Publications

4510

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.

In August 2008, unusual symptoms were observed in Mexican lime trees (Citrus aurantifolia (Christm.) Swing) in the municipality of Luperón, province of Puerto Plata on the north coast of the Dominican Republic. Symptoms observed in young and old trees included blotchy mottle on leaves, healthy-appearing larger branches with smaller side branches that displayed chlorotic leaves, abscised and lopsided fruit, and branch dieback, all symptoms similar to those of citrus huanglongbing associated with ‘Candidatus Liberibacter’ spp. (1). Symptoms were observed in an area of ~100 ha surrounding Luperón, where Mexican lime trees were grown as seedlings and no commercial plantings of other citrus were present. Symptomatic leaves were collected from 16 trees in September 2008, and DNA was extracted from petioles and midveins with a DNeasy kit (Qiagen, Gaithersburg, MD) or with chloroform/isoamyl alcohol (24:1). Real-time PCR with the16S rDNA primer/probe set specific to ‘Ca. Liberibacter asiaticus’ and performed as described (2) gave Ct values comparable with the positive control for five samples. Conventional PCR with the forward (5′-tcgagcgcgtatgcaatacg-3′) and reverse (5′-ctacctttttctacgggataacgc-3′) primers used in real-time PCR (2) amplified a 75-bp product from these five samples. Eleven to twelve clones were sequenced from each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ489643–FJ489647) showed 98% (e.g., EU921622, EU921618) to 100% identity (e.g., FJ236554, FJ263702) with 16S rDNA sequences of ‘Ca. Liberibacter asiaticus’. A larger 912-bp portion of the 16S rDNA gene was amplified from each sample by conventional PCR with sense (5′-gagcctaccaaggctacgat-3′) and antisense (5′-gcgttatcccgtagaaaaaggtag-3′) primers designed from the sequence of ‘Ca. Liberibacter asiaticus’ strain LJZ-4730 (GenBank Accession No. FJ263700). Nine to twelve clones were sequenced for each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ811891–FJ811895) showed 100% identity to ‘Ca. Liberibacter asiaticus’ (DQ471900, DQ4719010) from Florida and Brazil, respectively. To confirm this identification, 693 bp of the outer membrane protein gene (omp) were amplified from each of the five positive samples by conventional PCR using sense (5′-gtgattctgagggtgagcg-3′) and antisense (5′-cgaactcactgagaactgatc-3′) primers designed from nucleotides 15 to 33 and 687 to 707, respectively, of the omp gene from ‘Ca. Liberibacter asiaticus’ strain MZ (GenBank Accession No. EF580135). The consensus sequences from 12 clones from each sample (GenBank Accession Nos. FJ489638–FJ489642) showed 99% (e.g., FJ236564, AY842429) to 100% (e.g., FJ236566, EF580135) nucleotide identity and the predicted translation product showed 100% amino acid identity (e.g., AAX47433, AAX47431) with the omp gene from ‘Ca. Liberibacter asiaticus’. These results confirm the presence of and to our knowledge, this is the first report of ‘Ca. Liberibacter asiaticus’ in the Dominican Republic. Huanglongbing is a destructive disease of citrus (1) and its spread is expected to adversely affect citrus production in the Dominican Republic with subsequent negative effects predicted for the employment of thousands of people. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Summary Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite “ Candidatus Endonucleobacter bathymodioli” that invades the nuclei of deep‐sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur‐ and methane‐oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep‐sea ecosystems. We first discovered the intranuclear parasite “ Ca. E. bathymodioli” in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid‐Atlantic Ridge. Using primers and probes specific to “ Ca . E. bathymodioli” we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of “ Ca . E. bathymodioli” showed that the infection of a nucleus begins with a single rod‐shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by “ Ca . E. bathymodioli” were those of the gill bacteriocytes. These cells contain the symbiotic sulfur‐ and methane‐oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the “ Ca . E. bathymodioli” belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates.

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.

Zebra Chip (ZC), an emerging disease of potato (Solanum tuberosum L.) first documented in potato fields around Saltillo in México in 1994, has been identified in the southwestern United States, México, and Central America and is causing losses of millions of dollars to the potato industry (4). Recently, this damaging potato disease was also documented in New Zealand (3). This disease is characterized by a striped pattern of necrosis in tubers produced on infected plants, and fried chips processed from these infected tubers are commercially unacceptable (4). Recent studies conducted in the United States and New Zealand have associated ZC with a new species of ‘Candidatus Liberibacter’ vectored by the potato psyllid, Bactericera cockerelli Sulc (1,3,4). A bacterium designated ‘Candidatus Liberibacter psyllaurous’ has recently been identified in potato plants with “psyllid yellows” symptoms that resemble those of ZC (2). To investigate whether liberibacter is associated with ZC in México, 11 potato (cv. Atlantic) tuber samples exhibiting strong ZC symptoms and six asymptomatic tubers were collected from a ZC-affected commercial potato field near Saltillo City, Coahuila, México in September 2008 and tested for this bacterium by PCR. Total DNA was extracted from symptomatic and asymptomatic tubers with cetyltrimethylammoniumbromide (CTAB) buffer (4). DNA samples were tested by PCR using primer pair OA2/OI2c (5′-GCGCTTATTTTTAATAGGAGCGGCA-3′ and 5′-GCCTCGCGACTTCGCAACCCAT-3′, respectively) specific for 16S rDNA and primer pair CL514F/R (5′-CTCTAAGATTTCGGTTGGTT-3′ and 5′-TATATCTATCGTTGCACCAG-3′, respectively) designed from ribosomal protein genes (3). Seven of eleven (63.7%) ZC-symptomatic tubers and one of six (16.7%) asymptomatic potatoes yielded the expected 1,168-bp 16S rDNA and 669-bp CL514F/R amplicons, indicating the presence of liberibacter. Amplicons generated from symptomatic tubers were cloned into pCR2.1-Topo plasmid vectors (Invitrogen, Carlsbad, CA) and one clone of each amplicon was sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the ZC OA2/OI2c sequence (GenBank Accession No. FJ498806) showed 100% identity to liberibacter 16S rDNA sequences amplified from potato psyllids from Dalhart, TX and potato tubers from Garden City, KS (GenBank Accession Nos. EU921627 and EU921626, respectively). The ZC CL514F/R sequence (GenBank Accession No. FJ498807) was 98% identical to analogous rplJ and rplL liberibacter ribosomal protein gene sequences amplified from several solanaceous plants in New Zealand (GenBank Accession Nos. EU834131 and EU935005). The OA2/OI2c sequence was also identical to the 16S rDNA sequence (Genbank Accession No. EU812559) of ‘Ca. Liberibacter psyllaurous’ (2). To our knowledge, this is the first report of ‘Ca. Liberibacter psyllaurous’ associated with ZC-affected potatoes in México. References: (1) J. A. Abad et al. Plant Dis. 93:108, 2009. (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. J. Econ. Entomol. 100:656, 2007.

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.