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4474

Summary Knowledge concerning microbial infectious diseases in the current amphibian crisis is rudimentary and largely limited to ranavirosis and chytridiomycosis. The family Chlamydiaceae is gaining attention as a common cause of disease in amphibians and may harbour new and emerging amphibian pathogens. We identified a novel species of Chlamydiales (Candidatus Amphibiichlamydia ranarum ) with a prevalence of 71% in exotic invasive bullfrog tadpoles ( Lithobates catesbeianus ) from an introduced population in the Netherlands. The sequence of a 1474 bp 16S rRNA gene fragment showed that the novel taxon forms a well‐defined clade with ‘ Candidatus Amphibiichlamydia salamandrae’ within the Chlamydiaceae family. Although none of the tadpoles examined showed signs of clinical disease, urgent evaluation of its pathogenic potential for native amphibian species is required.

Two strains (JC85T and JC108) of Gram-stain-negative, motile bacteria were isolated from endolithic beach sand samples on an oligotrophic medium. Based on the 16S rRNA gene sequence analysis, both strains were identified as belonging to the genus Rhizobium . Strain JC108 had 16S rRNA gene sequence similarity of 100 % with Rhizobium pusense NRCPB10T and formed a cluster with this strain. Strain JC85T had 96.9 % 16S rRNA gene sequence similarity and was 18 % related (based on DNA–DNA hybridization) to Rhizobium borbori DN316T. With other strains of the genus Rhizobium , the 16S rRNA gene sequence similarity was less than 96.3 %. Strain JC85T could tolerate up to 3 % salinity, fix N2, was resistant to ampicillin (10 µg) and was positive for catalase and oxidase. The major fatty acid was C18 : 1ω7c (69 %) with minor amounts of C19 : 0 cyclo ω8c (8.9 %), C16 : 0 (6.9 %), C12 : 0 (5.7 %) and C19 : 1ω7c/C19 : 1ω6c (2.2 %). Polar lipids of strain JC85T include two unidentified aminophospholipids (APL1,2), two unidentified phospholipids (PL1,2), phosphatidylcholine and four unidentified lipids (L1–4). Q-10 is the major quinone of strain JC85T. Based on polyphasic taxonomic analysis, strain JC85T represents a novel species for which, the name Rhizobium subbaraonis JC85T is proposed. The type strain is JC85T ( = DSM 24765T = KCTC 23614T).

While performing a routine field survey on 2-year-old canes of Rubus fruticosus (cv. Evergreen Thornless) in the region of Plovdiv (central southern Bulgaria), severe stunting of single or grouped plants (3 to 4 in a row) was found in late August of 2009. It was noteworthy that the leaves of these plants were curved upwards and stayed green until the end of the season. The bushy aspect of the diseased plants led to the assumption of a phytoplasma origin; therefore, specific PCR and sequence based identification methods were applied on leaves, petioles, and stems from three infected Rubus plants grown in different rows of the field (midsummer, nine samples in total) and the same number of asymptomatic samples. Partial amplification of the 16S ribosomal RNA gene with generic phytoplasma primer pairs P1/P7 and fu5/ru3 (3), followed by a nested PCR specific for all members of the Phytoplasma stolbur subgroup by means of the stol11 primers (1), and an RFLP analysis of the tuf gene (elongation factor Tu) fragment produced with PCR primers tufAY/r tufAY (3), were used for the identification and characterization of the pathogen. All target amplicons were also sequenced by Macrogen (Seoul, South Korea) following gel purification (Nucleospoin Plant II, Macher-Nagel). Identical sequences were obtained from each of the P1/P7-derived amplicons (100% homology between samples) and a consensus 1,142 bp sequence was delineated and submitted to NCBI GenBank with accession no. JF293091. It had the highest similarity (99 to 100%) to sequences of ‘Bois noir’ phytoplasma (e.g. HQ589193; Candidatus Phytoplasma solani, position 29 to 1,171). The fu5/ru3 amplicons produced sequences that showed 99.5% homology to the Ca. Phytoplasma solani strains of a southern Russian and Romanian phytoplasma survey on different hosts (potato, tomato, Convolvulus) (GenBank Accession No. HM449999 to HM4450002). The stolbur specific primers also produced an amplicon in all samples and again the consensus sequence was identified (100% homology between the samples) and deposited in GenBank (JN561701). RFLP analysis of the tuf gene with the enzymes HindIII, HinfI, HpaII, and TaqI (Fermentas) produced the same profile types for the different samples and clearly allocated the phytoplasma in the tuf type-b (VKII), according to (2). This type is commonly reported as associated with bindweed (Convolvulus arvensis). Additionally, the sequenced tufAY fragment also confirmed a 100% correspondence with the submitted Tu elongation factor fragments of Ca. Phytoplasma solani strains in GenBank. No phytoplasma was detected in symptomless blackberry plants that were sampled from the same plot. In the molecular identification tests, a stolbur phytoplasma control (potato isolate), a Rubus stunt (EY subgroup, 16SrV) and an apple proliferation phytoplasma (AP subgroup, 16SrX) were used as controls. Based on the symptoms and the laboratory results, we concluded that the Rubus plants were infected by Ca. Phytoplasma solani, a species belonging to the stolbur subgroup (16SrXII-A). To our knowledge, this is the first report of Ca. Phytoplasma solani on Rubus fruticosus in Bulgaria. The disease is not likely to be an isolated case in the future because of the pathogen's spread on other hosts and the expected increase in blackberry fields. References: (1) X. Daire et al. Eur. J. Plant Pathol. 103:507, 1997. (2) M. Langer and M. Maixner. Vitis 43:191, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995.

Huanglongbing (HLB), also known as citrus greening, is one of the most destructive citrus diseases worldwide and is seen as a major threat to the multimillion dollar citrus industry in California. The vector of the two bacterial species associated with this disease, Candidatus Liberibacter asiaticus and Ca. L. americanus, is the Asian citrus psyllid (ACP), Diaphorina citri (4). ACP was detected in California in August of 2008 and has since been detected in nine counties in southern California. As part of a long term survey and testing program for the ACP carrying the HLB associated bacteria, groups of ACP nymphs and adults were submitted to the Jerry Dimitman Citrus Research Board/Citrus Pest and Disease Prevention Program Laboratory in Riverside, CA. In March 2012, DNA extracted using the Qiagen MagAttract 96 DNA plant kit (QIAGEN Inc., 27220 Turnberry Lane, Suite 200, Valencia, CA 91355) from a group of three ACP adults tested positive for Ca. L. asiaticus with the real-time PCR assay developed by Li et al. (4). ACP adults were collected from a residential citrus tree located in the Hacienda Heights area of Los Angeles County, California. The approximately 1.8 meter tall lemon tree had 23 graft unions, primarily of lemon (Citrus × meyeri) and pomelo (Citrus maxima) varieties. The tree was unthrifty, with yellow shoots and chlorotic leaves. Symptoms on the lemon and pomelo leaves included asymmetrical blotchy mottling, yellowing, and corking of the leaf veins, with the blotchy mottle more prominent in the pomelo leaves. Pomelo leaves appeared crinkled along the thickened veins. Lemon leaves had yellow veins and a few had islands of green tissue completely surrounded by yellow tissue. The entire tree was removed, cut into sections, bagged, and transported to the CDFA Plant Pest Diagnostics Lab for analysis. Two hundred milligrams of petiole and midrib tissue from leaves apical to each graft union was collected, and DNA from each sample was extracted using the Qiagen DNeasy plant mini kit. DNA extracted from both lemon and pomelo leaves tested positive for Ca. L. asiaticus using real-time PCR (4). A 1,160-bp fragment of the 16S ribosomal RNA gene was amplified from the insect and plant DNA extracts using conventional PCR with primers Ol1 and OI2c (2). A 703-bp fragment of the β-operon gene was amplified from the insect and plant extracts with primers A2 and J5 (1). The 16S rDNA fragments from the insect and plant respectively (GenBank Accession Nos. JX430434 and JX455745) and the β-operon fragments (JX430435 and JX455746) showed 100% identity with the corresponding regions of Ca. L. asiaticus (CP001677) strain psy 62. Our 16S rDNA sequence showed 98% identity with Ca. L. africanus (EU921620), 97% identity with Ca. L. solanacearum (HM246509), and 96% with Ca. L. americanus (FJ036892). In response to the detection of HLB, a 241 km2 quarantine area around the detection site was established. Surveys for ACP and symptomatic host plants within the HLB quarantine area are ongoing. To date, there have been no additional positive detections. In the United States, HLB was first detected in Florida in 2005 (4) and in Texas in January of 2012 (3). To our knowledge, this is the first confirmed report of Ca. L. asiaticus associated with HLB in California. References: (1) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) M. Kunta et al. Phytopathology 102:S4.66, 2012. (4) W. Li et al. J. Microbiol. Methods 66:104, 2006.

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described ‘Candidatus Phytoplasma ’ species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Candidatus Phytoplasma ’ species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1R as representative of a novel taxon, ‘Candidatus Phytoplasma pruni’. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the ‘Ca. Phytoplasma pruni’ lineage. We propose that the term ‘Ca. Phytoplasma pruni’ be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and - within the tolerance of a single base difference in one unique sequence - peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of ‘Candidatus Phytoplasma ’ taxa.

A phytoplasma was identified in naturally infected wild Balanites triflora plants exhibiting typical witches’ broom symptoms (Balanites witches’ broom: BltWB) in Myanmar. The 16S rRNA gene sequence revealed that BltWB phytoplasma had the highest similarity to that of ‘Candidatus Phytoplasma ziziphi’ and it was also closely related to that of ‘Candidatus Phytoplasma ulmi ’. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the BltWB phytoplasma clustered as a discrete subclade with Elm yellows phytoplasmas. RFLP analysis of the 16S rRNA gene including the 16S–23S spacer region differentiated the BltWB phytoplasma from ‘Ca. P. ziziphi ’, ‘Ca. P. ulmi ’ and ‘Candidatus Phytoplasma trifolii ’. Analysis of additional ribosomal protein (rp) and translocase protein (secY) gene sequences and phylogenetic analysis of BltWB showed that this phytoplasma was clearly distinguished from those of other ‘Candidatus Phytoplasma ’ taxa. Taking into consideration the unique plant host and the restricted geographical occurrence in addition to the 16S rRNA gene sequence similarity, the BltWB phytoplasma is proposed to represent a novel taxon, ‘Candidatus Phytoplasma balanitae’.