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1.
Fifty strains of Erwinia amylovora isolated in Bulgaria from different host plants and locations as well as in different years were analysed by RFLP analysis of the pEA29 PstI amplified fragment with HpaII. All the strains formed three well-resolved fragments (large—from 365 to 440 bp, medium—about 341 bp and small—about 180 bp).The strains were classified into three RFLP groups based on the polymorphism in the length of the largest fragment. This fragment was of intermediate size for 63% of the strains, and it was the longest (from 410 to 440 bp) for 29% of the strains. The variable region was sequenced for five strains. The DNA sequence analysis confirmed the different size of the largest fragment. Ten or more than ten SSRs were found for the strains in the group with the largest size of the largest fragment. Some correlation between the RFLP profiles and the origin of the strains was revealed. The RFLP profiles displayed stability in certain strains isolated from the same trees and orchards, but in different years. The number of SSRs was different in strains isolated from one and the same host plant, orchard and year, and also in strains isolated from the same host plant and orchard, but in different years. This could indicate that under natural conditions the fire blight symptoms might be caused by a mixture of E. amylovora strains with different SSR numbers, and so coexistence of distinguishable strains or a change in the population could be assumed.  相似文献   

2.
To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.  相似文献   

3.
Bacterial black spot disease of mango is caused by Xanthomonas campestris pv. mangiferaeindicae (Xcm), which consists of two genotypically and phenotypically distinct groups of strains. Monoclonal antibodies (MABs) were produced – 15 against CFBP 1717, a group I strain, and 9 against CFBP 2919 (yellow-pigmented), a group II strain – and were analyzed for their characteristics. On the avidin-biotin peroxidase complex enzyme-linked immunosorbent assay, the dilution limit of the MABs was between 100 and 200000 and was 10 times higher when measured on the corresponding ascitic fluid. All kinds of isotypes were represented among the MABs. All the Japanese Xcm strains, designated group I by hrp-restriction fragment length polymorphism (RFLP) analysis, reacted equally with MAB 1A7H12G3, which is the most specific for all but one worldwide group I strains, and to only one strain among group II. Also, to various extents, serological heterogeneity inside the two groups was consistently differentiated based on isozyme and RFLP analyses. MAB 1E2E1 against CFBP2919, because of its narrow specificity, and MAB 1A7H12G3 against CFBP1717, because of its broad specificity, will be useful for epidemiological studies or general control of the pathogen.  相似文献   

4.
Restriction fragment length polymorphism (RFLP) analysis of the PCR amplified fragments of recA, gyrA and rpoS genes was applied for the characterization of Erwinia amylovora and Erwinia strains, which cause fire blight and Asian pear blight in orchards. Primers, constructed on the basis of the published recA, gyrA and rpoS gene sequences of Erwinia carotovora, allowed us to amplify DNA fragments for RFLP differentiation of E. amylovora and E. pyrifoliae and finally to distinguish strains within these species and relate them to pear pathogens from Japan. Three to seven restriction endonucleases were applied for RFLP analysis of each gene fragment. The electrophoretic patterns generated after PCR–RFLP for each of the tested genes, were characteristic and specific for each species and allowed their differentiation. The data show that PCR–RFLP analysis of the recA, gyrA and rpoS gene fragments may be considered as a useful tool for the identification and differentiation of E. amylovora and E. pyrifoliae. Almost identical restriction patterns of the analyzed gene fragments indicated a high relationship of E. pyrifoliae strains from Korea and pear pathogens from Japan and a divergence to E. amylovora. For quick and effective differentiation of E. amylovora strains from Erwinia strains from Asia without nucleotide sequencing we recommend the amplification of recA and rpoS gene fragments and digestion of each of them with restriction endonuclease Hin6I.  相似文献   

5.
Candidatus Phytoplasma prunorum was detected for the first time in almond (Prunus dulcis Mill.) cv. ‘Abiod’ in Tunisia. Infected trees showed emergence of new growth during dormancy and leafed out before flowers opened in addition to early defoliation in summer. Phytoplasma was detected by nested polymerase chain reaction (PCR) using universal phytoplasma primer pairs P1/P7 and F2n/R2. A band with expected size was observed in samples collected from five symptomatic, but not symptomless almond trees. PCR products (1.2 kbp) were used for restriction fragment length polymorphism (RFLP) analysis after digestion with endonucleases RsaI and SspI. RFLP patterns obtained were similar to those reported previously for the European stone fruit yellows (ESFY, 16SrX-B). Identification has been further confirmed by PCR using ESFY specific primer pairs (ECA1/ECA2). This is the first report of Ca. Phytoplasma prunorum infecting almonds in Tunisia.  相似文献   

6.
The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.  相似文献   

7.
Differences in hrpZ sequences determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) were used to investigate the molecular epidemiology of Pseudomonas syringae pv. syringae (PSS) strains that were isolated from diseased barley and wheat plants in Okayama, Japan. PCR–RFLP using HhaI separated PSS strains into two groups (A and B). Although specific PCR–RFLP groups of PSS strains were not always isolated from specific cultivars or seeds produced in a specific area, many strains isolated from barley and wheat belonged to PCR–RFLP groups A and B, respectively.  相似文献   

8.
A disease on parthenium weed (Parthenium hysterophorus L.) was observed in June 2008 in Danzhou of Hainan Province. Infected weeds showed phytoplasma-like associated symptoms such as severe stunting, excessive proliferation of shoots, inflorescence-clustering, green petal, small leaves and witches’-broom. The original cause of phytoplasma was further confirmed by polymerase chain reaction (PCR). PCR products of 1.8 kb were obtained using the universal primers pair (P1/P7) designed to amplify the entire 16S rDNA and the 16/23S intergenic spacer region in a direct PCR assay. The primers pair R16F2n/R2 was used to amplify a PCR product of 1.2 kb. Restriction fragment length polymorphism (RFLP) was used to analyze the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA digested with five endonucleases (Kpn I, Hpa II, Taq I, Rsa I, EcoR I). The RFLP patterns of the strain were found to be identical with that of the reference peanut witches’-broom phytoplasma. Based on the RFLP data, it is suggested that the phytoplasma strain belongs to subgroup 16SrII-A. This is the first demonstration of a 16SrII-A group phytoplasma associated with parthenium weed.  相似文献   

9.
 柑桔衰退病毒(CTV)存在着许多生物学特性不同的株系。通过铲除感染强毒株植株或利用弱毒株交叉保护的方式来防治柑桔衰退病都需要对CTV株系进行准确、可靠的鉴定。本文根据对CTV衣壳蛋白基因(CPG)的限制性片段长度多态性(RFLP)分析,发现在重庆主栽柑桔品种的衰退病毒主要以CP/Hinf I RFLP第1、3和6组群为主,并且在田间以多株系混合感染为主。  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.
Isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot on tomato plants, were assessed for physiological and genetic characteristics using conventional and molecular techniques. All isolates were able to produce necrosis on tomato roots and classified into temperature group according to the optimal growth temperatures. Specific-PCR assays and DNA sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region confirmed the existence of both types (Type 1 and Type 2) of the pathogen among the isolates tested. All isolates were identified as Type 2 except for isolate Pl-4, which was classified as Type 1. Restriction fragment length polymorphism (RFLP) analysis with six enzymes resulted in three distinct banding patterns among the isolates depending on the length and restriction profiles of the rDNA intergenic spacer region. Inter-simple sequence-repeat analysis revealed a high level of genetic diversity among the isolates in agreement with the data of RFLP analysis. These results indicated that there were three different intraspecific groups among Turkish isolates of P. lycopersici. The presented study is the first attempting to characterize Turkish isolates of P. lycopersici. The results obtained will be useful in screening of tomato seedlings for resistance to P. lycopersici.  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.
Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.  相似文献   

14.
Grapevine virus A (GVA) is considered one of the viruses associated with rugose wood (RW), one of the most economically important diseases of grapevine. Thirty-seven GVA isolates collected from grapevine cultivars from Marche (central-eastern Italy), Apulia and Campania (southern Italy), were subjected to molecular characterization. The genetic and population diversity was studied in the coat protein (CP) gene by RT-PCR-RFLP analysis with three restriction enzymes (MseI, AluI, and AciI), and nucleotide sequencing. A new primer pair (CP1F/R) allowing amplification of the whole CP gene (621 bp) was developed. RFLP with AciI yielded the highest number of variants in GVA isolates, showing seven different ‘simple’ profiles (A, B, C, D, E, F, and G). ‘Complex’ profiles were also found, and the most common variant combination was A + B in 39% of isolates. The analysis of GVA sequences confirmed the presence of plants infected with more than one GVA variant and suggested that RT-PCR-RFLP is suitable for evaluating population diversity of GVA enabling a screening of different haplotypes. The distribution of RFLP profiles and the phylogenetic analysis were not correlated with the location of infected plants, showing the presence of a GVA population with genetic diversity in the average with those of RNA viruses.  相似文献   

15.
Novel primers for rep-PCR were developed with the original software and based on `ancient diverged periodical sequences'. Rep-PCR with these primers was applied to study genetic relationships among 51 Xanthomonas campestris strains. The strains were collected from different countries including Russia, Japan, UK, Germany and Hungary. Reference strains of three X. campestrispathovars and five other Xanthomonas species were included. Based on qualitative differences in amplification profiles, the strains were divided into four major groups. Two subgroups recognised within X. campestrispopulation were similar to RFLP haplotypes. The third subgroup included strains of two other pathovariants and Japanese isolates of X. campestris pv. campestriswhile the fourth group comprised the other species of Xanthomonas. The analysis of the diversity within X. campestris resulted in the conclusion that isolates belong to distinct clonal populations (subgroups). The differences between the subgroups of X. campestris were only slightly smaller than between species of Xanthomonas. A PCR fragment about 600 bp amplified by primer KRPN2 was found in nearly all tested strains of X. campestris.SCAR primers designed for this marker produced a single specific band for strains of X. campestris, but not for other Xanthomonas, Pseudomonas and Erwiniastrains tested. Application of the new primer set for rep-PCR offers a rapid, simple and reproducible method for identification of bacterial strains. The X. campestris-specific SCAR primers may be used in diagnostics of this important plant pathogen.  相似文献   

16.
Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.  相似文献   

17.
To detect Japanese yam mosaic virus (JYMV) and Yam mild mosaic virus (YMMV) in yam plants in Japan, we developed a duplex RT-PCR assay consisting of a tube-capture procedure followed by one-step RT-PCR with two primer pairs. A 241-bp fragment of the coat protein region of JYMV and a 174-bp fragment of the nuclear inclusion protein b region of YMMV were amplified, thus identifying the two viruses from yam plants cultivated in Yamaguchi Prefecture in 2007. All water yam plants examined were infected with YMMV alone. All the Japanese yam and Chinese yam plants were infected with either JYMV alone or both JYMV and YMMV, suggesting that YMMV and JYMV are prevalent among field-grown yam plants.  相似文献   

18.
Rapid diagnostic methods to detect known mutations in acetolactate synthase (ALS) genes that confer sulfonylurea (SU) resistance to Schoenoplectus juncoides were developed in this study. By using 11 SU‐resistant accessions (nine accessions with a Pro197 substitution in ALS1 or ALS2, one accession with an Asp376Glu substitution in ALS2 and one accession with a Trp574Leu substitution in ALS2), polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro197 substitutions, an Asp376Glu substitution and a Trp574Leu substitution, respectively. In each of the lanes where the detection of SU‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS1 or ALS2 from common templates containing both ALS1 and ALS2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU‐resistant S. juncoides by providing rapid and accurate diagnosis.  相似文献   

19.
The hyphal tip was isolated from 13 weakly or moderately virulent strains of Helicobasidium mompa to remove double-stranded (ds) RNAs and demonstrate their role as the hypovirulence factor. All of 829 hyphal tip subcultures retained dsRNAs. However, strain v670 containing two large fragments (10kb) and one small fragment (ca. 2.3kb) of dsRNA lost the largest fragment in 3 of 63 subcultures analyzed. One of the three subcultures (v670hti) was used to inoculate carrots to regain virulence compared to the parental strain v670. When isolate v670hti was paired with v670, the largest fragment was reintroduced to v670hti, and its virulence was diminished. Northern blot analysis with two probes hybridizing dsRNA fragments in most H. mompa strains revealed that the largest fragment involved in hypovirulence was different from two other fragments that are common in Japan. These results indicate that the largest dsRNA fragment in strain v670 is associated with hypovirulence in H. mompa.  相似文献   

20.
Molecular identification methods are widely used for the classification of organisms worldwide. Entomopathogenic nematodes are the most often isolated insect parasitic nematodes in the tropical and subtropical regions. In our investigation, PCR-RFLP (Polymerase Chain Reaction — Restriction Fragment Length Polymorphism) of the ITS region (Internal Transcribed Spacer) on the ribosomal (r) DNA of three entomopathogenic nematodes isolated from Ankara, Turkey, was analyzed for identification. The ITS region of rDNA was amplified by PCR and then digested with the following nine restriction enzymes: Alu I, Dde I, Hae III, Hha I, Hind III, Hinf I, Hpa II, Rsa I and Sau 3AI. The amplified and restricted sequences of the ITS regions were separated by agarose gel electrophoresis and the RFLP patterns of these three species were shown in this study. According to our results, these species were identified asSteinernema feltiae, Steinernema carpocapsae andHeterorhabditis bacteriophora. http://www.phytoparasitica.org posting Nov. 4, 2005.  相似文献   

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