拟轮枝镰孢菌EST-SSR信息分析与标记开发

为开发可用于拟轮枝镰孢菌Fusarium verticillioides及其近缘种遗传多样性分析的SSR引物,利用生物信息学方法和PCR技术,通过对从NCBI下载的87 086条拟轮枝镰孢菌的EST序列信息进行分析,设计EST-SSR引物,检测其在拟轮枝镰孢菌及其近缘种中的扩增情况,并用筛选出的多态性引物对15株拟轮枝镰孢菌进行SSR遗传多样性分析。结果表明,在EST序列中,共查找到11 952个SSR位点,592种重复基元,SSR出现频率为1.09%,重复基元出现数量最多的为三核苷酸(54.00%),其中(CAA/TTG)n基元出现频率最高。设计的25对EST-SSR引物在拟轮枝镰孢菌种内的有效扩增率和多态率分别为80.00%与32.00%,对5种近缘镰孢菌种的通用率和多态率分别为40.00%和8.00%。遗传多样性分析结果表明,在相似系数为0.664水平下,供试菌株可划分为4个SSR类群,但类群的划分与菌株的地理来源无关;不同菌株间存在明显的遗传分化。表明基于拟轮枝镰孢菌EST序列开发的SSR引物可用于拟轮枝镰孢菌及其近缘种的遗传多样性分析。     

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。     

尖孢镰刀菌及芬芳镰刀菌遗传多样性的ISSR分析

 为了明确镰刀菌属(Fusarium)美丽组（Section Elegans）中尖孢镰刀菌(F. oxysporum)和芬芳镰刀菌（F. redolens）2种菌的遗传差异性和亲缘关系,利用ISSR分子标记技术对这2种菌的35个菌株进行了遗传多样性分析。结果表明,用筛选的15条引物对35个供试菌株共扩增出231条条带,其中多态性条带220条,平均多态性比率为95.2%,平均每条引物产生条带为14.7条。聚类结果和遗传相似系数分析显示,35个菌株间的遗传相似系数范围为0.506~0.935,平均为0.661。在遗传相似系数为0.593时,供试的35株镰刀菌可明显的分成2个ISSR类群（IG）,其中IGⅠ包括1～23号菌株,全部为F. oxysporum;IGⅡ包括24～35号菌株,全部为F. redolens。ISSR类群划分与菌种分类之间存在一定相关性 (IGⅠ中23株F. oxysporum间的平均相似系数为0.720,IGⅡ中12株F. redolens间的平均相似系数为0.717),但与菌株的地理来源不存在相关性。而同一类群中,菌株之间的遗传相似性与菌株的地理来源存在一定的相关性。     

烟草疫霉SSR分子标记的开发与初步应用

 为开发烟草疫霉的SSR分子标记,利用MISA软件搜索烟草疫霉基因组序列中的 SSR位点,共发现1 311个SSR位点,优势SSR位点为二核苷酸和三核苷酸,分别占总 SSR 位点的56.45%和39.36%;根据分析到的SSR位点使用Primer 5.0软件设计48对SSR引物,以7株烟草疫霉的DNA 为模板对这些引物进行筛选,共获得扩增条带清晰且具有多态性的SSR引物20对,然后使用其中的6对SSR对32株烟草疫霉进行UPGMA聚类分析,遗传相似系数在 0.60~1.00 之间,在相似系数0.70水平上,可将其划分为3个类群,类群I包含29个菌株,类群II包含1个菌株,类群III包含2个菌株,显示出较低的遗传分化水平。这些SSR引物的开发将为研究我国烟草疫霉的遗传多样性、群体遗传结构以及遗传图谱构建等奠定基础。     

青藏高原地区青稞茎基腐病病原菌的群体遗传多样性、毒素化学型及其地理分布

为明确青藏高原沿线甘肃省甘南藏族自治州和青海省青稞茎基腐病燕麦镰孢菌Fusarium avenaceum的群体遗传多样性、毒素化学型及其地理分布,采用SSR分子标记法对6个地理种群91株燕麦镰孢菌菌株的遗传多样性进行分析。结果表明,6对SSR引物在91株燕麦镰孢菌中共检测到等位位点数14个,多态性位点数13个,多态性条带百分率为92.86%。6个地理种群的平均等位基因数为1.82,有效等位基因数为1.55,Nei’s基因多样性指数为0.32,Shannon信息指数为0.47,多态性位点数为11.50,多态位点百分率为82.15%。6个地理种群的Nei’s遗传相似度为0.83～0.99,遗传距离为0.01～0.18。种群间的遗传距离和地理距离、遗传相似度与海拔差距无显著相关性。燕麦镰孢菌地理种群聚为3个大类群,Group Ⅰ由甘肃省临潭县、合作市和卓尼县种群组成,Group Ⅱ由青海省互助土族自治县和刚察县种群组成,Group Ⅲ由青海省海晏县种群组成。燕麦镰孢菌种群的遗传变异主要来自种群内部,占总变异的93.63%。燕麦镰孢菌毒素化学型分为NIV、DON、3-AcDON三大类,没有15-AcDON毒素化学型,其中DON毒素化学型在6个地理种群中均有分布。表明燕麦镰孢菌地理种群的遗传多样性高。     

苹果炭疽病病原菌ISSR-PCR反应体系的优化及遗传多样性分析

为了从分子水平探讨苹果炭疽病病原菌的群体遗传多样性,采用L16 (45)正交设计对ISSRPCR反应体系中的Mg2浓度、dNTP浓度、Taq DNA聚合酶用量、引物浓度和DNA含量进行优化,并利用优化的体系进行引物筛选及苹果炭疽病菌遗传多样性分析.确立最优反应体系为2.0mmol/L Mg2+、0.2 mmol/L dNTP、2UTaqDNA聚合酶、1μmol/L引物和DNA 100 ng.筛选获得的10条引物对供试菌株共扩增出42条谱带,均为多态性条带.供试菌株在相似系数0.50处分为2个类群,分别与根据形态学鉴定的胶孢刺盘孢Colletotrichum gloeosporioides和尖孢刺盘孢C.acutatum 2个类群相一致,在相似系数0.74处,分为4个亚群,表明苹果炭疽病菌存在明显的种间及种内遗传分化.     

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。     

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%。     

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。     

京津冀设施大棚根际土壤中筛选出的防治根腐病的生防镰孢菌

 由致病性尖孢镰孢菌(Fusarium oxysporum)引起的根腐病严重危害果蔬生产,但非致病性镰孢菌可作为潜在的生防菌。为筛选防治根腐病的非致病生防镰孢菌,从京津冀设施大棚采集茄科、葫芦科果蔬78份根际土样中分离2 402株真菌,筛选出对致病性尖孢镰孢菌(F. oxysporum)具有拮抗效果的真菌173株。利用镰孢菌通用引物进行PCR扩增,从中筛选出28株候选镰孢菌;通过镰孢菌发酵液泡根进行安全性测试,筛选出对寄主黄瓜幼苗安全无害的镰孢菌菌株4株(1418、1441、1436和1473)。进一步通过镰孢菌测序通用引物TEF1αF/TEF1αR结合菌落和分生孢子的形态学特征,1418菌株和1441菌株被鉴定为尖孢镰孢菌(F. oxysporum)、1436菌株被鉴定为茄病镰孢菌(F. solani)。盆栽测试发现,除1441菌株外,1418菌株、1436菌株和1473菌株对黄瓜根腐病的防效均在50%以上,其中1418菌株的防效为70%,与杀菌剂咪酰胺的防效相当,具有很好的应用潜力。本研究筛选获得的具有生防潜力的镰孢菌不仅为镰孢菌致病力分化的研究提供了实验材料,也为新型生防产品的研制奠定了基础。     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

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。     

兰州百合枯萎病病原菌鉴定及罹病组织超微结构观察

为明确引起甘肃省兰州百合主产区百合枯萎病的致病镰孢菌种类,对从百合主产区枯萎病罹病植株上分离纯化的4株镰孢菌株进行形态学鉴定、分子生物学鉴定以及致病性测定,同时利用电子显微镜对尖孢镰孢菌Fusarium oxysporum侵入百合鳞片后的细胞超微结构进行观察。结果表明:4株镰孢菌菌株经鉴定分别为尖孢镰孢菌、茄病镰孢菌F. solani、三线镰孢菌F. tricinctum和燕麦镰孢菌F. avenaceum。4株镰孢菌菌株的致病力由强到弱的顺序依次是尖孢镰孢菌、燕麦镰孢菌、茄病镰孢菌、三线镰孢菌;尖孢镰孢菌侵入后,鳞片细胞壁、细胞质膜和细胞核结构被破坏,细胞核附近出现大量线粒体,细胞中淀粉粒数量减少。表明尖孢镰孢菌是兰州百合枯萎病防治的重点防控对象。     

Phylogenetic relationships and virulence assays of Fusarium secorum from sugar beet suggest a new look at species designations

Fusarium spp. are responsible for significant yield losses in sugar beet (Beta vulgaris) with Fusarium oxysporum f. sp. betae most often reported as the primary causal agent. Recently, a new species, F. secorum, was reported to cause disease in sugar beet but little is known on the range of virulence within F. secorum or how this compares to the virulence and phylogenetic relationships previously reported for Fusarium pathogens of sugar beet. To initiate this study, partial translation elongation factor 1-α (TEF1) sequences from seven isolates of F. secorum were obtained and the data were added to a previously published phylogenetic tree that includes F. oxysporum f. sp. betae. Unexpectedly, the F. secorum strains nested into a distinct group that included isolates previously reported as F. oxysporum f. sp. betae. These results prompted an expanded phylogenetic analysis of TEF1 sequences from genomes of publicly available Fusarium spp., resulting in the additional discovery that some isolates previously reported as F. oxysporum f. sp. betae are F. commune, a species that is not known to be a sugar beet pathogen. Inoculation of sugar beet with differing genetic backgrounds demonstrated that all Fusarium strains have a significant range in virulence depending on cultivar. Taken together, the data suggest that F. secorum is more widespread than previously thought. Consequently, future screening for disease resistance should rely on isolates representing the full diversity of the Fusarium population that impacts sugar beet.     

Fusarium Redolens f.sp asparagi, Causal Agent of Asparagus Root Rot, Crown Rot and Spear Rot

Two Fusarium species, F. oxysporum f.sp. asparagi and F. proliferatum, are known to be involved in the root and crown rot complex of asparagus. We have investigated reports on the involvement of F. redolens, a third species, which until recently was considered conspecific with F. oxysporum because of morphological similarities. RFLP analysis of the rDNA internal transcribed spacer region and AFLP fingerprinting identified eight strains from asparagus unambiguously as F. redolens. Four of these were tested and found to be pathogenic to asparagus either in this study (two strains) or in a previous one in which they were classified as F. oxysporum (three strains). Disease symptoms and disease development were the same as with F. oxysporum f.sp. asparagi and F. proliferatum. Present data and literature reports identify F. redolens as a host-specific pathogen involved in root, crown and spear rot of asparagus. The pathogen is formally classified as F. redolens Wollenw. f.sp. asparagi Baayen.     

Development of Strain-specific Primers for a Strain of Gliocladium catenulatum Used in Biological Control

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatum strain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatum strains and six other strains of two Gliocladium species, as well as from Trichoderma virens, and isolates of Nectria spp. and Fusarium spp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladium powder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5pg or more. Some variation between the Gliocladium strains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladium isolates. The 28S rDNA sequences and -tubulin sequences were identical or very similar in all Gliocladium strains. Thus, it is possible that the Gliocladium strains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladium species may be necessary.     

Morphological and molecular identification and PCR amplification to determine the toxigenic potential of Fusarium spp. isolated from maize ears in southern Poland

The average amount of precipitation in spring and summer 2010 and 2011 coupled with relatively high temperatures caused massive Fusarium spp. infection of maize and yield losses in southern Poland. In order to examine the cause of this disease outbreak, Fusarium spp. were isolated and fungal strains were identified based on morphological characters and species-specific PCR assays. A total of 200 maize samples were processed, resulting in the obtention of 71 strains, which belonged to five Fusarium species, F. poae being the predominant one (74.56%). Other isolates were identified as F. graminearum, F. oxysporum, F. verticillioides and F. proliferatum. PCR-based detection of mycotoxin-synthesis-pathway genes was also used to determine the potential of the analyzed strains to produce trichothecenes (DON and NIV) and fumonisins (FUM). Only 14 isolates revealed the potential to produce DON (11 strains) and FUM (3 strains). HPLC analyses of grain samples revealed the presence of DON only – other mycotoxins were not detected. Moreover, 57.1% of potentially mycotoxin-producing isolates indicated the toxicity in a biological test.