Abstract: Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2x102 cfu.
Abstract: Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II)
of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection
of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during
routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato
seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.
Abstract:Phytophthora fragariae var. fragariae is pathogenic to strawberry only and is an EPPO A2 quarantine pest. Seventy-five isolates of P. fragariae from all over the world have been characterized by amplified fragment length polymorphism (AFLP) analysis. However, a sensitive, specific and fast method of detection is now needed, and polymerase chain reaction (PCR)-based methods have now been set up. Based upon sequences of the ITS (internal transcribed spacer) regions of rDNA of several Phytophthora species, PCR primers specific for P. fragariae have been developed together with SCRI (Dundee, GB). These primers were tested on DNA extracted from water, soil and plant material. A nested PCR procedure has been included in the currently used bait test to improve sensitivity and reliability. Also several methods have been studied to detect the amplicon of the PCR reaction: gel electrophoresis, DIAPOPS (detection of immobilized amplified products in a one-phase system), PCR-ELIS A, TaqMan and molecular beacon. With the last two methods, using the ABI 7700 detection system, quantification of target DNA is possible.
Abstract: A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g-1 inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6(SE 14·8) and 100·9 (SE 22·6) CFU g-1 were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.
Abstract: Two immunodiagnostic detection assay procedures were compared with two conventional assays for their sensitivity in detecting propagules of Pythium ultimum var. sporangiiferum , Pythium Group F, Phytophthora cactorum and P. cryptogea in dilution series in sterile distilled water. The most sensitive assay for all four species was the zoospore trapping immunoassay (ZTI). Conventional membrane filtration-dilution plating gave similar results to ZTI with the two Phytophthora spp., but was less sensitive in Pythium detection. Immunodiagnostic dipstick assays and conventional bait tests showed similar sensitivities in the dilution series, and were generally about two orders of magnitude less sensitive than ZTI. The four techniques were also compared for their detection efficacy with water samples collected from horticultural nurseries and in in situ tests of infected root zones of Chamaecyparis , tomato and Chrysanthemum. In these comparisons, ZTI was again the most sensitive test for water samples, although membrane filtration-dilution plating proved to be a more consistent test. Dipstick and baiting assays were the best techniques for in situ testing, and dipsticks provided epidemiologically valuable, quantitative data on pathogen propagule numbers.
Abstract: New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein ( Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.
Abstract: The diseases pink rot, watery wound rot and gangrene are important storage rot diseases of potato associated predominantly with Phytophthora erythroseptica (P.), Pythium ultimum (Py.) and Phoma exigua ( Phoma) var. foveata respectively. Reliable molecular-based diagnostic tests are required that will not only allow unequivocal identification of symptoms but will further advance epidemiological studies of these potato diseases to increase our understanding and contribute to more effective management and control strategies to the potato industry. Primers and probes were designed in specific regions of the internal transcribed spacer (ITS) regions to develop conventional and real-time quantitative polymerase chain reaction (PCR) assays able to detect all possible fungal and oomycete pathogens causing pink rot, watery wound rot and gangrene. The specificity of each diagnostic assay was rigorously tested with over 500 fungal/oomycete plant pathogen isolates from potato and reference culture collections, and both conventional and real-time PCR methods produced similar results. In terms of sensitivity, the detection limits for real-time PCR went below ag DNA levels compared with pg DNA levels with conventional PCR. The real-time PCR assays developed to detect Phoma foveata and Py. ultimum on tubers were suitable for the comparative Ct method (^^Ct) of quantification using the cytochrome oxidase gene of potato as a normalizer assay; an advantage as the need for a standard curve is eliminated. Each assay detected Phoma species (var. foveata or exigua) from naturally infected tubers showing symptoms of gangrene, and P. erythroseptica or Py. ultimum were also detected following inoculation of Russet Burbank tubers. Each diagnostic assay developed could reliably detect and distinguish between the pink rot, watery wound rot and gangrene-causing potato pathogens.
Abstract: A polymerase chain reaction (PCR)-based protocol for detection of Phytophthora lateralis in plant tissues and water is described. Base-pair (bp) deletions in both of the ribosomal DNA internal transcribed spacer (ITS) regions in P. lateralis were used to design complementary PCR primer sequences that amplify a 738 bp fragment only if P. lateralisDNA is present in the sample. Universal control primers based on conserved sequences of the nuclear ribosomal small subunit are included in a multiplexed reaction, providing an internal check on the procedure. The universal primers amplify an approximately 550 bp fragment that is common to plants, protists, and true fungi. The procedure reliably detects P. lateralis in cedar stem tissues and in roots. Positive reactions were obtained with as few as 200P. lateralis zoospores in water.
Abstract: Isolates of alder Phytophthora were collected in the southern part of Belgium on riverbanks planted with Alnus glutinosa and A. incana. They were compared with strains isolated in other European countries in terms of maximum temperature for growth, oogonia shape, pathogenicity on Alnus seedlings and genetic traits. Using both molecular techniques [random amplified polymorphic DNA (RAPD) and random amplified microsatellite RAMS)], two groups of isolates were identified, the first group being further divided into two subgroups, Ia and Ib, using RAPD. Most of the Walloon alder Phytophthora isolates as well as the standard type from UK (formally designated P. alni subsp. alni) fell into group Ia. One isolate was classified in group Ib with the German and Dutch variants (P. alni subsp. multiformis), while three isolates were placed with the Swedish variant (P. alni subsp. uniformis) in group II. In terms of morphological properties, isolates from groups Ia and Ib developed colonies with a felt-like appearance and usually produced numerous oogonia, varying from wavy to warty after 1 week (group Ia) or 2–3 weeks (Ib) in darkness. In contrast, colonies from group II isolates were generally irregular, and smooth oogonia were produced in low quantities after approximately 1 month in culture. A polymerase chain reaction(PCR) using sequence-characterized amplification region (SCAR) primers derived from a polymorphic amplification product generated with a RAPD primer was developed for the specific detection of alder Phytophthora. The specificity and sensitivity of this test are discussed here.
Abstract: A PCR-based method was developed for the identification and detection of Phytophthora capsici in pepper plants. Three PCR primers (CAPFW, CAPRV1 and CAPRV2) specific for P. capsici were designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsiciDNA whereas CAPFW/CAPRV2 a 595 bp fragment; neither set amplifies DNA from pepper or several fungi pathogenic to pepper. In conventional (single-round) PCR, the limit of detection was 5 pg DNA for both primer sets, whereas in nested PCR the detection limit for both was of 0.5 fg. However, when the dilution series of target DNA were spiked with plant DNA, amplification declined two-fold in both conventional and nested PCR. The CAPFW/CAPRV2 set in conventional PCR was used to detect P. capsiciDNA in inoculated plants. Detection occurred as soon as 8 h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.
Abstract