Publications

4870

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.

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.

Two non-motile, rod-shaped gammaproteobacteria were isolated from marine sponges collected from the coast of Japan at Numazu. The isolates were oxidase- and catalase-positive facultative anaerobes that fermented carbohydrates. They required sodium ions for growth and were slightly halophilic, growing in the presence of 1.0–5.0 % (w/v) NaCl (optimum of 2.0 % NaCl). Under aerobic conditions, the major isoprenoid quinones were ubiquinone-9 and menaquinone-9 and the minor quinones were ubiquinone-8 and menaquinone-8. The major cellular fatty acids were C18 : 1ω7c, C16 : 1ω7c and C16 : 0 and the hydroxy acids were C10 : 0 3-OH and C12 : 0 3-OH. The DNA G+C content was 48.3–48.7 mol%. Phylogenetic analysis of 16S rRNA gene sequences placed the isolates within the radiation of the genus Endozoicomonas in a broad clade of uncultured clones recovered from various marine invertebrates. The isolates exhibited 96.5–96.9 % 16S rRNA gene sequence similarity with Endozoicomonas elysicola MKT110T and Endozoicomonas montiporae CL-33T, with which the isolates formed a monophyletic cluster with 100 % bootstrap support. The phenotypic features (carbohydrate fermentation, quinone system and some major cellular fatty acids) differed from those of members of the genus Endozoicomonas , which are aerobic, produce little or no menaquinone under aerobic conditions and possess different amounts of C14 : 0 and C18 : 1ω7c. Although some phenotypic differences were identified, the isolates should be assigned to the genus Endozoicomonas on the basis of congruity of phylogeny and should be classified as representatives of a novel species, for which the name Endozoicomonas numazuensis sp. nov. is proposed. The type strain is HC50T ( = NBRC 108893T  = DSM 25634T). An emended description of the genus Endozoicomonas is presented.

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’.

ABSTRACT Bemisia tabaci (Hemiptera: Aleyrodidae) is a species complex containing >28 cryptic species, some of which are important crop pests worldwide. Like many other sap-sucking insects, whiteflies harbor an obligatory symbiont, “ Candidatus Portiera aleyrodidarum,” and a number of secondary symbionts. So far, six genera of secondary symbionts have been identified in B. tabaci . In this study, we report and describe the finding of an additional bacterium in the indigenous B. tabaci cryptic species China 1 (formerly known as B. tabaci biotype ZHJ3). Phylogenetic analysis based on the 16S rRNA and gltA genes showed that the bacterium belongs to the Alphaproteobacteria subdivision of the Proteobacteria and has a close relationship with human pathogens of the genus Orientia . Consequently, we temporarily named it Orientia -like organism (OLO). OLO was found in six of eight wild populations of B. tabaci China 1, with the infection rate ranging from 46.2% to 76.8%. Fluorescence in situ hybridization (FISH) of B. tabaci China 1 in nymphs and adults revealed that OLOs are confined to the bacteriome and co-occur with “ Ca . Portiera aleyrodidarum.” The vertical transmission of OLO was demonstrated by detection of OLO at the anterior pole end of the oocytes through FISH. Quantitative PCR analysis of population dynamics suggested a complex interaction between “ Ca . Portiera aleyrodidarum” and OLO. Based on these results, we propose “ Candidatus Hemipteriphilus asiaticus” for the classification of this symbiont from B. tabaci .

ABSTRACT Neoehrlichiosis caused by “ Candidatus Neoehrlichia mikurensis” is an emerging zoonotic disease. In total, six patients have been described in Europe, with the first case detected in 2007. In addition, seven patients from China were described in a report published in October 2012. In 2009, we diagnosed the first human case of “ Ca . Neoehrlichia mikurensis” infection in the Zurich area (Switzerland). Here, we report two additional human cases from the same region, which were identified by broad-range 16S rRNA gene PCR. Both patients were immunocompromised and presented with similar clinical syndromes, including fever, malaise, and weight loss. A diagnostic multiplex real-time PCR was developed for specific detection of “ Ca . Neoehrlichia mikurensis” infections. The assay is based on the signature sequence of a 280-bp fragment of the “ Ca . Neoehrlichia mikurensis” 16S rRNA gene and incorporates a “ Ca . Neoehrlichia mikurensis” species, a “ Ca . Neoehrlichia” genus, and an Anaplasmataceae family probe for simultaneous screening. The analytical sensitivity was determined to be below five copies of the “ Ca . Neoehrlichia mikurensis” 16S rRNA gene. Our results show that the assay is suitable for the direct detection of “ Ca . Neoehrlichia mikurensis” DNA in clinical samples from, for example, blood and bone marrow. In addition, it allows for monitoring treatment response during antibiotic therapy. Using the same assay, DNA extracts from 1,916 ticks collected in four forests in close proximity to the patients' residences (<3 km) were screened. At all sampling sites, the minimal prevalence of “ Ca . Neoehrlichia mikurensis” was between 3.5 to 8% in pools of either nymphs, males, or females, showing a strong geographic association between the three patients and the assumed vector.