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1.
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   

2.
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   

3.
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   

4.
Crown gall disease of tobacco was found in Iwate Prefecture, Japan in 1995. Ten bacterial isolates, obtained from the galls of tobacco, were identified as Agrobacterium tumefaciens (Smith and Townsend 1907) Conn 1942 biovar 1 based on their ability to induce galls on the 14 tested plants, including tobacco after needle-prick inoculation, and on 12 cultural, physiological, and biological characteristics. The growth of the causal organism was not inhibited in vitro by agrocin of A. radiobacter strain K84. This report is the first on the natural occurrence of crown gall caused by A. tumefaciens on tobacco plants.  相似文献   

5.
Agrobacteria were previously isolated from tumors developing on branches and aerial and hypogeous roots of weeping fig plants in Italy and in The Netherlands. A representative group of 48 strains was analyzed by PCR–RFLP of 16S and 16S + IGS ribosomal regions, PCR–RFLP of six Ti plasmid (pTi) regions and characterized for plasmid content. Two groups of agrobacteria were separated by cluster analysis of PCR–RFLP profiles of rrs gene: seventeen strains were similar to the new species Agrobacterium larrymoorei, while the remaining strains were included within the agrobacterium biovar 1 group. Sixteen different plasmid profiles from one to five plasmids were observed. In addition, 21 ribotypes and 20 pTi structures were arranged in many different combinations, showing that fig agrobacteria were characterized by a wide heterogeneity. A general lack of correlation between strain ribotypes and plasmid content was observed.  相似文献   

6.
Ralstonia solanacearum is a known bacterial pathogen of eucalypt and potato plants in Africa. A survey was undertaken to detect this pathogen in eucalypt plantations in South Africa, the Democratic Republic of Congo, and Uganda. Numerous bacterial strains were isolated from trees with symptoms typical of bacterial wilt, but only seven were positively identified as R. solanacearum. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hrp (hypersensitive response and pathogenicity) gene region was used to determine and group the biovars of these R. solanacearum strains. The eucalypt isolates and one potato isolate formed a biovar 3 cluster, whereas the two other potato isolates formed a cluster that corresponded to biovar 2. Amplified fragment length polymorphism (AFLP) analysis confirmed these clusters. Therefore, PCR-RFLP can be used as a reliable diagnostic technique to enable researchers to rapidly identify the pathogen.  相似文献   

7.
We transformed Colletotrichum trifolii, the causal agent of alfalfa anthracnose, using Agrobacterium tumefaciens as a new tool for random insertional mutagenesis. Fungal spores of C. trifolii were transformed with T-DNA including the hygromycin phosphotransferase gene (hph). Southern analysis showed that every randomly selected transformant had a unique hybridization pattern of T-DNA, suggesting that the T-DNA was randomly integrated into the fungal genome. More significantly, about 75% of transformants had a single copy of the T-DNA. The results demonstrate that insertional mutagenesis via A. tumefaciens is a useful tool for studying the function of C. trifolii genes.  相似文献   

8.
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   

9.
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   

10.
Agrobacterium tumefaciens (AT) is the causal agent of crown gall, a major problem in the family Rosaceae and particularly for Prunus spp. Crown gall symptoms result from the bacterial infection of the cells damaged mechanically at the collar or by root parasitic nematodes. Myrobalan plum (P. cerasifera) is susceptible to AT and is not a host for the root-knot nematode (RKN), M. hapla. Some clones of this plum carry single Ma resistance genes that control M. arenaria, M. incognita and M. javanica. The four above mentioned RKN and Myrobalan progenies segregating for Ma were used in experiments aimed at obtaining a better knowledge of the interaction between AT and RKN in relation to the RKN resistance genes. Prunus rooted cuttings, naturally infected with the bacterium were repotted, grown and inoculated individually with RKN. In a first experiment, Prunus plants were (i) either inoculated with 10,000 juveniles (J2s) of M. arenaria to provide a short inoculum pressure (SIP) or (ii) inoculated by association with one M. arenaria-galled tomato root system that produced a high and durable inoculum pressure of the same nematode species. Four months after RKN inoculation, plants were rated for nematode and bacterial root galling symptoms. RKN and AT galls were more numerous and more homogenous under DIP than under SIP. Nevertheless, for both inoculum regimes, AT galls were present in the RKN-susceptible clones (= carrying none of the Ma genes) and absent in the RKN-resistant clones. Subsequent experiments, conducted under DIP with M. arenaria, M. incognita, M. javanica and M. hapla, also showed, for the three first species, the presence of AT galls only in RKN-susceptible clones whereas Prunus plants inoculated with M. hapla and nematode-free controls were free of AT galls. Consequently RKN act as a wound agent in the AT infection process of Myrobalan plum only when the plant develops a compatible reaction (i.e. when it lacks the Ma resistance genes). Considering that J2s do penetrate the roots of resistant plants, the absence of crown gall symptoms on this material even under durable inoculum pressure strengthens the hypothesis that this nematode stage has a very weak effect on plant cells during the infection process. This is the first evidence of the protective effect of a RKN resistance gene against the expression of root crown gall consecutive to RKN infection. The protective effect of Ma and presumably of other RKN resistance genes against AT is a strong argument for their introgression into Prunus and other Rosaceae or bacterium-susceptible crops.  相似文献   

11.
The 16S rDNA, endoglucanase, and hrpB genes were partially sequenced for Asian strains of Ralstonia solanacearum spp. complex, including 31 strains of R. solanacearum and two strains each of the blood disease bacterium (BDB) and Pseudomonas syzygii. Additional sequences homologous to these DNA regions, deposited at DDBJ/EMBL/GenBank databases were included in the analysis. Various levels of polymorphisms were observed in each of these DNA regions. The highest polymorphism (approximately 25%) was found in the endoglucanase gene sequence. The hrpB sequence had about 22% poly-morphism. The phylogenetic analysis consistently divided the strains into four clusters, as distinctly shown on the phylogenetic trees of 16S rDNA, hrpB gene, and endo-glucanase gene sequences. Cluster 1 contained all strains from Asia, which belong to biovars 3, 4, 5, and N2. Cluster 2 comprised the Asian strains of R. solanacearum (as biovars N2 and 1) isolated from potato and clove, as well as BDB and P. syzygii. Cluster 3 contained race 3 biovar 2 strains from potato, race 2 biovar 1 strains from banana, and race 1 biovar 1 strains isolated from America, Asia, and other parts of the world. Cluster 4 was exclusively composed of African strains. The results of the study showed the distribution and diversity of the Asian strains, which are present in three of the four clusters. The similarity of Asian strains to those in the other regions was also observed.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AY464950 to AY465050  相似文献   

12.
In this paper we analysed the basis of insecticide resistance in 59 Italian strains of the peach potato aphid Myzus persicae using both molecular and biochemical assays. Our data as a whole clearly indicate that most M. persicae strains (76.3%) have high or extremely high production of an esterase enzyme which sequester and detoxify insecticides with esteric group. Kdr genotypes conferring resistance towards pyrethoids are present in 57.7% of the analysed populations. Moreover, 26.5% of the kdr positive strains possess also the M918T mutation conferring super-kdr phenotype. Strains with modified AChE (MACE) are not so numerous (27.1%), although they can be found almost everywhere in Italy. Considering all the strains analysed, both MACE and kdr phenotypes are associated with high levels of esterase activity. In Central–Southern regions, kdr and MACE resistance mechanisms resulted in linkage disequilibrium. Bioassays performed in order to evaluate the efficacy of a pyrethroid insecticide against a strain possessing a F979S mutation within its para-type sodium channel gene suggests that this amino acid substitution could affect the sodium channel responsivity to pyrethroids.  相似文献   

13.
Olive mill waste water (OMW) and some of its indigenous bacterial strains were tested in vitro and in planta for their efficacy against crown gall disease caused by Agrobacterium tumefaciens. OMW and polyphenols displayed a high level of antibacterial activity, however the volatile fraction was less efficient as only a bacteriostatic effect was observed. In pot experiments, the percentage of bitter almond rootstock showing symptoms of crown gall was significantly reduced with the dosage rate of OMW 1% as compared to the control (highly natural infected soil treated with water). Five indigenous bacterial strains isolated from OMW exhibited an antagonistic effect against the bacterium. Based on the gene 16S rRNA sequence analysis, one isolate showed 99.2% similarity to known sequences of Bacillus subtilis, one isolate demonstrated high percentage similarities (99.3%) to the genera Bacillus pumilis, and two isolates were associated with Stenotrophomonas maltophilia and Pseudomonas putida 100% and 99.6% similarities respectively. Among these bacteria, the strain B1 proved efficient against the soil borne pathogen in vitro and pot experiments. Our study in controlled conditions suggested that the addition of OMW to soil exerts significant disease suppressiveness against A. tumefaciens. Thabet Yangui and Ali Rhouma contributed equally to this work and are regarded as joint first authors.  相似文献   

14.
为明确湖南省柑橘主产区的实蝇入侵为害现状,从该省7个市(自治州)22个地点收集柑橘蛆果中的幼虫,利用DNA条形码技术对其进行分子鉴定,并以DNA条形码序列作为分子标记探究橘大实蝇Bactrocera minax的20个中国地理种群(湖南省6个组群共16个种群、其它3省市4个种群)以及1个印度地理种群间的亲缘地理关系,分析橘大实蝇在我国的遗传进化关系。结果表明,仅采集自邵阳市城步苗族自治县柑橘蛆果中的5头幼虫被鉴定为蜜柑大实蝇B. tsuneonis,其余21个地点采集的595头幼虫均被鉴定为橘大实蝇。21个橘大实蝇地理种群的平均单倍型多样性为0.75,核苷酸多样性为0.0032,核苷酸差异数为2.13,中国所有地理种群均具有较高的遗传多样性;单倍型网络进化图显示湖南、重庆、贵州种群共享的单倍型H3为原始单倍型,表明其为比较原始的种群;AMOVA分析结果显示种群内个体间遗传变异占总体变异的59.04%,是遗传变异的主要来源;遗传分化结果表明湖南省6个组群间均出现了中度至高度的遗传分化,FST在0.0521~0.7795之间。表明DNA条形码技术可用于蜜柑大实蝇和橘大实蝇幼虫的分子鉴定及其种群遗传进化分析。  相似文献   

15.
Three evolutionary lineages of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici were found among a worldwide sample of isolates based on phylogenetic analysis of the ribosomal DNA intergenic spacer region. Each lineage consisted of isolates mainly belonging to a single or closely related vegetative compatibility group (VCG) and a single mating type (MAT). The first lineage (A1) was composed of isolates VCG 0031 and MAT1-1; the second (A2) included VCG 0030 and/or 0032 and MAT1-1; and the third (A3) included VCG 0033 and MAT1-2. Race 1 and race 2 isolates belonged to the A1 or A2 lineages, and race 3 belonged to A2 or A3 lineages, suggesting that there is no correlation between race and lineage. However, for the isolates from Japan, race 1 (with one exception), race 2, and race 3 isolates belonged to A2, A1, and A3 lineages, respectively. These results suggest that the races could have evolved independently in each lineage; and in Japan the present races were likely to have been introduced independently after they had evolved in other locations.  相似文献   

16.
Approximately 30,000 fluorescent bacterial strains isolated from tomato, lettuce, eggplant, Chinese cabbage, and Japanese pepper plants at seven different locations in Hyogo Prefecture, were screened for plant-growth-promoting (PGP) activity to induce disease resistance against bacterial wilt in tomato. The 37 strains that had higher PGP activity were subjected to molecular phylogenetic analyses using the sequences of the 16S rRNA, gyrB and rpoD genes. Most of the strains were identified as Pseudomonas fluorescens or its close relative, P. putida, while a few strains were grouped with more distantly related bacterial species such as Enterobacter and Stenotrophomonas. The phylogenetic relationships among tomato and lettuce isolates mostly coincided with the source locality and host plants, with a few exceptions. In contrast, isolates from Japanese pepper plants did not form their own cluster but represented several different bacterial species.  相似文献   

17.
为明确草地贪夜蛾Spodoptera frugiperda肠道细菌与环境适应性的关系,采集云南省嵩明(Songming,SM)、彝良(Yiliang,YL)、勐海(Menghai,MH)和澜沧(Lancang,LC)4个县的草地贪夜蛾种群,利用16S rDNA测序分析其肠道细菌的组成以及结构,对其差异性进行分析,并预测菌群的潜在功能。结果显示,4个草地贪夜蛾种群肠道共获得399个OTU序列,分布于17门25纲41目63科105属149种。在属水平上,草地贪夜蛾肠道优势细菌是苍白杆菌属、埃希氏菌属、乳杆菌属、荚膜菌属、鞘氨醇单胞菌属和肠球菌属,所占比例分别介于43.82%~91.75%、0.55%~21.14%、0.16%~21.52%、0.30%~6.50%、0.20%~4.20%和2.19%~3.64%之间。草地贪夜蛾SM种群和YL种群、LC种群和MH种群肠道菌群的相似性较高。草地贪夜蛾肠道细菌主要参与了物质代谢的过程,且行使代谢功能的肠道细菌在LC种群和MH种群中的相对丰度较高,SM种群参与代谢的相对丰度最低,这可能与当地气候条件有关。  相似文献   

18.
为有效防控由胶孢炭疽菌Colletorichum gloeosporioides引起的辣椒炭疽病,自辣椒上分离得到内生细菌,通过平板拮抗和辣椒离体生防试验筛选对胶孢炭疽菌有抑制作用的拮抗菌株,通过形态学特征、生理生化特征以及分子生物学技术对其进行鉴定,并于室内测定其对胶孢炭疽菌菌丝生长的影响、对辣椒炭疽病的防效及接种后辣椒内抗病活性物质含量以及防御酶活性。结果显示,从辣椒上共分离纯化获得46株细菌,其中菌株SQ-6对胶孢炭疽菌有明显的抑制作用,抑制率为61.11%,显著高于其他45株。结合菌株SQ-6的形态学特征、生理生化特征以及分子生物学特征,将该菌株鉴定为解淀粉芽胞杆菌Bacillus amyloliquefaciens。SQ-6菌株的50%无细胞滤液可引起胶孢炭疽菌菌丝畸形、断裂等,对其抑制率为57.87%。SQ-6菌株的10%、50%发酵液和10%、50%无细胞滤液均能显著降低由胶孢炭疽菌引起的辣椒炭疽病的发病率和病情指数,其中50%无细胞滤液的防效最好。SQ-6菌株能够提高辣椒内Vc、酚类和黄酮类物质含量,诱导辣椒内过氧化物酶(peroxidase,POD)、丙氨酸解氨酶(p...  相似文献   

19.
福建省烟粉虱不同地理种群遗传结构特征   总被引:1,自引:3,他引:1  
为明确福建省烟粉虱种群遗传结构特征,基于福建省烟粉虱不同地理种群中40个代表性的线粒体COI基因序列,分析了种群遗传多样性、遗传分化及分子变异情况,并构建了单倍型系统发育树与网络图。结果显示:在590 bp长度的mt COI基因序列中有效位点558个,其中187个核苷酸位点存在变异;序列核苷酸中A、T、C、G含量分别为42.32%、24.36%、20.25%、13.06%,其中A+T的含量为66.68%,表现出明显的A+T偏向性;共检测出11个单倍型,其中Hap3、4、7、9、11为特殊单倍型;种群多样性指数为0.838,核苷酸多样性指数为0.093,表明遗传多样性水平较高;AMOVA分析表明种群遗传变异主要来自种群内,总种群遗传分化系数仅为0.027,种群遗传分化较低。表明福建烟粉虱种群基因交流未受地理距离明显影响,种群遗传分化不显著。  相似文献   

20.
为探究光肩星天牛Anoplophora glabripennis对传统抗性树种白蜡的适应性机制,利用代谢组学分析不同白蜡品种木质部次生代谢产物,利用16S rRNA测序技术对取食白蜡后的光肩星天牛肠道细菌群落组成进行检测,并采用Pearson方法分析两者的相关性。结果表明,在洋白蜡Fraxinuspennsylvanica和中国白蜡F. chinensis木质部中共筛选出27类抗虫性差异代谢产物,其中黄酮苷、双黄酮及多黄酮、简单酚类、香豆素及其衍生物和木质素苷在中国白蜡中含量较丰富;而2-芳基苯并呋喃类黄酮、阿朴啡类、麦角林及其衍生物和有机氮化合物在洋白蜡中占优。光肩星天牛取食洋白蜡和中国白蜡2种寄主后肠道细菌群落组成存在明显差异,优势菌门分别为变形菌门和厚壁菌门;优势菌属分别为拉乌尔菌属Raoultella(55.79%)和乳球菌属Lactococcus(57.52%);筛选到21个差异菌属,其中13个菌属在中国白蜡中的相对丰度显著高于洋白蜡。相关性分析结果表明,尽管类黄酮、简单酚类、香豆素及其衍生物和木质素苷等酚类化合物在中国白蜡中含量更高,但取食中国白蜡后光肩星天牛肠道内一方面存在优势菌群参与代谢上述物质,如肠球菌属Enterococcus、拉乌尔菌属对类黄酮和木质素苷的代谢;另一方面中国白蜡通过一些特异菌属和共有菌属的增殖参与代谢,如乳杆菌属Lacto-bacillus和放线菌属Actinomycetes对类黄酮、乳球菌属对简单酚类、鞘氨醇杆菌属 Sphingobacterium对香豆素及其衍生物、放线菌属和肠球菌属对木质素苷的代谢。生物碱在中国白蜡中的含量低于洋白蜡,未发现特殊菌属降解该类物质。推测相对于洋白蜡,取食中国白蜡后光肩星天牛肠道菌群更具特异性和多样化,可以帮助光肩星天牛快速代谢相关次生物质,顺利完成其在中国白蜡上的生活史。  相似文献   

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