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。     

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。     

进境剪股颖草种中腥黑粉菌的检验鉴定

侵染剪股颖的腥黑粉菌是我国尚未发生的一种真菌病害，也是小麦矮腥黑穗病菌(TCK)的近似种。本文介绍了该病菌的检验鉴定情况，并从冬孢子形态、自发荧光现象及萌发生理特性几方面比较了两种病菌的差异。     

进境小麦中沙地牧草腥黑粉菌的鉴定

从上海口岸进境的澳大利亚小麦中发现一种类似小麦印度腥黑粉病菌的腥黑粉菌冬孢子，对该菌冬孢子进行了形态学特征和PCR检测，根据结果，将这种冬孢子鉴定为沙地牧草腥黑粉菌Tilletia ehghartaTle；本研究设计了T．ehrhartaea的特异引物Eh2/Eh4，结合引物Till／Til4建立了T．ehdmrtae的套式PCR检测方法。     

从进口草籽中截获绒毛草腥黑穗菌

本文介绍了小麦矮腥黑穗病菌（ＴＣＫ）的近似种－绒毛草腥黑穗菌（ＴＨＳ）的检验鉴定情况,并详细比较了两种黑粉菌,发现两种冬孢子萌发对温度条件要求不同。     

电子辐照灭活小麦矮腥黑穗菌

电子射线辐照处理小麦矮腥黑穗菌冬孢子,5kGy的剂量可使冬孢子丧失萌发力失去活性.     

水稻腥黑粉病菌的单孢检测

根据水稻腥黑粉菌两个聚类群与其它黑粉菌ITS序列的差异,分别设计了两个聚类群的特异引物Hor2/Hor9和Hm1/Hm5,结合ITS通用引物Til1/Til4建立了分别检测水稻腥黑粉菌两个聚类群单个冬孢子的套式(巢式)PCR检测方法,整个检测过程可以缩短至8h。     

小麦矮腥黑粉菌及其近缘种的RPB2基因片段序列分析

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。     

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。     

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.     

A Multiplex Real-Time Polymerase Chain Reaction (TaqMan) Assay for the Simultaneous Detection of Meloidogyne chitwoodi and M. fallax

ABSTRACT This study describes a multiplex real-time polymerase chain reaction (PCR) approach for the simultaneous detection of Meloidogyne chitwoodi and M. fallax in a single assay. The approach uses three fluorogenic minor groove binding (MGB) TaqMan probes: one FAM-labeled to detect M. chitwoodi, one VIC-labeled to detect M. fallax, and one NED-labeled to detect the internal amplification control (IAC) to monitor false negative results. One common primer set is used for the amplification of part of the internal transcribed spacer (ITS) region of M. chitwoodi and M. fallax and one primer set for the amplification of the IAC. The test enabled detection of M. chitwoodi and/or M. fallax in DNA samples extracted from batches of juveniles, from single juveniles, and from infected plant material. Compared with current assays to detect M. chitwoodi and M. fallax, the multiplex real-time PCR offers the following advantages: it is faster because the test can simultaneously detect both quarantine species without the need for post-PCR processing; and it is at least 10 times more sensitive than a comparable regular PCR also targeting the ITS sequence. Inclusion of the IAC facilitates the interpretation of the FAM and VIC cycle threshold (Ct) values and can prevent the scoring of false negative results when FAM, VIC, and NED Ct values are high. The test allows precise quantification when only one of the two species is present in the sample. However, experiments with mixtures of genomic DNA of M. chitwoodi and M. fallax revealed that the ability of the multiplex real-time PCR assay to detect small quantities of DNA of one species is reduced when large quantities of DNA of the other species are present.     

瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测

通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。     

Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction

ABSTRACT Plasmopara viticola is a strictly biotrophic oomycete that causes downy mildew, which is one of the most important grapevine diseases. Control of the disease is most often achieved by fungicide applications, which may have severe environmental consequences. Therefore, alternative control strategies based on biocontrol agents (BCAs) are currently in development. Thousands of potential BCAs have to be screened for their antagonist efficacy against Plasmopara viticola. Evaluation of their effect on the pathogen can be achieved by detecting the amount of P. viticola DNA in leaves treated with potential antagonists and infected with the pathogen. In this study, a rapid high-throughput method was developed for relative quantification of P. viticola DNA directly from Vitis vinifera leaves by means of multiplex real-time quantitative polymerase chain reaction (PCR) with TaqMan chemistry. This method allows simultaneous amplification, but independent detection, of pathogen and host DNA by using species-specific primers and TaqMan probes that are labeled with different fluorescent dyes. Including detection of V. vinifera DNA in the tests is fundamental because it provides an endogenous reference and allows normalization for variations caused by sample-to-sample differences in DNA extraction, PCR efficiencies, and pipetting volumes. The developed method allows highly sensitive and specific detection of P. viticola DNA (minimal detectable quantity of 0.1 pg). Moreover, high precision and reproducibility of TaqMan assays were observed over a linear range of four orders of magnitude, confirming the reliability of the developed PCR assay. Potential applications range from screening for BCA efficiency to evaluation of fungicide efficacy, or assessment of host resistance.     

A universal microarray detection method for identification of multiple Phytophthora spp. using padlock probes

The genus Phytophthora consists of many species that cause important diseases in ornamental, agronomic, and forest ecosystems worldwide. Molecular methods have been developed for detection and identification of one or several species of Phytophthora in single or multiplex reactions. In this article, we describe a padlock probe (PLP)-based multiplex method of detection and identification for many Phytophthora spp. simultaneously. A generic TaqMan polymerase chain reaction assay, which detects all known Phytophthora spp., is conducted first, followed by a species-specific PLP ligation. A 96-well-based microarray platform with colorimetric readout is used to detect and identify the different Phytophthora spp. PLPs are long oligonucleotides containing target complementary sequence regions at both their 5' and 3' ends which can be ligated on the target into a circular molecule. The ligation is point mutation specific; therefore, closely related sequences can be differentiated. This circular molecule can then be detected on a microarray. We developed 23 PLPs to economically important Phytophthora spp. based upon internal transcribed spacer-1 sequence differences between individual Phytophthora spp. Tests on genomic DNA of many Phytophthora isolates and DNA from environmental samples showed the specificity and utility of PLPs for Phytophthora diagnostics.     

Simultaneous and selective detection of two major soft rot pathogens of potato: Pectobacterium atrosepticum (Erwinia carotovora subsp. atrosepticum) and Dickeya spp. (Erwinia chrysanthemi)

Dickeya spp. and Pectobacterium atrosepticum are major pathogens of potato. Current methods to detect these soft-rotting bacteria require separate identification steps. Here we describe a simple method allowing simultaneous detection of both pathogens based on multiplex PCR. The sensitivity of the primer sets was first examined on purified genomic DNA of the type strains Dickeya chrysanthemi 2048^T and P. atrosepticum 1526^T. The specificity and detection limits of the primer sets were successfully tested on 61 strains belonging to various Dickeya and Pectobacterium species, on artificially inoculated and on naturally contaminated potato plants. This new method provides a gain in time and materials, the main advantages for large-scale processes such as pathogen-free seed certification.     

The Use of ARMS PCR in Detection and Identification of Xanthomonads Associated with Pistachio Dieback in Australia

Pistachio dieback occurs in the main pistachio growing areas of Australia. Xanthomonas strains belonging to the translucens group have been identified as the causal agent of the disease and two distinct groups, A and B, have been recognised within the pathogen population. In this study, specific primers for amplification of DNA of the pathogen were developed by sequencing the Internal Transcribed Spacer (ITS) region of rDNA from strains representing groups A and B, as well as from X. translucens isolated from wheat in Australia and one Xanthomonas translucens strain from orchard floor grasses. Primers were designed for amplification of DNA sequences specific to each group and a multiplex PCR test was developed that identified and differentiated strains of each group in a single PCR assay. To determine the specificity of the primers, PCR was carried out with DNA from 65 strains of the pistachio pathogen, 31 type and reference strains of Xanthomonas, and from 191 phytobacteria commonly found in and around pistachio orchards. In the multiplex PCR, a 331 bp fragment was amplified from all strains belonging to group A and a 120 bp fragment from all strains in group B. No PCR products were obtained from the other bacteria tested except for the type strain of X. translucens pv. cerealis, which has not been found in Australia. The assay was used to detect strains from both groups of the pathogen in pistachio plant material.     

The use of species-specific DNA probes for the identification of Mycosphaerella fijiensis and M. musicola, the causal agents of Sigatoka disease of banana

The polymerase chain reaction (PCR)-based technique of random amplification of polymorphic DNA (RAPD) was used to differentiate DNA from species of the genus Mycosphaerella. DNA from two pathogens which cause Sigatoka leafspot diseases of banana, M. fijiensis and M. musicola , and two other Mycosphaerella species which are commonly found on banana, M. musae and M. minima , gave distinct RAPD banding patterns with all PCR primers tested. PCR, using primer RC07, amplified a 1250bp RAPD fragment from all isolates of M. fijiensis obtained from 11 geographical origins. This fragment was absent from the other species of Mycosphaerella. In Southern blots of genomic DNA, this band hybridized exclusively to DNA from M. fijiensis , and the pattern of hybridization suggested that it was binding to repeated DNA. A RAPD band amplified with primer PM06 obtained from M. musicola was also found to be species-specific. Southern analysis suggested that the fragment hybridized to a single-copy sequence in the M. musicola genome. Total genomic DNA from M. musicola was found to be a species-specific hybridization probe. Dot-blots confirmed the specificity of these probes, and could be used to identify isolates of Mycosphaerella which cause Sigatoka disease of banana in south-east Asia.