共查询到19条相似文献,搜索用时 359 毫秒
1.
2.
小麦印度腥黑穗病菌PCR检测 总被引:6,自引:11,他引:6
应用PCR方法对小麦印度腥黑穗病菌及其近似种或相关种包括黑麦草腥黑粉菌、狼尾草腥黑粉菌、水稻腥黑粉菌等10种腥黑粉菌共14个菌株进行了检测研究。根据线粒体DNA的序列分别设计了扩增小麦印度腥黑穗病菌的特异性引物和扩增黑麦草腥黑粉菌的特异性引物,根据核糖体内转录区(ITS)DNA片段设计了扩增腥黑粉菌属真菌的引物,应用PCR方法能将小麦印度腥黑穗病菌与黑麦草腥黑粉菌及其它近似种或相关种加以区分。本方法稳定、可靠、重复性强,已分别在不同实验室的不同型号PCR仪上得到验证。 相似文献
3.
4.
小麦矮腥黑穗病菌与其近缘种的rDNA-ITS序列分析 总被引:5,自引:0,他引:5
本研究对小麦矮腥黑穗病菌(Tilletia controversa)及其近似种小麦网腥黑穗病菌(T.caries)、小麦光腥黑穗病菌(T. foetida)的rDNA-ITS进行了测序,并结合GenBank中登录的这3个种的其他菌株及腥黑粉属其他6个近缘种41条ITS序列进行了聚类分析。结果表明,所有菌株可以被划分为3个分支:第1个分支为印度腥黑穗病菌(T. indica)与其近似种T. walkeri;第2个分支主要是小麦矮腥黑穗病菌与其近似种T. caires和T. foetida及寄生在杂草上的一些腥黑粉菌(T.bromi 和T.fusca);第3个分支主要是寄生在杂草和水稻上的腥黑粉菌(T. barclayana和T. horrida)。第1分支与第2分支之间 ITS差异较大,同一分支内不同种之间ITS差异很小。rDNA-ITS序列只能用于腥黑粉菌属中部分种的区分。 相似文献
5.
小麦矮腥黑穗病菌(Tilletia controversa Kühn, 简称TCK)是小麦上的一种重要检疫性真菌。本研究利用内部简单重复序列(Inter-simple sequence repeat, ISSR)技术研究TCK及其近缘种的DNA多态性,开发了一种可靠而简单的方法用于TCK的分子鉴定。用ISSR引物P4从TCK中扩增出一条1 113 bp的特异性条带,据此设计了一对特异性引物TCKF/TCKR,在12个TCK菌株中均能扩增得到一条882 bp的特异性条带,而其他近缘种包括小麦网腥黑穗病菌(T. caries)和小麦光腥黑穗病菌(T. foetida)及相关黑粉菌的14个菌株均无扩增条带。用该特异性引物检测TCK的下限为25 μL反应体系中可检测到1 ng DNA模板。本研究开发的种特异性引物,可将TCK与其形态上相似的近缘种尤其是小麦网腥黑穗病菌准确区分开,本研究基于ISSR标记建立的小麦矮腥黑穗病菌的分子鉴定方法为腥黑粉菌的检疫提供了一种便捷的方法,是对现有分子鉴定方法的一个补充。 相似文献
6.
从进境美国小麦洗涤液中发现一种类似小麦印度腥黑穗病菌的冬孢子,冬孢子大小为25.0~33.8(29.5±2.3)μm,球形至近球形,淡黄褐色至暗红褐色,半透明至不透明,外胞壁具深褐色疣状突起,突起表面观为锥形至平截状,有时呈不完全脑纹状,外被淡色透明胶质鞘。冬孢子处理后经通用引物Til1/Til4和U2/U10扩增,其扩增产物用黑麦草腥黑穗病菌Tilletiawalkeri的特异性引物W1/W4再次扩增可以得到760bp的产物,产物测序得到的序列和GenBank中黑麦草腥黑穗病菌菌株YRG-001(AF218063)的序列相似性为99%(717/719),和GenBank中小麦印度腥黑穗病菌菌株Bpop(AF218060)的序列相似性为96%(694/719)。实时荧光检测的结果表明黑麦草腥黑穗病菌的探针TWAL能得到阳性扩增曲线。根据冬孢子形态特征、PCR分析、实时荧光检测和序列分析的结果,将这种腥黑穗病菌冬孢子鉴定为黑麦草腥黑穗病菌(T.walkeri)。 相似文献
7.
8.
9.
10.
1 名称、分类及分布学名 :TilletiawalkeriL .A .CastleburyetL .M .Carris.,英文名 :ryegrassbunt分类地位 :担子菌亚门 (Basidiomycoti na) ,冬孢菌纲 (Teliomycetes) ,黑粉菌目(Ustilaginales) ,腥黑粉菌科 (Tilletiaceae) ,腥黑粉菌属 (Tilletia)。分布 :美国 (东南部诸州、俄勒冈州 )、澳大利亚[1~ 2 ] 。寄主 :多花黑麦草(LoliummultiflorumL .)、黑麦草(L .PerenneL .) [1~2 ] 。2 … 相似文献
11.
应用聚合酶链反应技术鉴定印度腥黑穗病菌 总被引:7,自引:5,他引:2
用一对专化于印度腥黑穗病菌的引物T117M1(5'-TCCCCTTG-GATCAGAACGTA-3')和T117M2(5'-AGAAGTCTAACTCCCCCCTCT-3')可特异地扩增印度腥黑穗病菌产生一段825bp的产物,而稻粒黑粉病菌则不能被扩增。实验还表明,用聚合酶链反应(PCR)方法检测灵敏度可达到100个未萌发的冬孢子,这为进口粮印度腥黑穗病菌的检疫提供了有力工具。 相似文献
12.
13.
14.
麦麸中小麦矮腥黑穗病菌孢子的提取方法 总被引:1,自引:0,他引:1
对麦麸中小麦矮腥黑穗病菌孢子的提取方法进行了研究。通过建立的"过筛-α-淀粉酶降解"、"过筛-密度梯度离心"和"过筛-α-淀粉酶降解-密度梯度离心"等方法能从麦麸中提取TCK孢子。提取的TCK孢子数量与网筛、α-淀粉酶和密度梯度离心的应用情况有关,而提取的TCK孢子纯度则主要与密度梯度离心有关。选用双层纱布和5种不同规格的网筛或网筛组合进行过筛能弃除83.88%~94.09%的麦麸,应用"过筛-α-淀粉酶降解"方法能弃除95.01%~99.00%的麦麸,并能提取19.2%~51.7%的TCK孢子。在"过筛-密度梯度离心"实验中,只有60目+200目+300目+30μm+11μm该网筛组合处理能获取7.2%的纯孢子,密度梯度离心对孢子的网嵴高度、自发荧光和萌发没有影响;在"过筛-α-淀粉酶降解-密度梯度离心"实验中,60目+200目+300目和60目+200目+300目+30μm+11μm这2组网筛处理可分别得到18.8%和12.2%的纯孢子,该处理对孢子网嵴高度没有影响。 相似文献
15.
Qing Yuan Siji Nian Youping Yin Minhui Li Jun Cai Zhongkang Wang 《European journal of plant pathology / European Foundation for Plant Pathology》2009,124(4):585-594
Wheat dwarf bunt, one of the important international quarantine diseases, is caused by Tilletia controversa. Tilletia caries is a close relative species of T. controversa and the teliospore morphology and genomic structure of T. caries are very similar to those of T. controversa. In order to distinguish between them, a random amplified polymorphic DNA (RAPD) primer-mediated asymmetric-PCR (RM-PCR)
was developed to screen differential sequences between the two pathogens. By RM-PCR, a 1,322 bp DNA fragment (PR32) was selected
from 18 T. controversa and 29 T. caries strains. The PR32 genes were specific for T. controversa and almost had no homology to T. caries or other fungi in the present database. With primers designed from PR32, all 18 T. controversa strains were amplified, but no bands appeared in 29 T. caries strains by classical PCR. To identify T. controversa rapidly and accurately, SYBR Green I and TaqMan probe real-time PCR were established based on PR32. With TaqMan Real-Time
PCR, different T. controversa strains and T. caries strains were detected. The results showed that all T. controversa strains were amplified with Ct from 19–29 and amplified curves were obtained. In contrast, the amplification of all T. caries strains did not show any signals, without Ct and amplified curves. Moreover, the developed TaqMan real-time PCR was used
to detect T. controversa from asymptomatic wheat tissues successfully. 相似文献
16.
A quantitative real-time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat-seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log10 (number of spores per seed + 1) on mean log10 DNA ( µ g) produced a coefficient of determination ( R 2 ) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real-time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed. 相似文献
17.
烟草根黑腐病菌的PCR分子检测 总被引:6,自引:1,他引:5
根据烟草根黑腐病菌(Thielaviopsis basicola)与其它烟草病原真菌核糖体基因转录间隔区(internal transcribed spa-cer,ITS)序列间的差异,设计了一对特异性引物TB-5/TB-3,用于T. basicola的分子检测。利用该对引物对包括T. basicola在内的13个烟草病原菌菌株的基因组DNA进行PCR扩增,结果表明:只有T. basicola能扩增到一条400bp左右的特异性条带,其它菌株及阴性对照均无扩增产物。对烟草组织和土壤的检测结果也表明,该对引物能特异性的检测到T. basicola基因组DNA的存在。该引物对T. basicola基因组DNA检测的灵敏度为100fg/μL。 相似文献
18.
中美小麦矮腥黑穗病菌鉴定合作研究:I.自发荧光显微学特性研究 总被引:2,自引:0,他引:2
中美双方为探讨适用于进口粮检疫的小麦矮腥黑穗病菌(Tilletia controversa Kuehn,以下简称TCK)准确简便的鉴定方法,于1989~1992年进行联合试验。本文以美国不同时期和不同地域所收集的小麦矮腥黑穗病菌(TCK)和小麦网腥黑穗病菌(T.caries Tul.,以下简称TCT)共135个菌株为材料,对这两种病菌的自发荧光显微学特性,作了比较研究,建立了数学模型,并在人工污染样品中进行了应用研究,结果表明,自发荧光显微学法和LI-F方程,可以有效地鉴别TCK或TCT菌株,但如应用于鉴别进口粮中混合的或少量的TCK、TCT冬孢子,则其准确率随孢子含量的下降而降低。 相似文献
19.
小麦矮腥黑粉菌及其近缘种的RPB2基因片段序列分析 总被引:1,自引:0,他引:1
以小麦矮腥黑粉菌(Tilletia controversa Kühn)及其近缘种小麦网腥黑粉菌[T. caries (DC.)Tul.]、小麦光腥黑粉菌(T. laevis Kühn)和其他6种黑粉菌的DNA为模板,用RNA聚合酶II的第2亚基RPB2基因的通用引物RPB2-740F/RPB2-1365R进行PCR扩增。结果表明,3种小麦腥黑粉菌均能扩增出617 bp大小的DNA片段,供试的其他6种黑粉菌没有任何扩增产物。利用DNAMAN软件进行序列分析结果表明,3种小麦腥黑粉菌的RPB2蛋白基因序列的相似性为99.08%,存在17个碱基的差异。利用RPB2基因的通用引物作为小麦腥黑粉菌的内置对照引物,与小麦矮腥黑粉菌的特异引物CQUTCK2/CQUTCK3相结合可提高小麦矮腥黑粉菌检测的准确性。 相似文献