多年生黑麦草中小麦矮腥黑穗病菌PCR 检测方法的建立

寄生于多年生黑麦草的Tilletia属腥黑粉菌共有4种,即小麦矮腥黑穗病菌Tilletia controversa（TCK）、黑麦草腥黑粉病菌T.lolii、T.vankyi、黑麦草粒腥黑穗病菌T.walkeri。本研究分析了黑麦草上冬孢子形态非常相似的3种腥黑粉菌的DNA序列差异,设计了TCK的特异引物,成功建立了TCK菌丝基因组DNA的特异PCR检测方法和冬孢子的套式特异PCR检测方法。     

小麦矮腥黑穗病菌与其近缘种的rDNA-ITS序列分析

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。     

应用TaqMan MGB探针检测小麦印度腥黑穗病菌和黑麦草腥黑穗病菌

 以小麦印度腥黑穗病菌9个菌株和黑麦草腥黑穗病菌5个菌株及其近似种或相关种:稻粒黑粉菌、狼尾草腥黑粉菌、狗尾草腥黑粉菌、苏玛特腥黑粉菌、狐尾草腥黑粉菌、小麦网腥黑穗病菌和小麦矮化腥黑穗病菌共9种22个菌株为研究对象,通过序列比对分析,设计了检测小麦印度腥黑穗病菌及黑麦草腥黑穗病菌的TaqMan MGB实时荧光PCR引物和探针,优化了反应条件,筛选出特异性探针,分别建立了小麦印度腥黑穗病菌和黑麦草腥黑穗病菌实时荧光单重PCR和实时荧光双重PCR检测方法,其中实时荧光双重PCR检测方法实现了在同一PCR管中仅用5μL的反应体系,进行1次PCR反应就能特异性检测出小麦印度腥黑穗病菌或黑麦草腥黑穗病菌。本研究所建立的检测方法特异性强、结果可靠、检测速度快、成本明显降低,在文际应用中具有推广价值。     

黑麦草粒腥黑粉病菌与黑麦草的检疫问题

黑麦草既是某些外来危险性病害如小麦矮化腥黑穗病菌的重要寄主;本身也受到很多病菌包括其他腥黑粉病菌的侵害;美国农业部在1996年至1997年继续对小麦印腥病菌疫区分布的调查中,在俄勒冈州及美国东南部等州发现了一种在形态学上非常近似于小麦印腥,但其寄主是黑麦草的病菌,进一步研究证明,此病原菌是与小麦印腥病菌非常近似的种-黑麦草腥黑穗病菌(Tilletia walkeri Castlebury and Carris 1999).本文综合了国内外对黑麦草粒腥黑粉病菌的最新研究成果,并探讨了黑麦草的检疫性保护问题.     

套式PCR直接检测印度腥黑穗病菌冬孢子

用印度腥黑穗病菌冬孢子制备模板DNA ,利用印腥特异性引物T3 /T6,T3 /T4和套式PCR(nestPCR)扩增技术直接检测印腥冬孢子 ,检测的灵敏度可达 1个冬孢子。检测时间缩短为 1天。这种简单、快速、灵敏、实用和准确的PCR检测技术适用于口岸印腥检疫的需要 ,解决了常规PCR检测中DNA制备需要萌发冬孢子和检测时间长的难题。     

小麦印度腥黑穗病菌PCR检测

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。     

γ射线对小麦印度腥黑穗病菌的灭活效应

小麦印度腥黑穗病菌 Tilletia indicaMitra 是我国一类对外植物检疫对象,迄今对该病的防治仍无十分有效的措施和免疫的小麦品种。本试验应用钴—60γ射线处理小麦印度腥黑穗菌冬孢子。当照射剂量为50万拉德时,冬孢子相对死亡率为99.7%。采用将该菌冬孢子悬浮液直接涂抹在1.5%水琼脂平板上的方法,获得了21%的萌发率。     

黑麦草腥黑粉菌分子检测方法的研究

寄生于黑麦草属植物有4种腥黑粉菌,分别是小麦矮化腥黑穗病菌(Tilletia.Controversa(TCK))、黑麦草腥黑粉菌(T.lolli)、黑麦草粒腥黑粉菌(T.walkeri)和新种(T.vankyi)。其中TCK、T.lolli和T.vankyi冬孢子形态非常相似,难以区分。本研究以寄生于黑麦草上的这3种腥黑粉菌为研究对象,设计T.lolli的特异引物,成功建立了T.lolli冬孢子的套式特异PCR检测方法。     

进境美国小麦中黑麦草腥黑穗病菌的检测和鉴定

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)。     

小麦印度腥黑穗病菌鉴定方法的新进展

自1930年印度西北部Haryana邦的Kamal地区首次发现小麦印度腥黑穗病(Tilletia indicaMitra,以下简称印腥)以来,由于感病寄主的广泛种植和适宜环境条件的存在,该病已在世界范围内的不同生态区迅速传播蔓延,在70年左右的时间内从一个危害轻微的地方性病害发展为威胁世界小麦生产和贸易的危险性真菌病害.     

Identification and Differentiation of Tilletia indica and T. walkeri Using the Polymerase Chain Reaction

ABSTRACT Karnal bunt of wheat, caused by Tilletia indica, was found in regions of the southwestern United States in 1996. Yield losses due to Karnal bunt are slight, and the greatest threat of Karnal bunt to the U.S. wheat industry is the loss of its export market. Many countries either prohibit or restrict wheat imports from countries with Karnal bunt. In 1997, teliospores morphologically resembling T. indica were isolated from bunted ryegrass seeds and wheat seed washes. Previously developed PCR assays failed to differentiate T. indica from the recently discovered ryegrass pathogen, T. walkeri. The nucleotide sequence of a 2.3 kb region of mitochondrial DNA, previously amplified by PCR only from T. indica, was determined for three isolates of T. indica and three isolates of T. walkeri. There was greater than 99% identity within either the T. indica group or the T. walkeri group of isolates, whereas there was =3% divergence between isolates of these two Tilletia species. Five sets of PCR primers were made specific to T. indica, and three sets were designed specifically for T. walkeri based upon nucleotide differences within the mitochondrial DNA region. In addition, a 212 bp amplicon was developed as a target sequence in a fluorogenic 5' nuclease PCR assay using the TaqMan system for the detection and discrimination of T. indica and T. walkeri.     

Internal Transcribed Spacer Sequence-Based Phylogeny and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Differentiation of Tilletia walkeri and T. indica

ABSTRACT A polymerase chain reaction-restriction fragment length polymorphism assay to distinguish Tilleita walkeri, a rye grass bunt fungus that occurs in the southeastern United States and Oregon, from T. indica, the Karnal bunt fungus, is described. The internal transcribed spacer (ITS) region of the ribosomal DNA repeat unit was amplified and sequenced for isolates of T. indica, T. walkeri, T. horrida, and a number of other taxa in the genus Tilletia. A unique restriction digest site in the ITS1 region of T. walkeri was identified that distinguishes it from the other taxa in the genus. Phylogenetic analysis of the taxa based on ITS sequence data revealed a close relationship between T. indica and T. walkeri, but more distant relationships between these two species and other morphologically similar taxa.     

Ustilospores of Tilletia ehrhartae, a smut of Ehrharta calycina, are common contaminants of Australian wheat grain, and a potential source of confusion with Tilletia indica, the cause of Karnal bunt of wheat

Australian wheat consigned for export from Australian ports was surveyed in March 2004 using a national diagnostic protocol for detection and identification of Tilletia indica . No ustilospores of T. indica were detected, confirming previous surveys which have failed to detect T. indica in Australia. However, the survey detected moderate levels of the common smuts Tilletia caries (syn. Tilletia tritici ), Tilletia laevis and Urocystis agropyri , and very low levels (average fewer than six ustilospores per 150 g sample) of an unidentified dark, tuberculate-spored Tilletia in ≈ 60% of samples tested. Comparison with herbarium specimens enabled identification of the majority of the tuberculate ustilospores as Tilletia ehrhartae , a smut fungus known to infect only Ehrharta calycina (perennial veldt grass) and which is common in southern Australia. A smaller number of tuberculate smut ustilospores were identified as Tilletia walkeri , a smut of Lolium spp. recorded in Australia but apparently uncommon. Both T. ehrhartae and T. walkeri bear sufficient resemblance to T. indica for misidentifications to be possible where only a very few ustilospores are seen, although T. ehrhartae ustilospores are always <25  µ m in diameter. The frequent presence of ustilospores of both T. ehrhartae and T. walkeri as contaminants of Australian wheat grain exports has significance for diagnosticians testing Australian export wheat, as it demonstrates the potential for tuberculate ustilospores of species other than those covered in existing diagnostic protocols to be misidentified as T. indica . This paper describes T. ehrhartae in detail, and provides criteria for its differentiation from T. indica , T. walkeri and some other species.     

应用聚合酶链反应技术鉴定印度腥黑穗病菌

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。     

感染“中四”小麦条锈菌T4菌系SCAR检测标记的开发

正条锈病是由小麦条锈菌(Puccinia striiformis f.sp.tritici)引起的世界范围内小麦上最重要的病害之一。利用抗病品种是防治该病最经济、有效的措施。但是由于小麦条锈菌的高度变异性,品种抗病性很容易被条锈菌新小种所克服。因此,持续监测条锈菌生理小种的动态变化,及时发现新小种,对     

Modeling the Risk of Entry, Establishment, Spread, Containment, and Economic Impact of Tilletia indica, the Cause of Karnal Bunt of Wheat, Using an Australian Context

ABSTRACT Modeling techniques were developed to quantify the probability of Tilletia indica entering and establishing in Western Australia (WA), and to simulate spread, containment, and the economic impact of the pathogen. Entry of T. indica is most likely to occur through imports of bulk grain or fertilizer (0.023 +/- 0.017 entries per year and approximately 0.009 +/- 0.009 establishments per year). Entry may also occur through straw goods, new or second-hand agricultural machinery, and on personal effects of travelers who have visited regions with infected plants. The combined probability of entry and establishment of T. indica, for all pathways of entry, is about one entry every 25 years and one establishment every 67 years. Alternatively, sensitivity analysis does show that increases in quarantine funding can reduce the probability of entry to about one entry every 50 years and less than one establishment every 100 years. T. indica is spread efficiently through contaminated farm machinery, seed and soil, rain, air currents, and animals. Depending on the rate of spread of the pathogen and the amount of resources allocated for detection, the time until first detection could range from 4 to 11 years and the economic impact could range from 8 to 24% of the total value of wheat production in WA.     

翦股颖上腥黑粉菌近似种多重分子检测

匍匐翦股颖(Agrostis stolonifera)和细弱翦股颖(A. tenuis)是优良的牧草和草坪草。寄生于翦股颖上的腥黑粉菌共有3种,即小麦网腥黑穗菌(Tilletia caries,TCT)、翦股颖腥黑穗菌(T. sphaerococca)和苍白腥黑粉菌（T. pallida）。在形态、自发荧光和萌发生理3方面比较研究的基础上,选取冬孢子形态非常相似的2种腥黑粉菌TCT和T. sphaerococca为研究对象,依据序列特点设计4套引物,成功建立了TCT和T. sphaerococca菌丝基因组DNA的特异双重PCR检测方法和冬孢子的特异套式双重PCR检测方法。