A PCR-based 'molecular tool box' for in planta differential detection of Verticillium dahliae vegetative compatibility groups infecting artichoke

A multiplex-nested-PCR procedure was developed for in planta detection of Verticillium dahliae isolates infecting artichoke and assessment of their vegetative compatibility groups (VCGs). PCR markers were identified and assigned to V. dahliae VCGs, including: i) a 334 bp marker amplified from VCG1A or VCG2B³³⁴ isolates; ii) a 688 bp marker amplified from VCG2A or VCG4B isolates; and iii) a 688 bp and a 964 bp PCR marker amplified from VCG2B⁸²⁴ isolates. The infecting V. dahliae VCGs were identified in artichoke tissues according to specific patterns of amplified markers after two rounds of PCR. The PCR-based 'molecular tool box' was first optimized using DNA extracted from artichoke plants artificially inoculated with isolates representative of known VCGs. Thereafter, the efficiency of the molecular procedure was tested using DNA extracted from naturally-infected artichoke plants showing a range of symptom severity as well as from symptomless plants. The novel multiplex-nested-PCR assay was clearly superior in detecting the pathogen compared to conventional isolation procedures, and in addition was informative about the VCGs. Moreover, the PCR method allowed the detection and VCG identification of V. dahliae infections in symptomless but infected plants, which had yielded false negatives when checked by microbiological isolation procedures. This 'molecular tool box' has uncovered the presence of several V. dahliae VCGs infecting the same artichoke plants in the Comunidad Valenciana Region. In addition, it is useful for genetic and pathogenicity diversity studies of V. dahliae populations infecting artichoke, and may help in predicting the severity of verticillium wilt epidemics.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

豇豆疫霉LAMP检测方法的建立

本研究以豇豆疫霉Phytophthora vignae Purss核糖体内转录间隔区(internal transcribed spacer,ITS)序列为靶标片段,设计4条特异性引物,建立了环介导等温扩增(LAMP)检测方法。特异性检测结果表明:8株不同地理来源的豇豆疫霉菌株LAMP检测均为阳性(绿色),扩增产物用2.0%琼脂糖凝胶电泳出现特有的梯形条带,而其他11种卵菌近缘种及12种常见病原真菌和细菌共42个菌株均未观察到这些现象。灵敏度分析显示:该方法检测灵敏度在DNA水平上可达到100fg/25μL。采用LAMP方法对福建建瓯和宁德采集的62份疑似豇豆疫病病株样本进行检测,并用组织分离方法进行验证。结果表明,LAMP和组织分离方法的检出率分别为67.7%(42/62)和61.3%(38/62)。综合以上结果,LAMP方法具有特异性强、灵敏度高、快速高效、操作简单的特点,适合基层部门用于田间豇豆疫霉快速检测。     

芋疫霉重组聚合酶扩增结合侧流层析试纸条快速检测方法的建立及应用

为建立芋疫霉Phytophthora colocasiae快速准确的分子检测方法,基于Ypt1基因特异序列,设计芋疫霉的特异性引物与探针,建立一种快速、准确、可视化的芋疫霉重组聚合酶扩增结合侧流层析试纸条（recombinase polymerase amplification-lateral flow dipstick,LFD-RPA）检测方法,对该检测方法进行优化,评估其特异性与灵敏度,并对田间疑似样品进行检测。结果表明,优化后的芋疫霉LFD-RPA检测方法最适反应条件为39℃恒温反应30 min。LFD-RPA检测方法能够特异性地检测出芋疫霉,而对其他卵菌近缘种和常见植物病原真菌均未检出,且该检测方法对芋疫霉DNA的检测灵敏度达到1 pg/μL。对田间带病组织检测发现,LFD-RPA检测方法能够快速准确地从田间自然发病植株中检测出芋疫霉。表明本研究所建立的芋疫霉LFD-RPA快速可视化检测方法特异性好、灵敏度高、简单快捷,可用于芋疫病的田间快速诊断。     

Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection

Plant health regulations to prevent the introduction and spread of Phytophthora ramorum and P. kernoviae require rapid, cost effective diagnostic methods for screening large numbers of plant samples at the time of inspection. Current on-site techniques require expensive equipment, considerable expertise and are not suited for plant health inspectors. Therefore, an extensive evaluation of a commercially available lateral flow device (LFD) for Phytophthora species was performed involving four separate trials and 634 samples. The assay proved simple to use, provided results in a few minutes and on every occasion a control line reacted positively confirming the validity of the test. LFD results were compared with those from testing a parallel sample, using laboratory methods (isolation and real-time PCR). The diagnostic sensitivity of the LFD (87·6%) compared favourably with the standard laboratory methods although the diagnostic specificity was not as stringent (82·9%). There were a small number ( n  = 28) of false negatives, but for statutory purposes where all positive samples must be identified to species level by laboratory testing, overall efficiency was 95·6% as compared with visual assessment of symptoms of between 20-30% for P. ramorum and P. kernoviae . This work demonstrates the value of the LFD for diagnosing Phytophthora species at the time of inspection and as a useful primary screen for selecting samples for laboratory testing to determine the species identification.     

橡胶树胶孢炭疽菌复合群LAMP检测方法的建立及应用

由胶孢炭疽菌复合群(Colletotrichum gloeosporioides species complex)引起的炭疽病是我国橡胶树的主要病害之一。本研究以β-tubulin基因为靶标基因,设计橡胶树胶孢炭疽菌复合群特异性引物,以SYBR Green I为指示剂,建立了特异性强、灵敏度高的环介导等温扩增(LAMP)检测方法,进行了室内侵染和田间自然发病样品的检测验证。结果表明,该方法能在63℃恒温条件下60 min内检测出病原菌,且能特异性识别我国主要植胶区的橡胶树胶孢炭疽菌。该方法最低检测限为1 pg DNA或100个分生孢子。室内和田间样品检测结果表明,该方法可同时检测出潜伏侵染期和发病期的胶孢炭疽菌。本研究建立的LAMP检测方法为橡胶树胶孢炭疽菌的快速鉴定提供了新技术。     

Development of a recombinant antibody ELISA test for the detection of Polymyxa betae and its use in resistance screening

An ELISA test was developed for the quantitative detection of the obligate parasite Polymyxa betae , the vector of Beet necrotic yellow vein virus (BNYVV), in infected sugarbeet roots. The test used monoclonal and polyclonal antibodies raised to a recombinantly expressed glutathione-S-transferase (GST) from P. betae . A close correlation was found between the number of P. betae zoospores in serially diluted suspensions and absorbance values in the ELISA test. Time-course studies of plants grown in naturally infested soils in controlled environment tests demonstrated the value of the ELISA test in screening for P. betae resistance. In preliminary tests, P. betae -resistant accessions of the wild sea beet ( Beta vulgaris ssp. maritima ), which might be used to restrict the transmission of BNYVV, were identified.