Publications

4629

ABSTRACT The characterization of transcriptional start sites of 14 genes encoded by the extremely AT-rich genome of “ Candidatus Blochmannia floridanus” revealed a high degree of conservation with the RpoD promoter consensus sequence of the free-living relative Escherichia coli . Moreover, in agreement with the presence of the alternative heat shock sigma factor RpoH in “ Ca . Blochmannia,” typical RpoH-dependent promoters were identified. However, no heat shock response resembling that of E. coli could be detected in “ Ca . Blochmannia.”

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at –20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with ‘Candidatus Liberibacter’ sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of ‘Ca. L. solanacearum’ previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of ‘Ca. L. solanacearum’ with potatoes affected by ZC in the United States, Mexico, and Guatemala.

Bamboos are ecologically and economically valuable plants. Young shoots of almost all species are edible, either raw or cooked, and are major components of Asian cuisine while culms are used for furniture or handicrafts as well as fuel wood. Symptoms indicative of phytoplasma disease were observed on Sasa fortunei (van Houtte) Fiori during a survey in Yangling, Shaanxi Province, China during 2007. Symptoms included internode shortening, a mosaic pattern on leaves of diminished size, stunted growth, and death of entire plants. Total nucleic acids were extracted from leaf veins of 10 diseased plants and 6 symptomless plants with a modified cetyltrimethylammoniumbromide (CTAB) method (1). Phytoplasma infection of plants was demonstrated by a nested PCR assay employing primer pair R16mF2/R16mR1 followed by R16F2n/R16R2 (2), which generated a 16S rDNA product of approximately 1.2 kb from all symptomatic plants only. Restriction fragment length polymorphism analysis by digestion of nested PCR products with restriction enzymes AluI and MseI indicated that S. fortunei plants contained group 16SrI (aster yellows), subgroup B phytoplasmas. Sequencing and phylogenetic analysis of phytoplasma 16S rDNA from S. fortunei (GenBank Accession No. FJ501958) revealed this strain to be very similar (99.7 and 99.6%, respectively) to phytoplasmas previously associated with Henon bamboo (Phyllostachys nigra) witches'-broom (GenBank Accession No. AB242433) (4) and sasa (S. borealis) witches'-broom (GenBank Accession No. AB293421) in Korea but less so (98.6%) to bamboo (Phyllostachys spp.) witches'-broom (GenBank Accession No. AY635145) disease in China (3). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’-related strain infecting S. fortunei in China. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) C. Hong et al. Plant Prot. 31:39, 2005. (4) H. Jung et al. J. Gen. Plant Pathol. 72:261, 2006.

ABSTRACT Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191 T is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 μm long and 0.17 to 0.3 μm wide). The isolate grows heterotrophically on sugars and ferments d -galactose, d -glucose, d -fructose, d -glucosamine, and N -acetyl- d -glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191 T belongs to the “intestinal cluster,” a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called “endomicrobia” lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name “ Elusimicrobium minutum ” gen. nov., sp. nov. (type strain, Pei191 T = ATCC BAA-1559 T = JCM 14958 T ) for the first isolate of this deep-branching lineage and the name “ Elusimicrobia ” phyl. nov. for the former TG1 phylum.

Biosulfidogenesis (the generation of hydrogen sulfide by microorganisms) in acidic liquors was investigated using two metabolically-distinct bacteria. One was a novel acidophilic sulfate-reducing bacterium (isolate CL4) that grew at pH 3.0 and above using glycerol as electron donor, and the other was the type strain of Acidithiobacillus ferrooxidans which was grown at pH 2.5 using hydrogen (derived from dissolution of metallic iron) as electron donor and elemental sulfur as electron acceptor. Both bacteria were grown in pH-controlled bioreactors. Isolate CL4 mediated the selective precipitation of zinc in situ, while the At. ferrooxidans bioreactor operated as an off-line system, generating hydrogen sulfide that precipitated copper in a separate reaction vessel. The potential of using acidophilic sulfidogens for the selective recovery of metals from acidic waste streams is discussed.

A disease that severely affects processing potatoes (Solanum tuberosum L.), termed zebra chip (ZC), has been identified in several locations in the United States (Texas, Nebraska, Colorado, Kansas, New Mexico, Arizona, and Nevada), Mexico, and Central America (4). The disease name comes from the rapid oxidative darkening of freshly cut tubers and the dark stripes and blotches that occur in chips processed from infected tubers. Recently, the disorder has been associated with a new ‘Candidatus Liberibacter’ species in New Zealand (3). Also, a bacterium designated ‘Candidatus Liberibacter psyllaurous’ has been identified recently in potato plants with “psyllid yellows” symptoms that resemble foliar symptoms of ZC (2). In the fall of 2008, 10 tubers were received at the Prosser laboratory from a commercial potato grower and five had symptoms characteristic of ZC. The tubers, cv. Dakota Pearl, were grown near Lancaster in southern California. The tubers showed rapid oxidation upon slicing and the sunken stolon attachment characteristic of ZC (4). Nucleic acid was extracted from symptomatic tubers (1) and tested by PCR for ‘Ca. Liberibacter’ species with primer pairs OA2/OI2c (5′-GCGCTTATTTTTAATAGGAGCGGCA-3′ and 5′-GCCTCGCGACTTCGCAACCCAT-3′) and CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′ and 5′-TATATCTATCGTTGCACCAG-3′), which amplify from the 16S rDNA and rplJ and rplL ribosomal protein genes, respectively (3). Four of the five tubers with distinct ZC symptoms yielded the expected amplicons with both primer pairs. Two tubers with mild internal discoloration yielded correctly sized amplicons but in lesser amounts than from the severely affected tubers. Nucleic acid from healthy potato tubers yielded no product with these primers. One clone of the 1,168-bp OA2/OI2c amplicon and two clones of the 669-bp CL514F/R amplicon from a strongly positive sample were sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST alignments of the consensus sequences of the OA2/OI2c and CL514F/R amplicons (GenBank Accessions Nos. FJ498802 and FJ498803, respectively) revealed 100% identity with analogous ‘Ca. Liberibacter’ sequences reported from ZC-symptomatic potatoes in New Zealand (GenBank Accession Nos. EU849020 and EU919514). The OA2/OI2c amplicon was also identical to a sequence of ‘Ca. Liberibacter psyllaurous’ (GenBank Accession No. EU812559) from psyllid yellows-affected potatoes in the United States (2) and also showed a 99% identity with sequences from a ‘Ca. Liberibacter’ species reported in ZC tubers from Kansas (GenBank Accession No. EU921626). Potato crops with symptoms of ZC have been observed previously in California (4), but this is the first identification of ‘Ca. Liberibacter psyllaurous’ from diseased potatoes grown in California. Since ZC was first reported in the mid- to late-1990s, information from potato growers and processors suggests that ZC is becoming more important. The disease has caused millions of dollars in losses, particularly in south Texas (4). The identification of ‘Ca. Liberibacter psyllaurous’ in California provides additional evidence that the disease is increasing in importance in other potato-growing regions. References: (1) J. M. Crosslin et al. Plant Dis. 90:663, 2006. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 92:1474, 2008. (4) J. E. Munyaneza et al. Subtrop. Plant Sci. 59:30, 2007.