Determination of Ralstonia (Pseudomonas) solanacearum rDNA subgroups by PCR tests

Rapid and sensitive polymerase chain reaction (PCR) methods are described for determination of the two 16 S rDNA subgroups of Ralstonia solanacearum, the causal agent of bacterial wilt. A third subgroup consisting of Indonesian R. solanacearum isolates belonging to Division II, the blood disease bacterium and Pseudomonas syzygii can also be identified. Primers were designed to sequences within R. solanacearum 16 S rDNA (equivalent to Escherichia coli 16 S rDNA positions 74–97, 455–475, 1454–1474), and the internal transcribed spacer region between the 16 S and 23 S rDNA genes. Different combinations of forward and reverse primers allowed selective PCR amplification of (a) R. solanacearum Division I (biovars 3, 4 and 5), (b) Division II (biovars 1, N2, and 2) including the blood disease bacterium and P. syzygii , or (c) amplification of Division II only except for five biovar 1, 2 or N2 isolates of R. solanacearum from Indonesia, P. syzygii and the BDB. A total of 104 R. solanacearum , 14 blood disease bacterium and 10 P. syzygii isolates were tested. Simultaneous detection of species and subdivision was achieved by designing a multiplex PCR test in which a 288-base pair (bp) band is produced by all R. solanacearum isolates, and an additional 409-bp band in Division I strains.     

芝麻细菌性青枯病病原菌及其生化变种鉴定

芝麻细菌性青枯病是我国南方芝麻生产上的重要病害。调查研究表明,芝麻青枯病除前人报道的萎蔫、顶梢常有溃疡裂缝等典型症状外,还有植株畸形、茎秆表皮泡状突起、溃疡裂缝延伸至茎秆中下部、折断茎秆可见菌脓形成的透明细丝等症状。分离获得的病原菌菌株经菌体形态、菌落形态、培养性状、致病性、烟草过敏性反应及16S rRNA基因和16S～23S rRNA基因间区ITS序列测定,证实了芝麻青枯病是由青枯菌Ralstonia solanacearum引起。江西省15个县(市)的22个代表性菌株的生化变种鉴定显示,20株菌属于生化变种Ⅲ,占90.91%,2株属于生化变种Ⅳ,占9.09%。说明生化变种Ⅲ菌群是诱发芝麻青枯病流行的优势种群。     

12种寄主来源的茄科雷尔氏菌16S-23SrDNA间隔区序列比较

应用PCR方法,获得了分离自广东番茄、茄子、辣椒、烟草、空心菜、沙姜、姜、马铃薯、花生、菊花、桑树和藿香等12种作物21个茄科雷尔氏菌菌株的16S 23S rDNA 间隔区序列（ITS）。序列分析结果表明,除HZ 1菌株外,其余20个茄科雷尔氏菌菌株ITS序列长均为503 bp,序列间相似性99.2%~100%,序列间差异仅1~4 bp;而HZ 1菌株的ITS序列长为498 bp,与其他菌株的ITS序列相似性为95.4%~95.6%。这些结果说明,这21株来源于12种不同寄主的茄科雷尔氏菌菌株的16S 23S rDNA ITS序列比较保守。系统进化分析显示,仅菌株HZ 1聚类于茄科雷尔氏菌区组2中,其余20个菌株均聚类于茄科雷尔氏菌区组1中。     

双重PCR检测马铃薯晚疫病菌和青枯病菌方法的建立及应用

 利用真菌通用引物ITS1和ITS4扩增马铃薯晚疫病菌转录间隔区并进行序列测定,通过序列比较,设计了1对马铃薯晚疫病菌的特异引物INF1/INF2,并对15种不同真菌、细菌和7种疫霉属和腐霉属卵菌基因组DNA进行PCR扩增,结果只有不同来源的马铃薯晚疫病菌株可获得324 bp的特异带。将引物INF1/INF2与卵菌通用引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达30 fg。运用设计的引物与马铃薯青枯病菌特异引物结合建立了双重PCR体系,能从马铃薯晚疫病菌和马铃薯青枯病菌总基因组DNA以及人工接种和自然发病的马铃薯植株中分别或同时扩增到324 bp和281 bp的特异片段。实现了同时对马铃薯晚疫病菌和马铃薯青枯病菌的快速可靠检测。     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

Isolation and characterization of <Emphasis Type="Italic">Serratia rubidaea</Emphasis> from dark brown spots of tomato fruits

Bacterial contamination of fresh tomato fruits is of great concern. From naturally infected tomato fruits showing dark brown irregularly shaped spots, 36 bacterial isolates were recovered and identified on phenotypic characteristics and sequences of the gene encoding the 16S rRNA. Five isolates showing spots on tomato fruits in the pathogenicity test with healthy tomato fruits belong to the genus Serratia on the basis of phenotypic characteristics. One representative isolate of these has been further identified as a Serratia rubidaea by sequencing of the 16S rRNA gene. This is the first evidence showing that a S. rubidaea strain can cause spots on tomato fruits. Virulence of the S. rubidaea was also confirmed by the production and secretion of a large variety of enzymes capable of degrading the complex polysaccharides of the plant cell wall and membrane constituents. Nineteen bacterial isolates of the 36 did not induce any spot symptoms in a pathogenicity test on artificially infected tomato fruits although these are known as phytopathogenic bacteria. Five of these 19 bacterial isolates were identified as Ralstonia species on the basis of biochemical tests. Sequencing of the 16S ribosomal gene of one representative isolate revealed that the isolate is closely related to Ralstonia solanacearum. Six isolates of the 19 were related to Xanthomonas vesicatoria on the basis of biochemical tests and eight were related to the Enterobacteriaceae. One representative isolate of the Enterobacteriaceae could be identified by the 16S rRNA gene as Enterobacter cloacae subsp. dissolvens. The 12 other strains were related to Proteus mirabilis based on the 16S RNA gene sequence of one representative isolate. The isolates related to P. mirabilis did not produce any symptoms on artificially infected tomato fruits. The nucleotide sequences of S. rubidaea strain E9, E. cloacae strain E23, P. mirabilis strain E11, and R. solanacearum strain E15 have been deposited in the GenBank nucleotide sequence database under accession numbers HM585373 to HM585376.     

Detection and identification of the maize bushy stunt phytoplasma in corn plants in Brazil using PCR and RFLP

Plants of corn (Zea mays L.) exhibiting symptoms of stunting and leaf reddening were assayed for the presence of phytoplasma gene sequences through the use of phytoplasma rRNA and ribosomal protein gene and maize bushy stunt (MBS) phytoplasma-specific oligonucleotide primers in polymerase chain reactions (PCR). Polymorphisms in 16S rDNA amplified from diseased plants were those characteristic of phytoplasmas classified in the16S rRNA gene group 16SrI, subgroup IB, of which MBS phytoplasma is a member. Amplification of ribosomal protein (rp) gene sequences in PCR primed by phytoplasma-specific primers confirmed presence of a phytoplasma in the diseased plants. Restriction fragment length polymorphism (RFLP) patterns of the amplified phytoplasma rp gene sequences were similar or identical to those observed for a known strain of MBS phytoplasma. In separate PCR, an MBS-specific oligonucleotide pair primed amplification of a MBS-characteristic DNA from templates derived from the diseased corn. Our data provide the first firm evidence for the presence of maize bushy stunt phytoplasma in corn in Brazil.     

Identification and characterization of Agrobacterium spp. isolated from apricot in Serbia

Crown gall, caused by tumorigenic strains of Agrobacterium spp., is considered one of the most important diseases in stone fruit nurseries throughout the world. Since the crown gall disease has not been studied extensively in Serbia for more than 30 years, the objective of this study was to isolate, identify and characterize the bacterium associated with crown gall symptoms on one-year-old apricot trees. Samples were collected from a nursery in central Serbia and subjected to laboratory analysis. Bacteria were isolated from tumour tissue on yeast mannitol agar (YMA) and six gram-negative isolates were selected for further study. PCR assay using primers specific for virD2, virC, ipt and tms2 pathogenicity-associated plasmid genes revealed that isolates harbour Ti plasmids. All studied strains carrying Ti plasmids were classified as nopaline-type based on further molecular analysis. Using a multiplex PCR assay, targeting 23S rRNA gene sequences, and physiological and biochemical tests, five strains were assigned as A. rhizogenes/biovar 2 and the remaining one as A. tumefaciens/biovar 1. Identity of the strains was confirmed by sequence analysis of the 16S rRNA gene. In pathogenicity assay, all six strains caused tumour formation on inoculated carrot root discs, young tomato and sunflower plants.     

福建及贵州等地烟草青枯菌系统发育分析

[目的]探寻烟草上青枯菌的系统发育.[方法]采用演化型分类框架对福建及贵州等地的62个烟草青枯病菌株进行鉴定分析.[结果]基于内切葡聚糖酶基因系统发育学的分析结果表明:所有参试菌株均归属于青枯菌亚洲分支的4个序列变种,分别为序列变种15、17、34和44;尚未发现归属于美洲或非洲分支的烟草青枯病菌株.其中序列变种15和17为优势菌系,序列变种34的菌株都来自福建省,只发现3个菌株属于序列变种44.基于avrA基因的氨基酸序列比对结果表明4个序列变种的avrA基因都属于RS1000类型.[结论]本研究表明福建及贵州等地烟草上的青枯菌存在一定的遗传分化.     

Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

Use of terminal restriction fragment length polymorphism (T-RFLP) for identification of phytoplasmas in plants

A terminal restriction fragment analysis (T-RFLP) technique was developed for the simple and rapid detection and diagnosis of phytoplasmas in plants. The selected primers amplified part of the 23S rRNA gene to provide improved resolution between the taxonomic groups compared to conventional restriction enzyme analysis of the 16S rRNA. Using the restriction enzymes Bsh 12361 and Mse I on the PCR products, and fragment analysis in the range 68–640 bp, the technique was tested on 37 isolates from 10 of the 16Sr groups. Distinct and unambiguous T-RFLP profiles were produced for nine of the 10 taxonomic groups, such that almost all isolates within a group shared the same profile and could be distinguished from isolates in other groups. The technique also identified the presence of mixtures of phytoplasmas from different groups in samples. Furthermore, the primers were devised to amplify a terminal restriction fragment (TRF) product of a specific defined size (461 bp) from the host plant chloroplast DNA, so that there was a built-in internal control in the procedure to show that the absence of a phytoplasma peak in a sample was the result of no detectable phytoplasma being present, not the result of PCR inhibition. This method offers the possibility of simultaneously detecting and providing a taxonomic grouping for phytoplasmas in test samples using a single PCR reaction.     

Partial characterization of phytoplasmas associated with lettuce and wild lettuce phyllodies in Iran

Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.     

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.     

Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species

Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.     

Occurrence of black rot of cultivated mushrooms (Flammulina velutipes) caused by Pseudomonas tolaasii in Korea

From black spots on winter mushroom (Flammulina velutipes), fluorescent bacteria were repeatedly isolated during surveys at places of production in the years 2009–2010 in Korea. From these lesions three bacterial strains (designated CHM13, CHM16, CHM17) were isolated which, following inoculation of mushroom stipes and caps, yielded characteristic black spots and sunken lesions, which developed into a severe black rot. Results of Gram stain and biochemical tests preliminarily identified these isolates as Pseudomonas tolaasii. This was confirmed by pathogenicity to winter mushroom, physiological and biochemical properties, analysis of the 16S rRNA and rpoB gene sequences, fatty acids profile, specific and sensitive PCR assays and, lipopeptide detection. This is the first report of the isolation of Pseudomonas tolaasii from cultivated winter mushroom in Korea.     

云木香内生细菌的分离鉴定及其对大丽轮枝菌的抑制作用

从药用植物云木香中分离得到41株内生细菌, 采用细菌16S rRNA基因测序的方法对这些菌株进行鉴定, 结果显示, 这些云木香内生细菌分别属于6个属, 其中假单胞菌属Pseudomonas和拉恩氏菌属Rahnella为优势菌属。采用对峙培养方法对这些菌株进行筛选, 发现其中12株内生细菌对大丽轮枝菌Verticillium dahliae具有抑制作用, 其中有2株属于拉恩氏菌属Rahnella, 7株属于假单胞菌属Pseudomonas, 3株属于沙雷氏菌属Serratia。12株内生细菌对棉花黄萎病的温室防效评估结果表明, 假单胞菌属菌株R1-6、R1-13和S1-2对棉花黄萎病的温室防效达到55%以上, 具有潜在的生产利用价值。     

来源于湖北省枣疯病植原体分离物16S rDNA和rp基因的序列分析

以田间采集的来源于我国湖北省枣树产业主产区随州市随县种植的表现为"枣疯病"症状的枣树分离株为试材,对其16S rDNA和核糖体蛋白(ribosomal protein,rp)基因采用Nested-PCR进行扩增以及序列分析。结果表明,湖北JWB-Hubei植原体分离物16S rDNA基因的核苷酸序列与我国山东、河南等地的分离株一致率均为99%以上,在进化树中位于同一亚组的不同进化分支;虚拟RFLP图谱分析表明,JWB-Hubei属于16SrV-B亚组一个成员,与其进化树分组结果一致。JWBHubei分离株rp基因的核苷酸序列也与我国山东、陕西等地区的分离株一致率均为99%以上,在进化树中聚为同一亚组,与报道的基于RFLP分类属于rpV-C亚组的中国枣疯病分离物(JWB)聚集于同一亚组不同分支。该研究结果明确了湖北省枣疯病植原体的分类地位以及与来源于我国不同地区枣疯病分离株之间的遗传进化关系,为进一步研究植原体的株系划分、基因遗传变异研究提供了理论基础。     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

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.     

Molecular identification of a phytoplasma associated with Russian olive witches’ broom in Iran

Russian olive trees (Elaeagnus angustifolia) showing witches’ broom symptoms typical of phytoplasma infection were observed in the Urmia region of Iran. A phytoplasma named Russian olive witches’ broom phytoplasma (ROWBp-U) was detected from all symptomatic samples by amplification of the 16S rRNA gene and 16S/23S rDNA spacer region using the polymerase chain reaction (PCR) which gave a product of expected length. DNA from symptomless plants used as a negative control yielded no product. The sequence of the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U showed 99% similarity with the homologous genes of members of the aster yellows group. We also detected a phytoplasma in neighboring alfalfa plants (AlWBp-U) showing severe witches’ broom symptoms. An 1107 bp PCR product from the 16S rRNA gene showed 99% homology with the corresponding product in ROWBp-U, suggesting the presence of the same phytoplasma actively vectored in the area. Further observations showed that Russian olive trees with typical ROWB symptoms were present in an orchard near Tehran which is located over 530 km south-east of the original Urmia site. The corresponding sequence of this phytoplasma (ROWBp-T) showed 99% homology to that of the ROWBp-U. A sequence homology study based on the 16S rRNA gene and 16S/23S rDNA spacer region of ROWBp-U and other phytoplasmas showed that ROWBp-U is most closely related to the 16SrI group. To our knowledge, this is the first report of a phytoplasma infection in a member of the Elaeagnaceae.