Publications

4498

A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria ; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria . The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.

Mung bean (Vigna radiata (L.) R. Wilczek) is an important edible legume grown in Asia, particularly in the Indian subcontinent, where it is used for human and animal consumption. In September 2013, 10% of a group of 90 mung bean breeding lines in experimental plots of S. V. Agricultural College, Tirupati, Andhra Pradesh, India, exhibited symptoms typical of a phytoplasma infection, including stunting, extensive proliferation of branches, reduction in leaf size, phyllody, and longitudinal splitting of green pods followed by germination of green seeds producing small plants. These symptoms have been associated with mung bean phyllody (1,3). While the severity of infection varied within each line, on average, 20% of symptomatic plants did not produce seeds at all. Leaf samples from two symptomatic plants and one asymptomatic plant were collected and DNA was extracted from leaves following a CTAB DNA extraction procedure (2). Direct PCR and nested PCR assay was performed using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (4). Both of the symptomatic samples produced 1.8 kb and 1.2 kb size amplicons after direct and nested PCR cycles, respectively. No amplicons were produced with DNA from the asymptomatic sample. Nested PCR products (1.2 kb) from the two symptomatic samples corresponding to the F2nR2 region of 16S rDNA were directly sequenced on an ABI 3730 XL automated sequencer at McLab sequencing services (McLab, CA). Both samples were 100% identical and the representative sequence was designated as APMBP and deposited in GenBank with the accession number KF811205. BLAST analysis revealed a 100% sequence identity with 16SrII group ‘Candidatus Phytoplasma aurantifolia’ phytoplasmas that include sesame phyllody phytoplasmas isolated from sesame and tomato in India (KF429485 and JX104335, respectively). Subgroup identification was performed using the iPhyClassifier online tool (5). The samples were identified as 16SrII-D subgroup phytoplasma based on their 100% identity to the reference strain (GenBank Accession No. Y10097) and virtual RFLP profiles. Phylogenetic analysis based on the F2nR2 sequences with the representative sequences were placed the APMBP in a single distinct cluster with the 16SrII-D reference strain Y10097. Although occurrence of phyllody on mung bean in India was first reported in 1988 (3), the report was based on the appearance of symptoms. This pathogen was recently reported as associated with mung bean phyllody in Pakistan (1). To our knowledge, this is the first report of ‘Ca. P. aurantifolia’ strain infecting mung bean in India. Phytoplasmas belonging to subgroup 16SrII-D are known to have a wide host range, including chickpea, peanuts, sesame, and tomato, which are commonly grown in this region. Mung bean plants infected early failed to produce normal seeds indicating of the potential of this 16SrII-D phytoplasma to become a production constraint for mung bean and other host crops in the area. References: (1) K. P. Akhtar et al. Plant Pathol. 59:399, 2010. (2) J. J. Doyle and J. L. Doyle. Phytochem. Bull. 19:11, 1987. (3) P. Lakshmanan et al. Curr. Sci. 57:809, 1988. (4) I. M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (5) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.