR Super Mix为8 μL、引物浓度为0.6 μmol/L、DNA模板浓度为56 ng/μL,循环次数为36次,退火温度为54℃。在选用的18条ISSR引物中有13条对30株镰刀菌扩增出84条条带,大小多分布在250~2 000 bp之间,其中多态性条带为82条,多态性比例平均为98.3%。30株镰刀菌的遗传相似系数在0.349~0.976之间,在0.478水平上可划分为2个类群,其中类群I包括尖孢镰刀菌Fusarium oxysporum、木贼镰刀菌F.equiseti、茄病镰刀菌F.solani和锐顶镰刀菌F.acuminatum;类群II全部为接骨木镰刀菌F.sambucinum;在0.976水平上30株镰刀菌可被全部区分开。ISSR类群划分与菌种分类之间存在一定相关性,同一类群不同菌株之间的遗传相似性与菌株地理来源存在一定的相关性;分离自同一地区的同一菌种,其菌株间也存在一定的遗传差异性。     

尖镰孢菌EST-SSR遗传多样性分析及通用性评价

为了解38株尖镰孢菌的遗传多样性并开发可在近缘镰孢菌种中通用的尖镰孢菌EST-SSR标记,利用设计和筛选的18对多态性EST-SSR引物对38株尖镰孢菌和5种近缘镰孢菌进行SSRPCR扩增,经6%非变性聚丙烯酰胺凝胶分离扩增产物,并用NTSYS软件分析供试尖镰孢菌的PCR扩增结果。结果表明,18对EST-SSR标记引物在38株尖镰孢菌中检测到75条多态性条带,多态性比率达92.6%,平均每对引物可扩增4.2条;各菌株间的遗传相似系数介于0.565~0.946之间,平均为0.721;来源于同科寄主植物群体的菌株间的平均遗传相似系数以葫芦科最大,锦葵科最小,依次为葫芦科兰科豆科亚麻科茄科锦葵科。在相似系数为0.756时,供试38株菌有35株按照不同科寄主植物聚为不同的类群,说明尖镰孢菌SSR类群的划分与其寄主来源具有一定的相关性。18对EST-SSR引物在近缘镰孢菌种中均能有效扩增的引物数及通用性比率为10对和55.6%,均显示多态性的引物有2对,占供试引物总数的11.1%。表明尖镰孢菌ESTSSR区域遗传多样性丰富,基于尖镰孢菌EST序列开发镰孢菌通用SSR标记是可行的。     

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。     

西瓜枯萎病菌遗传多样性的相关序列扩增多态性分析

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。     

陕西省小麦禾谷镰刀菌的遗传多样性研究

为了明确陕西省小麦禾谷镰刀菌混合群(Fusarium graminearum species complex)的遗传多样性,利用4对E coRⅠ和M seⅠ引物对来自陕西省19个区县的162株小麦禾谷镰刀菌菌株进行了AFLP扩增。结果表明,4对引物均能扩增出数量不等的多态性条带,最少的6条,最多的20条,大部分扩增片段在100～750bp之间。利用NTSYS-2.1软件聚类分析表明,不同地区禾谷镰刀菌可分为两大类群,即类群A和类群B。这两大类群的分化和地理来源有明确相关性,类群A主要分布在关中地区,类群B主要分布在陕南地区。初步判断可能与两个地区生态环境和小麦主栽品种差异有关。各类群内的菌系与地理位置间的关系较为复杂,一些菌系与地理来源存在明确关系,而个别菌系与地理来源间的关系尚不能完全明确。还需进一步研究以明确各菌株与地理来源之间的关系。     

利用UP-PCR、ISSR和AFLP标记分析玉米丝黑穗病菌遗传多样性

利用UP-PCR、ISSR和AFLP分子标记方法研究了我国主要玉米产区34株玉米丝黑穗病菌的遗传多样性。从供试引物中筛选获得具多态性的UP-PCR引物9个、ISSR引物11个和AFLP引物组合22对,分别扩增出113、72和293条谱带,多态性条带比率分别为91.15%、84.7%和83.27%。聚类分析表明,玉米丝黑穗病菌存在丰富的遗传变异,与地理来源无明显相关性。3种分子标记的遗传相似系数矩阵相关性分析表明,UP-PCR与AFLP具有较高的相关性,相关系数为0.698;UP-PCR与ISSR、ISSR与AFLP的相关系数分别为0.659和0.633。从多态性水平、稳定性和可操作性可以看出,UP-PCR技术更适于分析玉米丝黑穗病菌遗传多样性。此外,UP-PCR、ISSR和AFLP标记划分的类群与鉴别寄主划分的致病类型之间存在一定的相关性,吻合率分别为50.0%、60.0%和47.6%。     

我国玉米灰斑病菌遗传多样性的ISSR分析

为明确我国发生的玉米灰斑病菌地理差异及遗传结构,利用简单序列重复区间(ISSR)对玉米灰斑病菌遗传多样性进行了分析,并利用尾孢菌特异引物对分离自四川、云南、湖北、贵州等西南地区的16个玉米灰斑病菌菌株进行了分子鉴定。结果显示,通过ISSR标记筛选出10个扩增多态性好且稳定的通用引物,共扩增出81条DNA条带,均为多态性条带,扩增片段大小在200~2 000 bp之间,菌株遗传相似系数为0.19~1.00。在遗传相似系数为0.19时,供试菌株被聚为2大类群,来自西南地区和东北地区的菌株各自聚为一组,在DNA水平上表现出明显差异,认为是2类不同的致病类群。分子鉴定结果显示引起西南各地区玉米灰斑病的主要致病菌均为玉米尾孢菌Cercospora zeina。表明我国玉米灰斑病菌存在丰富的遗传多样性,ISSR标记可揭示出玉米灰斑病菌株间的亲缘关系及遗传差异性,可用于其遗传多样性研究。     

陕西棉花黄萎病菌致病力分化及其遗传多样性

采用生物学培养性状、致病力测定和ISSR分子标记方法研究15个棉花黄萎病代表菌株的遗传变异.结果表明,供试菌株的生长速率、孢子产量与致病力的强弱呈正相关.致病力测定结果显示,致病力强的Ⅰ型有7个菌株,占46.67%,平均病情指数大于36.1;致病力弱的Ⅱ型有3个菌株,占20.00%,平均病情指数在21以下;致病力中等的Ⅲ型有5个菌株,占33.33%,平均病情指数在20～28之间.用4条ISSR引物对这些菌株进行PCR扩增,共得到623个条带,具多态性的有425条.聚类分析和相似系数分析结果显示,在0.55遗传相似水平下,供试菌株分为2个遗传类型,遗传类型与菌株致病力类型存在明显的相关性,与菌株地理来源也具有一定的相关性.     

甘肃省大麦条纹病病原菌致病力分化、rDNA-ITS及遗传多样性分析

为明确甘肃省大麦条纹病病原菌麦类核腔菌Pyrenophora garminea的致病力分化、r DNA-ITS序列特征及变异,采用"三明治"法测定甘肃省大麦条纹菌的致病力,通过r DNA-ITS序列分析菌株间变异类型,并对菌株进行ISSR遗传多样性分析。结果表明,共筛选到43株大麦条纹病菌,生长7 d后的菌落直径为2.60~7.93 cm,其中菌株TB生长速度最快,菌株JT生长速度最慢,强、中等和弱致病力菌株分别为2、21和20株;43株菌的核糖体DNA-ITS序列与麦类核腔菌菌株SMCD2015同源性在99%以上;在9个菌株中检测到15个变异位点,共17种变异类型;8个ISSR标记从43个菌株中扩增出41条带,平均每个标记5.13条带,90.24%片段具有多态性,遗传相似系数为0.44~1.00,平均值为0.71,当遗传相似系数为0.68时,可将供试菌株划分为4个类群。表明甘肃省大麦条纹病病原菌存在致病力分化,菌株核糖体DNA-ITS序列变异丰富,且菌株间遗传结构复杂。     

美国大豆中镰刀菌的分离鉴定

为加强对美国输华大豆真菌病害的监测力度,降低外来生物入侵风险,本文通过对美国进境大豆病菌分离,共得到32个菌株,并对其中3株镰刀菌进行了形态学和分子生物学鉴定,确认了它们分别是尖孢镰刀菌(Fusarium oxysporum)、木贼镰刀菌(F.equiseti)和拟枝孢镰刀菌(F.sporotrichioides)。本研究从32个分离菌株中得到的3株镰刀菌分属于不同种,不仅证实了美国大豆中镰刀菌的多样性,也可为港口的植物检疫工作提供借鉴。     

黄瓜枯萎病菌遗传多样性的AFLP分析

黄瓜枯萎病是由半知菌亚门尖孢镰刀菌黄瓜专化型(Fusarium oxysporumf.sp.cucumainum Owen)侵染引起的一种土传病害,是影响黄瓜生产的最主要病害之一[1].近年来随着分子生物学技术的迅速发展,国内外学者对于病原真菌的遗传多样性做了大量的研究,Wang等[2]对影响黄瓜枯萎病菌AFLP技术体系的多种因素作了探讨,得到了1种适合于黄瓜枯萎病菌AFLP分析的优化体系;Duan等[3]应用RAPD、ISSR和AFLP标记揭示出了西瓜枯萎病菌株在分子水平上的遗传多样性.     

基于Histone H3、EF-1α和β-tubulin基因部分序列的芒果畸形病菌鉴定与系统发育研究

<正>芒果畸形病是芒果生产中重要的病害之一,主要表现为花畸形和芽畸形,受害果园常因畸形花絮无法挂果而造成减产,我国攀西地区的部分果园株发病率高达100%,给当地的芒果产业带来严重经济损失。该病自1891年首次在印度发现后,陆续在世界芒果产区发生危害~([1])。引起芒果畸形病的病原菌有Fusarium mangiferae、F.mexicanum、     

Assessing new SSR markers for utility and informativeness in genetic studies of brown rust fungi on wheat,triticale, and rye

The aim of the present study was to validate new simple-sequence repeat (SSR) markers and use them to assess genetic variability among 24 isolates of Puccinia triticina collected from wheat (Pt-wheat) and triticale (Pt-triticale), and 15 isolates of P. recondita f. sp. secalis (Prs) collected from rye. The Pt and Prs isolates were tested for virulence on a set of 35 Thatcher wheat near-isogenic lines, eight rye lines with known resistance genes, and 53 triticale cultivars with uncharacterized leaf rust resistance. Molecular genotypes were determined using a newly developed set of 34 SSR microsatellite primer pairs. All SSR markers tested on Pt isolates successfully amplified fragments of appropriate size. When tested on the Prs isolates, 21 out of the 34 Pt SSRs amplified expected fragments. Sixteen of these 21 SSRs were polymorphic, providing for the first time microsatellite markers to study genetic variation in Prs. Based on virulence data, variation among Prs isolates was low, probably due to the small number of rye differential lines available. Much higher variation for virulence was observed within the collection of Pt isolates from wheat and triticale, and two separate groups were established with mixed host origin. Substantial genetic variation was detected among the isolates studied with the SSR markers, assuming two different models of SSR evolution (infinite alleles model and stepwise mutation model). The newly developed set of SSR markers proved their effectiveness in detecting genetic variation and should be useful in further population genetics investigations of the two pathogens.     

PCR detection of Fusarium oxysporum f.sp. gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms

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.     

Genetic Variability and Population Structure of the Wheat Foot Rot Fungus, Fusarium culmorum, in Tunisia

The random amplified polymorphic DNA (RAPD) method was used to investigate the genetic variability and population structure of Fusarium culmorum isolated from wheat stem bases. A total of 108 isolates, representing seven geographically distinct populations, was collected from five climatic regions in Tunisia. Pseudo-allelic frequencies were estimated at each of the 25 putative RAPD loci analyzed by scoring for the presence or absence of amplified fragments; 92 haplotypes were found among the 108 strains. The analysis of the population structure did not reveal any trend with regard to geographic origin. Total gene diversity (H_T ^* = 0.318) was mostly attributable to diversity within populations (H_S ^* = 0.308). Analysis of molecular variance confirmed that most of the genetic variability was within populations. Genetic differentiation among populations was low to moderate (G_ST ^* ranged from 0 to 0.190 and averaged 0.041 over all loci). Cluster analysis with UPGMA using genetic distances did not reveal any spatial clustering of the isolates collected from the different geographic regions. Based on these results, we conclude that the F. culmorum isolates recovered from different regions in Tunisia might be part of a single population pool.     

柑橘叶点霉属(Phyllosticta spp.)真菌的遗传多样性分析

利用筛选的11条ISSR引物对从我国柑橘主产区和国外收集的135株叶点霉属真菌菌株进行扩增,扩增产物进行凝胶电泳,扩增图谱用NTSYS-pc2.10软件进行群体聚类分析。结果显示,共扩增出116个DNA条带,其中多态性位点为112个,多态率为96.55%;平均每个引物可以扩增出10.55个条带,扩增产物大小在250~3 000 bp。聚类分析显示,中国柑橘叶点霉属真菌可以分为4个种,即P.citricarpa、P.citriasiana、P.capitalensis和P.citrichinaensis。以遗传相似性系数0.97为阈值时,柑橘黑斑病病原菌(P.citricarpa)种群可分为5个亚类,subclade-I的菌株来自中国的本地早、温州蜜柑、槾橘和南丰蜜橘,subclade-II的菌株均来自中国的砂糖橘,subclade-III的菌株来自非洲莫桑比克柠檬和葡萄柚、佛罗里达甜橙、南非甜橙和来自杭州市场上进口的澳橘,subclade-IV的菌株来自中国的甜橙,subclade-V的菌株来自中国的柠檬,表明我国柑橘黑斑病病原菌具有丰富的遗传变异,其遗传变异与寄主相关;我国与国外的柑橘黑斑病病原菌在起源上可能存在差异。P.citriasiana、P.capitalensis和P.citrichinaensis种群内也存在遗传变异,但其遗传变异与地理分布和寄主未发现相关性。该研究结果为研究柑橘叶点霉属真菌的遗传多样性奠定了基础。     

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。     

淡紫拟青霉诱变菌株的RAPD分析

选用6个随机引物对淡紫拟青霉IPC菌株及其15株突变菌株的全基因组DNA进行随机多态性扩增,共扩增出114条250～2500bp的DNA片段,多态检测率为83．3％。利用UPGMA方法对扩增的DNA片段聚类分析,结果表明：供试菌株具有丰富的遗传多样性,应用RAPD技术可以快速准确地鉴定菌株是否突变及突变程度;参照突变菌株表型特性上的改变,该方法可以作为确定其遗传性上是否发生分化的依据。     

Development of sequence tagged site markers to identify races of Fusarium oxysporum f. sp. lactucae

By random amplified polymorphic DNA (RAPD) analysis of the representative isolates of each race of Fusarium oxysporum f. sp. lactucae, RAPD fragments of 0.6, 1.6, and 2.9kb were obtained. The 0.6-kb RAPD fragment was common to the representative isolates of all three races. Amplification of the 1.6- and 2.9-kb fragments were unique to the isolates of races 1 and 2, respectively. Sequence tagged site (STS) marker FLA0001, FLA0101, and FLA0201 were generated from the 0.6-, 1.6-, and 2.9-kb RAPD fragments, respectively. Polymerase chain reaction (PCR) analysis showed that FLA0001 was common to all 49 isolates of F. oxysporum f. sp. lactucae. FLA0101 was specifically generated from all 23 isolates of race 1 but not from races 2 or 3. FLA0201 was specifically amplified from all 12 isolates of race 2 but not from races 1 or 3. In two isolates of F. oxysporum f. sp. lactucum, PCR amplified FLA0001 and FLA0101 but not FLA0201. On the other hand, these STS markers were not detected from isolates of five other formae speciales. Because these STS markers were not generated from isolates of other plant pathogenic fungi, bacteria, or plant materials examined in this study, PCR analysis combined with the three STS markers should be a useful means for rapid identification of races of F. oxysporum f. sp. lactucae.     

香蕉枯萎病菌生理小种鉴定及其SCAR标记

 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。