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1.
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   

2.
A biovar 3-specific primer set Ab3-F3/Ab3-R4 was designed based on the comparison of sequences of the 16S rDNA region of agrobacteria and related rhizobia for rapid identification of Agrobacterium biovar 3 strains. A 570-bp 16S rDNA fragment was amplified from cell lysates of Agrobacterium biovar 3 strains by polymerase chain reaction (PCR) using Ab3-F3/Ab3-R4 primers. Discrimination of Agrobacterium tumefaciens biovar 3 from Agrobacterium radiobacter biovar 3 and of Agrobacterium biovar 3 strains from other Agrobacterium strains was done simultaneously using multiplex PCR with a mixture of two primer sets (Ab3-F3/Ab3-R4 and VCF3/VCR3) previously designed for the virC region of Ti-plasmid and Ri-plasmid.  相似文献   

3.
Fifty-six tumorigenic Spanish grapevine strains of Agrobacterium spp. were tested for biovar classification, pathogenicity on several hosts, opine utilization, 16S rRNA gene sequencing and PCR amplifications using five primer sets targeting chromosomal and Ti plasmid genes. Fifty of them belonged to A. vitis (biovar 3), three to A. tumefaciens (biovar 1) and three to A. rhizogenes (biovar 2). All strains were tumorigenic on grapevines. Most A. vitis strains were also pathogenic on tomato and tobacco plants, while the three A. tumefaciens strains were only pathogenic on grapevine. Although most A. vitis strains used octopine, 12 utilized neither octopine nor nopaline. 16S rRNA gene sequencing clearly distinguished between strains belonging to the three species. Those of A. vitis could be further divided into three chromosomal backgrounds according to their 16S ribosomal RNA gene sequences. No universal primer pair was found for the detection of all three Agrobacterium species isolated from grapevine. DNA from all A. vitis strains was amplified with the chromosomally-encoded pehA primer pair. In both A. vitis and A. tumefaciens a correlation was observed between the amplifications obtained using the tmr and the virA Ti-plasmid-targeting primer pairs. Three types of Ti plasmid were found in A. vitis strains according to their PCR amplifications and opine utilization profiles. A given chromosomal background harboured only one type of Ti plasmid within the strains from each analysed sample, showing a strong association between chromosomal backgrounds and Ti plasmids in A. vitis .  相似文献   

4.
In the Sidi M’djahed nursery (Algeria), over 60,000 eucalyptus (Eucalyptus occidentalis) plantlets exhibited tumour-like growths localized at the crown of the plants that resembled crown galls caused by Agrobacterium tumefaciens. Bacteria colonizing the galls were isolated and purified. Most (22 out of 24) of the isolates had cultural and biochemical characteristics similar to those of strains of the biovar 1 of A. tumefaciens. Twenty out of 22 Agrobacterium isolates induced tumour formation on various test plants. In PCR experiments, DNA extracted from these virulent strains yielded an amplification signal corresponding to a 247-bp fragment located within the virulence region of nopaline type Ti plasmid. Consistent with this, the opine nopaline was detected in the tumours induced on test plants – but not on eucalyptus plants. Nopaline was degraded by the 20 pathogenic isolates that were also sensitive to agrocin 84, indicating the presence of a nopaline-type pTi in these strains. The chromosomal region encoding the 16S rRNA was analyzed in a sub-population of the pathogenic agrobacterial isolates. The analyzed strains were found to belong to the ribogroup of the reference strain B6. Interestingly, Eucalyptus camaldulensis and Eucalyptus cladocalyx grown in the same nursery and in the same soil substrate developed no galls.  相似文献   

5.
Six strains of crown gall bacteria were isolated from flowering cherry. It was revealed by Otten paper electrophoresis that of the six strains, only BYH18-4 possessed the octopine type Ti plasmid, the remainder having nopaline type Ti plasmid. BYH5-1 was identified by physiological and biochemical tests to be Agrobacterium tumefaciens (originally biovar 1). The other five were A. rhizogenes (originally biovar 2). It was demonstrated with Stonier's method of double layer medium that flowering cherry crown gall bacteria exhibited different sensitivities to agrocin produced by biocontrol strain K1026. Strain K1026 on greenhouse-grown sunflower seedlings exerted a relatively potent inhibitory action on flowering cherry crown gall bacteria. Artificial inoculation showed that K1026 produced 67–99% inhibition of flowering cherry crown gall disease, compared with the treatment of inoculation with crown gall bacteria only.  相似文献   

6.
Crown gall was previously reported on grape in Israel but the pathogen was not isolated and characterized. The three recognized biovars ofAgrobacterium tumefaciens can be tumorigenic on grape, but biovar 3 is the most important world wide. A single occurrence of tumors in a vineyard yielded bacteria which incited galls on grape,Nicotiana glauca and tomato, but not on bryophyllum. The bacteria were confirmed asA. tumefaciens because they contained DNA which hybridized with T-DNA from a Ti plasmid. Biochemical and physiological tests, octopine production and utilization, and agrocin 84 insensitivity conformed with those of bv. 3. Subsequent occurrences of the grape disease have not been found, but the presence ofA. tumefaciens bv. 3 in Israel is a potential threat to nurseries and vineyards.  相似文献   

7.
Novel PCR primers were developed to amplify a 243-bp fragment of an intergenic region between gene 5 and tms2 on the T-DNA of Agrobacterium tumefaciens biovar 1. These primers exhibit 100% positive correlation with strain virulence, 100% negative correlation with avirulence, and did not generate extraneous bands, thus facilitating robust real-time PCR detection.  相似文献   

8.
Phialophora gregata f. sp. adzukicola, a causal agent of brown stem rot in adzuki beans, produces phytotoxic compounds: gregatins A, B, C, D, and E. Gregatins A, C, and D cause wilting and vascular browning in adzuki beans, which resemble the disease symptoms. Thus, gregatins are considered to be involved in pathogenicity. However, molecular analyses have not been conducted, and little is known about other pathogenic factors. We sought to isolate nonpathogenic and gregatin-deficient mutants through Agrobacterium tumefaciens-mediated transformation (ATMT) for cloning of pathogenicity-related genes. The co-cultivation of P. gregata and A. tumefaciens for 48 h at 20°C with 200 μM acetosyringone resulted in approximately 80 transformants per 106 conidia. The presence of acetosyringone in the A. tumefaciens pre-cultivation period led to an increase in T-DNA copy number per genome. Of 420 and 110 transformants tested for their pathogenicity and productivity of gregatins, one nonpathogenic and three gregatin-deficient mutants were obtained, respectively. The nonpathogenic mutant produced gregatins, whereas the gregatin-deficient mutants had pathogenicity comparable to the wild-type strain. This is the first report of ATMT of P. gregata. Further analysis of these mutants will help reveal the nature of the pathogenicity of this fungus including the role of gregatin in pathogenesis.  相似文献   

9.
Several polymerase chain reaction (PCR) primers were designed from the internal transcribed spacer (ITS) regions of the rDNA genes of Rosellinia necatrix to develop a PCR-based identification method. Screening the primers against two isolates of R. necatrix and six other Rosellinia species resulted in the amplification of a single specific product from R. necatrix for most of the primer pairs. Two primer pairs (R2-R8 and R10-R7) confirmed their specificity when tested against 72 isolates of R. necatrix and 93 other fungi from different hosts and geographic areas. The R10 primer was modified to obtain a Scorpion primer for detecting a specific 112bp amplicon by fluorescence emitted from a fluorophore in a self-probing PCR assay. This assay specifically recognised the target sequence of R. necatrix over a large number of other fungal species. In conventional PCR, with primer pairs R2-R8 and R10-R7, 10-fold dilutions of R. necatrix DNA indicated a detection limit of 10pgul-1 using a single set of primers and 10fgl-1 in nested-PCR. For Scorpion-PCR, the detection limit was 1pgl-1 and 1fgl-1 in nested Scorpion-PCR, i.e. 10 times more sensitive than conventional PCR. A simple and rapid procedure for DNA extraction directly from soil was modified and developed to yield DNA of purity and quality suitable for PCR assays. Combining this protocol with the nested Scorpion-PCR procedure it has been possible to specifically detect R. necatrix from artificially inoculated soils in approximately 6h.  相似文献   

10.
A technique based on the use of specific primers for polymerase chain reaction (PCR) was developed for the identification of the stem and bulb nematode belonging to the Ditylenchus dipsaci species complex. The internal transcribed spacer region ITS1 and ITS2, the gene 5.8 S and part of genes 18 S and 26 S of twenty populations of the D. dipsaci species complex belonging to both D. dipsaci sensu stricto and Ditylenchus sp. B (corresponding to populations of giant individuals associated to Vicia faba) and three congeneric species were amplified with two universal ribosomal primers. PCR-amplified DNA samples were digested with five restriction enzymes in order to reveal some polymorphism allowing the identification of D. dipsaci populations associated with Fabaceae seeds. The polymorphism among species was confirmed by the sequencing of the PCR products. A primer (DdpS2) was designed in a region conserved in all populations of both D. dipsaci sensu stricto and D. sp. B studied in the present work. The other Anguinidae species (except a few species from Central Asia associated to Astereaceae and D. sp. G associated to Plantago maritima) differ in two to four nucleotides at the 3′ extremity of this region. This sequence portion coincides with a TspEI restriction site. In combination with a primer located in the ribosomal region, this first primer is a good candidate for identification by PCR of populations of the D. dipsaci species complex found in Fabaceae seeds. A second primer (DdpS1) was designed in a similar way and was specific to D. dipsaci sensu stricto. The utility of these two sets of primers is discussed against the background of quarantine regulation.  相似文献   

11.
Eighty stone fruit nurseries located in different regions of Poland were examined for the presence of crown gall affected plants. The disease was observed in 39 nurseries, and galls were sampled for bacterial isolation. Out of 1213 isolates, 409 were pre‐identified as Agrobacterium/Rhizobium spp. with 23S rDNA‐based multiplex PCR, and out of these, 315 were pathogenic when tested on sunflowers. Sequence analysis of three housekeeping genes (fusA, recA, rpoD) revealed that 366 strains belonged to Rhizobium rhizogenes, 23 to Agrobacterium tumefaciens species complex, and the rest of the strains were allocated to new phylogenetic lineages. Of these, the most numerous was the lineage allocated in the Pararhizobium genus. Positive results obtained from pathogenicity tests were generally in agreement with results obtained by PCR with primers complementary to T‐DNA except for two strains, which were positive for PCR but negative for the pathogenicity test. All detected Ti plasmids were nopaline‐type. Independent of their pathogenicity, 59% of tested strains were not sensitive to agrocin 84 in in vitro tests. Analysis of biochemical and physiological features distinguished 50 groups with different phenotypic profiles, but the tested traits were not consistent for strains classified to one taxon. This finding shows limited value of biochemical tests in identification procedures. The bacteria causing tumours were heterogeneous and strains classified to different taxa were found even in a single tumour.  相似文献   

12.
Serious outbreaks of grapevine crown gall disease were observed in major Serbian viticultural regions during the last five years. Tumorigenic Agrobacterium vitis was identified as a causal agent by using conventional bacteriological and molecular tests. The 36 studied strains of A. vitis showed homogeneous biochemical and physiological characteristics, but were heterogeneous in their pathogenic properties, especially on tomato and sunflower. Furthermore, genetic differences related to chromosomal and plasmid DNA were observed. The Ti plasmid of 35 strains was classified as the octopine/cucumopine (O/C) type, whereas one was classified as the vitopine (V) type. The O/C strains were further divided into O/C-1 and O/C-2 groups based on PCR analysis. Moreover, the sequence analysis of the 16S-23S rRNA ITS region provided robust and precise delineation of studied strains. Although a high level of genetic diversity in A. vitis strains from Serbia was revealed by using this approach, their genotypic relatedness with the strains from other countries suggested their common origin. Also, association between the chromosomal and plasmid DNA was determined for some phylogenetic groups and clusters.  相似文献   

13.
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   

14.
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   

15.
Agrobacterium tumefaciens is a Gram‐negative bacterium. It causes plants to produce crown gall disease because of the transfer, integration and expression of oncogenes encoded by the T‐DNA (transferred DNA) region of the tumour‐inducing (Ti) plasmid. A set of transferred genes directs the production of bacterial nutrients, called opines, formed by condensation of an amino acid and a keto acid or a sugar. Transformed cells synthesize and secrete substantial quantities of particular opines, which A. tumefaciens then uses as a carbon and sometimes nitrogen source. Agrobacterium tumefaciens strains are usually classified on the basis of the opines they can catabolize. Because of the ability to transfer DNA between different kingdoms, A. tumefaciens is also frequently used to generate transgenic plants. This study analyses five poorly characterized wildtype Agrobacterium strains, 1D1108, 1D1460, 1D132, 1D1478 and 1D1487, isolated from Euonymus, cane, cherry, Salix and apple, respectively. Partial Ti‐plasmid sequence analysis demonstrated that the five strains harbour the nopaline‐type Ti plasmid. Tumorigenesis and transient transformation assays of the five analysed and six wildtype A. tumefaciens strains were performed with selected plant species, including two or three species of Brassicaceae, Asteraceae, Solanaceae, Apiaceae and Leguminosae. The A. tumefaciens strains 1D1108, 1D1460 and 1D1478 showed higher transformation efficiencies than the previously characterized A. tumefaciens strains with several economically important crops. These data suggest the potential use of these newly characterized wildtype A. tumefaciens strains in transient transformation assays with certain plant species.  相似文献   

16.
月季根癌病病原菌分离及抗病资源初步筛选   总被引：3，自引：0，他引：3  
从月季品种金玛丽、曼海姆、杏花村、梅郎口红的根部肿瘤组织中纯化了9株分离物,根据其在MW选择性培养基上的单菌落形态初步判断其为根癌土壤杆菌。以根癌土壤杆菌高度保守的virD2和ipt基因的部分序列设计引物对分离的菌株进行PCR检测,其中有6株菌株能扩增出virD2和ipt基因片段,为根癌土壤杆菌毒性菌株。采用针刺涂抹法接种向日葵、荷花蔷薇幼茎,6株菌株均能在供试植株上形成肿瘤,一个月后肿瘤大小有显著差异,说明分离获得的根癌土壤杆菌株毒性有差异。以强毒性菌株J-5-1在田间接种了部分蔷薇属野生资源,以鉴定其对根癌病的抗性。根据发病率、肿瘤大小、肿瘤干质量以及感病后植株生长状况将植物的根癌病抗性分为高度抗病、中度抗病、中度感病、高度感病4个类型。  相似文献   

17.
A partial sequence of Oat mosaic virus (OMV) has been obtained for four isolates of the virus from four European countries. This represents the first available sequence data for this important disease of winter-sown oats. The longest clone of 1699 nucleotides was obtained from infected English oats using a degenerate primer, designed to members of the Potyviridae family. Alignment of the predicted amino acid sequence with members of the Potyviridae showed closest identity with viruses of the Bymovirus genus. The predicted amino acid sequence has one open reading frame corresponding to part of the NIb and capsid protein, with a 3 untranslated region of 351 nucleotides, followed by a poly(A) tail. PCR primers were designed to the coat protein and NIb gene of members of the Bymovirus genus and used to obtain partial sequences of 1441 nucleotides at the 3 end of infected oats from both Wales and France. A specific primer set designed to the English isolate was used to generate a product of 701 nucleotides from OMV-infected oat leaves from Ireland. All four isolates are highly conserved at the amino acid level.The first two authors contributed equally to the work  相似文献   

18.
Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.  相似文献   

19.
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   

20.
Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 106 conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.  相似文献   

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