Simultaneous monitoring and quantification of Melampsora allii‐populina and Melampsora larici‐populina on infected poplar leaves using a duplex real‐time PCR assay

The rust fungi Melampsora larici‐populina (Mlp) and Melampsora allii‐populina (Map) are the main phytosanitary constraints for commercial poplar cultivation in Europe. Although Mlp is more aggressive and prevalent than Map, the two species may co‐infect the same poplar tree or even the same poplar leaf, making the epidemiological surveys of each species difficult to achieve. In this study, a new duplex real‐time PCR assay targeting each species was developed, based on single‐copy genes. This test proved to be specific, inclusive and was successfully used to detect and quantify each species, starting from urediniospore samples or directly from infected poplar leaves, with or without visible symptoms. This new molecular tool was also assessed for comparative studies of time‐course infection experiments on artificially inoculated poplar leaf discs. These studies showed that the growth dynamics of Map were significantly slower when the two species were co‐inoculated on the same leaf disc, confirming that Map is less aggressive than Mlp.     

A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed

Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.     

长孢轮枝菌Taq〖KG-*4〗Man-MGB探针实时荧光PCR快速检测方法

长孢轮枝菌是一种在我国局部地区新近出现且危害性极大的植物病原真菌?根据长孢轮枝菌及其近似种的actin序列差异, 设计并合成特异性引物和探针, 建立了长孢轮枝菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能特异性检测长孢轮枝菌; 灵敏度试验结果表明, 最低检测限量为10 μL反应体系中总DNA含量10 pg; 实时荧光PCR优化反应条件为引物终浓度0.8 μmol/L, 探针终浓度0.8 μmol/L, 优化后的整个反应过程约1 h?实际样品检测结果表明, 该方法可用于疑似受长孢轮枝菌侵染的萝卜样品检测与初筛?此方法快速?灵敏, 检测过程完全闭管, 无需PCR后续处理, 为早期快速检测长孢轮枝菌提供了重要参考?     

基于TaqMan MGB探针的黑白轮枝菌检测方法

根据黑白轮枝菌(Verticillium albo-atrum)及其近似种β-微管蛋白基因(β-tubulin)序列差异,设计并合成1对引物和1条Taq Man-MGB探针,建立了黑白轮枝菌的实时荧光PCR检测方法。对供试黑白轮枝菌及其近似种实验表明,该方法特异性强,只有黑白轮枝菌可被检出。通过对反应体系的优化,确定了最佳反应条件:引物终浓度为1.0μmol/L,探针终浓度为0.7μmol/L。灵敏度试验结果显示,最低检测限量为总DNA含量10 pg(20μL反应体系)。此方法快速灵敏,为快速检测黑白轮枝菌提供了重要参考。     

基于TaqMan MGB探针的可可花瘿病菌快速检测方法

可可花瘿病菌是一种我国进境植物检疫性真菌?本文根据可可花瘿病菌EF1α基因的保守序列, 设计并合成1对特异性的实时荧光PCR引物和1条TaqMan MGB探针, 建立了可可花瘿病菌的实时荧光PCR检测方法?特异性试验结果表明, 该检测方法能够特异性检出可可花瘿病菌; 实时荧光PCR优化反应条件为引物终浓度0.2 μmol/L, 探针终浓度0.6 μmol/L; 灵敏度试验结果表明, 20 μL反应体系中可可花瘿病菌DNA含量最低检测限为10 pg; 重复性试验结果表明, 该检测方法的重复性和稳定性良好; 接种试验样品检测结果表明, 该方法可用于疑似携带可可花瘿病菌样品的检测与初筛?本文建立的方法具有良好的灵敏性?特异性和应用性, 为可可花瘿病菌早期快速检测提供了一种有效手段?     

Quantitative detection and monitoring of Colletotrichum acutatum in strawberry leaves using real-time PCR

Real-time PCR assays for Colletotrichum acutatum , one of the most important pathogens of strawberry worldwide, were developed using primers designed to the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) and the β-tubulin 2 gene. Using TaqMan technology, the ITS-based assay could reliably detect as little as 50 fg genomic DNA, 100 copies of target DNA, or 25 conidia. The β-tubulin-based assay was c . 66 times less sensitive, and therefore less suitable for detection purposes. The TaqMan-ITS assay recognized all C. acutatum isolates tested from various intraspecific molecular groups, while no amplification was observed with several other Colletotrichum species or other strawberry pathogens, indicating the specificity of this assay. Detection and quantification of C. acutatum was demonstrated in artificially and naturally infected strawberry leaves. First, C. acutatum was detected in plant mixes of which only 0·001% of the tissue was infected by C. acutatum . Secondly, real-time PCR analysis of leaf samples taken at various times after inoculation indicated that the assay allowed monitoring of growth progression of C. acutatum . This real-time PCR-mediated monitoring of the pathogen was well-correlated with microscopic data, and confirmed that leaf age may play a role in the extent of C. acutatum infection. Finally, the assay allowed detection of C. acutatum in naturally infected and symptomless strawberry leaves collected from production fields and planting material.     

A new large scale soil DNA extraction procedure and real-time PCR assay for the detection of Sclerotium cepivorum in soil

A real-time PCR assay specific for Sclerotium cepivorum, the causal agent of white rot in onions, was developed for use with a new DNA extraction method capable of processing up to 1?kg of soil in weight. The assay was specific for S. cepivorum when tested against 24 isolates representative of 14 closely related species and other pathogens of onion. The assay was highly sensitive when used with soil DNA extracted using the new DNA extraction procedure. In three different field soils tested, a good relationship between cycle threshold (Ct) and number of sclerotia was observed (R2?=?0.89). Twenty-nine soil samples from onion and leek crops were obtained and the pathogen was detected in four samples. All four positive samples were associated with current or past outbreaks of white rot of onion. Additional assays were also used on the 29 field soil samples, Botrytis aclada and Rhizoctonia solani AG8 were also detected, in one soil sample each. Rhizoctonia solani AG2-1 was more widespread and was detected in eight different soil samples. The method is therefore suitable to quantify levels of S. cepivorum in soil samples, with the added advantage that the technique allows other soil pathogens of interest to be assayed from the same DNA sample. The bulk soil DNA extraction method described here has the potential to be used to detect soil-borne pests and pathogens for other crops in a wide range of soil types.     

An optimized duplex real-time PCR tool for sensitive detection of the quarantine oomycete Plasmopara halstedii in sunflower seeds

Plasmopara halstedii, the causal agent of downy mildew of sunflower, is an oomycete listed as a quarantine pathogen. This obligate parasite resides in a quiescent state in seeds of sunflower and can be spread from seed production areas to areas of crop production by international seed trade. To prevent the spread or the introduction of potentially new genotypes or fungicide-tolerant strains, an efficient method to detect P. halstedii in sunflower seed is required. This work reports the optimization of a real-time detection tool that targets the pathogen within sunflower seeds, and provides statistically validated data for that tool. The tool proved to be specific and inclusive, based on computer simulation and in vitro assessments, and could detect as few as 45 copies of target DNA. A fully optimized DNA extraction protocol was also developed starting from a sample of 1,000 sunflower seeds, and enabled the detection of <1 infected seed/1,000 seeds. To ensure reliability of the results, a set of controls was used systematically during the assays, including a plant-specific probe used in a duplex quantitative polymerase chain reaction that enabled the assessment of the quality of each DNA extract.     

A generic RT‐PCR assay for the detection of Luteoviridae

This study, using RT‐PCR, is the first comprehensive assessment since 1991 of a generic detection method for the Luteoviridae. Thirteen Luteoviridae species were detected using three separate sets of low‐degeneracy generic primers with RT‐PCR to amplify 68‐, 75‐ and 129/156‐bp regions of the Luteoviridae coat‐protein gene. Species detected include all members of the genus Luteovirus [Barley yellow dwarf virus (BYDV)‐PAV, BYDV‐PAS, BYDV‐MAV (129 and/or 156 bp amplicons), Soybean dwarf virus, Bean leafroll virus (68 bp amplicon)] and eight of nine species from the genus Polerovirus [Beet western yellows virus, Beet chlorosis virus, Beet mild yellowing virus, Turnip yellows virus, Potato leafroll virus, Cucurbit aphid‐borne yellows virus, Cereal yellow dwarf virus‐RPV (68‐bp amplicon) and Sugarcane yellow leaf virus (75‐bp amplicon)]. These primers were not able to detect Carrot red leaf virus, Sweet potato leaf speckling virus (both belong to unassigned Luteoviridae) and Pea enation mosaic virus‐1 (genus Enamovirus). A synthetic positive control containing all primer sequence priming sites was designed to facilitate this method as a generic tool for use with a variety of host plants. The Luteoviridae primers described in this study present a simple infection‐detection tool of benefit to biosecurity authorities in nursery‐stock surveillance, disease management or outbreak prevention, and may also be useful in detection of as‐yet undiscovered species within the Luteovirus and Polerovirus genera.     

向日葵黑茎病菌TaqMan-MGB探针实时荧光快速检测方法

 向日葵黑茎病菌是我国进境检疫性有害生物名录中的一种检疫性真菌。根据向日葵黑茎病菌及其近似种的ITS序列差异,设计并合成特异性引物和探针,建立了向日葵黑茎病菌的实时荧光PCR检测方法。特异性试验结果表明,该检测方法能特异性检测向日葵黑茎病菌;灵敏度试验结果表明,最低检测限量为20 μL反应体系中总DNA含量0.1 pg;实时荧光PCR优化反应条件为引物终浓度0.6 μmol·L^-1,探针终浓度0.3 μmol·L^-1。实际样品检测结果表明,该方法可用于疑似携带向日葵黑茎病菌样品的检测与初筛。此方法快速、灵敏,整个反应过程约1 h,检测过程完全闭管,无需PCR后续处理,为早期快速检测向日葵黑茎病菌提供了重要参考。     

The development of a validated real-time (TaqMan) PCR for detection of Stagonosporopsis andigena and S. crystalliniformis in infected leaves of potato and tomato

Stagonosporopsis andigena and S. crystalliniformis are serious foliage pathogens on potato (Solanum tuberosum) and tomato (Solanum lycopersicum). As both species have been recorded only in the Andes area, S. andigena is listed as an A1 quarantine organism in Europe. The actin region of isolates of Stagonosporopsis and allied species of Boeremia, Didymella, Peyronellaea and Phoma was amplified using generic primers. DNA sequence differences of the actin gene were utilised to develop species-specific real-time (TaqMan) PCR assays for the detection of S. andigena and S. crystalliniformis in leaves of potato or tomato. The specificity of the TaqMan PCR assays was determined on genomic DNA extracted from two S. andigena and two S. crystalliniformis isolates and 16 selected isolates of Stagonosporopsis, Phoma and Boeremia, which are the closest relatives. The validation of the methods developed included the DNA extraction and the TaqMan PCR assays. The performance criteria specificity, analytical sensitivity, reproducibility, repeatability and robustness of the TaqMan PCR assays demonstrated the reliability of both methods for the detection of S. andigena and S. crystalliniformis in leaf material. The TaqMan PCR assays were tested on symptomatic leaves of potato and tomato that were obtained after artificial inoculation of detached leaves with both pathogens under quarantine conditions. In the artificial inoculation experiments both S. andigena and S. crystalliniformis caused leaf infections on potato and tomato.     

A PCR diagnostic assay for rapid detection of plant pathogenic pseudomonads

Molecular detection of phytopathogens is increasingly being applied to identify regulated organisms at the border in many parts of the world. However, even with molecular tests, complete phenotyping and identification of a strain is often time consuming and sometimes inconclusive. In this study, a leaf-based pathogenicity test was used to separate pseudomonads into two groups, Group A containing pathogens, and Group B containing saprotrophs. Comparative genomics of 56 pseudomonad genomes from different plant hosts (including 29 strains from kiwifruit) agreed with kiwifruit pathogenicity test results, placing pathogens into Group A and saprotrophs into Group B. Sixteen loci were found unique to Group A. A PCR assay was developed for amplification of one of these loci, the trehalose phosphatase gene. The generation of this 655 bp amplicon was associated with production of water-soaked lesions on inoculated kiwifruit leaves by pseudomonads in Group A. This test was validated for further strains from all seven pathogenic Pseudomonas phylogroups, non-pathogenic pseudomonads, and other bacterial genera. The sensitivity of the PCR was comparable to the limit of recovery of pseudomonads by culturing. This simple PCR assay could be used as part of a testing pipeline at the border and for general surveillance for screening plants with and without symptoms, offering the potential to detect uncharacterized pseudomonads that may pose a biosecurity risk. The method was shown to be able to rapidly identify pathogens cultured from plant material with symptoms, or, more importantly, to detect pathogens directly from plant tissue.     

Quantitative real-time PCR assay for rapid detection of Pseudomonas amygdali pv. lachrymans in cucumber leaf rinse

Journal of Plant Diseases and Protection - Pseudomonas amygdali pv. lachrymans (Pal) is a bacterium that causes angular leaf spot disease of cucumbers. There is no direct protection against this...     

A multiplex real-time PCR assay enables simultaneous rapid detection and quantification of bacteria associated with acute oak decline

Acute oak decline (AOD) is a syndrome affecting mature oak trees and is characterized by stem bleeds from vertical fissures on trunks, and inner bark necrosis caused by a polybacterial consortium, in which Gibbsiella quercinecans and Brenneria goodwinii, and to a lesser extent Rahnella victoriana and Lonsdalea britannica, play key roles. Here we report a novel multiplex real-time PCR assay that enables simultaneous and rapid detection and quantification of these four bacterial species from stem bleed swabs. Experiments with axenic cultures were performed to determine specificity and sensitivity of the multiplex quantitative PCR (qPCR). Whilst the primer/probe set for B. goodwinii was species-specific, primer/probe sets for the other three species were able to identify other members of their respective genera. There was no cross detection of genera within the multiplex qPCR, and non-target bacteria were not detected. The multiplex AOD assay had differential sensitivity for each bacterial species. The assay was evaluated on swab samples collected from stem bleeds of declining oak trees at a site in south-east England and was able to detect all four bacterial species. Absolute quantification of the bacteria from swab samples was possible through the inclusion of a standard curve prepared from dilutions of gene copy standards. This diagnostic tool will facilitate rapid detection of AOD-associated bacteria from samples that can easily be taken by non-specialists without specific training, and will also find application in other experimental work such as pathogenicity and control trials.     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

基于TaqMan MGB探针的葡萄茎枯病菌实时荧光PCR检测方法

 葡萄茎枯病菌是我国进境植物检疫性有害生物。带菌植物材料是病害传播的重要载体,准确、灵敏、快速的检测方法是严格执行口岸检疫措施及研究病害防控措施的有力工具。根据葡萄茎枯病菌及其近似种的细胞骨架蛋白(Actin)基因序列差异,设计并合成1对引物和1条特异性TaqMan-MGB探针,建立了葡萄茎枯病菌的实时荧光PCR检测方法。通过对反应体系的优化,确定了葡萄茎枯病菌的实时荧光PCR最佳反应条件:引物终浓度为0.6 μmol·L^-1,探针终浓度为0.6 μmol·L^-1。灵敏度试验结果显示,最低检测限为总DNA含量20 pg(20 μL反应体系)。此方法快速灵敏,整个反应1 h即可完成,检测过程完全闭管,无需PCR产物后续处理,为快速检测葡萄茎枯病菌提供了重要参考。该方法用于口岸疑似菌株检测,可成功检测出葡萄茎枯病菌。本研究建立的基于TaqMan MGB探针的荧光定量PCR检测方法为葡萄茎枯病菌的早期快速检测监测提供了有力工具。     

红火蚁实时荧光分子检测方法

根据GenBank上发表的火蚁属(Solenopsis)COI基因序列,手工设计了检测红火蚁(Solenopsis invicta)的引物与探针.实时荧光PCR结果表明:红火蚁样品可被成功地检测到,而近似种热带火蚁没有被检测到,同种不同虫态的个体得到一致结果.     

Effects of pre- and post-inoculation photoperiod on the severity of Melampsora medusae leaf rust of Populus species

Disease severity, based on six parameters (time from inoculation to flecking, and eruption of uredinia, uredinial density and the amounts of urediniospoies produced), was assessed on leaf discs cut from four cultivars of Populus spp. raised with either a long (15 h) or a short (10 h) photoperiod. The discs were inoculated with race 4 of Melampsora medusae and subsequently incubated with either a long (15 h) or a short (10 h) photoperiod. While disease severity, based on most parameters, was lower in discs from the continuing long photoperiod (15 h pre- and 15 h post-inoculation) than from the continuing short photoperiod (10 h pre- and 10 h post-inoculation), maximum severity was developed by discs given a combination of a short pre- and a long post-inoculation photoperiod. The relative contribution of the cultivar, the pre- and the post-inoculation photoperiod and their second- and third-order interactions to variation in disease severity depended on the parameter employed to assess severity. The parameters used are elements of disease monocycles and their possible significance in modelling epidemics is discussed.