Publications

4433

ABSTRACT A recently described clinical isolate, “ Candidatus Bartonella ancashi,” was obtained from a blood sample of a patient presenting with verruga peruana in the Ancash region of Peru. This sample and a second isolate obtained 60 days later from the same patient were molecularly typed using multilocus sequence typing (MLST) and multispacer sequence typing (MST). The isolates were 100% indistinguishable from each other but phylogenetically distant from Bartonella bacilliformis and considerably divergent from other known Bartonella species, confirming their novelty.

The objective of this work was to evaluate the reaction of four sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' (Las) infection, a bacterium associated to huanglongbing (HLB) disease. Transgenic sweet orange plants of Hamlin, Natal, Pêra, and Valência cultivars, as well as nontransgenic controls received inocula by grafting budwood sections of HLB-infected branches. Disease progression was evaluated through observations of leaf symptoms and by polymerase chain reaction (PCR) analysis, eight months after inoculation. A completely randomized design was used, with four experiments (one for each cultivar) performed simultaneously. Bacteria title was estimated by quantitative PCR (qPCR). HLB symptoms and Las titers were present in nontransgenic and transgenic plants expressing the attacin A gene of the four sweet orange cultivars, eight months after bacteria inoculation. Five transgenic lines (transformation events) of 'Pêra' sweet orange expressing the attacin A gene have significantly lower Las titers in comparison with nontransgenic plants of this cultivar.

ABSTRACT “ Candidatus Cardinium hertigii” ( Bacteroidetes ) is a maternally inherited endosymbiont known from several arthropods. Its mechanisms for persistence in host populations are mostly reproductive manipulation, though it has been occasionally reported to improve fitness parameters in several hosts. In Culicoides (Diptera: Ceratopogonidae) biting midges, the prevalence of “ Candidatus Cardinium” infection was documented as moderate, with no detectable sex bias. We therefore investigated whether “ Candidatus Cardinium” affects important fitness parameters, such as survival and body size, in Culicoides imicola , a dominant vector species. Field-collected midges were trapped and analyzed for survival under different environmental conditions and antibiotic treatment, taking into account “ Candidatus Cardinium” infection status and parity status (i.e., parous or nulliparous). Additionally, wing lengths were measured as a proxy parameter for body size and analyzed together with “ Candidatus Cardinium” infection data. The findings revealed no difference in survival of Culicoides infected with “ Candidatus Cardinium” and that of uninfected midges in both parity states and under all tested conditions: optimal, starvation, heat, and antibiotic treatment. Beyond survival, no wing length difference was found for “ Candidatus Cardinium”-infected versus uninfected midges. In aggregate, these findings support our conclusion that “ Candidatus Cardinium” does not have an overt effect on the survival and size of adult C. imicola midges. “ Candidatus Cardinium” may affect immature stages or may alter adult reproductive performance.

A new species of Hassallia (Cyanobacteria, Nostocales, Microchaetaceae) from a supralittoral tropical marine biotope is described. Hassallia littoralis is a false-branched nostocalean cyanobacterium with caespitose free filaments or with fasciculated individual filaments not in a common sheath. Filaments are mainly heteropolar, bearing mono- and bi-pored heterocysts and isopolar or heteropolar hormogonia. The sheath is often widening, with pronounced rounded terminals. This new species is defined according to molecular, morphological and ecological criteria, considering data from different stages of its life cycle as well as the 16S rRNA partial gene sequence.

A novel anaerobic bacterium that could ferment amino acids and organic acids was isolated from an anaerobic, propionate-oxidizing enrichment culture originating from soil of a rice field in Japan. Cells of the isolate, designated strain 4F6ET, were Gram-staining-negative, oxidase- and catalase-negative, non-spore-forming, vibrio-shaped, motile rods (0.8×2.0–2.5 µm) with two or three lateral flagella. Growth occurred at 20–42 °C (optimum at 37–40 °C), at pH 6.4–8.4 (optimum at pH 7.3) and at 0–1.5 % (w/v) NaCl (optimum at 0–0.5 %). Good growth occurred on glycine, serine, cysteine, pyruvate and citrate, whereas poor growth was observed on threonine, glutamine, l-malate, α-ketoglutarate, peptone and Casamino acids. In co-culture with the hydrogen-utilizing methanogen Methanobacterium formicicum JCM 10132T, strain 4F6ET oxidized alanine, valine, leucine, isoleucine, methionine, aspartate, glutamate, histidine, asparagine and fumarate. Yeast extract was required for growth. The G+C content of genomic DNA was 61.9 mol%. A phylogenetic analysis based on comparison of the 16S rRNA gene sequence showed that the type strains of Fretibacterium fastidiosum , Aminobacterium colombiense and Aminobacterium mobile , members of the family Synergistaceae , were the closest relatives of strain 4F6ET, with low sequence similarities (89.3, 89.5 and 86.2 %, respectively). Strain 4F6ET contained iso-C13 : 0 (24.43 %), iso-C15 : 0 (16.47 %) and C19 : 1ω11c/C19 : 1ω9c (16.32 %) as the major fatty acids, which differed from those of F. fastidiosum , Aminobacterium colombiense and Aminobacterium mobile . On the basis of phenotypic, chemotaxonomic and phylogenetic differences between strain 4F6ET and the type strains of F. fastidiosum and Aminobacterium species, we propose that strain 4F6ET represents a novel genus and species, Aminivibrio pyruvatiphilus gen. nov., sp. nov. The type strain of Aminivibrio pyruvatiphilus is strain 4F6ET ( = JCM 18417T = DSM 25964T).

In April of 2012, tobacco (Nicotiana tabacum L.) plants with symptoms resembling those associated with viral infection were observed in commercial fields in the Department of El-Paraíso, Honduras. Symptoms on affected plants included apical leaf curling and stunting, overall chlorosis and plant stunting, young plant deformation with cabbage-like leaves, wilting, and internal vascular necrosis of stems and leaf petioles. All cultivars grown were affected, with disease incidence ranging from 5 to 80% of symptomatic plants per field. The fields were also heavily infested with the psyllid Bactericera cockerelli. This psyllid is a serious pest of solanaceous crops in the United States, Mexico, Central America, and New Zealand and has been shown to transmit the bacterium “Candidatus Liberibacter solanacearum” to potato, tomato, and other solanaceous species (2,3). Tobacco (cv. Habano criollo) plant samples were collected from one field in the municipality of Trojes. Initial testing of the plant samples for viruses, including Tobacco mosaic virus, Impatiens necrotic spot virus, Cucumber mosaic virus, and Potato virus Y, using Immunostrips (Agdia, Elkhart, IN) were negative. Total DNA was then extracted from leaf tissues of a total of 13 plants, including eight symptomatic plants and five asymptomatic plants with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,4). The DNA samples were tested by PCR using specific PCR primer pairs OA2/OI2c and OMB 1482f/2086r, to amplify a portion of 16S rDNA and the outer membrane protein (OMB) gene of “Ca. L. solanacearum,” respectively (2). All eight (100%) symptomatic plant samples were positive for “Ca. L. solanacearum” with both sets of primer pairs. “Ca. L. solanacearum” was not detected in the asymptomatic plants. The 16S rDNA and OMB gene amplicons of two plant samples each were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the consensus sequences confirmed “Ca. L. solanaeacrum” in the tobacco samples. The 16S rDNA consensus sequences (1,168 bp) of all amplicons were identical and showed 100% identity with several 16S rDNA sequences of “Ca. L. solanacearum” in GenBank (e.g., Accession Nos. HM245242, JF811596, and JX559779). The consensus sequence of the OMB amplicon (605 bp) showed 97 to 100% homology with a number of “Ca. L. solanacearum” OMB sequences in GenBank, including Accession Nos. CP002371, FJ914617, JN848754 and JN848752. The tobacco-associated consensus 16S rDNA and OMB sequences from this study were deposited in GenBank as Accession Nos. KC768320 and KC768328, respectively. To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tobacco in Honduras, where this cash crop is economically important. This bacterium has also caused millions of dollars in losses to potato, tomato, and several other solanaceous crops in North and Central America and New Zealand, particularly in regions where B. cockerelli is present (3). Furthermore, “Ca. L. solanacearum” has caused significant economic damage to carrot crops in Europe, where it is transmitted by the psyllids Trioza apicalis in northern Europe (4) and B. trigonica in the Mediterranean region (1). References: (1) A. Alfaro-Fernandez et al. Plant Dis. 96:581, 2012. (2) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.