Publications

4785

Cauliflower stunt, caused by a phytoplasma of the group 16SrIII-J, was reported in the beginning of 2012 and has occurred with high incidences of infected plants (up to 90%) in crops located in the state of São Paulo in the southeast region of Brazil (3). Diseased plants exhibit general stunting, malformation of inflorescence, reddening leaves, and vessel necrosis (3). Further investigations with plants displaying identical symptoms collected in Nova Bassano, state of Rio Grande do Sul, Brazilian south region, have revealed the presence of a phytoplasma distinct from 16SrIII-J subgroup. Four symptomatic plus four asymptomatic samples were assayed from a field, and the presence of phytoplasma was evidenced by nested PCR assays performed with primers P1/Tint followed by R16F2n/16R2 in three affected plants, which amplified genomic fragments of 1.2 kb from the 16S rRNA gene. No amplification occurred in non-affected samples. Nested PCR products analyzed by conventional RFLP (2) using the enzymes AluI, RsaI, KpnI, HpaII, MseI, HhaI, MboI, and BstUI pointed to the presence of a phytoplasma belonging to group 16SrXV-A in all three phytoplasma-positive samples. Virtual RFLP analysis based on restriction patterns, derived from in silico digestion with 17 endonucleases (4), confirmed the previous results obtained from those samples by conventional RFLP. The 16S rDNA sequences of this phytoplasma identified in cauliflower (GenBank Accession No. JN818845) shared 99% sequence similarity with the reference phytoplasma for subgroup 16SrXV-A (Hibiscus witches'-broom phytoplasma, AF147708), designated ‘Candidatus Phytoplasma brasiliense.’ Analysis of putative restriction sites showed excellent identity between the phytoplasma studied here and the reference phytoplasma. In addition, the arrangement of branches of a phylogenetic tree constructed with phytoplasmas representing diverse 16Sr groups and subgroups supported that the phytoplasma found in cauliflower is closed related to the representative of the subgroup 16SrXV-A. Association of distinct phytoplasmas with the same kind of disease is not rare and the present pathosystem constitutes a new example. Members of this subgroup have been described almost exclusively in Brazil and previously reported in Sida sp., periwinkle, and hibiscus (1). In some European countries, as well as in the United States and Canada, phytoplasmas belonging to group 16SrI has been associated with this type of disease, which has been reported for various species of the genus Brassica, as published in previous works (3). However, a representative of the group 16SrVI was described in infected plants in Iran (3). Although the 16SrIII-J phytoplasma is currently the most important agent of cauliflower stunt in Brazil, and members of 16SrI are prevalent in other countries, this study revealed that a 16Sr XV-A phytoplasma may be also associated with this important disease of brassicas. Besides, the findings here reported expand the natural host range, including cauliflower as new host for phytoplasmas affiliated with 16SrXV-A. References: (1) B. Eckstein et al. Plant Dis. 95:363, 2009. (2) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) M. C. C. Rappussi et al. Eur. J. Plant. Pathol. 133:829, 2012. (4) Wei et al. Int. J. Syst. Evol. Microbiol. 57:1855, 2007.

Abstract Chemoheterotrophic marine bacteria of the SAR11 clade are Earth’s most abundant organisms. Following the first cultivation of a SAR11 bacterium, ‘Candidatus Pelagibacter ubique’ strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional requirements were identified for reduced sulfur compounds and glycine or serine. These requirements were linked to genome streamlining resulting from selection for efficient resource utilization in nutrient-limited ocean habitats. Here we report the first successful cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an additional requirement for pyruvate or pyruvate precursors. Optimal growth was observed with the collective addition of inorganic macro- and micronutrients, vitamins, methionine, glycine and pyruvate. Methionine served as the sole sulfur source but methionine and glycine were not sufficient to support growth. Optimal cell yields were obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid cycle intermediates. Moreover, both glycine betaine and serine could substitute for glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We propose that this is the result of the use of glycolate, a product of phytoplankton metabolism, as both a carbon source for respiration and as a precursor to glycine. These findings are important because they provide support for the hypothesis that some micro-organisms are challenging to cultivate because of unusual nutrient requirements caused by streamlining selection and gene loss. Our findings also illustrate unusual metabolic rearrangements that adapt these cells to extreme oligotrophy, and underscore the challenge of reconstructing metabolism from genome sequences in organisms that have non-canonical metabolic pathways.

ABSTRACTThree cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined forChlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the orderChlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported “CandidatusPiscichlamydia salmonis” (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35ChlamydiaandChlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the orderChlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as “CandidatusParilichlamydia carangidicola” and that the new family be known as “CandidatusParilichlamydiaceae.”

ABSTRACT The bacteria that cause necrotizing hepatopancreatitis in Penaeus vannamei adversely affect penaeid shrimp cultured in the western hemisphere. 16S rRNA and gyrase B gene analyses determined the taxonomic position of these bacteria. The name “ Candidatus Hepatobacter penaei” is proposed for these pathogenic bacteria, which are members of the Rickettsiales order.

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.