Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems

A PCR-based 'molecular tool box', based on a region of the ras-related protein gene Ypt 1, was developed for the identification of 15 Phytophthora species that damage forests and trees: P. cactorum , P. cambivora , P. cinnamomi , P. citricola , P. europaea , P. inundata , P. lateralis , P. megasperma , P. nemorosa , P. kernoviae , P. pseudosyringae , P. psychrophila , P. quercina , P. ramorum and P. ilicis . Most primers proved highly specific in blast analyses and in tests with DNA from 72 isolates of 35 species of Phytophthora and nine species representative of Pythium . Exceptions were primers designed for P. cactorum and P. ilicis , which cross-reacted with P. idaei and P. nemorosa , respectively. Amplification with Phytophthora -genus-specific primers before amplification with the various species-specific primers (nested PCR) increased the sensitivity of detection over amplification with species-specific primers only: detection limits ranged between 100 and 10 pg target DNA µ L⁻¹ in the latter, compared with 100 fg µ L⁻¹ in nested PCR. Using existing methods for rapid extraction and purification of DNA, single-round amplification was appropriate for detection of target Phytophthora species in leaves, but nested PCR was required for soil and water samples. The quarantine pathogens P. ramorum and P. kernoviae were detected in a number of naturally infected leaves collected in England and Wales, whereas P. citricola was commonest in water and soil samples from natural Scottish ecosystems.     

基于Ypt1基因为靶标的木香疫病病组织及病田土壤分子检测技术

A species-specific PCR assay was established for rapid and accurate detection of the oomycete pathogen Phytophthora tentaculata in diseased plant tissues and infected soil.A pair of species-specific primers Pt1/Pt2 were designed on the basis of Ras-related protein(Ypt1) gene sequences of the Phytophthora species.PCR amplification with the Pt primers resulted in a 386 bp product only from isolates of P.tentaculata.The detection threshold with Pt primers was 100 pg of genomic DNA.A nested PCR procedure was developed using Ypt1F/Ypt1R as the first-round amplification primers and Pt1/Pt2 as the second-round primers,which increased the detection sensitivity 100-fold to 1 pg.PCR using these Pt primers can also be used to detect P.tentaculata in naturally infected plant tissues and soil.The PCR-based method developed in this study provides a rapid and sensitive tool for detection of P.tentaculata.     

Rapid detection of Phytophthora cinnamomi using PCR with primers derived from the Lpv putative storage protein genes

Phytophthora cinnamomi is an ecologically and economically important pathogen. In this study, PCR assays were developed with primer pair LPV2 or LPV3 for rapid detection and identification of this organism. Both primer pairs were selected from putative storage protein genes. The specificity of these primer pairs was evaluated against 49 isolates of P. cinnamomi , 102 isolates from 30 other Phytophthora spp., 17 isolates from nine Pythium spp. and 43 isolates of other water moulds, bacteria and true fungi. PCR with both primer pairs amplified the DNA from all isolates of P. cinnamomi regardless of origin. The LPV3 primers showed adequate specificity among all other species tested. The LPV2 primers cross-reacted with some species of Pythium and true fungi, but not with any other Phytophthora species. PCR with the LPV3 primers detected the pathogen at levels of a single chlamydospore or 10 zoospores in repeated tests. The PCR assay was at least 10 times more sensitive than the plating method for detection of the pathogen from artificially infested soilless medium, and, to a lesser extent, from naturally infected plants. PCR with LPV3 primers can be a useful tool for detecting P. cinnamomi from soilless media and plant tissues at ornamental nurseries, whereas the LPV2 primers can be an effective alternative for identification of this species from pure culture. Applications of these assays for detection of P. cinnamomi in other environments were also discussed.     

Molecular detection of Phytophthora capsici in infected plant tissues, soil and water

A species-specific PCR assay was developed for rapid and accurate detection of the pathogenic oomycete Phytophthora capsici in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Phytophthora spp. and other oomycetes, one pair of species-specific primers, PC-1/PC-2, was synthesized. After screening 15 isolates of P. capsici and 77 isolates from the Ascomycota, Basidiomycota, Deuteromycota and Oomycota, the PC-1/PC-2 primers amplified only a single PCR band of c . 560 bp from P. capsici . The detection sensitivity with primers PC-1/PC-2 was 1 pg genomic DNA (equivalent to half the genomic DNA of a single zoospore) per 25- µ L PCR reaction volume; traditional PCR could detect P. capsici in naturally infected plant tissues, diseased field soil and artificially inoculated irrigation water. Using ITS1/ITS4 as the first-round primers and PC-1/PC-2 in the second round, nested PCR procedures were developed, increasing detection sensitivity to 1 fg per 25- µ L reaction volume. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guiding plant disease management.     

Development of a One-Step Real-Time Polymerase Chain Reaction Assay for Diagnosis of Phytophthora ramorum

ABSTRACT Phytophthora ramorum is a recently described pathogen causing bleeding cankers, dieback, and leaf blight on trees and shrubs in parts of Europe and North America, where the disease is commonly known as sudden oak death. This article describes the development of a single-round real-time polymerase chain reaction (PCR) assay based on TaqMan chemistry, designed within the internal transcribed spacer 1 region of the nuclear ribosomal (nr)RNA gene for detection of P. ramorum in plant material. Unlike previously described methods for the molecular detection of P. ramorum, this assay involves no post amplification steps or multiple rounds of PCR. The assay was found to have a limit of detection of 10 pg of P. ramorum DNA, and could detect P. ramorum in plant material containing 1% infected material by weight within 36 cycles of PCR. The assay also was used to test DNA from 28 other Phytophthora spp. to establish its specificity for P. ramorum. A quick and simple method was used to extract DNA directly from host plant material, and detection of P. ramorum was carried out in multiplex with an assay for a gene from the host plant in order to demonstrate whether amplifiable DNA had been extracted. Amplifiable DNA was extracted from 84.4% of samples, as demonstrated by amplification of host plant DNA. The real-time protocol was used to test 320 plant samples (from 19 different plant species) from which DNA extraction had been successful, and was shown to give results comparable with a traditional isolation technique for diagnosis of P. ramorum in plant material from common U.K. hosts.     

雪松疫霉(Phytophthora lateralis)的快速分子检测

由雪松疫霉(Phytophthora lateralis)引起的疫病是一类植物检疫性病害。为建立该病原菌的快速检测技术,本文比较分析了雪松疫霉和其他疫霉的tRNA序列,在此基础上设计了一对检测雪松疫霉的特异性引物T1/T2,该对引物从雪松疫霉中扩增得到1条192 bp的条带,而其他15种疫霉和其他真菌菌株均无扩增条带,表明该对引物对雪松疫霉具有特异性。在25μL PCR反应体系中,引物T1/T2检测灵敏度为10 pg基因组DNA;而以引物T3/T4和T1/T2进行巢式PCR扩增,能够检测到1 fg基因组DNA,使检测灵敏度提高了10 000倍。该检测体系对灭菌水中游动孢子的检测灵敏度可达0.5个游动孢子,对人工接种发病的植物组织能够特异性地检测到该病原菌。此外,进一步建立了该病原菌的实时荧光定量PCR检测体系。     

Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

ABSTRACT Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), beta-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using beta-tubulin and seemed to be more sensitive.     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Specific detection of Phytophthora cactorum in diseased strawberry plants using nested polymerase chain reaction

Validated protocols for DNA purification and PCR amplification are reported for detection of Phytophthora cactorum in diseased strawberry plants. To remove PCR inhibitors, necrotic strawberry tissues were soaked in 5% alconox solution for >12 h before DNA extraction, and the extracted genomic DNA was embedded in an agarose gel chamber and subjected to electrophoresis. The purified DNA was amplified reliably by PCR. Nested PCR was used to detect a portion of the rRNA gene of P. cactorum in samples. In the first round of PCR, primers ITS1 and ITS4 amplified fragments of varying sizes from total genomic DNA from diseased strawberry plants. In the second round of PCR, a 1:25 dilution of the first-round PCR products was used as template with two P. cactorum- specific primer pairs (BPhycacL87FRG and BPhycacR87RRG, which amplified a 340-bp fragment and a 480-bp fragment from the rRNA gene; and BPhycacL89FRG and BPhycacR176RRG, which amplified a 431-bp fragment). Validation tests using culture-based isolations as a standard for comparison indicated that the DNA purification and PCR primers and amplification protocols were reliable and specifically amplified a portion of the rRNA gene of P. cactorum from necrotic root, crown and petiole tissues of strawberry naturally infected by the pathogen.     

TaqMan Chemistry for Phytophthora ramorum Detection and Quantification, with a Comparison of Diagnostic Methods

ABSTRACT The choice of detection method for phytopathogens can be critically important in determining the success or failure of pest regulation systems. We present an assay for Phytophthora ramorum that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify the pathogen from diseased tissue, and include a universal primer and probe set for an internal positive control. This method is sensitive, detecting as little as 15 fg of target DNA when used in a nested design or 50 fg when used in a single round of polymerase chain reaction. None of the 17 other Phytophthora spp. tested was amplified by this assay. A comparison of the nested and non-nested TaqMan assays, and of one other nested assay, showed nested methods to be significantly more sensitive than nonnested and showed that host substrate significantly affected sensitivity of all assays. The nested TaqMan protocol was successfully field-tested; P. ramorum was detected in 255 of 874 plants in California woodlands, whereas the single-round TaqMan protocol detected significantly fewer positive samples. Finally, we documented increases in the quantity of pathogen DNA in Umbellularia californica leaves in initial stages of infection.     

降香黄檀黑痣病菌的快速分子检测

<正>黄檀黑痣菌(Phyllachora dalbergiicola)能引起降香黄檀黑痣病,该病菌在世界分布十分广泛,且寄主植物种类繁多,除了降香黄檀以外,还可侵染大果紫檀、檀香紫檀等多种紫檀属植物。该菌所致病害会在寄主表面形成黑色凸起的盾片,严重影响寄主光合作用,致使其提前落叶~([1])。通过对黄檀黑痣菌进行早期检测,可以及时采取防治措施,控制病害的进一步发展。而黄檀黑痣菌是活体营养真菌,不能离体培养,使用常规的病害诊断技术     

Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

ABSTRACT A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10(-5) dilution of extracted tissue of artificially infected rhododendron 'Cunningham's White', and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.     

Rapid and Sensitive Detection of Phytophthora sojae in Soil and Infected Soybeans by Species-Specific Polymerase Chain Reaction Assays

ABSTRACT Root and stem rot caused by Phytophthora sojae is one of the most destructive diseases of soybean (Glycine max) worldwide. P. sojae can survive as oospores in soil for many years. In order to develop a rapid and accurate method for the specific detection of P. sojae in soil, the internal transcribed spacer (ITS) regions of eight P. sojae isolates were amplified using polymerase chain reaction (PCR) with the universal primers DC6 and ITS4. The sequences of PCR products were aligned with published sequences of 50 other Phytophthora species, and a region specific to P. sojae was used to design the specific PCR primers, PS1 and PS2. More than 245 isolates representing 25 species of Phytophthora and at least 35 other species of pathogens were used to test the specificity of the primers. PCR amplification with PS primers resulted in the amplification of a product of approximately 330 bp, exclusively from isolates of P. sojae. Tests with P. sojae genomic DNA determined that the sensitivity of the PS primer set is approximately 1 fg. This PCR assay, combined with a simple soil screening method developed in this work, allowed the detection of P. sojae from soil within 6 h, with a detection sensitivity of two oospores in 20 g of soil. PCR with the PS primers could also be used to detect P. sojae from diseased soybean tissue and residues. Real-time fluorescent quantitative PCR assays were also developed to detect the pathogen directly in soil samples. The PS primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in soil and infected soybean tissue.     

Detection of Colletotrichum coccodes from soil and potato tubers by conventional and quantitative real-time PCR

Colletotrichum coccodes is the causal agent of the potato blemish disease black dot. Two PCR primer sets were designed to sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a nested PCR. The genus-specific outer primers (Cc1F1/Cc2R1) were designed to regions common to Colletotrichum spp., and the species-specific nested primers (Cc1NF1/Cc2NR1) were designed to sequences unique to C . coccodes . The primer sets amplified single products of 447 bp (Cc1F1/Cc2R1) and 349 bp (Cc1NF1/Cc2NR1) with DNA extracted from 33 European and North American isolates of C. coccodes. The specificity of primers Cc1NF1/Cc2NR1 was confirmed by the absence of amplified product with DNA of other species representing the six phylogenetic groups of the genus Colletotrichum and 46 other eukaryotic and prokaryotic plant pathogenic species. A rapid procedure for the direct extraction of DNA from soil and potato tubers was used to verify the PCR assay for detecting C. coccodes in environmental samples. The limit of sensitivity of PCR for the specific detection of C. coccodes when inoculum was added to soils was 3·0 spores per g, or the equivalent of 0·06 microsclerotia per g soil, the lowest level of inoculum tested. Colletotrichum coccodes was also detected by PCR in naturally infested soil and from both potato peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed to perform quantitative real-time (TaqMan) PCR to obtain the same levels of sensitivity for detection of C. coccodes in soil and tubers during a first-round PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR diagnostic assay allows an accurate estimation of tuber and soil contamination by C. coccodes .     

利用基因YKT6鉴定疫霉属的不同种

 利用分子标记或对特异位点的碱基序列进行分析是植物病原物分子检测的基础,可以在属和种的水平上对物种进行区分和鉴定。对疫霉属的不同种已有一系列的分子检测方法。SNARE蛋白相关基因YKT6拥有保守的侧翼编码区,适于设计疫霉属特异性的PCR引物,同时其内含子所具有的多态性可开发出几乎所有疫霉种的分子标记。利用疫霉属特异性引物对P-YKT6-F/P-YKT6-R可在31个疫霉种中特异地扩增出一条约600 bp的条带,而在腐霉或其他真菌中不能扩增出该条带。利用大豆疫霉的引物对Ps-YKT6-F/ Ps-YKT6-R和辣椒疫霉的引物对Pc-YKT6-F/Pc-YKT6-R,能分别从大豆疫霉菌株和辣椒疫霉菌株中扩增出一条399 bp和282 bp的条带,常规PCR和巢式PCR的灵敏度分别达到100 pg和10 fg。利用这些引物也可从土壤和病组织中检测到目标病原菌。此外,利用上述特异性引物开发出了大豆疫霉和辣椒疫霉的实时定量PCR检测方法。基于YKT6基因的分子标记和检测方法可用于疫霉种的调查检测和法定定量检测。     

Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

ABSTRACT The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.     

南方镰孢Fusarium meridionale特异性PCR检测方法的建立与应用

为建立快速、稳定的南方镰孢Fusarium meridionale特异性检测方法,对已报道的镰孢属reductase-like基因部分序列进行比对分析,寻找特异性SNP位点,设计出特异性检测引物F-Fm/R-Fm3。利用该引物对包括南方镰孢在内的30株镰孢的基因组DNA进行PCR扩增。结果显示仅在7株南方镰孢中均扩增出400 bp左右的特异性条带。PCR灵敏度试验结果表明该方法的检测灵敏度达到500 pg基因组DNA。     

A Species-Specific Polymerase Chain Reaction Assay for Rapid Detection of Phytophthora nicotianae in Irrigation Water

ABSTRACT Phytophthora nicotianae is a common and destructive pathogen of numerous ornamental, agronomic, and horticultural crops such as tobacco, tomato, and citrus. We have developed a species-specific polymerase chain reaction (PCR) assay for rapid and accurate detection of this pathogen in irrigation water, a primary source of inoculum and an efficient means of propagule dissemination. This PCR assay consists of a pair of species-specific primers (PN), customization of a commercial soil DNA extraction kit for purification of DNA from propagules in irrigation water, and efficient PCR protocols for primer tests and sample detection. The PN primers proved adequately specific for P. nicotianae in evaluations with 131 isolates of P. nicotianae, 102 isolates from 15 other species of Phytophthora, and 64 isolates from a variety of other oomycetes, true fungi, and bacteria. These isolates originated from a wide range of host plants, three substrates (plant tissue, soil, and irrigation water), and numerous geographic locations. The detection sensitivity is between 80 and 800 fg DNA/mul. The assay detected the pathogen in naturally infested water samples from Virginia and South Carolina nurseries more rapidly and accurately than standard isolation methods. Use of this PCR assay can assist growers in making timely disease management decisions with confidence.     

番石榴焦腐病菌的ITS分析及PCR检测

番石榴焦腐病是台湾入境大陆水果的重要植物病害,由葡萄座腔菌Botryosphaeria rhodina引起.为建立该病原菌快速、灵敏的检测技术,比较分析了葡萄座腔菌和葡萄座腔菌属其它种的ITS序列,在此基础上设计了1对检测番石榴焦腐病菌的特异性引物BF1/BR1,利用此引物从葡萄座腔菌中特异性扩增出287bp条带,而其余参试的菌株未能获得扩增条带.将真菌通用引物ITS1/ITS4和BF1/BR1进行巢式PCR扩增后,检测灵敏度提高1 000倍,可检测到葡萄座腔菌1pg的基因组DNA.结合快速碱裂解法提取发病组织的DNA,采用该PCR检测技术可从自然感染焦腐病果实中检测到葡萄座腔菌.     

Rapid Diagnosis of Pseudomonas syringae pv. papulans, the Causal Agent of Blister Spot of Apple, by Polymerase Chain Reaction Using Specifically Designed hrpL Gene Primers

ABSTRACT The identification and detection of Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, on apple leaves and fruit was achieved by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL(1) and hrpL(2) were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 37 of the 57 pseudomonads strains tested. These strains belonged to genomospecies 1 and 2, as described by Gardan et al. (8). The amplicon obtained from 30 of these strains was digested with eight restriction enzymes. Three different restriction patterns were produced from strains belonging to genomospecies 1, resulting in A1 and A2 patterns, while strains belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22-24. Group A2 consisted solely of P. syringae pv. papulans strains. The hrpL gene in P. syringae pv. papulans strain CFBP3323 was sequenced. Two primer sets, Pap1/Pap2 and Pap1/Pap3, were designed and tested for specificity to P. syringae pv. papulans. These primers amplified expected fragments of 242 and 303 bp, respectively. Pap1/Pap2 amplified a fragment only with P. syringae pv. papulans DNA, while Pap1/Pap3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Pap1/Pap2 primer set was successful for the detection of P. syringae pv. papulans in diseased fruit and artificially inoculated leaves.