Development of Species-Specific PCR Primers and Sensitive Detection of the Tylenchulus semipenetrans in China

Tylenchulus semipenetrans is the most economically important and widespread nematode pest of citrus in China.rDNA-ITS of 14 populations of T.semipenetrans which were collected from different citrus groves or Chinese fir（Cunninghamia lanceolata） plantations in China were amplified and sequenced.The species-specific primers were designed for the first time to diagnosis T.semipenetrans based on the sequences of rDNA-ITS regions of geographic population above.The primers were sensitive to amplify the expected band size（297 bp） from DNA template of a single second-stage juvenile（J2） or different life stages of T.semipenetrans.No specific band was amplified from 15 non-target nematode species which were commonly found in citrus groves.Specificity and reliability of the primers were validated by further PCR amplification of 16 extra populations of T.semipenetrans collected from 4 provinces of China.The primers successfully detected a single J2 of T.semipenetrans within a whole nematode community comprising a large numbers of non-target nematode.The developed diagnostic technique can be used for accurate identification of T.semipenetrans and also as a decision tool for nematode management for citrus or Chinese fir in China.

Species-specific COI primers for rapid identification of a globally significant invasive pest, the cassava mealybug Phenacoccus manihoti Matile-Ferrero

The globally invasive cassava mealybug Phenacoccus manihoti Matile-Ferrero is a pernicious pest of cassava,and its recent introduction into Asia has raised considerable alarm.To slow or prevent further invasion,an accurate,simple,and developmental-stage-independent detection method for P.manihoti is required.In the present study,a PCR method based on a species-specific mitochondrial DNA cytochrome oxidase I(SS-COI)marker was developed for rapid identification of P.manihoti.One pair of SS-COI primers(PMSSZW-1F and PMSSZW-1R)was designed based on sequence variations in the COI gene among P.manihoti and related mealybug species.Specificity of the primer pair was validated on 21 closely related species.Sensitivity tests were performed on four immature developmental stages and female adults.Efficacy tests demonstrated that at the relatively low concentration of(135.2±14.7)pgresuspended DNA,the specific fragment was detected in all replicates.Furthermore,the SS-COI primer pair was assayed on three populations of P.manihoti from major exporting countries of cassava.The PCR assay was proved to be a rapid,simple,and reliable molecular measure for the identification of P.manihoti.This tool will be useful for quarantine,monitoring,and management of this invasive pest.

A Rapid Identification Method of Bactrocera cilifera (Hendel) with Species-Specific Primers(SS-COI)

[Objective]The paper was to establish a rapid identification method of Bactrocera cilifera(Hendel)with species-specific primers(SS-COI).[Method]Using B.cilifera(Hendel)as the positive control,and 19 species of fruit flies such as B.diaphora(Coquillett)and B.dorsalis(Hendel)as the negative controls,a pair of species-specific primers,YF290 and YR511,were designed and screened for accurate identification of B.cilifera,based on mitochondrial DNA COI sequence.[Result]The PCR products were amplified and detected by electrophoresis.Only a clear and single band was observed at about 222 bp in the positive control,while no bands were found in the other negative controls.[Conclusion]The established rapid identification method with species-specific primers(SS-COI)is of great practical significance for rapid identification of fruit flies intercepted from import and export fruits and vegetables at ports,and for rapid clearance and early warning of import fruits and vegetables at ports.

Species-specific PCR-based assays for identification and detection of Botryosphaeriaceae species causing stem blight on blueberry in China

Botryosphaeriaceae species are important causal agents of blueberry stem blight worldwide. Blueberry stem blight has become an important disease, potentially affecting the quality and production of blueberries in China. It is difficult and time-consuming to identify at the species level using morphological methods. The aim of this study was to develop polymerase chain reaction（PCR） assays for the diagnosis and early detection of latent infections of blueberry stems by Botryosphaeria spp. Species-specific primers, based on the ribosomal DNA internal transcribed spacer region and β-tubulin gene, were designed and selected for use in PCR assays. Three primer pairs, Lt347-F/R for Lasiodiplodia theobromae, Np304-F/R for Neofusicoccum parvum and FaF/Bt2b for Botryosphaeria dothidea, successfully amplified specific PCR fragments of different sizes on pure cultures or from blueberry stems inoculated and naturally infected blueberry plants with three pathogens, respectively. These primers did not amplify any PCR fragments from other blueberry stem disease-associated pathogens, such as Phomopsis spp. and Pestalotiopsis spp. This PCR protocol could detect as low as 1 00 pg to 1 ng of purified fungal DNA. This PCR-based protocol could be used for the diagnosis and detection of these pathogens from pure cultures or from infected blueberry plants.

基于种特异性线粒体细胞色素氧化酶Ⅰ引物快速鉴定茄二十八星瓢虫和马铃薯瓢虫

【目的】茄二十八星瓢虫Henosepilachna vigintioctopunctata和马铃薯瓢虫Henosepilachna vigintioctomaculata都是茄科作物上的重要害虫,对茄科作物造成极大的经济损失。这2种瓢虫由于外形非常相似,无法通过体表特征区分,因此很有必要开发一种准确且快速的区分方法。【方法】基于这2种瓢虫线粒体细胞色素氧化酶I(Mitochondrial cytochrome oxidase I,mtCOI)的物种特异性,利用种特异性(Species-specific,SS)PCR引物建立了一种分子鉴定技术,即以茄二十八星瓢虫和马铃薯瓢虫之间的mtCOI基因序列变异为基础,设计了2对SS-mtCOI引物Hvp和Hvm。【结果】用这2对引物进行PCR扩增,均出现物种特异性扩增现象。在不同的DNA质量浓度下对该SS-mtCOI引物进行敏感性检测,结果表明,Hvp引物在茄二十八星瓢虫DNA质量浓度为3.13 mg/L时仍可检测到扩增条带,Hvm引物在马铃薯瓢虫DNA浓度为2.43 mg/L时仍能检测到扩增条带。此外,Hvp和Hvm引物也能准确鉴定马铃薯瓢虫和茄二十八星瓢虫的卵和1龄幼虫,从6个不同省份采集的田间种群也能通过Hvp引物准确鉴定。【结论】这2对SS-mtCOI引物能快速、准确、灵敏地鉴别茄二十八星瓢虫和马铃薯瓢虫。

LSQP‑DB:a species‑specific quantitative PCR primer database for 307 Lactobacillaceae species

Quantitative real-time PCR is widely used to determine absolute abundance of microbes in food fermentation.However,it remains challenges in the application for quantification at the species level due to the difficulty in designing species-specific primer sets.This work,using Lactobacillaceae,a dominant family within the lactic acid bacteria that involved in diversity food fermentations,as a case,presents an extendable strategy to design species-specific primer sets for microbial quantita-tive analysis.136,257 species-specific genes were obtained from all 307 species within Lactobacillaceae family through comparative genomics analysis.A total of 130,521 primer sets were designed using species-specific genes.Among them,81,710 primer sets had 100%interspecific specificity and 100%intraspecific coverage,and were reserved to quantify all 307 individual Lactobacillaceae species.These primer sets had uniform melting temperature(57–63℃)and product size(100–300 bp),that allowed simultaneously quantify different Lactobacillaceae species with the same qPCR condition.We then established a Lactobacillaceae species quantitation primer database(LSQP-DB,http://lsqp-db.com)containing all 81,710 species-specific primer sets.The database would facilitate a fast and easy absolute quantitation analysis of all indi-vidual Lactobacillaceae species.This work represented the first ever large-scale integration of species-specific primer sets for microorganism,it can be extended to other bacterial and fungal genera to advance development of microbial absolute quantification.

Phyllosticta species associated with citrus diseases in China

Phyllosticta species associated with diseases of four commercial Citrus species grown in China are reported.Totally,496 Phyllosticta strains were isolated from mandarins(Citrus reticulata),pomeloes(C.maxima),oranges(C.sinensis)and lemons(C.limon)in the main citrus producing regions across China,and 74 strains were selected for phylogenetic analysis.Analyses inferred from the sequences of internal transcribed spacer region(ITS1,5.8S nrDNA and ITS2),partial translation elongation factor 1-alpha(TEF1)and partial actin gene(ACT),showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known,and one undescribed species.The newly resolved taxon,Phyllosticta citrichinaensis was isolated from leaves and fruits of all four Citrus species and is introduced in this paper.This taxon caused minor damage,showing irregular spots or freckles.Phyllosticta citriasiana,associated with tan spot of pomeloes,was isolated only from pomeloes,and never from lemons,mandarins and oranges.Phyllosticta citricarpa,the citrus black spot pathogen,which is presently subjected to phytosanitary legislation in the EU and United States,was isolated from lemons,mandarins and oranges,but never from pomeloes.The isolates of P.citricarpa clustered in two subclades,one from mandarins,the other from oranges and lemons.P.capitalensis was isolated from all four Citrus species as an endophyte,causing false melanose,or together with P.citricarpa or P.citriasiana.Morphological,cultural and biochemical characters were consistent with the results of phylogenetic analysis.In addition,a specific primer pair Pca8/ITS4 was designed and selected,and its corresponding PCR procedure was developed for the detection of P.citriasiana in this study.

Shoot rot of Zizania latifolia and the first record of its pathogen Pantoea ananatis in China

The aquatic grass Zizania latifolia grows symbiotically with the fungus Ustilago esculenta producing swollen structures called Jiaobai,widely cultivated in China.A new disease of Z.latifolia was found in Zhejiang Province,China.Initial lesions appeared on the leaf sheaths or sometimes on the leaves near the leaf sheaths.The lesions extended along the axis of the leaf shoots and formed long brown to dark brown streaks from the leaf sheath to the leaf,causing sheath rot and death of entire leaves on young plants.The pathogen was isolated and identified as the bacterium Pantoea ananatis,based on 16 S ribosomal RNA(r RNA)gene sequencing,multilocus sequence analysis(atp D(β-subunit of ATP synthase F1),gyr B(DNA gyrase subunit B),inf B(translation initiation factor 2),and rpo B(β-subunit of RNA polymerase)genes),and pathogenicity tests.Ultrastructural observations using scanning electron microscopy revealed that the bacterial cells colonized the vascular tissues in leaf sheaths,forming biofilms on the inner surface of vessel walls,and extended between vessel elements via the perforated plates.To achieve efficient detection and diagnosis of P.ananatis,species-specific primer pairs were designed and validated by testing closely related and unrelated species and diseased tissues of Z.latifolia.This is the first report of bacterial sheath rot disease of Z.latifolia caused by P.ananatis in China.

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.

Molecular authentication of the traditional Chinese medicine Tongren Dahuoluo Wan and its alternative formulation

Tongren Dahuoluo Wan has been a popular traditional Chinese medicine in international pharmaceutical markets for hundreds of years. Leopard bone powder is the key element in its formulation. However, the leopard has been listed for wildlife conservation, which limits the use of the leopard bone supplies. Therefore, an alternative formulation which substitutes leopard bone with zokor bone in the formula of Tongren Dahuoluo Wan is now manufactured. To develop a simple and reliable molecular method for authenticating the two patent medicines,mitochondrial nucleotide polymorphic sites of 12 S rRNA,COI and Cytb genes were screened in leopard and zokor bones, and nine pairs of species-specific primers were verified for discriminating the two species. For the patent medicine authentication, we set up a molecular diagnostic assay to resolve the difficulties of low concentration of target DNAs and presence of PCR-inhibitory substances in this complex medicine, and successfully confirmed leopard or zokor content using the nine pairs of species-specific primers. We recommend a common technical strategy for authentication of species origins in traditional Chinese medicine, and discuss the experimental solutions for technical problems of molecular diagnostic assays.