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1.
Silver scurf is an economically important blemish disease of potato caused by the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions of H. solani. Nested PCR was used to increase the specificity and sensitivity of single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soils and potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g–1 of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.  相似文献   

2.
The potential of Cartapip, an albino Ophiostoma piliferum, as a biocontrol agent against sapstain in logs has been tested in Germany. To detect the albino strain in field-tested wood, the usefulness of the -tubulin gene as a target region for developing PCR-based assays was evaluated with 102 strains of O. piliferum and 31 strains of other wood-inhabiting species. A partial -tubulin gene sequence of O. piliferum strains from different geographic origins was amplified by PCR and analyzed by restriction enzyme digestions and DNA sequencing. Variation in size and nucleotide sequences was found in intron regions indicating that intraspecific variation is present in the -tubulin gene. Consequently, -tubulin gene-derived PCR methods using PCR–RFLP patterns generated by HinfI and SpeI and sequence-specific primers Cat1 and Cat2, were developed and their specificity for Cartapip was accessed with field-tested logs and lumber. The -tubulin gene-based PCR methods were found to be valuable tools for rapid and reliable identification of Cartapip in field-tested logs and lumber in Germany. Specificity tests against other wood-inhabiting species and wild type O. piliferum strains from diverse nations showed that the Cat1 and Cat2 primers have potential to be used in other European countries, New Zealand, Alberta and British Columbia.  相似文献   

3.
Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l-1 for P. nicotianaeand 100 pg l–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.  相似文献   

4.
Genetic variability within Septoria carvi isolates obtained from various organs of caraway cultivated in south-eastern and central Poland was studied using the RAPD-PCR technique. The tests were performed using randomly selected primers. The DNA profiles obtained using four primers proved useful in determining genetic variability among the genotypes of Septoria carvi isolates. The present study characterized the differences in the nucleotide sequence within the internal transcribed spacer region of rDNA (ITS1, 5.8S, ITS2) of selected S. carvi isolates and reference strains of Septoria spp. Moreover, eight isolates were sequenced for three loci: actin, calmodulin and translation elongation factor 1-alpha, and the obtained sequences were compared with the sequences of Septoria reference strains affecting other plants of the family Apiaceae. Phylogenetic analysis showed distinct differences of the tested isolates, which allowed to treat them Septoria carvi species affecting the above-ground organs of caraway Carum carvi L. This study is the first report on the genetic characteristics of the species S. carvi.  相似文献   

5.
The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.  相似文献   

6.
Fifty-five isolates of Rosellinia necatrix, the cause of common avocado white root rot disease, were collected from south-east Spain and characterised according to their virulence behaviour and their molecular patterns to assess broader levels of genetic diversity. Virulence properties were revealed by in vitro inoculation on avocado plants. Differences in reaction types showed variability among these isolates. No sequence differences were observed when the internal transcribed spacer 1 (ITS1) and ITS2 regions and DNA fragments of the β-tubulin, adenosine triphosphatase and translation elongation factor 1 genes were explored in representive isolates from five virulence groups. Random amplified polymorphic DNA (RAPD) amplifications were also performed for each isolate using 19 random primers. Four of these primers revealed polymorphism among isolates and repetitive and discriminative bands were used to build an unweighted pair group with arithmetic mean tree. However, RAPD clustering showed low stability, and no correlation between RAPD and virulence groups was observed, possibly indicating high levels of sexual recombination.  相似文献   

7.
Population structure of Eleusine isolates of Pyricularia oryzae (Magnaporthe oryzae) was examined using DNA markers. On the basis of rDNA sequences, Eleusine isolates were divided into two groups. One group clustered with Triticum isolates, while the other clustered with Eragrostis isolates. This grouping was supported by DNA fingerprinting with three repetitive elements: MGR586, MGR583, and grasshopper. These results suggest that the population of Eleusine isolates is composed of at least two groups that evolved independently from the original population of P. oryzae. Most of the isolates that were collected just after an outbreak of finger millet blast in the 1970s had almost identical fingerprint profiles although they were collected in distant prefectures. This result supports the idea that the outbreak was caused by seed transmission of a particular strain of Eleusine isolates.  相似文献   

8.
In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.  相似文献   

9.
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   

10.
Aureobasidium isolated from Vitis vinifera (cv Chardonnay) grapevine tissues were characterised using morphological and molecular techniques. Species level identification of 29 isolates was accomplished by partial amplification and sequencing of the ITS region (ITS1–5.8S–ITS2) using universal primers ITS1 and ITS4. A comparison of nucleotide sequences using BLAST followed by phylogenetic analysis revealed that all isolates examined were Aureobasidium pullulans. Strain level discrimination of a total of 100 epiphytic Aureobasidium isolates including three reference strains was successfully carried out using two inter simple sequence repeat (ISSR) primers, (AAC)5 and (GTG)5 and the Intron Splice Junction R1 (ISJ-R1) primer in which 24, 24 and 15 scorable bands were produced for each primer, respectively. The high level of genetic variation recorded among the isolates further highlighted the high levels of strain diversity among A. pullulans residing on grapevines. Thirty-two epiphytic Aureobasidium isolates were examined for their ability to inhibit the growth of Greeneria uvicola, responsible for bitter rot of grapes. Using an in-vitro dual-culture antagonism assay, all isolates inhibited the growth of G. uvicola (Isolates DAR 77272 and DAR 77273) with inhibition ranging from 15 to 85%. Three Aureobasidium isolates were then examined for their ability to inhibit G. uvicola when co-inoculated onto detached berries, leaves and grape bunches growing on potted vines in a glass house. All isolates reduced the severity of bitter rot infection. The results indicate that A. pullulans has the potential to suppress bitter rot of grapes.  相似文献   

11.
Ten types of plant baits were tested in the laboratory to assess their capacity to detect pathogenic Pythium species. These were orange tree leaves, tomato leaves, pepper leaves, geranium leaves, Bermuda grass leaves, pine needles, immature carnation petals, hemp-seed cotyledons, pepper and cucumber fruits. The Pythium spp. tested were P. aphanidermatum, P. irregulare and Pythium group F from hydroponic market garden crops in the Poniente region of Almería (south-east Spain). The test consisted of observing the velocity at which five baits were colonized and the day of colonization of the first bait. Results indicated that the slowest baits to be infected were immature carnation petals and pine needles. These two, together with Bermuda grass leaves, were also the baits infected in lowest number, such that practically no further infection was produced in the baits after the fifth day of contact with the inoculated water. The other plant baits tested were equally suitable for detection of Pythium spp. over the first two days, although only orange leaves and hemp-seed cotyledons were infected on the first day.  相似文献   

12.
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   

13.
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   

14.
Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.  相似文献   

15.
In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg−1 nivalenol and 2,400 to 17,000 μg kg−1 moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.  相似文献   

16.
The complex of Diaporthe (asexual morph) species occurring on soybean constitutes an important pathogenic group associated with diseases such as pod and stem blight, seed decay and stem canker. Stem canker, caused by Diaporthe aspalathi, has been reported as the most aggressive form of canker and its occurrence has limited soybean crop productivity in the southern United States. The main form of pathogen control is the use of stem canker resistant soybean varieties. In this study, strains of Diaporthe and Phomopsis were isolated from stem and seeds of soybean in different locations in South America during the years 1989–2014. Genomic DNA from 26 isolates were analyzed by PCR-restriction fragment length polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP) techniques, and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA. The molecular analysis of ITS sequences by alignment with those of ex-type strains deposited in GenBank and morphological characteristics allowed the identification of Phomopsis longicolla, D. phaseolorum var. sojae, D. caulivora and D. aspalathi. An analysis of the pathogenicity of 13 isolates of D. aspalathi inoculated in soybean genotypes carrying different resistance genes to stem canker (Rdm1, Rdm2, Rdm3, Rdm4, Rdm5 and Rdm?) enabled us to identify the occurrence of at least three races of D. aspalathi occurring in Brazil. Among the isolates identified as D. aspalathi, both molecular and phenotypic analyses showed clustering depending on the date of collection and pathogenicity, which revealed the existence of variability of the pathogen.  相似文献   

17.
Shot hole disease of stone fruits caused by Thyrostroma carpophilum has become a major threat to stone fruit industry of Jammu and Kashmir, India because of the failure in its management with fungicides. To understand the diversity in shot hole pathogen, a combination of conventional (morphological, cultural and pathological) and molecular (ISSR and ITS markers) approaches were employed to discern variability in 25 isolates of T. carpophilum isolated from peach, plum, apricot, almond and cherry leaves collected from Srinagar, Ganderbal, and Baramulla districts of Jammu and Kashmir, India. The studies revealed a high level of variability among the pathogen. Based on the morpho-cultural and pathological studies, the isolates were grouped into different categories based on colony growth, texture, margin and colour besides change in media colour, incubation period, leaf area infected, etc. Using ISSR markers, a high level of polymorphism in different isolates of T. carpophilum was observed which indicated that these markers are suitable for studying the genetic diversity in this pathogen. Based on dendrogram, the isolates were grouped irrespective of their geographical origin or host species. Phylogenetic analysis of the 25 sequences based on ITS region showed maximum similarity with T. carpophilum (Syn. Wilsonomyces carpophilus) sequences retrieved from NCBI and grouped them in a single clade which proved it as a powerful tool for authentic identification. The pathogen was highly variable based on morpho-cultural, pathological and molecular (ISSR) characterisation.  相似文献   

18.
We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)  相似文献   

19.
Colletotrichum isolates isolated from cowpea in the Hangzhou area of China were identified as C. destructivum based on morphological characteristics, pathogenicity tests, sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal DNA and the infection process. The ability of the C. destructivum isolates to infect Arabidopsis thaliana was investigated under laboratory conditions and showed a two-phase hemibiotrophic infection process. In addition, the sequences of the rDNA ITS region of C. destructivum isolates from cowpea were identical with 100% similarity to that of isolates of C. higginsianum originating from cruciferous plants. This article presents new evidence in support of C. higginsianum as a synonym of C. destructivum.  相似文献   

20.
Fusarium oxysporum f. sp. melongenae (Fomg), causal agent of Fusarium wilt of eggplant, is a serious pathogen in open fields and greenhouses. Inter-simple sequence repeat (ISSR) banding profiles, sequence analyses of inter-transcribed-spacer (ITS), translation elongation factor 1-alpha (TEF-1α), and actin (actA) DNA regions were employed in this study to determine genetic diversity and population structure of Fomg isolates obtained from Turkey. For ISSR study, (ACTG)5, (GACAC)3, (GACA)4, (GATA)4, HVH(TG)7 and (CA)8RG primers were selected from a set of 16. Discriminative ability of the primers revealed with various indices including polymorphic information content (PIC), and mean PIC value was calculated as 0.26. The ISSR data revealed 31 loci belonging to 202 Fomg isolates and 14 of them were found to be polymorphic. The isolates on neighbor joining ISSR tree were grouped into two major clusters which separated Fomg and outgroup isolates. Population structure was investigated based on bayesian modeling and results indicated five subpopulations (K = 5, ?K = 205.42). Mean genetic and geographical distances among sampling locations revealed only a weak and insignificant correlation (r = 0.583, P = 0.06). Phylogenetic analyses were carried out with ITS, TEF-1α and actA DNA regions with a selected subset of 30 Fomg, along with one non-host and one outgroup isolates. Since ITS region were not able to provide a meaningful separation, TEF-1α and actA sequences of each organism were concatenated individually to build a dendrogram. The clustering tree successfully separated the Fomg, non-host and outgroup isolates in which all Fomg were located on the same branch, forming a monophyletic group in the dendrogram.  相似文献   

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