LAMP assay and rapid sample preparation method for on‐site detection of flavescence dorée phytoplasma in grapevine

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on‐site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real‐time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real‐time PCR. The whole procedure of sample preparation and testing was designed and optimized for on‐site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.     

Rapid and reliable detection of phytoplasma by loop-mediated isothermal amplification targeting a housekeeping gene

Phytoplasmas are plant pathogenic bacteria that infect more than 700 plant species. Because phytoplasma-resistant cultivars are not available for the vast majority of crops, the most common practice to prevent phytoplasma diseases is to remove infected plants. Therefore, developing a rapid, accurate diagnostic method to detect a phytoplasma infection is important. Here, we developed a phytoplasma detection assay based on loop-mediated isothermal amplification (LAMP) by targeting the groEL gene and 16S rDNA. We designed 19 primer sets for the LAMP assay and evaluated their amplification efficiency, sensitivity, and spectra to select the most suitable primer sets to detect Candidatus Phytoplasma asteris. As a result, DNA was efficiently amplified by one of the primer sets targeting the groEL gene, and LAMP assay sensitivity with this primer set was 10-fold higher than that of the polymerase chain reaction. Moreover, the groEL gene was successfully amplified from several strains of Ca. Phytoplasma asteris by this primer set, indicating that the groEL gene can be used as a LAMP assay target gene for a broad range of phytoplasma strains. Additionally, a simple DNA extraction method that omits the homogenizing and phenol extraction steps was combined with the LAMP assay to develop a simple, rapid, and convenient diagnostic method for detecting phytoplasma.     

Development and evaluation of a one‐hour DNA extraction and loop‐mediated isothermal amplification assay for rapid detection of phytoplasmas

A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas.     

Test performance study of isothermal amplification tests for the detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’

This test performance study (TPS) was carried out on DNA samples from grapevine, clematis, fungi and bacteria to compare and validate loop‐mediated isothermal amplification (LAMP) tests for detection of Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (Grapevine Bois noir phytoplasma). Two LAMP tests, for Grapevine flavescence dorée phytoplasma and ‘Candidatus Phytoplasma solani’ (as developed by Kogov?ek and colleagues), with proven applicability for rapid laboratory or on‐site detection were included in this study. They were performed in 10 laboratories. In addition, the commercial Qualiplante/Hyris isothermal amplification test for Grapevine flavescence dorée phytoplasma was performed in three laboratories. The accuracy of the three tests was shown to be over 98%. Moreover, the high accuracy of these tests, which used different devices across different laboratories, confirmed their reproducibility.     

Fast and sensitive on-site isothermal assay (LAMP) for diagnosis and detection of three fruit tree phytoplasmas

Over the years, real-time PCR outflanked endpoint PCR in phytopathogen diagnostics, mainly because of the increase in sensitivity and timesaving aspects of the technique. However, a time consuming 16S rRNA-based nested PCR method is still the gold standard for phytoplasma diagnosis. This is also the case for phytoplasma detection in Malus, Pyrus and Prunus, the three main host plants of apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY) phytoplasma, respectively. The last decade, loop-mediated isothermal amplification (LAMP) (Notomi et al. 2000) is gaining a lot in significance and is also for phytoplasmas expected to become a widely used reliable diagnostic tool. High specificity and sensitivity which also requires a less stringent need for DNA purification, and the short analysis time and the limited equipment requirements makes the LAMP method a fast and affordable alternative with great point-of-care diagnostic potential. In this paper, we present a LAMP primer set for the ribosomal group 16SrX, containing the important fruit tree phytoplasmas AP, PD and ESFY. The primers were developed and validated for fast and sensitive detection and general use for diagnosis. We foresee that the LAMP technique will also have its application in on-site diagnosis of the fruit tree phytoplasmas during inspections and surveys.     

Development of a grower‐conducted inoculum detection assay for management of grape powdery mildew

Management of grape powdery mildew (Erysiphe necator) and other polycyclic diseases often relies on calendar‐based pesticide application schedules that assume the presence of inoculum. An inexpensive, loop‐mediated isothermal amplification (LAMP) assay was designed to quickly detect airborne inoculum of E. necator to determine when to initiate a fungicide application programme. Field efficacy was tested in 2010 and 2011 in several commercial and research vineyards in the Willamette Valley of Oregon from pre‐bud break to véraison. In each vineyard, three impaction spore traps were placed adjacent to the trunk. One trap was maintained and used by the grower to conduct the LAMP assay (G‐LAMP) on‐site and the other two traps were used for laboratory‐conducted LAMP (L‐LAMP) and quantitative PCR assay (qPCR). Using the qPCR as a gold standard, L‐LAMP was comparable with qPCR in both years, and G‐LAMP was comparable to qPCR in 2011. Latent class analysis indicated that qPCR had a true positive proportion of 98% in 2010 and 89% in 2011 and true negative proportion of 96% in 2010 and 64% in 2011. An average of 3·3 fewer fungicide applications were used when they were initiated based on spore detection relative to the grower standard practice. There were no significant differences in berry or leaf incidence between plots with fungicides initiated at detection or grower standard practice plots, suggesting that growers using LAMP to initiate fungicide applications can use fewer fungicide applications to manage powdery mildew compared to standard practices.     

喜树丛枝植原体的分子鉴定及TaqMan探针实时荧光定量PCR检测方法的建立

 为确定在云南文山地区喜树上发生的疑似丛枝病的病原种类及快速检测喜树丛枝病,本研究利用植原体16S rDNA基因通用引物P1/P7和R16F2n/R16R2对感病喜树总DNA进行常规PCR和巢式PCR扩增、克隆和测序,通过系统进化分析,明确了喜树丛枝植原体属于16SrXXXII组。然后根据喜树丛枝病植原体16S rDNA基因保守区域设计并合成特异性引物和TaqMan探针,制备了喜树丛枝病植原体标准质粒,确定了最优引物浓度和最佳探针浓度,制作的标准曲线有极好的线性关系,决定系数(R²)达到0.999,建立的实时荧光定量PCR检测方法能够特异性地检测喜树丛枝植原体。本研究首次明确了喜树丛枝植原体的分类地位,优化和建立了喜树丛枝植原体TaqMan探针qPCR检测方法,为快速检测喜树丛枝病植原体提供参考。     

Spatiotemporal distribution of flavescence dorée phytoplasma in grapevine

In this study quantitative real‐time PCR was used to follow the seasonal changes of flavescence dorée phytoplasma (FDp) titre in grapevines of cv. Modra frankinja (syn. Blaufränkisch) and cv. Refo?k (syn. Refosco'd'Istria) from two vineyards located in climatically different vine‐growing regions of Slovenia. Besides its known presence in the leaf veins, FDp was also detected in flowers, berry tissues and tendrils. In plants with high concentrations of FDp in tissues with symptoms, phytoplasma was also detected in symptomless tissues. A trend of decreasing FDp titre in all examined symptomless tissues from June to July and an increasing one throughout the growing season in tissues with symptoms was recorded. Accordingly, FDp was present in detectable amounts in flowers, petioles and veins of almost all infected plants in the late spring, and was detected in all examined tissue types in summer, with the highest titre in berries in August. The study showed that in the absence of plant health measurements an FDp infection may spread exponentially by a factor of 40 per year.     

Visual detection of <Emphasis Type="Italic">Didymella bryoniae</Emphasis> in cucurbit seeds using a loop-mediated isothermal amplification assay

A method was developed using a Loop-mediated isothermal amplification assay (LAMP) for detecting Didymella bryoniae in cucurbit seeds. The LAMP primers were designed based on the DNA-dependent RNA polymerase II RPB140 gene (RPB2) from D. bryoniae. Calcein was used as an indicator for the endpoint visual detection of DNA amplification. The LAMP assay was conducted in isothermal (65 °C) conditions within 1 h. The detection threshold of the LAMP assay was 10 pg of genomic DNA and D. bryoniae was detected in 100 % of artificially infested seedlots with 0.05 % infestation or greater. With the LAMP assay, 16 of 60 watermelon and muskmelon seedlots collected from Xinjang province were determined to be positive for D. bryoniae. In contrast, a real-time PCR assay determined that 11 of the 60 seedlots from Xinjiang province were positive for the pathogen. These results showed that the LAMP technique was simple, rapid and well suited for detecting D. bryoniae DNA, especially in seed health testing.     

Development of a loop-mediated isothermal amplification assay for sensitive and specific detection of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">cucumerinum</Emphasis> Owen

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum Owen (FOC), is a destructive disease affecting cucumber production worldwide. Developing an accurate and reliable method for detection of FOC is important for disease prediction and control. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for specific and sensitive detection of FOC. Four LAMP primers were designed based on the sequence of the FOC-specific random amplified polymorphic DNA (RAPD) marker OPZ-12₈₆₅. LAMP reactions were performed at different temperatures and for different durations, and the optimal temperature and duration were 63 °C for 60 min, respectively. Hence, a LAMP assay for detection of FOC was established. The specificity of the LAMP method was evaluated against 119 isolates of FOC and other pathogens, and only FOC isolates yielded positive results. In sensitivity tests, the lowest concentration of genomic DNA required for the LAMP assay was 10 fg in a 25 μL reaction. The LAMP assay was successfully applied to detect FOC in cucumber tissues and soil from infested fields, and the positive ratios of LAMP, PCR, and traditional tissue isolation for detecting FOC from diseased cucumber root samples were100%, 86.6 and 83.3%, respectively. Therefore, the LAMP assay developed herein should serve as a simple, cost-effective, rapid, highly specific, and sensitive tool for the visual detection of FOC and contribute to improved disease management.     

Real-time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.     

Development of a TaqMan allelic discrimination assay for the distinction of two major subtypes of the grapevine yellows phytoplasma Bois noir

Bois noir is a grapevine yellows disease that is gaining importance in many regions of Europe. The Bois noir phytoplasma (“Candidatus Phytoplasma solani”) was shown to be transmitted by the planthopper Hyalesthes obsoletus, which normally feeds on herbaceous weeds, and occasionally also on grapevines. Three subtypes of the Bois noir phytoplasma have been described and were shown to be associated with distinctive host plants. In this study, we developed a novel and rapid real-time PCR allelic discrimination assay for the distinction of the two major Bois noir phytoplasma subtypes, VK type I and II. Two TaqMan probes carrying different fluorescent dyes were designed to specifically bind to a polymorphism characteristic for the two Bois noir phytoplasma subtypes, thereby allowing discriminative amplification in a single-tube and single-step assay. A total of 259 bois noir-positive grapevine samples collected over 5 years were analysed using the conventional PCR-RFLP method and our newly developed TaqMan allelic discrimination assay. 257 out of 259 samples could be typed with the TaqMan method, compared to 200 out of 259 samples when using the conventional method. The overall concordance of the two methods was 100%. Our newly developed TaqMan assay represents a useful tool for fast and reliable determination of Bois noir phytoplasma subtypes in infected grapevine, insect vector, and host plant samples. The test is suitable for high-throughput analysis and will thereby facilitate further characterisation of Bois noir epidemiology.     

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Erwinia amylovora based on chromosomal DNA

A reliable and rapid pathogen detection protocol that utilizes loop-mediated isothermal amplification (LAMP) was developed for detection of Erwinia amylovora, the casual agent of fire blight. The six LAMP primers applied were derived from the highly conserved fragment of the chromosomally amsH gene. Despite the proposed LAMP as well as nested PCR presenting equal values of sensitivity (2?×?10¹?CFU/ml or more) for pure cultures, as compared with conventional PCR (2?×?10³?CFU/ml), both methods were together superior. The specificity assay also showed that the LAMP protocol is species-specific for detection of E. amylovora even in inter-species analysis. Meanwhile, when all 208 naturally infected samples were examined, the specificity value of LAMP was 84%, while conventional and nested PCR could detect only 59% and 73% of the whole collection. Significantly, an independent behaviour versus host plant as well as each strain origin was also observed regarding the current LAMP method as well as other two PCR-based methods. All the results, overall, indicated that the LAMP offers an interesting novel and convenient assay format for the quick and specific chromosomal detection and diagnostic tool of recognition of E. amylovora and therefore presents an alternative to PCR-based assays.     

甘蔗白叶病植原体巢式PCR检测方法的建立及初步应用

甘蔗白叶病(sugarcane white leaf,SCWL)是由植原体引起的重要甘蔗病害[1],广泛分布在印度、泰国等许多国家[1,2].我国甘蔗产区的栽培品种也有SCWL的发生[3].甘蔗是无性繁殖作物,植原体可通过繁殖种苗进行传播,台湾斑纹叶蝉(Matsumuratetlix hiroglyhious)通过咬食感染甘蔗植株的韧皮部可引起该病害[4].     

A loop-mediated isothermal amplification (LAMP) assay based on unique markers derived from genotyping by sequencing data for rapid in planta diagnosis of Panama disease caused by Tropical Race 4 in banana

The socio-economic impact of Fusarium odoratissimum, which is colloquially called tropical race 4 (TR4), is escalating as this fungal pathogen spreads to new banana-growing areas. Hence, the development of simple, reliable and rapid detection technologies is indispensable for implementing quarantine measures. Here, a versatile loop-mediated isothermal amplification (LAMP) assay has been developed that is applicable under field and laboratory conditions. DNA markers unique to TR4 isolates were obtained by diversity arrays technology sequencing (DArTseq), a genotyping by sequencing technology that was conducted on 27 genotypes, comprising 24 previously reported vegetative compatibility groups (VCGs) and three TR4 isolates. The developed LAMP TR4 assay was successfully tested using 22 TR4 isolates and 45 non-target fungal and bacterial isolates, as well as on infected plants under greenhouse and field conditions. The detection limit was 1 pg µL⁻¹ pure TR4 DNA or 10² copies plasmid-localized TR4 unique sequence (SeqA) per reaction, which was not affected by background DNA in complex samples. The LAMP TR4 assay offers a powerful tool for the routine and unambiguous identification of banana plants infected with TR4, contributing to advanced diagnosis in field situations and monitoring of fusarium wilt.     

Rapid detection of Fusarium oxysporum f. sp. lactucae on soil,lettuce seeds and plants using loop‐mediated isothermal amplification

Fusarium oxysporum f. sp. lactucae (FOL) is a soil‐ and seedborne pathogen and the causal agent of fusarium wilt on lettuce. Four races have been identified within FOL, with different worldwide distribution. Several molecular techniques have been used to detect and identify this pathogen; however, not all of them have the optimal characteristics in terms of sensitivity to perform FOL detection in plant and seed material. A loop‐mediated isothermal amplification (LAMP) assay was developed based on the sequence‐characterized amplified region (SCAR) obtained in a previous rapid amplification of polymorphic DNA (RAPD) study. The LAMP assay has been validated according to the EPPO standard PM7/98. The LAMP assay was tested with lettuce seeds, soil and plant material, and can be used successfully to amplify DNA from each of these matrices. In seed lots artificially inoculated with FOL, the detection limit of the LAMP test was 0.004% infected seed.     

Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers

A loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the rDNA ITS sequence of P. aphanidermatum was developed. Results of a specificity test using 57 strains of Pythium spp. indicated that the LAMP assay gave no cross reactions in other 39 Pythium species, 11 strains of Phytophthora spp. and eight other soil borne pathogens. The detection limit was 10 fg of genomic DNA, which was ten times the sensitivity of the polymerase chain reaction. The LAMP assay was applied to hydroponic solution samples from tomato fields, and the results were compared to those of the conventional plating method. LAMP was observed to be effective for the specific detection of P. aphanidermatum. Furthermore, P. aphanidermatum was detected directly in tomato roots infected with P. aphanidermatum without DNA extraction. The LAMP method established in this study is a simple, sensitive and rapid tool for the detection of P. aphanidermatum.     

玉蜀黍黑粉菌环介导等温扩增检测体系的建立及应用

 本文根据玉蜀黍黑粉菌(Ustilago maydis )的UmPep1、UmPit2和UmSee1基因各设计4套环介导等温扩增(LAMP)引物,从中筛选出1套引物对LAMP反应体系进行3因素(Bst DNA聚合酶浓度、Mg²⁺浓度、内外引物浓度比)3水平的优化试验。并对优化的U. maydis LAMP反应体系进行特异性、灵敏度及田间检测可行性试验。特异性试验表明,该方法能特异性检测U. maydis,而与其他病原菌的DNA没有交叉反应;灵敏度试验表明,该反应体系的最低检出限为44 fg·μL^-1 pEasy-Pep质粒DNA,制作的标准曲线可对U. maydis进行定量分析。该方法也适用于在U. maydis侵染前或侵染早期对田间样品进行检测,对现场采集的172份田间样品进行检测,其中140个样品显示为阳性。本研究所建立的LAMP体系具有特异性好、灵敏度高、重复性好的特点,并能在45 min内完成对田间样品的检测,是快速、定量检测U. maydis的有效手段。     

SYBR Green real-time quantitative PCR for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in field samples from New Zealand

Diseases of solanaceous crops caused by the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), vectored by the tomato potato psyllid Bactericera cockerelli, pose a major economic threat to crop production. Lso is yet to be cultured and, therefore, effective control strategies depend heavily on the early detection of the pathogen via polymerase chain reaction (PCR) assays. In this study, two new assays for the detection of Lso in New Zealand field samples were developed, and compared with previously available assays. Firstly, a single-tube semi-nested gel-based PCR assay was developed for the genus-specific detection of liberibacter species, and shown to provide increased sensitivity over standard and nested PCR. Secondly, a single-tube semi-nested SYBR Green real-time PCR (qPCR) assay was developed for the specific detection of Lso in field samples from New Zealand, with a limit of detection of five copies of the target gene per reaction. Semi-nested qPCR showed similar sensitivity compared with TaqMan qPCR with the primer-probe combination LsoF-HLBpr and was 10- to 50-fold more sensitive than the conventional PCR assays tested. Quantification of titre in Lso-affected tubers by SYBR Green qPCR revealed a positive relationship between pathogen titre and the discolouration of fried tuber slices, a symptom indicative of Lso infection. Quantification of Lso in field samples of potato and tomato also revealed many samples with titres below the limit of detection of conventional PCR. The observation of low-titre samples demonstrated the utility of SYBR Green qPCR for detection of Lso, as in addition to increased sensitivity melt-curve analysis enables confirmation of qPCR data by identifying false positive results.