共查询到17条相似文献，搜索用时 279 毫秒
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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。 相似文献
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雪松疫霉(Phytophthora lateralis)的快速分子检测 总被引：1，自引：0，他引：1
由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。 相似文献
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冬生疫霉(Phytophthora hibernalis)的快速分子检测 总被引：4，自引：1，他引：3
由冬生疫霉(Phytophthora hibernalis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了冬生疫霉和其它疫霉的ITS序列,在此基础上设计了一对检测冬生疫霉的特异性引物751F/752R,该对引物从冬生疫霉中扩增得到一条616bp的条带,而其它19种疫霉和其它真菌菌株均无扩增条带,表明该对引物对冬生疫霉具有特异性。在25μL PCR反应体系中,引物751F/752R检测灵敏度为10龟基因组DNA;而以卵菌ITS区通用引物ITS1/ITS4和751F/752R进行套式PCR扩增,能够检测到10ag的基因组DNA,使检测灵敏度提高了1000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子。结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术,在1个工作日内即可从人工接种发病的植物组织中特异性的检测到该病原菌。表明本研究建立的检测方法可用于冬生疫霉的快速分子检测。 相似文献
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小麦光腥黑粉菌冬孢子总DNA提取方法比较 总被引：6，自引：1，他引：6
以小麦光腥黑粉菌冬孢子为试验材料,比较分析了改良CTAB法、氯化苄法、SDS法对小麦光腥黑粉菌冬孢子总DNA提取的效果。结果表明,SDS法无论在DNA纯度(R=A260 nm/A280 nm)和产量上均优于氯化苄法和CTAB法。利用SDS法能获得186 ng/mg的DNA产率,而氯化苄法和CTAB法只能分别获得145 ng/mg和112 ng/mg。采用SDS法提取的DNA纯度较高,-RSDS=1.485,氯化苄法(BC)和CTAB法所得DNA纯度无明显差异,分别为-RCTAB=1.254和-RBC=1.264。 相似文献
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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
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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
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Frederick RD Snyder KE Tooley PW Berthier-Schaad Y Peterson GL Bonde MR Schaad NW Knorr DA 《Phytopathology》2000,90(9):951-960
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. 相似文献
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Tilletia indica Mitra is a fungal pathogen causing Karnal bunt of wheat. Tilletia indica is a quarantine pest in many countries worldwide. In the European Union, imported wheat grain from countries where the fungus is present must be checked for the presence of T. indica teliospores. The inspection services at the borders need rapid, sensitive and reliable detection tests to identify T. indica spores on wheat grain. In this work, validation was carried out according to EPPO Standard PM 7/98 to evaluate the multiplex real‐time PCR test described in ISPM 27 Diagnostic Protocol for regulated pests (Annex 4 Tilletia indica Mitra) by means of a test performance study with nine participating laboratories, and the performance characteristics of the test were established. The original protocol was modified with regard to the extraction of DNA from the pellet obtained from the ‘washing test’ and the enrichment PCR step in order to increase the amount of template DNA for the real‐time PCR. The optimized test still has five teliospores as the limit of detection for the contaminated pellet but has an increased analytical sensitivity and had positive results with three teliospores in 93% of cases instead of 43% for the original test. The two closest Tilletia species, Tilletia horrida and Tilletia walkeri, were used to evaluate analytical specificity (exclusivity) and no cross‐reactions were obtained. Diagnostic sensitivity, diagnostic specificity, accordance and concordance were also evaluated. 相似文献
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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
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《EPPO Bulletin》2018,48(1):7-31
Specific scope
This Standard describes a diagnostic protocol for Tilletia indica. 1 It should be used in conjunction with PM 7/76 Use of EPPO diagnostic protocols.Specific approval and amendment
This Standard was originally developed under the EU DIAGPRO Project (SMT 4‐CT98‐2252) by a partnership of contractor laboratories and interlaboratory comparison in European countries. First approved as an EPPO Standard in 2003–09. First revision approved in 2007–09. Second revision approved on 2017–11. Although this EPPO Diagnostic Standard differs in terms of format it is in general consistent with the content of the IPPC Standard adopted in 2014 on Tilletia indica (Annex 4 to 2006 ) with the following exceptions. (1) In the EPPO region, as the pest is not present, a higher confidence in the results is required, a sieve wash test should be carried out (optional in the IPPC protocol). (2) When fewer than 10 teliospores are found the options should allow testing the (<10) teliospores with conventional or real‐time PCR (this was not an option in the IPPC protocol flow chart, although it was stated that direct real‐time PCR could be used on individual teliospores in the text). (3) The method for extracting teliospores from untreated seed or grain by size‐selective sieving is slightly different based on the experience in the region (European Union test performance study). The EPPO Diagnostic Standard also includes a test for a direct real‐time PCR for use on pellets (developed in 2016). Some additional information on methods for morphological identification, from the former version of the EPPO Standard, which are not in the IPPC protocol are included in this protocol in Appendix 3 as they were considered useful by the members of the Panel on Diagnostics in Mycology. 相似文献17.
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. 相似文献