Rapid Detection of Phytophthora nicotianae in Infected Tobacco Tissues and Soil Samples Based on Its Ypt1 Gene

This paper describes the development of a polymerase chain reaction (PCR) assay for the detection of Phytophthora nicotianae , the causal agent of Phytophthora blight of tobacco and other plants. The PCR primers were designed based on a Ras-related protein ( Ypt 1) gene, and 115 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers. PCR amplification with species-specific (Pn) primers resulted in a product of 389 bp only from isolates of P. nicotianae . The detection sensitivity with Pn primers was 1 ng of genomic DNA. Using Ypt 1F/ Ypt 1R as first-round amplification primers, followed by a second round using the primer pair Pn1/Pn2, a nested PCR procedure was developed, which increased the detection sensitivity 100-fold to 10 pg. PCR with the Pn primers could also be used to detect P. nicotianae from naturally infected tobacco tissues and soil. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management.     

Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences

Fifty-eight isolates representing 39 Pythium species and 17 isolates representing nine Phytophthora species were chosen to investigate intra- and intergeneric relationships with sequence analysis of three genomic areas. The internal transcribed spacer regions (ITS1 and ITS2), including the 5.8S gene of the ribosomal DNA were PCR amplified with the universal primers ITS1 and ITS4. On the other hand 563 bp of the cytochrome oxidase II (cox II) gene was amplified with the primer pair FM66 and FM58 for Pythium and FM75 and FM78 for Phytophthora. The 658 bp partial beta-tubulin gene was amplified with the forward primer BT5 and reverse primer BT6. Maximum parsimony analysis of the three DNA regions revealed four major clades, reflective of sporangial morphology. Clade 1 was composed of Pythium isolates that bear filamentous to lobulate sporangia. Clade 2 represents Pythium isolates that bear globose to spherical zoosporangia or spherical hyphal swellings. Meanwhile Phytophthora isolates were lumped into Clade 3 wherein the papillate, semipapillate and nonpapillate species occupied separate subclades. Lastly, Clade 4 was composed of Pythium species that bear subglobose sporangia resembling the papillate sporangia observed in Phytophthora. Hence a number of species (Ph. undulata, P. helicoides, P. ostracodes, P. oedochilum and P. vexans) have been proposed to be the elusive intermediate species in the Pythium-to-Phytophthora evolutionary line.     

Specific molecular detection of Phytophthora sojae using conventional and real-time PCR

Phytophthora rot, caused by Phytophthora sojae, is one of the most damaging diseases of soybean (Glycine max) worldwide. This disease can be difficult to diagnose and other Phytophthora species can infect soybean. Accurate diagnosis is important for management of Phytophthora rot. The objective of this study was to evaluate polymerase chain reaction (PCR) methods for rapid and specific detection of P. sojae and diagnosis of Phytophthora rot. PCR assays using two sets of primers (PS and PSOJ) that target the ITS region were evaluated for specificity and sensitivity to P. sojae. Genomic DNA extracted from 11 species of Phytophthora and 19 other species of fungal and oomycete pathogens were used to test the specificity of each primer set. The previously published PS primers amplified DNA from P.?sojae and from four other Phytophthora species using conventional PCR, indicating they are not specific for P. sojae. The new PSOJ primers amplified DNA only from P. sojae using conventional and real-time PCR and not from Phytophthora sansomeana, which has been found in soybean production areas, indicating that they are specific for P. sojae. The PSOJ primers were also used to detect P. sojae in diseased soybean tissue and infested soil. The PCR assays based on the PSOJ primers are specific, rapid, and sensitive tools for the detection of P. sojae.     

大豆疫霉菌ITS分子检测程序的建立及其应用

依据GenBank中登录的大豆疫霉菌(Phytophthora sojae)、近缘种及相似种rDNA的ITS区序列差异,进行多重比较后设计合成一对大豆疫霉菌特异引物,并在PCR反应体系和扩增条件优化的基础上,对包括大豆疫霉菌在内的共140个菌株进行PCR检测。结果表明,电泳后只有大豆疫霉菌扩增出一条288bp的特异性条带。运用设计的大豆疫霉菌专用引物(专利申请号200610089105.4)及建立的检测程序对大豆疫霉菌纯培养游动孢子、接种于土壤中的游动孢子和卵孢子以及接种发病的大豆染病组织进行了检测应用,结果显示该检测程序对接种于土壤中的大豆疫霉菌游动孢子和卵孢子的检测理论精度分别达0.3和0.06个孢子,对染病组织检测也表现出了较高的灵敏度。     

PCR-based DNA Markers for Identifying Hybrids within Phytophthora alni

Two pairs of oligonucleotide primers were designed for the polymerase chain reaction (PCR)‐based detection and differential identification of naturally occurring interspecific hybrid types (subspecies) of Phytophthora alni, all of which cause collar rot of alder trees. Primer pairs were derived from randomly amplified polymorphic DNA (RAPD) fragments that were unique to various subspecies of this alder pathogen. The primer pair set, SAP1/SAP2 (SAP), was derived from a 0.93‐kb RAPD fragment amplified from P. alni ssp. alni. The primer pair set, SWAP1/SWAP2 (SWAP), was derived from a 1.13‐kb fragment amplified from P. alni ssp. uniformis. Patterns of SAP and SWAP amplification enabled distinction among the three subspecies. No PCR products were amplified from isolates of 31 other Phytophthora spp. examined, including P. cambivora and P. fragariae, the suspected progenitors of P. alni. The SAP and SWAP primer sets were able to detect a minimum of 10 pg of DNA from pure cultures or DNA extracted from as few as 10 zoospores. Pathogen DNA could also be amplified directly from bark lesions of artificially inoculated and naturally infected common alders and from lesions developed on common cherry‐laurel leaves used in baiting the pathogen from infested soil. Direct detection of pathogen DNA from alder tissue using SAP and SWAP primer sets should prove useful in developing measures for effective quarantine and management of P. alni.     

Development of a real-time PCR assay for the detection of Cladosporium fulvum in tomato leaves

Aims: The aim of this study was to develop a sensitive real-time polymerase chain reaction (PCR) assay for the rapid detection of Cladosporium fulvum in tomato leaves. Methods and Results: Three PCR primer pairs were designed based on the nucleotide sequences of: (i) the internal transcribed spacer regions of ribosomal RNA; (ii) a microsatellite region amplified by the microsatellite primer M13; and (iii) the β-tubulin gene of C. fulvum. Each primer pair amplified the expected target DNA fragment from geographically diverse isolates of C. fulvum. No PCR products were amplified with these primer pairs from DNA of other fungal species. Among the three pairs of primers, the primer pair CfF1/CfR1 developed based on the microsatellite region was the most sensitive. Using this sensitive primer pair, a real-time PCR assay was developed to detect early infection of C. fulvum in tomato leaves. Significance and Impact of the Study: DNA regions amplified by the microsatellite primer M13 have a high potential for developing highly sensitive species-specific PCR primers for the detection of phytopathogenic fungi. The real-time PCR assay developed in this study is useful in monitoring early infection of C. fulvum, and can help growers make timely decisions on fungicide application.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.     

Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Detection and Identification of Decay Fungi in Spruce Wood by Restriction Fragment Length Polymorphism Analysis of Amplified Genes Encoding rRNA

We have developed a DNA-based assay to reliably detect brown rot and white rot fungi in wood at different stages of decay. DNA, isolated by a series of CTAB (cetyltrimethylammonium bromide) and organic extractions, was amplified by the PCR using published universal primers and basidiomycete-specific primers derived from ribosomal DNA sequences. We surveyed 14 species of wood-decaying basidiomycetes (brown-rot and white-rot fungi), as well as 25 species of wood-inhabiting ascomycetes (pathogens, endophytes, and saprophytes). DNA was isolated from pure cultures of these fungi and also from spruce wood blocks colonized by individual isolates of wood decay basidiomycetes or wood-inhabiting ascomycetes. The primer pair ITS1-F (specific for higher fungi) and ITS4 (universal primer) amplified the internal transcribed spacer region from both ascomycetes and basidiomycetes from both pure culture and wood, as expected. The primer pair ITS1-F (specific for higher fungi) and ITS4-B (specific for basidiomycetes) was shown to reliably detect the presence of wood decay basidiomycetes in both pure culture and wood; ascomycetes were not detected by this primer pair. We detected the presence of decay fungi in wood by PCR before measurable weight loss had occurred to the wood. Basidiomycetes were identified to the species level by restriction fragment length polymorphisms of the internal transcribed spacer region.     

Single- and double-SSR primer combined analyses in rice

Polymerase chain reaction (PCR) is the foundation of SSR molecular marker technology. We used sib rice varieties J518, XD1 and SD23 as experimental materials, selecting 30 pairs of SSR primers, including RM127, RM337 and RM5172, covering the rice genome, and performed single- and double-SSR primer combined analyses. We found that under the same PCR system and conditions, a single primer of the SSR primer pairs could amplify the same fragments as double primers do. The sequencing results demonstrated that some amplified fragments that we previously believed to come from double primers were actually produced by a single primer. The use of this kind of primer, such as the RM127 primer pair, for marker-assisted breeding will therefore be misleading. Additionally, using the same PCR system and conditions, some single primers that are part of SSR primer pairs can amplify many more specific fragments than double-SSR primers. For instance, in the case of the RM5172 primer pair, a single primer P1 amplified approximately three times the number of fragments as the double primer. This information can contribute to research on genetic diversity of species, understanding of genetic relationships and identification of germplasm resources. Accordingly, combined analyses of single- and double-primer amplification products not only can remove single-primer amplification fragments and false-positives from double-primer amplification products in order to improve test accuracy, but also can facilitate research on genetic diversity, exploration of phylogenetic relationships and identification of germplasm resources. We define this method as "single- and double-SSR primer combined analyses".     

Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase II gene

For PCR-based identification of Aspergillus species, a common primer of the DNA topoisomerase II genes of Candida, Aspergillus and Penicillium, and species-specific primers of the genomic sequences of DNA topoisomerase II of A. fumigatus, A. niger, A. flavus (A. oryzae), A. nidulans and A. terreus were tested for their specificities in PCR amplifications. The method consisted of amplification of the genomic DNA topoisomerase II gene by a common primer set, followed by a second PCR with a primer mix consisting of 5 species-specific primer pairs for each Aspergillus species. By using the common primer pair, a DNA fragment of approximately 1,200 bp was amplified from the Aspergillus and Penicillium genomic DNAs. Using each species-specific primer pair, unique sizes of PCR products were amplified, all of which corresponded to a species of Aspergillus even in the presence of DNAs of several fungal species. The sensitivity of A. fumigatus to the nested PCR was found to be 100 fg of DNA in the reaction mixture. In the nested PCR obtained by using the primer mix (PsIV), the specific DNA fragment of A. fumigatus was amplified from clinical specimens. These results suggest that this nested PCR method is rapid, simple and available as a tool for identification of pathogenic Aspergillus to a species level.     

Variation Within and Between Phytophthora Species from Rubber and Citrus Trees in China, Determined by Polymerase Chain Reaction Using RAPDs

Variation among 39 isolates of Phytophthora of six morphological species (P. citrophthora. P. parasitka, P. capsici, P. palmivora and P. meadii. from rubber and citrus trees, and P. colocasiae from taro) was studied using random amplified polymorphic DNA (RAPD) analysis. Ten randomly-chosen 10-mer primers were used. Generally, the banding patterns were similar within species and different between species, but no one primer was able to distinguish all six species from one another. Cluster analysis on pooled data from all the primers gave six groups of isolates corresponding to the six morphological species. The group corresponding to P. citrophthora was divided further into subgroups that were related to host species and geographical location. This work confirmed the existing morphological classification of Phytophthora isolates from rubber and citrus trees in tropical China and showed the validity of using RAPDs to study the taxonomy of Phytophthora.     

Molecular and microscopical detection of aster yellows phytoplasma associated with infected parsnip

Typical phytoplasma yellows symptoms were observed in parsnip (Pastinaca sativa L.) plants grown around Edmonton, Alberta, Canada. Examination of ultrathin sections of leaf midribs by electron microscopy revealed numerous phytoplasma bodies localized in the phloem cells. DNA extracted from the infected leaves was amplified with a 16S rDNA universal primer pair P1/P6 giving the expected PCR product of 1.5 kb. The phytoplasma was confirmed as a member of the aster yellows (AY) group by amplification with the specific primer pair R16(1)/F1/R1 that was designed on the basis of AY phytoplasma 16S rDNA sequences. In the nested PCR assays, the expected DNA fragment of 1.1 kb was amplified with this specific primer set. Similar restriction patterns were found for the 1.1 kb PCR products of the phytoplasma isolated from parsnip and an AY phytoplasma control after digestion with restriction endonucleases AluI, HhaI, KpnI and RsaI. This is the first reported observation of aster yellows in parsnip in Canada.     

Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses.

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism wee used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600-bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC-infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

16S rRNA gene-based detection of tetrachloroethene-dechlorinating Desulfuromonas and Dehalococcoides species

Members of the genera Desulfuromonas and Dehalococcoides reductively dechlorinate tetrachloroethene (PCE) and trichloroethene. Two primer pairs specific to hypervariable regions of the 16S rRNA genes of the Dehalococcoides group (comprising Dehalococcoides ethenogenes and Dehalococcoides sp. strain FL2) and the acetate-oxidizing, PCE-dechlorinating Desulfuromonas group (comprising Desulfuromonas sp. strain BB1 and Desulfuromonas chloroethenica) were designed. The detection threshold of a nested PCR approach using universal bacterial primers followed by a second PCR with the Desulfuromonas dechlorinator-targeted primer pair was 1 x 10(3) BB1 cells added per gram (wet weight) of sandy aquifer material. Total community DNA isolated from sediments of three Michigan rivers and six different chloroethene-contaminated aquifer samples was used as template in nested PCR. All river sediment samples yielded positive signals with the BB1- and the Dehalococcoides-targeted primers. One chloroethene-contaminated aquifer tested positive with the Dehalococcoides-targeted primers, and another contaminated aquifer tested positive with the Desulfuromonas dechlorinator-targeted primer pair. Restriction fragment analysis of the amplicons could discriminate strain BB1 from other known Desulfuromonas species. Microcosm studies confirmed the presence of PCE-dechlorinating, acetate-oxidizing Desulfuromonas and hydrogenotrophic Dehalococcoides species in samples yielding positive PCR signals with the specific primers.     

利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧

扶桑绵粉蚧Phenacoccus solenopsis Tinsley是我国近年新发现的一种严重威胁农林业生产的重要外来入侵害虫。针对扶桑绵粉蚧与其他粉蚧类昆虫难以准确快速识别且适生区广泛的问题, 以扶桑绵粉蚧为靶标, 以我国常见的其他7种粉蚧为参照, 采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ (mtDNA COI) 基因序列的种特异性(species-specific COI, SS-COI) PCR方法, 研究其快速分子检测技术。通过已知粉蚧的COI基因序列设计通用型引物1对, 获得扶桑绵粉蚧及其他7种粉蚧包括康氏粉蚧Pseudococcus comstocki Kuwana、 南洋臀纹粉蚧Planococcus lilacius Cockerell、 木槿曼粉蚧Maconellicoccus hirsutus (Green)、 甘蔗红粉蚧Saccharicoccus sacchari (Cockerell)、 新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley、 番石榴粉蚧Planococcus minor Maskel和石蒜绵粉蚧Phenacoccus solani Ferris的COI基因序列, 根据测序结果及数据库中已知粉蚧的COI基因序列设计SS COI引物1对(PSZTF1/PSZTR1), 其扩增片段大小为546 bp。种特异性检验结果表明, 该引物只对扶桑绵粉蚧的COI基因具有扩增能力, 对其他7种粉蚧不具有扩增效果; 该引物不仅对成虫具有良好的扩增能力, 对不同虫态的扶桑绵粉蚧以及来自我国不同省市的14个地理种群和口岸截获的来自巴基斯坦的扶桑绵粉蚧亦具有同样的扩增效能。这些结果表明, 该技术体系完全可用于扶桑绵粉蚧的准确识别及其检测监测, 对有效阻截其进一步扩张蔓延意义重大。     

PYTHIUM SYLVATICUM鉴定及其专一性PCR引物

从杭州采集的水稻、棉花和大豆猝倒苗中分离到国内新记录腐霉种Pythium sylvaticum。该腐霉为异宗配合种,菌丝膨大体球形或柠檬形,雄器异丝生,藏卵器光滑,每个藏卵器上1~3个雄器,雄器常在接近藏卵器处形成二叉状分枝,卵孢子不满器。测定了该种4 个菌株的核糖体内转录间隔区(ITS)的序列,根据与59种腐霉ITS序列的比较,设计了P. sylvaticum种专一性引物PSF1和PSR2。实际结果表明:该引物能从11种共14株腐霉DNA中特异性地扩增P. sylvaticum,从而与其它10种腐霉区分。     

A molecular method to assess Phytophthora diversity in environmental samples

Current molecular detection methods for the genus Phytophthora are specific to a few key species rather than the whole genus and this is a recognized weakness of protocols for ecological studies and international plant health legislation. In the present study a molecular approach was developed to detect Phytophthora species in soil and water samples using novel sets of genus-specific primers designed against the internal transcribed spacer (ITS) regions. Two different rDNA primer sets were tested: one assay amplified a long product including the ITS1, 5.8S and ITS2 regions (LP) and the other a shorter product including the ITS1 only (SP). Both assays specifically amplified products from Phytophthora species without cross-reaction with the related Pythium s. lato, however the SP assay proved the more sensitive and reliable. The method was validated using woodland soil and stream water from Invergowrie, Scotland. On-site use of a knapsack sprayer and in-line water filters proved more rapid and effective than centrifugation at sampling Phytophthora propagules. A total of 15 different Phytophthora phylotypes were identified which clustered within the reported ITS-clades 1, 2, 3, 6, 7 and 8. The range and type of the sequences detected varied from sample to sample and up to three and five different Phytophthora phylotypes were detected within a single sample of soil or water, respectively. The most frequently detected sequences were related to members of ITS-clade 6 (i.e. P. gonapodyides-like). The new method proved very effective at discriminating multiple species in a given sample and can also detect as yet unknown species. The reported primers and methods will prove valuable for ecological studies, biosecurity and commercial plant, soil or water (e.g. irrigation water) testing as well as the wider metagenomic sampling of this fascinating component of microbial pathogen diversity.