Abstract: The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1 pg µl-1DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1 fg µl-1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3 h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.
Abstract:Phytophthora nicotianae and P. palmivora are the most important soil-borne pathogens of citrus in Florida. These two species were detected and
identified in singly and doubly infected plants using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer (ITS) regions of ribosomal DNA. The sensi-
tivity of the PCR-RFLP was analyzed and the usefulness of the method evaluated as an alternative or supplement to serological methods and recovery
on semi-selective medium. In a semi-nested PCR with universal primers ITS4 and ITS6, the detection limit was 1 fg of fungal DNA, which made it 1000· more sensitive than a single-step PCR with primers
ITS4 and DC6. The sensitivity of detection for P. nicotianae was shown to be ten-fold lower than for P. palmivora, limiting its detection with restriction profiles in plants infected by both fungal species. Phytophthora nicotianae was detected with species-
specific primers in all samples inoculated with this species despite the absence of species-specificpatterns in RFLP. In contrast, the incidence of
detection of P. palmivora in the presence of P. nicotianae was considerably lower using plating and morphological detection methods. Due to its high sensitivity, PCR amplification of ribosomal ITS regions is a valuable tool for detecting and identifying Phytophthora spp. in citrus roots, provided a thorough knowledge of reaction conditions for the
target species is established prior to the interpretation of data.
Abstract: Six primers based on the sequence of the flanking and coding regions of the elicitin gene ParA1 of Phytophthora nicotianae were tested for specific detection of the fungus by the polymerase chain reaction (PCR).One combination, ILM/b>7/IL8, with IL7 in a flanking region and IL8 in a coding region of the gene, gave an intense 378 bp signal with a diverse collection of isolates of P. nicotianae, that included some from black shank disease of tobacco and others froma variety of hosts. The sequence of the amplification product obtained with an isolate that produces elicitin and one that does not, was homologous with the known sequence of the ParA1 gene. The same primer combination gave no signal with sixteen other Phytophthora species tested except for two isolates P. palmivora with which it gave a weak 800 bp signal. It gave no signal with DNA from healthy tobacco and tomato plants but P. nicotianae was detected in inoculated tobacco and tomato plants. Small numbers of zoospores (>100) trapped onto a nitrocellulose membrane after filtration from suspension were also detected after two successive rounds of PCR.
Abstract: The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and
taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Realtime polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is
well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security,biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multidisciplinary studies.
Abstract: A conventional PCR and a SYBR Green real-time PCR assays for the detection and quantification of Phytophthora cryptogea, an economically important pathogen, have been developed and tested. A conventional primer set (Cryp1 and Cryp2) was designed from the Ypt1geneof P. cryptogea. A369bp product was amplified on DNA from 17 isolates of
P. cryptogea. No product was amplified on DNA from 34 other Phytophthora spp., water moulds, true fungi and bacteria. In addition, Cryp1/Cryp2 primers were successfully adapted to real-time PCR. The conventional PCR and real-time PCR assays were compared.The PCR was able to detect the pathogen on naturally infected gerbera plants and on symptomatic artificially infected plants collected 21 days after pathogen inoculation. The detection limit was 5×103P. cryptogea zoospores and 16 fg of DNA. Real-time PCR showed a detection limit 100 times lower (50 zoospores, 160 ag of DNA) and the possibility of detecting the pathogen in symptomless artificially infected plants and in the recirculating nutrient solution of closed soilless cultivation systems.
Abstract: In four neighbouring regions of southern Italy, Basilicata, Campania, Apulia and Calabria, pepper and zucchini plants showing Phytophthora blight symptoms, tomato plants with either late blight or buckeye rot symptoms, plants of strawberry showing crown rot symptoms and declining clementine trees with root and fruit rot were examined for Phytophthora infections by means of polymerase chain reaction (PCR) assays, using primers directed to nuclear ribosomal DNA (rDNA) repeat sequences. All diseased plants and trees examined tested positive. The detected fungal-like organisms were differentiated and characterized on the basis of primer specificity as well as through extensive restriction fragment length polymorphism (RFLP) and sequence analysis of PCR-amplified rDNA. Phytophthora capsici was identified in diseased pepper and zucchini plants, P. infestans was identified in tomato with late blight symptoms whereas buckeye rot-affected tomatoes and diseased strawberry plants proved to be infected by P. nicotianae and P. cactorum, respectively. Declining clementine trees were infected with P. citrophthora and P. nicotianae in about the same proportion. Also, thirty-one pure culture-maintained isolates of Phytophthora which had previously been identified in southern Italy by traditional methods but were never examined molecularly,were examined by RFLP and sequence analysis of PCR-amplified nuclear rDNA. Among these, an isolate from gerbera which had previously been identified by traditional methods only at genus level, was assigned to P. tentaculata. For the remaining pure culture-maintained isolates examined, the molecular identification data obtained corresponded with those delineated by traditional methods. Most of the diseases examined were already known to occur in southern Italy but the pathogens were molecularly detected and fully characterized at nuclear rDNA repeat level only from other geographic areas, very often outside Italy. A new disease to southern Italy was the Phytophthora blight of zucchini. This is also the first report on the presence and molecular identification of P. tentaculata from Italy.
Abstract: Random amplified polymorphic DNA (RAPD)analysis using the OPG-06 primer generated specific patterns for Japanese genotypes US-1, JP-1, and a new A1(JP-2, JP-3, and JP-4) of Phytophthora infestans. N605, a specific RAPD fragment, was cloned and sequenced. PCR primers BD1/BD2 were constructed based on the N605 sequence and were used to clarify the genotypes. PCR products using the BD1/BD2 primers (N605ab marker)easily distinguished the new A1 from US-1 and JP-1. This technique provides a simple and effective method for rapid genotype discrimination that can be used in ecological experiments and forecasts for the occurrence of late blight.
Abstract:Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR ampliconwas detected by severalmethods, including fluorescent labelled probes (TaqManTM and Molecular BeaconTM). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives.
Abstract: In New Jersey, Phytophthora cinnamomi is the pathogen most commonly isolated from diseased roots and runners of the cultivated cranberry (Vaccinium macrocarpon). A second distinct species of Phytophthora has been isolated from dying cranberry plants and surface irrigation water. This species is homothallic with paragynous antheridia and ellipsoid–limoniform, nonpapillate sporangia. It was tentatively identified as P. megasperma in an earlier report. Laboratory experiments demonstrate that the cardinal temperatures for vegetative growth are between 5 and 300C with an optimum near 250C. Sporangia are produced at temperatures between 10 and 200C with the majority of sporangia produced at 10 and 150C. In pathogenicity tests, no growth effect was observed on cranberry plants (cv. Early Black) when tests were conducted at 250C; however, significant reductions in plant growth occurred when tests were conducted at 150C. This species was insensitive to metalaxyl but was sensitive to buffered phosphorous acid. Sequence analysis of the internal transcribed spacer 1 (ITS1), 5.8S rDNA, and ITS2 regions place these isolates in Phytophthora clade 6 with greatest similarity to Phytophthora taxon raspberry. To our knowledge, this is the first report of isolates of this affiliation in North America. However, the observation of low temperature preferences makes this species unique in an otherwise high temperature clade. The isolates described in this study are tentatively classified as Phytophthora taxon cranberry.
Abstract: 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-µL 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-µL 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.
Abstract