Detection of Fiber-Digesting Bacteria in the Ceca of Ostrich Using Specific Primer Sets

The purpose of this study was to detect three fibrolytic bacteria, Fibrobacter succinogenes, Ruminococcus flavefaciens, and Ruminococcus albus, in the cecal digesta of the ostrich (Struthio camelus) by PCR using a species-specific primer set for each 16S ribosomal RNA gene (16S rDNA). Although amplified DNA fragments obtained from each primer set had the expected size, the clone library derived from the amplimer contained non-specific sequences. The F. succinogenes-specific primer set recovered a partial 16S rDNA sequence of an uncultivated Fibrobacter with low similarity (<95%) and distantly related phylogenetic positioning to Fibrobacter sequences deposited in the databases, indicating a novel species of Fibrobacter. The sequence was considered to be identical to a clone detected in our previous experiment. Thus, we confirm that the gastrointestinal tract of the ostrich is one of the habitats of Fibrobacter species. The clone library derived from the R. flavefaciens-specific primer set contained a 16S rDNA sequence with 97% similarity to R. flavefaciens, indicating it could be one of a major fibrolytic bacterium in the ostrich ceca. No R. albus 16S rDNA sequence was found in the clone library of the R. albus-specific primer set.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

A sex determination protocol for the Iberian desman (<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>) based on a three primer amplification of <Emphasis Type="Italic">DBX</Emphasis> and <Emphasis Type="Italic">DBY</Emphasis> fragments with non-invasive samples

We have sequenced partial fragments of DBX and DBY genes of the endangered Iberian desman (Galemys pyrenaicus). The sequences were used to design a sex determination protocol for non-invasive samples based on a PCR reaction, using only three primers. This protocol allows the simultaneous amplification of two fragments, one corresponding to the DBX gene and the other to the DBY gene, both differing in size. To increase sensitivity on the detection of positive amplifications and on the determination of fragment size we use a fluorescently labelled primer. The protocol has been tested in DNA samples from hair and stool, revealing major difficulties in sexing faecal samples, but unambiguous sexing of hair samples.     

白蜡虫体内杀雄菌属(Arsenophonus)共生菌的分子检测

[目的]研究白蜡虫体内杀雄菌属(Arsenophonus)共生菌的含量与白蜡虫性比之间的关系.[方法]采用16S rDNA文库的方法对白蜡虫雄虫体内的共生菌进行分析,利用杀雄菌属特异的2条16S rDNA引物以及23S rDNA引物进行PCR检测.对昭通、昆明、金口河、杭州、长春、江华6个不同地理种群白蜡虫体内的杀雄菌属共生菌进行半定量分析,并采用荧光定量PCR对昭通、昆明、金口河白蜡虫体内的杀雄菌属共生菌进行绝对定量分析.[结果]在白蜡虫体内首次发现杀雄菌属共生菌.在白蜡虫雌雄虫体内均扩增出杀雄菌属的两条不同长度的16S rDNA序列,分别为445bp和1462bp,并扩增得到长度为582bp的23S rDNA序列.杭州和江华地理种群白蜡虫的一些个体不含杀雄菌属共生菌.昭通地理群的杀雄菌属共生菌含量显著高于金口河和昆明,而昆明和金口河白蜡虫的杀雄菌属含量无显著差异.[结论]白蜡虫体内杀雄菌属共生菌的含量与白蜡虫性比无关.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

A new primer set for sex identification in the genus Sorex (Soricidae,Insectivora)

In order to develop a new accurate method for sexing in Sorex species (Soricidae, Insectivora), we synthesized a polymerase chain reaction (PCR) primer set to amplify a part of Sry HMG box in the long‐clawed shrew, Sorex unguiculatus. When the primers were applied to the samples of known sex, PCR products were successfully obtained for males as a clear, single band on 3% agarose gels after electrophoresis in Sorex unguiculatus and five other Sorex species, but not for females of these six species. Thus, PCR amplification using the primer set may be applicable to discern sex in the six Sorex species.     

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.     

CHARACTERIZATION OF GENETIC MARKERS LINKED TO SEX DETERMINATION IN THE HAPLOID‐DIPLOID RED ALGA GRACILARIA CHILENSIS1

Bulk segregant analysis, random amplified polymorphic DNA (RAPD), and sequence characterized amplified region (SCAR) methods were used to identify sex‐linked molecular markers in the haploid‐diploid rhodophyte Gracilaria chilensis C. J. Bird, McLachlan et E. C. Oliveira. One hundred and eighty 10 bp primers were tested on three bulks of DNA: haploid males, haploid females, and diploid tetrasporophytes. Three RAPD primers (OPD15, OPG16, and OPN20) produced male‐specific bands; and one RAPD primer (OPD12), a female‐specific band. The sequences of the cloned putative sex‐specific PCR fragments were used to design specific primers for the female marker SCAR‐D12‐386 and the male marker SCAR‐G16‐486. Both SCAR markers gave unequivocal band patterns that allowed sex and phase to be determined in G. chilensis. Thus, all the females presented only the female band, and all the males only the male band, while all the tetrasporophytes amplified both male and female bands. Despite this sex‐specific association, we were able to amplify SCAR‐D12‐386 and SCAR‐G16‐486 in both sexes at low melting temperature. The differences between male and female sequences were of 8%–9% nucleotide divergence for SCAR‐D12‐386 and SCAR‐G16‐486, respectively. SCAR‐D12‐386 and SCAR‐G16‐486 could represent degenerated or diverged sequences located in the nonrecombining region of incipient sex chromosomes or heteromorphic sex chromosomes with sequence differences at the DNA level such that PCR primers amplify only one allele and not the other in highly specific PCR conditions. Seven gametic progenies composed of 19 males, 19 females, and the seven parental tetrasporophytes were analyzed. In all of them, the two SCAR markers segregated perfectly with sexual phenotypes.     

Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran

Evaluation of phytoplasmas infection was conducted in the pistachio-growing areas of Iran (Rafsanjan in Kerman province) in early autumn of 2011. A total of 30 pistachio trees collected from a pistachio orchard in Rafsanjan showing Psylla damage symptoms and 10 samples with different abnormal symptoms from miscellaneous orchards were tested for the presence of phytoplasma. By using nested PCR with primer pairs P1/P7 and internal primer sets R16F2N/R16R2 and fU3-rU5, amplified fragment of expected size was observed in some trees with deformation and yellowing symptoms. On the basis of nucleotide sequence analysis of 16S?rDNA amplified by PCR, this phytoplasma was classified in group 16S?rII. In addition, we observed false positive reaction in three trees of Ahmadaghaei cultivar by using primer sets R16F2N/R16R2 and sequence analysis of ~1250bp PCR product indicated that amplified fragment was related to Schinus terebinthifolius; a species of flowering plant in the Anacardiaceae family.     

Detection of Phytoplasma from deferent infected hosts in Jordan based on PCR amplification of the 16S rDNA sequences

Sixteen leaf samples, both healthy and Phytoplasma diseased, were collected from different plants such as grape, peach, almond, tomato, paper, squash, apple and pear in northern Jordan. Extracted DNA from diseased grape, peach, almond, tomato, paper and squash plus from infected periwinkle (Catharanthus roseus) samples were amplified with the Phytoplasma universal 16S rDNA sequences primer pairs. Extracted DNA samples from healthy and diseased apple and pear plants were not amplified with the same primer pairs. All the PCR-amplified DNA samples show a common band with size of 558 bp, indicating Phytoplasma pathogens as a disease-causative agent for grape, peach, almond, tomato, paper and squash plants. The restriction fragment length polymorphisms of Alu1 enzyme for the amplified 16S rDNA sequences shows the same DNA fragment patterns indicating no or a limited diversity among the DNA of the detected Phytoplasma pathogens.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

A novel method of caenophidian snake sex identification using molecular markers based on two gametologous genes

Sex identification provides important information for ecological and evolutionary studies, as well as benefiting snake conservation management. Traditional methods such as cloacal probing or cloacal popping are counterproductive for sex identification concerning very small species, resulting in difficulties in the management of their breeding programs. In this study, the nucleotide sequences of gametologous genes (CTNNB1 and WAC genes) were used for the development of molecular sexing markers in caenophidian snakes. Two candidate markers were developed with the two primer sets, and successfully amplified by a single band on the agarose gel in male (ZZ) and two bands, differing in fragment sizes, in female (ZW) of 16 caenophidian snakes for CTNNB1 and 12 caenophidian snakes for WAC. Another candidate marker was developed with the primer set to amplify the specific sequence for CTNNB1W homolog, and the PCR products were successfully obtained in a female‐specific 250‐bp DNA bands. The three candidate PCR sexing markers provide a simple sex identification method based on the amplification of gametologous genes, and they can be used to facilitate effective caenophidian snake conservation and management programs.     

Use of 16S rDNA Sequences as Signature Characters to Identify Xylella fastidiosa

The nucleotide sequences of 16S rDNAs (coding for the small subunit ribosomal RNAs) were used to identify Xylella fastidiosa, a nutritionally fastidious plant pathogenic bacterium. The near-complete 16S rDNAs from nine strains of Xyl. fastidiosa, including seven pathotypes and one strain of Xanthomonas campestris pv. campestris, were amplified through PCR with two conserved primers (forward primer 5′-AGA GTT TGA TCC TGG CTC AG-3′ and reverse primer 5′-AAG GAG GTG ATC CAG CC-3′) and sequenced. The 16S sequences were compared with all eukaryote and prokaryote DNA entries in GenBank database. A Xyl. fastidiosa 16S rDNA sequence, M26601, was determined to be the most similar to all the near-complete (1537 bp) and partial 5′ end sequences from Xyl. fastidiosa, but not those from the Xanthomonas strain. A 20-bp oligonucleotide (5′-TTG GTA GTA ATA CCA TGG GT-3′) was found to be highly characteristic of Xyl. fastidiosa. Since the 16S rDNA of Xyl. fastidiosa strains are highly homologous and characteristically different from other bacteria, including the most closely related Xanthomonas, 16S rDNA sequences can be used as signature characters to identify this bacterium. Received: 22 June 1999 / Accepted: 2 August 1999     

First report on molecular detection of a stolbur phytoplasma (Group16SrXII-A) associated with kenaf (Hibiscus cannabinus L.) in India

Infection of stolbur phytoplasma was detected in kenaf (Hibiscus cannabinus) plants at CRIJAF research farm, Barrackpore, India. The infected plants formed profuse short branches at the top with bushy and bunchy top appearance. PCR with universal 16S rDNA phytoplasma primers P1/P7 yielded amplicons of 1.5 kb from all symptomatic leaf samples. Nested PCR with 16S-rDNA-specific nested primer pair R16F2n/R2 generated an amplicon of 1241 bp confirming the presence of a phytoplasma. The nested PCR products were sequenced and BALSTn analysis revealed 100% identity with 16S rRNA gene of phytoplasma. Phylogenetic analysis showed kenaf phytoplasma having 99% identity with both “Bois noir” stolbur phytoplasma 16SrXII group (Accession no: JQ181540). The RFLP data also supported the phylogenetic analysis. Multi-locus sequence characterisation assay was conducted by using different locus-specific primers viz. tuf, rpsC-rplV, rplF-rplR, map-SecY and uvrB-degV. The infected phytoplasma samples amplified only SecY gene and generated 1224 bp product which was deposited at NCBI (accession no: KC508636).     

Systematic Design of 18S rRNA Gene Primers for Determining Eukaryotic Diversity in Microbial Consortia

High-throughput sequencing of ribosomal RNA gene (rDNA) amplicons has opened up the door to large-scale comparative studies of microbial community structures. The short reads currently produced by massively parallel sequencing technologies make the choice of sequencing region crucial for accurate phylogenetic assignments. While for 16S rDNA, relevant regions have been well described, no truly systematic design of 18S rDNA primers aimed at resolving eukaryotic diversity has yet been reported. Here we used 31,862 18S rDNA sequences to design a set of broad-taxonomic range degenerate PCR primers. We simulated the phylogenetic information that each candidate primer pair would retrieve using paired- or single-end reads of various lengths, representing different sequencing technologies. Primer pairs targeting the V4 region performed best, allowing discrimination with paired-end reads as short as 150 bp (with 75% accuracy at genus level). The conditions for PCR amplification were optimised for one of these primer pairs and this was used to amplify 18S rDNA sequences from isolates as well as from a range of environmental samples which were then Illumina sequenced and analysed, revealing good concordance between expected and observed results. In summary, the reported primer sets will allow minimally biased assessment of eukaryotic diversity in different microbial ecosystems.     

Isolation of Female-Specific AFLP Markers and Molecular Identification of Genetic Sex in Half-Smooth Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

The sex-specific molecular marker is a useful gene resource for studying sex- determining mechanisms and controlling fish sex. Artificially produced male and female half-smooth tongue sole (Cynoglossus semilaevis) were used to screen sex-specific amplified fragment length polymorphism (AFLPs) molecular markers. The phenotypic sex of 28 tongue soles was determined by histological sectioning of gonads. The AFLP analysis of 15 females and 13 males via 64 primer combinations produced a total of 4681 scorable bands, of which 42.11% and 43.39% of bands were polymorphic in females and males, respectively. Seven female-specific AFLP markers were identified and designated as CseF382, CseF575, CseF783, CseF464, CseF136, CseF618, and CseF305, respectively. One female-specific AFLP marker (CseF382) was amplified, recovered from the gels, cloned, and sequenced (accession no. DQ487760). This female-specific AFLP marker was converted into a single-locus polymerase-chain reaction (PCR) marker of a sequence-characterized amplified region (SCAR). A simple PCR method of using the specific primers was developed for identifying genetic sex of half-smooth tongue sole. PCR products demonstrated that the initial 15 females produced the female-specific band of about 350 bp, but the initial 13 male individuals failed to produce the band. We also investigated the applicability of the PCR primers in other tongue sole individuals. The same female-specific fragment of about 350 bp was found in the additional 59 female individuals, but not in the additional 58 male individuals. This AFLP-based molecular sexing technique may have great application potential in elucidation of sex determination mechanisms and sex control in half-smooth tongue sole.     

A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Detection and Molecular Characterization of a 16SrII‐A* Phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing‐like Symptoms in China

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)‐infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for ‘Candidatus Liberibacter asiaticus’ and 5 for both phytoplasma and ‘Ca. L. asiaticus’. Sequence analysis indicated that seven 881‐bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNA^Ile gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII‐A*) of phytoplasma subgroup 16SrII‐A. As phytoplasmas were only detected in blotchy‐mottle leaves, the 16SrII‐A* phytoplasma identified was related to HLB‐like symptoms.