Publications

4938

Two Gram-negative, facultatively anaerobic, rod-shaped bacteria, strains EF212Tand PS125T, were isolated from the octocoralsEunicea fuscaandPlexaurasp., respectively. EF212Twas isolated from a specimen ofE. fuscacollected off the coast of Florida, USA, and PS125Twas isolated from a specimen ofPlexaurasp. collected off the coast of Bimini, Bahamas. Analysis of the nearly full-length 16S rRNA gene sequences showed that these novel strains were most closely related toEndozoicomonas montiporaeCL-33T,E. elysicolaMKT110TandE. numazuensisHC50T(EF212T, 95.6–97.2 % identity; PS125T, 95.1–96.4 % identity). DNA–DNA hybridization values among EF212T, PS125T,E. montiporaeLMG 24815TandE. elysicolaKCTC 12372Twere far below the 70 % cut-off, with all values for duplicate measurements being less than 35 %. Both EF212Tand PS125Trequired NaCl for growth and showed optimal growth at 2–3 % NaCl, 22-30 °C and pH 8.0. The predominant cellular fatty acids were summed feature 3 (C16 : 1ω6cand/or C16 : 1ω7c), summed feature 8 (C18 : 1ω6cand/or C18 : 1ω7c), C16 : 0and C14 : 0. The DNA G+C content of EF212Twas 48.6 mol% and that of PS125Twas 47.5 mol%. In addition to the genotypic differences observed between the two novel strains and related type strains, phenotypic and chemotaxonomic experiments also revealed differences between strains. Thus, strains EF212Tand PS125Trepresent novel species of the genusEndozoicomonas, for which the namesEndozoicomonas euniceicolasp. nov. andEndozoicomonas gorgoniicolasp. nov., respectively, are proposed. The type strains are EF212T( = NCCB 100458T = DSM 26535T) forEndozoicomonas euniceicolasp. nov. and PS125T( = NCCB 100438T = CECT 8353T) forEndozoicomonas gorgoniicolasp. nov. An emended description of the genusEndozoicomonasis also provided to encompass differences observed in the results of genotypic, chemotaxonomic and phenotypic tests compared from the original and amended genus descriptions.

During a survey carried out in Jordanian vineyards in August and October 2012, grapevine (Vitis vinifera L.) plants showing typical grapevine yellows (GY) disease symptoms, including leaf discoloration and curling, berry shriveling, and irregular maturation of wood, were observed. In the same vineyards, bindweed (Convolvulus arvensis L.) plants showing stunting and leaf chromatic alteration were found, suggesting the involvement of phytoplasmas in the disease etiology. Using a CTAB method, total DNA was extracted from leaf veins of 25 symptomatic and two asymptomatic grapevines, and from five symptomatic and two asymptomatic bindweeds for PCR analysis. DNAs from periwinkle (Catharanthus roseus (L.) G. Don) plants infected by ‘Ca. Phytoplasma asteris’ strain SAY (group 16SrI), ‘Ca. Phytoplasma solani’ strain STOL (group 16SrXII), and ‘Ca. Phytoplasma ulmi’ strain EY1 (group 16SrV), were used as positive controls. DNAs from healthy periwinkle and reactions without template DNA were employed as negative controls. 16S rDNA nested PCRs, carried out using the primer pairs P1/P7, followed by R16F2n/R16R2 (1), yielded an amplicon of the expected size (1,250-bp) in three grapevine and in five bindweed samples, and in positive controls. Amplicons were not produced with DNA from 22 symptomatic grapevines (probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform [2]); nor from asymptomatic plants and negative controls. PCR products were sequenced by commercial services in Italy (Primm, Milan) and Korea (Macrogen Inc., Soul). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank with accessions KC835139 (from grapevine) and KC835140 (from bindweed). The 16S rDNA nucleotide sequences of phytoplasmas identified in grapevine and bindweed in Jordan shared >99.5% sequence identity with ‘Ca. Phytoplasma solani’ reference strain STOL (AF248959), and carried identical STOL-unique signature sequences and distinguishing sequence blocks (3). Phylogenetic and in silico RFLP analyses confirmed the affiliation of phytoplasma strains identified in grapevine and bindweed in Jordan to the species ‘Ca. Phytoplasma solani’ (subgroup 16SrXII-A), opening an avenue to future studies on the dissemination and impact of Bois noir (BN) in Jordan. These studies may add new information about BN, previously reported in neighboring countries (4). Further studies will investigate the role of Hyalesthes obsoletus Signoret, a polyphagous Cixiidae responsible for the BN phytoplasma transmission in Europe, and other possible insect vector(s) in the BN spread in Jordan. References: (1) I.-M. Lee et al. Int. J. Syst. Bact. 48:1153, 1998. (2) F. E. Constable et al. Plant Pathol. 52:267, 2003. (3) F. Quaglino et al. Int. J. Syst. Evol. Microb. 63:2879. (4) E. Choueiri et al. Plant Dis. 86:697, 2002.

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.

Tomato (Lycopersicum esculentum) crops grown in several departments of Honduras and heavily infested with the psyllid Bactericera cockerelli were observed in April of 2012 with plant symptoms suggestive of “Candidatus Liberibacter solanacearum” infection. B. cockerelli is a serious pest of potato, tomato, and other solanaceous plants and a vector of “Ca. L. solanacearum” (1,2,3,4). The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (2,3). Disease incidence ranged from 5 to 50% symptomatic plants per field. Tomato (cv. Pony) plant samples were collected from two psyllid-infested commercial fields in the municipalities of Danli and Comayagua in the departments of El-Paraiso and Comayagua, respectively. Total DNA was extracted from leaf tissues of 50 and 20 symptomatic and asymptomatic plants, respectively, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1,3). The DNA samples were tested for “Ca. L. solanacearum” by PCR with primer pairs specific for 16S rDNA (OA2 and OI2c) and the outer membrane protein gene (OMB 1482f and 2086r) of the bacterium (1,2). Ten (20%) of the 50 symptomatic tomato samples were positive for “Ca. L. solanacearum” using both primer pairs and the remaining samples were negative for the bacterium with both primer sets. None of the 20 asymptomatic plants tested positive for “Ca. L. solanacearum”. Amplicons from DNA of two plant samples (one plant/municipality) with each primer pair were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences from the clones (deposited in GenBank as Accession Nos. KC768321 and KC768322) were identical for both locations and showed 99 to 100% identity to several “Ca. L. solanacearum” sequences in GenBank (e.g., JN848753, JN84856, and HM246509). The OMB consensus sequences from the two tomato plants (deposited in GenBank as KC768329 and KC768330) were 100% identical to OMB sequences of Lso in GenBank (CP002371 and JN48754, respectively). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” associated with tomato crops in Honduras. This bacterium has caused millions of dollars in losses to the tomato industry in the United States, Mexico, and New Zealand (2,3,4). Serious damages to tomato crops due to “Ca. L. solanacearum” could expand throughout Central America, especially in those countries where B. cockerelli occurs. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009. (4) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012.

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).

Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation ‘Melainabacteria’. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut.