Publications

4697

ABSTRACT “ Candidatus Neoehrlichia mikurensis” was detected by PCR in 4.0% (34/841) of the rodents tested in this study. The 34 rodents represented nine species from seven regions of China. Phylogenetic analyses based on the partial groEL and nearly entire 16S rRNA gene sequences of the agent revealed genetic diversity, which was correlated with its geographic origins.

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.

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by ‘Ca. L. asiaticus’, the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the ‘Ca. L. asiaticus’ bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the ‘Ca. L. asiaticus’ bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of ‘Ca. L. asiaticus’, while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate ‘Ca. L. asiaticus’ bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of ‘Ca. Liberibacter’-infected plants in nursery and greenhouse settings.

A novel bacterial strain designated HA-01T was isolated from a freshwater terrestrial hot spring located at Hot Springs National Park, Arkansas, USA. Cells were Gram-negative-staining, rod-shaped, aerobic, chemo-organotrophic, oxidase- and catalase-positive, non-spore-forming and motile by means of a single polar flagellum. Growth occurred at 37–60 °C, with an optimum between 45 and 50 °C, and at pH 6.5–8.5, with an optimum between pH 6.5 and 7.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the closest relatives of strain HA-01T were Solimonas aquatica NAA-16T (93.8 %), Solimonas flava CW-KD 4T (94.1 %), Solimonas soli DCY12T (93.1 %), Solimonas variicoloris MN28T (94.0 %), Nevskia ramosa Soe1T (91.2 %) and Hydrocarboniphaga effusa AP103T (91.1 %). Major fatty acids consisted of C16 : 0, iso-C16 : 0, C16 : 1ω5c and summed feature 8 (C18 : 1ω9c, C18 : 1ω7c and C18 : 1ω6c). Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the major isoprenoid quinone was Q-8. The DNA G+C content was 64.4 mol%. Based on phylogenetic, phenotypic and chemotaxonomic evidence, it is proposed that strain HA-01T represents a novel species in a new genus for which the name Fontimonas thermophila gen. nov., sp. nov. is proposed. The type strain of the type species is HA-01T ( = DSM 23609T = CCUG 59713T). A new family, Solimonadaceae fam. nov., is also proposed to replace Sinobacteriaceae Zhou et al. 2008.