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1.
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 Ypt1 gene of P. cryptogea. A 369 bp 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 × 103 P. 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 re-circulating nutrient solution of closed soilless cultivation systems.  相似文献   

2.
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.  相似文献   

3.
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 1pgl–1 DNA 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 1fgl–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 2–3h. 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.  相似文献   

4.
Tumour tissue samples were collected from vines grown in various regions of Italy and other parts of Europe and extracted for detection of Agrobacterium vitis. Fifty strains were isolated on agar plates and screened by PCR with consensus primers from the virD2 gene. They were confirmed as A. vitis with a species-specific monoclonal antibody. The isolates were further analyzed by PCR for their opine synthase genes and ordered into octopine, nopaline and vitopine strains. Primers designed on the octopine synthase gene did not detect octopine strains of Agrobacterium tumefaciens. For quantitative PCR, virD2 fragments were sequenced: two classes of virD2 genes were found and two primer sets designed, which detected octopine and nopaline strains or only vitopine strains. For simultaneous identification of all opine-type strains, multiplex real-time PCR with either primer pair and SYBR Green was performed: the combined sets of primers gave signals with DNA from any A. vitis strain. Specificity of the new primers for real-time PCR was evaluated using several unidentified bacterial isolates from grapevines and other plant species. An elevated level of non-specific background was observed when the combined primer sets were used in multiplex PCR assays. The real-time PCR protocol was also used to detect A. vitis cells directly from grapevine tumours; avoiding direct isolation procedures a sensitivity in the range of one to ten cells per assay was found. Inhibition of the PCR reaction by plant material was overcome by treating tumour extracts with a DNA purification kit as a step for the isolation of nucleic acids.  相似文献   

5.
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 sensitivity 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-specific patterns 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.  相似文献   

6.
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7.
A real-time PCR assay was designed to quantify seed-borne infection of Pyrenophora graminea in barley (Hordeum vulgare). Conventional tests such as the freezing blotter method cannot distinguish P. graminea from the closely related P. teres. The seed infection threshold for P. graminea is lower than the one for P. teres and is therefore applied for both species although P. graminea may be absent. This results in unnecessary rejections of seed lots. PCR primers and a TaqMan probe were designed to target a P. graminea-specific DNA sequence. The potential of the real-time PCR assay for quantifying seed-borne infection of P. graminea was investigated by examining seed lots harvested from P. graminea-infected fields. The major part (84%) of the variation in the amount of P. graminea DNA measured by real-time PCR could be attributed to variation between seed lots while only about 8% was due to variation within seed lots. DNA quantities of P. graminea were positively correlated with seed infection incidence detected by the freezing blotter method as well as with the infection incidence of plants examined in the greenhouse. Both correlations were highly significant (P < 0.001) but the DNA quantities accounted only for 59% (R 2 = 0.59) and 56% (R 2 = 0.56), respectively, of the variation in the results obtained by the two conventional methods. Seed lots of varieties resistant to P. graminea contained considerable amounts of P. graminea DNA but showed no or only few leaf symptoms in the greenhouse test suggesting that the recommended seed infection thresholds could be raised for resistant varieties.  相似文献   

8.
Thirty six isolates of Phoma tracheiphila from Italy, the causal agent of the mal secco disease on Citrus species, were characterised by different molecular tools in comparison with representative isolates of other phytopathogenic Phoma species. These included analysis of the distribution of RAPD and microsatellite markers and sequencing of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The results obtained with 12 RAPD primers (92 markers) and 7 microsatellite primers (56 markers) suggest that Italian isolates of P. tracheiphila are genetically homogeneous, leading to identical patterns upon amplification with all the tested primers. Accordingly, ITSI-5.8S-ITS2 sequences were highly conserved (98–100% identity along a 544-characters alignment) among all the isolates of P. tracheiphila. A neighbor-joining analysis of ITS sequences of P. tracheiphila in comparison with those of other Phoma species, as well as with alignable sequences from anamorphic and teleomorphic taxa retrieved in BLAST searches, revealed a close relationship between P. tracheiphila and Leptosphaeria congesta. A pair of P. tracheiphila-specific primers was designed on the consensus sequence (555 residues) obtained from the alignment of the newly generated P. tracheiphila ITS sequences. A PCR-based specific assay coupled to electrophoretic separation of amplicons made it possible to detect P. tracheiphila in naturally infected Citrus wood tissue collected from both symptomatic and symptomless plants. The limit of detection was 10 pg of genomic DNA and 5 fg of the ITS target sequence.  相似文献   

9.
Three citrus scions were evaluated to determine seasonal changes in susceptibility to infections by Phytophthora citrophthora and Phytophthora nicotianae. In a period of 24 months, the Clementine mandarin cv. Hernandina, the hybrid Fortune mandarin and the sweet orange cv. Lane-Late were branch-inoculated under field and laboratory conditions. Field studies showed that the cultivars inoculated with P. citrophthora developed the highest lesion areas during March–June (spring) and September–October (autumn) and with P. nicotianae from June to August (summer). However, lesion areas on detached citrus branches did not show a definite pattern of infection because lesion sizes fluctuated irregularly during the study. The lesion area caused by P. nicotianae in different citrus scions correlated significantly with the monthly mean maximum values of temperature, relative humidity, and the percentage of the relative water content in the 24-month period of inoculations. In contrast, there was no correlation between these variables and the extent of colonisation by P. citrophthora. Nevertheless, a significant relationship was observed between lesion areas caused by P. citrophthora from October to May of each year and the same variables that were significant in inoculations with P. nicotianae. Seasonal changes in the susceptibility of citrus cultivars to P. citrophthora and P. nicotianae may facilitate timing of disease control measures to coincide with periods when disease development is greatest.  相似文献   

10.
Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).  相似文献   

11.
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 2 × 102cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757  相似文献   

12.
Epidemiological studies were conducted in five cocoa growing districts in the Eastern Region of Ghana solely infected by Phytophthora palmivora and five districts in the Ashanti and Brong Ahafo Regions prevalently infected by Phytophthora megakarya to determine the natural incidence, the vertical distribution on trees and the probable sources of stem canker infections, and to isolate and identify the causal pathogens. The incidence of canker in the solely P. palmivora infected area was higher (between 0% and 16.0%) than in the area mainly infected with P. megakarya (0.5–8.0%). Differences were found in the natural height distribution of cankers in the two areas, whilst the areas solely infected with P. palmivora showed a near normal curve, those prevalently infected with P. megakarya were positively skewed. Most of the cankers caused by P. megakarya were found at the base or near the base of the tree trunks (1–40cm above ground level), while those of P. palmivora were concentrated between 41 and 100cm from the ground level. The majority (71.8%) of cankers in the solely P. palmivora infected area were cushion-borne, followed by 24.3% from unknown sources and only 3.9% from the soil. In contrast, a significantly large proportion (32.6%) of the cankers in the prevalently P. megakarya infected area were soil-borne, although cushion-borne cankers formed the majority (48.4%) due to the presence of P. palmivora infection whilst those of unknown sources constituted 19.0%. Phytophthora megakarya was frequently isolated from all the three sources of canker infections, indicating P. megakarya readily causes stem canker on cocoa. These results emphasise the importance of different reservoirs as sources of primary inoculum for diseases caused by the two Phytophthora species particularly pod rot infection on cocoa.  相似文献   

13.
The polymerase chain reaction (PCR) is a rapid, precise method for detecting and identifying pathogenic bacteria. In addition to the published primers for identification of Agrobacterium tumefaciens up to species level, two sets of primers were designed to identify the nopaline and octopine types of Agrobacterium tumefaciens. The RBF-RBR primer set designed based on the nopaline type T-DNA right border detected the nopaline type A208 and R225f strains, and the ocsF-ocsR primer set derived from the ocs gene of the octopine type A. tumefaciens detected the octopine type A348 strain. After polymerase Chain reaction (PCR) amplification by the RBF-RBR primers, the A208 and R225f strains could be differentiated from each other by restriction fragment length polymorphism digestion using the restriction enzymes DraI and XbaI. Multiple colonies can be screened at one time in a single PCR tube with satisfactory efficiency, thereby allowing rapid detection of pathogenic A. tumefaciens. Following a rough screening by classical biovar medium and -methyl-d-glucoside medium, the developed PCR system was introduced to identify isolates collected from soil and crown gall samples. Of 42 isolates determined to be A. tumefaciens, 7 were found to be octopine type; all the rest were R225f type.  相似文献   

14.
Disease on fruit trees in Bulgaria caused by Phytopthora cactorum and P. citrophthora was found in the period 1998–1999. Leaves of some trees become reddish during July, and later in the season fall off. Infected trees die during the same season, or the next season. Observations on symptom development and spread of Phytophthora root and crown rot of fruit trees was undertaken from 1999 to 2009. Disease incidence is between 2% and 14% in some gardens and nurseries. The disease was registered in the regions of Plovdiv, Kjustendil, Sliven, Yambol, Karnobat, Bourgas and Svishtov. Samples from infected plant tissues were taken and isolations were done on selective PARP media, or by applying a baiting bioassay. Based on morphological and cultural characteristics and temperature requirements the following Phytophthora species have been identified: Phytophthora cactorum, P. citrophthora, P. drechsleri, P. cryptogea, hybrid and Pythium. Pathogenicity of the isolates was tested on green apple fruits or one-year-old apple rootstocks. Laboratory studies of the effect of temperature on mycelia growth showed that most isolates can grow from 5° up to 30°C, with an optimum from 18° to 25°C. Only three strains grew at 35–36°C, two developed slowly, one grew well. The optimal pH for mycelia development was tested. Aiming at control of disease, in vivo pot trials have been carried out for studying resistance of rootstocks to P. cactorum. At the end of the growing season a good level of resistance has been shown in the rootstocks M29C, Gizela 6, and MAXMA 14.  相似文献   

15.
Pepper mottle virus, genus Potyvirus, was first identified in Japan based on particle morphology, host range, aphid transmission, and molecular classification using the nucleotide sequence of the coat protein gene and 3-untranslated region.  相似文献   

16.
Strains of Xanthomonas campestris pv. vesicatoria Dye 1978 (Xcv), the causal agent of bacterial spot, have been classified into two groups based on their ability to hydrolyze starch. Three monoclonal antibodies (MAbs), 7AH10, 5HB3, and 4AD2, were produced immunized against the living bacteria and were specific to and could distinguish Xcv strains able or unable to hydrolyze starch (Amy+ or Amy–). The MAb 7AH10, obtained against strain UPB141(Amy–) reacted in an enzyme-linked immunosorbent assay with all the Amy– strains (n = 19) and 1 of 11 Amy+ strains. Against Xcv 2625, an Amy– unusual phenotype strain, MAb 5HB3, recognized 97% of our worldwide collection of Xcvs (n = 30). Also against that strain, the MAb 4AD2 reacted with none of the homologous Amy– phenotypes and with 90% (n = 11) of the heterologous Amy+ phenotypes. For all the MAbs, cross reactions with other pathovars or species were less than 4% (n = 67). By assaying a Japanese collection of strains against the three MAbs, the Amy+ strains were distinguished from the Amy– strains, and their relation with other world strains could be demonstrated. All the MAbs reacted with the lipopolysaccharide fraction of the bacterial cell wall during immunoblotting.  相似文献   

17.
Leptosphaeria maculans,a fungal pathogen of Brassica napus, secretes large amounts of a 28kDa protein (SP2) in liquid culture. This protein shows high sequence similarity to secreted serine proteases from other ascomycetes and is the major component of culture filtrate with protease activity, as analysed on casein zymogels. The sp2 gene is expressed during infection of B.napuscotyledons when L. maculans hyphae are growing between mesophyll cells, as well as at later stages when the fungus invades the vascular tissue.  相似文献   

18.
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. capsiciwere designed based on the sequence of its internal transcribed spacer regions. CAPFW/CAPRV1 amplify a 452 bp product from P. capsici DNA 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. capsici DNA in inoculated plants. Detection occurred as soon as 8h post-inoculation in stem samples from infected but still symptomless plants. The method was also tested to detect fungal DNA in infected soils.  相似文献   

19.
Using a leaf disc method, 19 isolates of the poplar rust, Melampsora larici-populina , and one isolate of M.populnea from England were inoculated on to 25 poplar clones belonging to Populus nigra and P.trichocarpa, and hybrids between P. deltoides and P. nigra, P. deltoidesand P. trichocarpa, P.tacamahaca and P.trichocarpa, and P. alba and P. tremula. Disease was scored based on the pustule area and inoculum density. In terms of whether sporulating uredinia formed, the 19 isolates showed seven different patterns to the tested poplar clones. The majority of the rust isolates infected P. nigra P3090 and Vereecken, P.nigra×P. deltoides Casale and Tasman, P. tacamahaca×trichocarpa 36 and Balsam Spire, and P.trichocarpa Blom. Populus trichocarpa×P. deltoides 69039/4 was infected by only three isolates collected from southern England. No visible symptoms appeared on P. alba ×P. tremulaTower and P.trichcarpa×P. deltoides×P. deltoides76028/5 in inoculations with M. larici-populina isolates. Populus alba×P.tremula Tower was infected only by M. populnea. When M. larici-populina isolates were tested using AFLP, no differences were found either between isolates from different geographical regions or between those having narrow spectrum of virulence and those showing wide spectrum of virulence on the tested clones. The results suggest that the UK rust populations possess virulences which were found in races E1, E2, E3 and E4 in continental Europe and that rust having virulence patterns similar to race E4 has occurred in UK poplar plantations since 1996.  相似文献   

20.
Environmental factors influencing sporocarp formation in Typhula ishikariensis were studied under controlled conditions. Sporocarp formation in T. ishikariensis was divided into two stages: stipe elongation from the sclerotium and fertile head development at the tip of the stipe. Factors required for each stage differed. At the stipe elongation stage, low temperature (10°/5°C; day/night) and high humidity were important, but light was not required. In contrast, at the fertile head stage, light and moderate day length (8h/day) were essential. Fertile heads developed at 46µEm–2s–1; and high intensity (137µEm–2s–1) did not suppress development. Moreover, adding unsterilized soil to the sea sand medium accelerated sporocarp formation. These findings imply that the sclerotium of T. ishikariensis recognizes several physical factors for sporocarp formation. Sporocarps of T. ishikariensis developed within 4 weeks after incubation under optimal conditions. The sporocarp produced basidiospores, and differential mating incompatibility was confirmed among monokaryons derived from basidiospores produced under artificial conditions. This method should be useful for obtaining monokaryons for genetic studies of T. ishikariensis.  相似文献   

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