Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Chromosomal and Ti plasmid characterization of tumorigenic strains of three Agrobacterium species isolated from grapevine tumours

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 .     

Biological control of crown gall of grapevine, rose, and tomato by nonpathogenic Agrobacterium vitis strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent for crown gall of grapevine (Vitis vinifera). When roots of grapevine, rose (Rose multiflora), and tomato (Lycopersicon esculentum) were soaked in a cell suspension of antagonists before planting in soil infested with tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens, respectively, treatment with VAR03-1 significantly reduced the number of plants with tumors and disease severity in the three plant species. The inhibitory effects of treatment with VAR03-1 and the nonpathogenic A. rhizogenes strain K84 on crown gall of rose and tomato were almost identical, and the inhibitory effect of VAR03-1 on grapevine was superior to that of K84. Moreover, VAR03-1 greatly controlled crown gall of grapevine due to tumorigenic A. vitis in the field. VAR03-1 established populations averaging 10(6) colony forming units (CFU)/g of root in the rhizosphere of grapevine and persisted on roots for 2 years. VAR03-1 was bacteriocinogenic, producing a halo of inhibition against those three species of Agrobacterium. This is the first report that a nonpathogenic strain, VAR03-1, can effectively control crown gall caused by tumorigenic A. vitis, A. rhizogenes, and A. tumefaciens.     

Mutations affecting chemotaxis of Agrobacterium vitis strain E26 also alter attachment to grapevine roots and biocontrol of crown gall disease

Crown gall in grapevine, caused by tumorigenic Agrobacterium vitis strains, can cause severe losses in most viticulture regions worldwide. The only effective means of control is through cultivation practices. One non-tumorigenic A. vitis strain, E26, can prevent crown gall infection when applied to wounds on grapevine prior to or simultaneous with inoculation of tumorigenic strains. ME19, a Tn5 mutant of strain E26, was impaired in terms of its ability to be chemo-attracted by grapevine root tissue extracts and its attachment to grapevine roots, and had reduced biological control activity; it did not significantly differ from the wild-type strain of E26 in phenotypes of agrocin production, growth in minimum medium or swarming activity. Complementation of ME19 with the cosmid clone of CP1543 from an E26 DNA library restored the chemotaxis, attachment, and biocontrol phenotypes. A 7·3-kb Kpn I fragment from CP1543 was cloned and sequenced, and sequence analysis revealed that the Tn 5 insertion occurred in a region that shares a significant homology with genes coding for methyl-accepting chemotaxis proteins (MCPs) in many bacteria. Complementation of the mcp gene mutants restored the affected phenotypes to the level of wild-type E26. An in-frame deletion mutant of the mcp gene was generated and was determined to have the same phenotypes as the original Tn5 mutant.     

Molecular Typing and Presence of Genetic Markers Among Strains of Banana Finger-Tip Rot Pathogen, Burkholderia cenocepacia, in Taiwan

ABSTRACT Burkholderia cenocepacia (genomovar III of B. cepacia complex), the causal agent of banana finger-tip rot, is a common plant-associated bacterium but also an important opportunistic pathogen of humans. To better understand the nature of B. cenocepacia from banana, the genetic variation among B. cenocepacia isolates from various banana-growing regions in southern Taiwan was examined. Forty-four serial isolates recovered from diseased banana stigmata from three banana-growing regions during the periods ranging from 2002 to 2004 were investigated. All B. cenocepacia isolates picked from quinate-yeast extract tetracycline-polymyxin semiselective medium could cause onion maceration and were polymerase chain reaction (PCR) positive for bcscV, which is a type III secretion gene present in all members of the B. cepacia complex except B. cepacia (formerly genomovar I). Genetic diversity was assessed using recA PCR restriction fragment length polymorphism, recA nucleotide sequence analysis, and pulsed-field gel electrophoresis assays. The assays revealed the genetic variability among the isolates and also allowed us to trace the relationship among isolates. The isolates all were assigned to genomovar III and consisted of two groups, A and B, which corresponded to recA lineage IIIA and IIIB. The group B strains were separated into B1 and B2 subgroups and the B1 strains were further divided into distinct lineages. The B1 strains were the most frequently detected and occurred in all regions tested. There was no significant difference between strains from each subgroup in the virulence on banana fingers of cv. Cavendish. PCR assays were further used to determine whether B. cenocepacia from banana contained the cable pilus subunit gene (cblA), IS1356, and B. cepacia epidemic strain marker (BCESM), which are DNA markers associated with epidemic B. cepacia clinic strains. The results indicated that cblA and IS1356 were absent but the BCESM was found in all isolates. The present study revealed that banana is a natural reservoir of genetically diversified B. cenocepacia strains.     

Biological control of grapevine crown gall by nonpathogenic <Emphasis Type="Italic">Agrobacterium vitis</Emphasis> strain VAR03-1

A nonpathogenic strain of Agrobacterium vitis VAR03-1 was tested as a biological control agent against crown gall of grapevine (Vitis vinifera L.). A mixture of the nonpathogenic strain VAR03-1 and a tumorigenic strain G-Ag-27 of A. vitis at cell ratios of 1 : 1, 3 : 1, 9 : 1, and 99 : 1 significantly inhibited gall formation and size on stems of tomato (Lycopersicon esculentum Mill.). Strain VAR03-1 also inhibited gall formation on stems of both tomato and grapevine at a 1 : 1 cell ratio with several tumorigenic A. vitis strains isolated from different fields of grapevine in Japan. In biological control tests, when roots of grapevine and tomato seedlings were soaked in a cell suspension of strain VAR03-1 for 24 h before a 1-h soaking in a cell suspension of the pathogen and subsequent planting in pots of infested soil, strain VAR03-1 significantly reduced the incidence of gall formation on both plants.     

Virulence and pCM1 plasmid carriage are related to BOX-PCR fingerprint type in strains of Clavibacter michiganensis subsp. michiganensis that cause bacterial wilt and canker of tomato in Argentina

Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker in tomato, producing important economic losses worldwide. Its virulence has been related to several putative virulence factors present on a chromosomal pathogenicity island and on plasmids pCM1 and pCM2, in strain NCPPB382. We genotypically characterized a collection of Cmm isolates from the main greenhouse tomato-producing areas of Argentina by BOX-PCR fingerprinting and screened for the presence of genes and plasmids involved in pathogenicity by PCR. In addition, we evaluated in vitro cellulolytic activity and virulence in planta of selected strains. BOX-PCR fingerprinting clustered strains into four groups. Group II was dominant and included the most virulent strains, while Group III was the smallest and had the least virulent strains. All local strains exhibited similar cellulolytic activity. Most of the examined strains carry two plasmids of similar size to those of NCPPB382, although there were strains with one or three plasmids. By PCR amplification of repA, pCM1 was detected only in strains belonging to Group III, which includes local strains closely related to reference strain NCPPB382. All analysed pathogenicity genes were widespread among strains, and so in strains belonging to Groups I and II, celA found on pCM1 in NCPPB382 could be found in the chromosome or in plasmids other than pCM1. This study contributes to a better understanding of the diversity of Cmm genetic profiles and virulence of strains present in Argentina. Such information could be useful for the selection of strains for screening of host resistance and development of resistant tomato varieties.     

PCR–RFLP identifies differences in hrpZ sequences to distinguish two genetic groups of Pseudomonas syringae pv. syringae strains from barley and wheat with bacterial black nod

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.     

Application of RFLP analysis of recA, gyrA and rpoS gene fragments for rapid differentiation of Erwinia amylovora from Erwinia strains isolated in Korea and Japan

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.     

Genetic diversity of Xanthomonas arboricola pv. pruni strains in Japan revealed by DNA fingerprinting

Xanthomonas arboricola pv. pruni (Xap) strains collected from peach orchards in six areas in Japan were fingerprinted by inter-simple sequence repeat (ISSR)-polymerase chain reaction (PCR) and repetitive sequence-based (rep)-PCR. Although 148 strains were differentiated into four fingerprint groups (A–D), the genetic diversity among the Xap population was low. The pathogenicity of strains belonging to different fingerprinting groups was very similar. These results show that the causal agent of bacterial spot on peach in Japan is genetically nearly homogeneous.     

中国4省猕猴桃细菌性溃疡病菌的生物型检测和遗传多样性分析

由丁香假单胞菌猕猴桃致病变种Pseudomonas syringae pv. actinidiae (Psa)侵染引起的猕猴桃细菌性溃疡病(kiwifruit bacterial canker)是全球猕猴桃生产上最具毁灭性的细菌病害。为探明福建、安徽、四川和陕西4省Psa菌株的生物型和遗传多样性,用5对PCR特异性引物PsaJ-F/-R、PsaK-F/-R、Tac-F/-R、Con002-F/-R和avrRps4-F1/-R2检测Psa菌株的生物型;用4对PCR引物27F/1492R、PsaF1/PsaR2、gapA-Fps/Rps和rpoD+364s/-1222ps分别扩增16S rRNA、ITS、gapA和rpoD基因,进行多基因联合分析Psa菌株的遗传多样性。结果表明,特异性引物Tac-F/-R从47株Psa菌株中均能扩增出一条545 bp的特异条带,其他4对引物未扩增出任何条带,说明供试Psa菌株的生物型均为biovar 3。多基因联合分析表明,4省Psa存在丰富的遗传多样性,4个群体共检测出27个单倍型,单倍型多样性为0.955。安徽、福建、四川和陕西群体的单倍型数差异较大,分别为1、8、12个和12个。4个群体的多态性位点数、核苷酸多样性和平均核苷酸差异数差异极显著(P<0.01),其中福建群体的多态性最丰富,而安徽群体的多态性最低。AMOVA分析表明,3.6%的遗传变异来源于种群间,而96.4%的遗传变异来源于种群内,说明种群内变异是遗传变异的主要来源。遗传分化分析表明,安徽省Psa群体与其他3个群体间的遗传分化极高(Fst>0.175),福建、四川和陕西群体间的遗传分化水平较低(Fst<0.017)。研究结果有利于了解福建省Psa的来源,为阻断Psa的传播和猕猴桃细菌性溃疡病的长期可持续控制提供了理论参考。     

Phylogenetic study of Japanese Dickeya spp. and development of new rapid identification methods using PCR–RFLP

Forty-one representative Japanese Dickeya spp. (Erwinia chrysanthemi) strains isolated from 24 plants in Japan were investigated using multilocus sequence analysis of recA, dnaX, rpoD, gyrB and 16S rDNA; PCR–RFLP (restriction fragment length polymorphism) of recA, rpoD and gyrB genes; PCR genomic fingerprinting; and biochemical tests. Based on the recA, dnaX, rpoD, gyrB and 16S rDNA sequences and PCR genomic fingerprinting, the strains were essentially divided into six groups (I–VI). Group I corresponded to D. chrysanthemi, group II corresponded to D. dadantii, group III to D. dianthicola and group IV to D. zeae. Meanwhile, group V and group VI could not be assigned to any existing Dickeya species, and they were deduced to be two putative new species. The PCR–RFLP analysis of gyrB, rpoD and recA clearly differentiated the six groups of Dickeya strains. From the results of the biochemical tests, the strains were assigned to biovars 1, 3, 5, 8 and 9; only one strain (SUPP 2525) was not assignable to the existing biovars. We also showed that the PCR–RFLP analysis of rpoD, gyrB and recA can be used as a rapid technique to identify Japanese Dickeya strains.     

Genetic, phenotypic and pathogenic diversity of Xanthomonas arboricola pv. corylina strains question the representative nature of the type strain

A collection of 31 Xanthomonas arboricola pv. corylina strains isolated from Corylus maxima and C. avellana of different countries were assessed by means of repetitive PCR using ERIC, BOX and REP primer sets and analysis of whole-cell protein extracts; pathogenicity tests to three hazelnut ( C. avellana ) cultivars; and some key biochemical tests. From these studies, the X. arboricola pv. corylina strains were clustered into five and three groups by repetitive PCR and protein analysis, respectively, and by using UPGMA cluster analysis, with two strains forming an outlier group to these. The groups showed a high degree of similarity. Strain membership between the groups designed by the two methods exhibited a high degree of congruence, and diversity between the groups was low. Surprisingly, the two strains originating from C. maxima , that include the type strain NCPPB 935, formed the most distinctive group. No relationship to geographic origin of the strains was evident. All strains proved pathogenic towards three different hazelnut cultivars, although the strains obtained from C. maxima did not incite any significant symptoms on buds and twigs. No other relationships between rep-PCR and whole-cell protein groups and pathogenicity were evident. The distinctiveness of the C. maxima strains was supported further by atypical negative gelatin liquefaction test and reduced quinate metabolism results.     

Migration of biological control agent Rhizobium vitis strain ARK-1 in grapevine stems and inhibition of galls caused by tumorigenic strain of R. vitis

Journal of General Plant Pathology - Nonpathogenic strain ARK-1 of Rhizobium vitis has antagonistic activity against a tumorigenic (Ti) strain of R. vitis and works as a biocontrol agent, but the...     

Detection of tumorigenic Agrobacterium strains from infected apple saplings by colony PCR with improved PCR primers

With the colony polymerase chain reaction (PCR), the specificity of newly prepared primer sets VCF2/VCR2, VCF3/VCR3, VCF4/VCR4, and VCF5/VCR5 to Ti or Ri plasmids in Agrobacterium strains were compared to that of the conventional set VCF/VCR. At first, control strains, which consisted of a nonpathogenic strain and phytopathogenic strains carrying Ti or Ri plasmid, were used. VCF3/VCR3 and VCF5/VCR5 were highly specific to all the phytopathogenic strains, whereas the others were not. These two primer sets were superior to VCF/VCR in their specificity with colony PCR to tumorigenic Agrobacterium strains isolated from apple saplings.     

瓜类尖孢镰刀菌的营养体亲和群研究

 应用营养亲和性方法研究了尖孢镰刀菌菌株抗氯酸盐突变体和nit突变体的诱发规律及分布特性,以及菌株营养体亲和群(VCG)的划分。研究表明,不同寄主(黄瓜、甜瓜和西瓜)分离的尖孢镰刀菌菌株形成的抗氯酸盐突变体数目差异不显著,平均为每个接种点产生0.89~0.98个;但寄主不同部位(根部、茎基部和茎中部)分离的菌株间差异显著,形成的数目分别为1.27、0.75及0.76个。菌株产生的nit1突变体比例(75.40%)显著高于nitM突变体比例(13.17%);nit1突变体数目会因菌株的寄主及菌株寄主部位的不同而有差异,寄主为黄瓜、甜瓜和西瓜的菌株产生的比例依次为67.73%、83.71%和77.50%,根部、茎基部及茎中部分离菌株产生的比例依次为81.82%、78.48%和68.64%,而在致病菌株与非致病菌株间无显著差异,分别为74.43%和79.63%;nitM突变体数目受菌株寄主影响较小,所占比例在11.17%~13.92%之间;而在寄主不同部位分离的菌株及致病菌株与非致病菌株间差异显著,分离自茎基部的菌株所占比例最高为15.97%,茎中部菌株所占比例最低为9.87%,致病菌株与非致病菌株所占比例分别为14.08%和9.26%。供试菌株分为7个VCGs,其特点为来源于不同寄主的尖孢镰刀菌菌株互不亲和,同一寄主的致病菌株与非致病菌株均不亲和,同一寄主不同部位分离的菌株可亲和。     

我国部分地区土壤杆菌的数值分类及DNA同源性分析

 本研究对我国42株土壤杆菌待测菌株和28株参比菌进行了数值分类和DNA同源性分析,结果将41株土壤杆菌与2株根瘤菌分在3个土壤杆菌种内,其中23株来自内蒙等地葡萄的菌株分在葡萄土壤杆菌种内,10株来自山东等地樱桃、桃树的菌株为发根土壤杆菌,8株来自4个地区4种植物的菌株及分自宁夏刺果甘草的2株根瘤菌菌株参比菌分在根癌土壤杆菌种内。1株土壤杆菌和1株根瘤菌的分类地位有待进一步研究。     

Phylogenetic Analysis of 16S rRNA Genes and PCR Analysis of the nec1 Gene from Streptomyces spp. Causing Common Scab, Pitted Scab, and Netted Scab in Finland

ABSTRACT The sequences of the 16S rRNA genes (nucleotides 29 to 1,521) from various Streptomyces strains pathogenic to potato were compared. These included 10 pathogenic Streptomyces strains isolated from potato scab lesions in Finland, the type strains of S. aureofaciens NRRL 2209(T) and S. lydicus ATCC 25470(T), 'S. griseus subsp. scabies' ATCC 10246, and two S. griseus strains that were originally deposited to the collection as pathogens. The nucleotide sequence (>94.5% sequence identity [SI]) and length (1,469 to 1,481 nucleotides) of the analyzed region varied. Phylogenetic analysis of 16S rRNA genes placed Finnish strains into three species, supported by previously characterized morphological and physiological traits. Six Finnish strains, including two strains that deviated from the others in one trait (no spiral sporophores or D-xylose utilization), had identical 16S rRNA genes and were identified as S. scabies (99.9% SI to S. scabies ATCC 49173). Three Finnish strains were identified as S. turgidiscabies, a species previously described only in Japan (99.9% SI to S. turgidiscabies ATCC 700248). Finnish strain 317 and S. aureofaciens NRRL 2209 (99.8% SI) were placed in a distinct phylogenetic cluster together with Kitosatospora spp., which suggests that S. aureofaciens may belong to the recently revived genus Kitosatospora. In pathogenicity tests, S. scabies caused characteristic symptoms of common scab, S. turgidiscabies caused mainly pitted scab, and S. aureofaciens caused netted scab and necrotic lesions on stolons of potato cultivars Bintje and Matilda in the greenhouse. The nec1 gene and the intergenic region between nec1 and the 5' transposase pseudogene ORFtnp were successfully amplified by polymerase chain reaction from S. scabies ATCC 49173 and the pathogenic Finnish strains of S. scabies, but not from a nonpathogenic strain of S. scabies, three pathogenic and two nonpathogenic strains of S. turgidiscabies, and S. aureofaciens.