Qiyong Weng

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

Development and evaluation of ITS- and aflP-based LAMP assays for rapid detection of Aspergillus flavus in food samples

Aspergillus flavus is a common filamentous fungus that produces aflatoxins and presents a major threat to agriculture and human health. Previous studies focused mainly on the detection of A. flavus or aflatoxin separately. Here, we developed internal transcribed spacer (ITS)- and aflP-based rapid detection of A. flavus in food samples using the loop-mediated isothermal amplification (LAMP) method. The ITS1-5.8S-ITS2 rDNA region of A. flavus and the aflatoxin-encoding gene aflP were used as target regions. The detection limits of A. flavus and aflP were 10 fg and 1 pg pure DNA, respectively, which allows aflatoxin-contaminated samples to be differentiated from infected samples and reduces false-negative or false-positive results. For specificity testing, DNA extracted from 7 A. flavus, 5 different Aspergillus spp., and 21 other fungi were used, and our results showed that A. flavus strains are detected by ITS-based detection and aflatoxigenic A. flavus strains are detected by aflP-based detection. Furthermore, the ITS- and aflP-based LAMP assays were used for detection analysis of DNA from food samples artificially and naturally contaminated with A. flavus. Our results showed that the detection rate of A. flavus based on the multi-ITS-based LAMP detection is 100% and that the aflatoxigenic strains in all A. flavus are detected by the aflP-based LAMP assay. The LAMP protocol described in our study represents a rapid and highly specific and sensitive diagnostic method for A. flavus detection, which can be used as a diagnostic tool that simplifies A. flavus monitoring and guarantees the quality and safety of foods.

The L-type Ca2+ Channel Blocker Nifedipine Inhibits Mycelial Growth, Sporulation, and Virulence of Phytophthora capsici

The oomycete vegetable pathogen Phytophthora capsici causes significant losses of important vegetable crops worldwide. Calcium and other plant nutrients have been used in disease management of oomycete pathogens. Calcium homeostasis and signaling is essential for numerous biological processes, and Ca2+ channel blockers prevent excessive Ca2+ influx into the fungal cell. However, it is not known whether voltage-gated Ca2+ channel blockers improve control over oomycete pathogens. In the present study, we compared the inhibitory effects of CaCl2 and the extracellular Ca2+ chelator EDTA on mycelial growth and found that calcium assimilation plays a key role in P. capsici mycelial growth. Next, we involved the voltage-gated Ca2+ channel blockers verapamil (VP) and nifedipine (NFD) to analyze the effect of Ca2+ channel blockers on mycelial growth and sporulation; the results suggested that NFD, but not VP, caused significant inhibition. Ion rescue in an NFD-induced inhibition assay suggested that NFD-induced inhibition is calcium-dependent. In addition, NFD increased P. capsici sensitivity to H2O2 in a calcium-dependent manner, and extracellular calcium rescued it. Furthermore, NFD inhibited the virulence and gene expression related to its pathogenicity. These results suggest that NFD inhibits mycelial growth, sporulation, and virulence of P. capsici.

Loop-mediated isothermal amplification assay for sensitive and rapid detection of Phytophthora capsici

Abstract Phytophthora capsici is an oomycete plant pathogen that infects peppers, tomatoes, and other agronomic and ornamental crops of the Solanaceae and Cucurbitaceae families worldwide. Early and accurate detection of P. capsici is essential for controlling this disease in the field. The internal transcribed spacer (ITS) regions of nuclear-encoded ribosomal DNA genes are widely used to detect Phytophthora nicotianae, Pythium helicoides and Peronospora variabilis. In this study, a novel and highly sensitive loop-mediated isothermal amplification (LAMP) assay was developed for the specific detection of P. capsici using calcein as a fluorescent indicator. Four LAMP primers were designed based on the ITS sequence of P. capsici. A total of 23 isolates of P. capsici from geographically distinct counties in China yielded positive results in the LAMP assay. No cross-reaction was observed with other oomycetes or fungal pathogens. The detection limit of P. capsici by LAMP was 100 fg genomic DNA per 25 μL reaction. The LAMP assay developed in this study is simple, fast, sensitive and specific, and can be used in the field to detect P. capsici in infected plant tissue.

Genetic analysis of Phytophthora sojae populations in Fujian, China

&NA; Phytophthora sojae is a destructive soilborne pathogen causing seedling damping‐off and root rot of soybean (Glycine max). The goal of this study was to determine the genetic structure of P. sojae populations in Fujian, China. Nine microsatellite markers were used to investigate the genetic variation in 19 P. sojae populations, sampled from Fujian Province and northeastern China (Jilin and Heilongjiang Provinces) between 2002 and 2013. Overall, a low genetic diversity, Hardy‐Weinberg disequilibrium, and an Symbol index (an index of association) that was significantly different from zero were detected in populations; these results were consistent with self‐fertilization and clonal modes of reproduction for this pathogen. However, using Bayesian Markov chain Monte Carlo approach, principal component analysis and neighbour joining (NJ) algorithm, the Fujian P. sojae populations clustered into three distinct groups, one of which included most isolates of the northeast populations. What is more, significant estimates of pairwise fixation indices (FST) were detected between most populations, especially in different clusters. It is hypothesized that the cropping system used, the limited dispersal ability, and human‐mediated gene flow may account for the observed genetic structure of P. sojae populations in Fujian, China. In addition, a high virulence frequency of the pathogen on different cultivars carrying known major R genes for resistance, and a rapid increase in virulence frequency, indicated that these major R genes should not be used to manage seedling damping‐off and root rot diseases of soybean (Glycine max). Symbol. No caption available.

Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

Early blight (EB), caused by the pathogen Alternaria solani, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the histidine kinase gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified A. solani artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure A. solani culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting A. solani in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.

Construction of Monitoring System of Dangerous and Harmful Species of Import Taiwan Fruits and Vegetables Based on GIS

In order to improve the efficiency of dangerous and harmful species monitoring of import Taiwan fruits and vegetables and achieve informatization, networking and visualization of monitoring, this paper uses geography information system technology to develop monitoring system of import Taiwan fruits and vegetables dangerous and harmful species on the basis of the analysis of monitoring business needs. The system realizes some useful functions, such as showing monitoring data in map, map operation, monitoring data report and monitoring data management. This paper supply informatization supporting platform to dangerous and harmful species monitoring of import Taiwan fruits and vegetables and provide information support for scientific decision-making of alien invasive biological prevention and control department.In order to improve the efficiency of dangerous and harmful species monitoring of import Taiwan fruits and vegetables and achieve informatization, networking and visualization of monitoring, this paper uses geography information system technology to develop monitoring system of import Taiwan fruits and vegetables dangerous and harmful species on the basis of the analysis of monitoring business needs. The system realizes some useful functions, such as showing monitoring data in map, map operation, monitoring data report and monitoring data management. This paper supply informatization supporting platform to dangerous and harmful species monitoring of import Taiwan fruits and vegetables and provide information support for scientific decision-making of alien invasive biological prevention and control department.