Publications

4358

Prunus persica (L.) Bastch (family Rosaceae) is currently represented by 83 accessions at the Canadian Clonal Genebank. Approximately 3,200 ha are devoted to peach cultivation in Canada where Ontario Province accounts for 82% of the national production. The clonal peach accessions, also located in Ontario, are monitored routinely for symptoms of phytoplasma infection, including rosette-like symptoms (3) that are characterized by new shoots with very short internodes, loss of older shoot leaves leaving only bunches of young leaves on the tips of naked shoots, and flowers that rarely set fruit. From June to August 2009, peach accessions PRU0382 and PRU0445 showed typical peach rosette symptoms, while another 14 accessions exhibited either short internodes or no symptoms. Leaf midrib samples were collected from 16 peach accessions, including 17 symptomatic (from which 8 corresponded to accession PRU0382, 6 for PRU0445, 1 for PRU0335, 1 for PRU0179, and 1 for PRU0451) and 16 asymptomatic (from which 5 corresponded to a representative of each accession PRU0382, PRU0445, PRU0335, PRU0179, and PRU0451 and 11 to other peach accessions). Total DNA was extracted (DNeasy Plant Extraction Mini Kit, QIAGEN, Valencia, CA) from 100 mg of each sample and used as a template in a nested PCR with phytoplasma universal primers R16mF2/R1 (1) and fU5/rU3 (2). Nested PCR products of the expected size (~880 bp) were obtained from all symptomatic samples (14 of 14) of accessions PRU0382 (peach-almond cv. Kando from the Czech Republic) and PRU0445 (peach cv. HW271 from Canada) only. All other plants with or without symptoms yielded no PCR products. Amplicons were purified (Wizard PCR Clean-up, Promega, Madison, WI), cloned in pGEM-T Easy Vector (Promega), and sequenced (Robarts Institute, London, Canada). The resulting 16S rDNA sequences were identical; one of each was archived in GenBank as Accession No. GU223904. BLAST analysis determined that the P. persica phytoplasma sequence shared 99% identity with 16S rDNA sequences of ‘Candidatus Phytoplasma asteris’-related strains. This relationship was also supported by restriction fragment length polymorphism analysis (RFLP) of rDNA amplicons using AluI, RsaI, and MseI endonucleases that yielded fragment profiles indicative of phytoplasmas belonging to group 16SrI (Aster Yellows), subgroup B (16SrI-B). Among phytoplasma diseases, those attributed to group 16SrI strains are most numerous and affect the widest plant host range. They include peach rosette in the United States and Europe (3) as well as diseases of various horticultural crops in Canada, including grapevine (4). To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma affecting peach in Canada. Early detection of phytoplasmas by PCR in accessions with both European and Canadian origins underscores the importance of prompt identification of infected plants for subsequent thermotherapy treatment to maintain the health of the collection and prevent further disease spread. References: (1) D. E Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:1441, 1996. (2) K. H. Lorenz et al. Phytopathology 85:771, 1995. (3) C. Marcone et al. Acta Hortic. 386:471, 1995. (4) C. Y. Olivier et al. Plant Dis. 93:669, 2009.

ABSTRACT Recently, a new genus of Anaplasmataceae termed “ Candidatus Neoehrlichia” was discovered in ticks and rodents. Here, we report on two patients who suffered from febrile bacteremia due to “ Candidatus Neoehrlichia mikurensis” associated with thrombotic or hemorrhagic events. 16S rRNA and groEL gene sequencing provided evidence of three groups of sequence variants.

ABSTRACT To investigate the fine-scale diversity of the polyphosphate-accumulating organisms (PAO) “ Candidatus Accumulibacter phosphatis” (henceforth referred to as “ Ca. Accumulibacter”), two laboratory-scale sequencing batch reactors (SBRs) for enhanced biological phosphorus removal (EBPR) were operated with sodium acetate as the sole carbon source. During SBR operations, activated sludge always contained morphologically different “ Ca . Accumulibacter” strains showing typical EBPR performances, as confirmed by the combined technique of fluorescence in situ hybridization (FISH) and microautoradiography (MAR). Fragments of “ Ca. Accumulibacter” 16S rRNA genes were retrieved from the sludge. Phylogenetic analyses together with sequences from the GenBank database showed that “ Ca. Accumulibacter” 16S rRNA genes of the EBPR sludge were clearly differentiated into four “ Ca. Accumulibacter” clades, Acc-SG1, Acc-SG2, Acc-SG3, and Acc-SG4. The specific FISH probes Acc444, Acc184, Acc72, and Acc119 targeting these clades and some helpers and competitors were designed by using the ARB program. Microbial characterization by FISH analysis using specific FISH probes also clearly indicated the presence of different “ Ca. Accumulibacter” cell morphotypes. Especially, members of Acc-SG3, targeted by probe Acc72, were coccobacillus-shaped cells with a size of approximately 2 to 3 μm, while members of Acc-SG1, Acc-SG2, and Acc-SG4, targeted by Acc444, Acc184, and Acc119, respectively, were coccus-shaped cells approximately 1 μm in size. Subsequently, cells targeted by each FISH probe were sorted by use of a flow cytometer, and their polyphosphate kinase 1 ( ppk1 ) gene homologs were amplified by using a ppk1 -specific PCR primer set for “ Ca. Accumulibacter.” The phylogenetic tree based on sequences of the ppk1 gene homologs was basically congruent with that of the 16S rRNA genes, but members of Acc-SG3 with a distinct morphology comprised two different ppk1 genes. These results suggest that “ Ca. Accumulibacter” strains may be diverse physiologically and ecologically and represent distinct populations with genetically determined adaptations in EBPR systems.

ABSTRACTThe orderRickettsiales(Alphaproteobacteria) is a well-known group containing obligate endocellular prokaryotes. The order encompasses three families (Rickettsiaceae,Anaplasmataceae, andHolosporaceae) and a fourth, family-level cluster, which includes only one candidate species, “CandidatusMidichloria mitochondrii,” as well as several unnamed bacterial symbionts. The broad host range exhibited by the members of the “CandidatusMidichloria” clade suggests their eventual relevance for a better understanding of the evolution of symbiosis and host specificity ofRickettsiales. In this paper, two new bacteria belonging to the “CandidatusMidichloria” clade, hosted by two different strains of the ciliate protistEuplotes harpa, are described on the basis of ultrastructural observations, comparative 16S rRNA gene sequence analysis, and an estimation of the percentage of infection. Ultrastructure of these bacteria shows some unusual features: one has an electron-dense cytoplasm, and the other one lacks a symbiosomal membrane. The latter was up to now considered an exclusive feature of bacteria belonging to the familyRickettsiaceae. 16S rRNA gene phylogenetic analysis unambiguously places the new bacteria in the “CandidatusMidichloria” clade, although their phylogenetic relationships with other members of the clade are not clearly resolved. This is the first report of a ciliate-borne bacterium belonging to the “CandidatusMidichloria” clade. On the basis of the data obtained, the two bacteria are proposed as two new candidate genera and species, “CandidatusAnadelfobacter veles” and “CandidatusCyrtobacter comes.”

ABSTRACT The complete genome sequence of “ Candidatus Puniceispirillum marinum” IMCC1322, the first cultured representative of the SAR116 clade in the Alphaproteobacteria , is reported here. The genome contains genes for proteorhodopsin, aerobic-type carbon monoxide dehydrogenase, dimethylsulfoniopropionate demethylase, and C 1 compound metabolism. The genome information proposes the SAR116 group to be metabolic generalists in ocean nutrient cycling.

The citrus industries of North and South America are endangered by Huanglongbing (HLB), also known as citrus greening, a devastating disease associated with ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’, two species of fastidious phloem-limited bacteria spread by the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama. The first reports of HLB from the Americas were from Brazil in 2004 followed by Florida in 2005 (3). The ACP was found in Belize in 2005 (S. Williams, personal communication) and is now present throughout Central America. On the basis of the report that the HLB-associated bacteria can be easily detected in the ACP vector (4), an initial sampling of ACP from 67 locations was collected in February 2009 from trees in the Belize, Corozal, Orange Walk, Stann Creek, and Toledo Districts of Belize, and shipped in 95% ethanol to Riverside, CA for analysis. DNA was extracted from lots containing three to five psyllids from each of the 67 samples with Fast DNA kits (MP Biomedicals, Solon, OH) and analyzed by multiplex qPCR for ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ with a Stratagene MX3005P thermocycler with primers and Taqman probes to detect the 16sRNA gene of ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’ and a psyllid gene, wingless, as an internal control target (4). Nine of the sixty-seven psyllid extractions were clearly positive for ‘Ca. L. asiaticus’ with cycle threshold values of 24 to 29. ‘Ca. L. americanus’ was not detected in any of the samples. From the districts previously sampled for ACP, leaves and fruit peduncles were collected from Citrus sinensis and C. aurantifolia plants showing HLB symptoms of asymmetrical leaf mottle and lopsided fruit with aborted seeds. DNA extracted from 10 of the 12 plant samples with a Qiagen Plant DNeasy kit (Qiagen Inc., Valencia, CA) was positive for ‘Ca. L. asiaticus’ with the qPCR procedure of Li et al (3). The presence of ‘Ca. L. asiaticus’ in the positive plant and ACP samples was corroborated by amplification, cloning, and sequencing of a 1,168-bp region of the 16S rRNA gene (2) with SpeedSTAR HS DNA polymerase (TaKaRa Bio Inc., Shiga, Japan). Consensus sequences obtained from three clones each from psyllids (Accession No. GQ502291) and plants (Accession No. GU061003) showed >99% identity to corresponding regions of ‘Ca. L. asiaticus’ in GenBank. The presence of ‘Ca. L. asiaticus’ was further indicated by amplification of a 227-bp fragment from the same 10 positive plant samples using primers for the ‘Ca. L. asiaticus’ preprotein translocase subunit SecE gene (nucleotides 31418 to 31644 of the genomic DNA) (1). Presence of trees with HLB symptoms and the detection of the associated ‘Ca. L. asiaticus’ confirm the disease in the Cayo, Corozal, Stann Creek, and Toledo districts in Belize. Analyses of psyllids from limited surveys conducted from 2006 to 2008 had not detected ‘Ca. L. asiaticus’ or ‘Ca. L. americanus’. Confirmation of HLB in Belize has significant implications to the citrus industries in Central America. References: (1) T. H. Hung et al. J. Phytopathol. 147:599, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) K. L. Manjunath et al. Phytopathology 98:387, 2008.

A moderately thermophilic chemoheterotrophic bacterium, strain Mat9-16T, was isolated from microbial mats developed in hot spring water streams from Yumata, Nagano, Japan. Cells of strain Mat9-16Twere strictly anaerobic, Gram-stain-negative, non-sporulating, non-motile and short to long rods (2.0–15.5 μm in length). Strain Mat9-16Tgrew fermentatively with optimum growth at 45 °C, pH 7.0–7.5 and 1 % NaCl (w/v). Phylogenetic analysis based on the 16S rRNA gene revealed that strain Mat9-16Twas affiliated with an uncultivated lineage, and the nearest cultivated neighbours were green sulfur bacteria belonging to the classChlorobeawith 77–83 % sequence similarity. However, strain Mat9-16Tcould not grow phototrophically and did not possess light-harvesting structures, morphologically and genetically, such as the chlorosomes of green sulfur bacteria. On the basis of phenotypic features and phylogenetic position, a novel genus and species are proposed for strain Mat9-16T, to be namedIgnavibacterium albumgen. nov., sp. nov. (=NBRC 101810T=DSM 19864T). We also propose to place the cultivated bacterial lineage accommodating the sole representative Mat9-16Tin a novel class,Ignavibacteriaclassis nov. In addition, we present a formal description of the phylum-level taxon‘Chlorobi’asChlorobiphyl. nov.