Publications

4481

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.

In March of 2014, carrot plants (Daucus carota L. var. Mascot) exhibiting symptoms of yellowing, purpling, and curling of leaves, proliferation of shoots, formation of hairy secondary roots, general stunting, and plant decline were observed in commercial fields in the Gharb region of Morocco. The symptoms resembled those caused by phytoplasmas, Spiroplasma citri, or ‘Candidatus Liberibacter solanacearum’ infection (1,2,3). About 30% of the plants in each field were symptomatic and plants were infested with unidentified psyllid nymphs; some psyllids are known vectors of ‘Ca. L. solanacearum.’ A total of 10 symptomatic and 2 asymptomatic plants were collected from three fields. Total DNA was extracted from petiole and root tissues of each of the carrots, using the CTAB buffer extraction method (3). The DNA samples were tested for phytoplasmas and spiroplasmas by PCR (3) but neither pathogen was detected in the samples. The DNA extracts were tested for ‘Ca. L. solanacearum’ by PCR using specific primer pairs OA2/OI2c, Lso adkF/R, and CL514F/R, to amplify a partial fragment of the 16S rDNA, the adenylate kinase gene, and rpIJ/rpIL50S rDNA ribosomal protein genes, respectively (1,2,5). DNA samples from all 10 symptomatic carrots yielded specific bands; 1,168 bp for the 16S rDNA fragment, 770 bp for the adk fragment, and 669 bp for rpIJ/rpIL, indicating the presence of ‘Ca. L. solanacearum.’ No ‘Ca. L. solanacearum’ was detected in asymptomatic plants. DNA amplicons of three plant samples (one plant/field) for each primer pair were directly sequenced (Macrogen Inc., Amsterdam). Sequencing results identified two distinct products for the OA2/OI2c primer pair (GenBank Accession Nos. KJ740159 and KJ740160), and BLAST analysis of the 16S rDNA amplicons showed 99 and 100% identity to ‘Ca. L. solanacearum’ (KF737346 and HQ454302, respectively). Two different sequences of the adk amplicon were obtained (KJ740162 and KJ740163), both of which were 98% identical to ‘Ca. L. solanacearum’ (CP002371). Sequencing results also identified two distinct products for the CL514F/R primer pair (KJ754506 and KJ754507), and BLAST analysis of the 50S rDNA ribosomal protein showed 99 and 100% identity to ‘Ca. L. solanacearum’ (KF357912 and HQ454321, respectively). The differences in our 16S and 50S rDNA sequences identified the presence of both ‘Ca. L. solanacearum’ haplotypes D and E (4). To our knowledge, this is the first report of the occurrence of ‘Ca. L. solanacearum’ in Morocco and Africa, suggesting a wider distribution of the bacterium in carrot crops in the Mediterranean region, including North Africa. ‘Ca. L. solanacearum’ has caused economic damages to carrot and celery crops in the Canary Islands and mainland Spain, France, Sweden, Norway, and Finland (3). This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in the United States, Mexico, Central America, and New Zealand (1,2,5). Given the economic impact of ‘Ca. L. solanacearum’ on numerous important crops worldwide, it is imperative that preventive measures be taken to limit its spread. References: (1) L. W. Liefting et al. Plant Dis. 93:208, 2009. (2) J. E. Munyaneza et al. Plant Dis. 93:552, 2009. (3) J. E. Munyaneza et al. J. Plant Pathol. 93:697, 2011. (4) W. R. Nelson et al. Eur. J. Plant Pathol. 135:633, 2013. (5) A. Ravindran et al. Plant Dis. 95:1542, 2011.

Potatoes are a major crop in the Columbia Basin of Oregon and Washington, representing an annual farm gate value of almost $750 million. Zebra chip disease (ZC), a new and economically important disease of potato, was first reported in Oregon and Washington in 2011 (1). The disease is caused by the bacterium ‘Candidatus Liberibacter solanacearum’ (Lso, also referred to as ‘Ca. L. psyllaurous’), which is vectored by the potato psyllid (Bactericera cockerelli Sulc) (1,2). Identifying alternative hosts for Lso may facilitate management of ZC disease, which has increased potato production costs in the region. The perennial weed, bittersweet nightshade (Solanum dulcamara L.), is a year-round host of the potato psyllid (3) and is also a suspected host of Lso. However, little is known about the role of this weed in ZC epidemiology. Naturally occurring bittersweet nightshade plants (n = 21) were sampled at six different locations near Hermiston, Oregon, between May and October in 2012. These plants exhibited several symptoms associated with Lso, ranging from asymptomatic to slight purpling, chlorosis, or scorching of the foliage. However, S. dulcamara exhibits similar symptoms under a variety of environmental conditions (drought stress, etc.); therefore, it was difficult to identify potentially infected plants based solely on symptomology. Leaf and stem tissue (n = 21) was analyzed with high-fidelity PCR using species-specific primers for the 16S rDNA gene, CLipoF, and OI2c (2,4). Approximately 27.3% of the plants tested positive for Lso using these primers, including plants from the following locations on 16 April, 16 May, and 24 May, respectively: Hat Rock, OR (45°55.033′ N, 119°10.495′ W), Irrigon, OR (45°54.560′ N, 119°24.857′ W), and Stanfield, OR (45°46.971′ N, 119°13.203′ W). Three plants were selected for further PCR analysis with primers for the outer membrane protein gene, 1482f and 2086r (1). Amplicons obtained with both sets of PCR primers were directly sequenced. A BLAST analysis showed that the 16S rDNA gene sequence (993 to 1,000 bp) shared 99 to 100% identity with several Lso accessions, including JN848751.1 (from Washington) and JN848753.1 (from Oregon). Likewise, the outer membrane protein gene sequence (600 to 601 bp) shared 99 to 100% identity with ‘Ca. L. solanacearum’ accession KC768330.1 (from Honduras). All six sequences were deposited in GenBank (Accession Nos. KJ854199 to KJ854204). According to these findings, bittersweet nightshade may be an important annual source of Lso in the region, particularly since it serves as a host for the potato psyllid. Potato psyllids were also detected at two of the locations with infected S. dulcamara: Irrigon, OR, and Stanfield, OR. A subsample of the psyllids collected in 2012 were analyzed with PCR and Lso was detected in a sample from Stanfield, OR (5). Identifying perennial hosts of Lso promotes a better understanding of both ZC disease epidemiology and management. To our knowledge, this is the first report of Lso causing natural infections in S. dulcamara in the United States. References: (1) J. M. Crosslin et al. Plant Dis. 96:452, 2012. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) A. F. Murphy et al. Am. J. Pot. Res. 90:294, 2013. (4) G. A. Secor et al. Plant Dis. 93:574, 2009. (5) K. D. Swisher et al. Am. J. Pot. Res. 90:570, 2013.

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.