Publications

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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.

Metagenomics has provided access to genomes of as yet uncultivated microorganisms in natural environments, yet there are gaps in our knowledge—particularly for Archaea—that occur at relatively low abundance and in extreme environments. Ultrasmall cells (<500 nm in diameter) from lineages without cultivated representatives that branch near the crenarchaeal/euryarchaeal divide have been detected in a variety of acidic ecosystems. We reconstructed composite, near-complete ~1-Mb genomes for three lineages, referred to as ARMAN (archaeal Richmond Mine acidophilic nanoorganisms), from environmental samples and a biofilm filtrate. Genes of two lineages are among the smallest yet described, enabling a 10% higher coding density than found genomes of the same size, and there are noncontiguous genes. No biological function could be inferred for up to 45% of genes and no more than 63% of the predicted proteins could be assigned to a revised set of archaeal clusters of orthologous groups. Some core metabolic genes are more common in Crenarchaeota than Euryarchaeota , up to 21% of genes have the highest sequence identity to bacterial genes, and 12 belong to clusters of orthologous groups that were previously exclusive to bacteria. A small subset of 3D cryo-electron tomographic reconstructions clearly show penetration of the ARMAN cell wall and cytoplasmic membranes by protuberances extended from cells of the archaeal order Thermoplasmatales . Interspecies interactions, the presence of a unique internal tubular organelle [Comolli, et al. (2009) ISME J 3:159–167], and many genes previously only affiliated with Crenarchaea or Bacteria indicate extensive unique physiology in organisms that branched close to the time that Cren - and Euryarchaeotal lineages diverged.

Summary Here we report the first metatranscriptomic analysis of gene expression and regulation of ‘ Candidatus Accumulibacter’‐enriched lab‐scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. RNA samples were collected at the early stage of anaerobic and aerobic phases (15 min after acetate addition and switching to aeration respectively). We detected the expression of a number of genes involved in the carbon and phosphate metabolisms, as proposed by EBPR models (e.g. polyhydroxyalkanoate synthesis, a split TCA cycle through methylmalonyl‐CoA pathway, and polyphosphate formation), as well as novel genes discovered through metagenomic analysis. The comparison between the early stage anaerobic and aerobic gene expression profiles showed that expression levels of most genes were not significantly different between the two stages. The majority of upregulated genes in the aerobic sample are predicted to encode functions such as transcription, translation and protein translocation, reflecting the rapid growth phase of Accumulibacter shortly after being switched to aerobic conditions. Components of the TCA cycle and machinery involved in ATP synthesis were also upregulated during the early aerobic phase. These findings support the predictions of EBPR metabolic models that the oxidation of intracellularly stored carbon polymers through the TCA cycle provides ATP for cell growth when oxygen becomes available. Nitrous oxide reductase was among the very few Accumulibacter genes upregulated in the anaerobic sample, suggesting that its expression is likely induced by the deprivation of oxygen.

A novel halophilic actinomycete, designated strain YIM 93223T, was isolated from a salt lake in Xinjiang Province, north-west China, and was subjected to a polyphasic taxonomic study. The isolate grew at 25–45 °C, at pH 6–8 and in the presence of 1–15 % (w/v) NaCl; no growth was observed in the absence of NaCl. Strain YIM 93223T contained meso-diaminopimelic acid, glutamic acid and alanine as cell-wall amino acids, and glucose and galactose as major whole-cell-wall sugars. Major fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/iso-C15 : 0 2-OH. MK-8(H4) was the predominant menaquinone. The genomic DNA G+C content was 66.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 93223T belongs to the genus Amycolatopsis. It shared highest 16S rRNA gene sequence similarity with Amycolatopsis palatopharyngis 1BDZT (96.6 %) and Amycolatopsis marina Ms392AT (96.4 %), but lower values (94.5–96.2 %) with the type strains of other recognized species of the genus Amycolatopsis. On the basis of the data from this polyphasic study, strain YIM 93223T is considered to represent a novel species of the genus Amycolatopsis, for which the name Amycolatopsis halophila sp. nov. is proposed. The type strain is YIM 93223T (=DSM 45216T =KCTC 19403T).

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of ‘Candidatus Liberibacter asiaticus’ from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted ‘Ca. L. asiaticus’ into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of ‘Ca. L. asiaticus’ from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of ‘Ca. L. asiaticus’ in infected Murraya.

Pear decline (PD) is a serious disease of pear (Pyrus communis L.) caused by ‘Candidatus Phytoplasma pyri’, which belongs to the subgroup 16SrX-C of the apple proliferation (AP) group of phytoplasmas (3). Pear seedlings from the Agriculture and Agri-Food Canada (AAFC) pear breeding program, which have been selected for advanced test and grower trials, are routinely submitted to the Canadian Food Inspection Agency (CFIA) Sidney Laboratory (formerly, CFIA Centre for Plant Health, Saanichton, BC) for virus testing at the same time that propagation is initiated to produce trees for further evaluations. In early 2007, the CFIA reported that samples of two seedling selections submitted in 2005 tested positive for phytoplasmas by a nested PCR assay with phytoplasma universal primers P1/P7 (1), followed by phytoplasma universal primers fU5/rU3 (2) and real time PCR with universal phytoplasma primers developed by the CFIA-Sidney (personal communication). Phytoplasmas present in both selections were subsequently identified as ‘Ca. P. pyri' strains by nested PCR with the P1/P7 primers followed by PD/peach yellow leaf roll (PYLR)-specific primers fPD/rPDS (2,4). These were the first PD-positive results from many samples submitted over the years for testing. Following PD-positive diagnoses for the seedling trees, others propagated from these seedling trees were removed from the nursery. When tested by PD-specific nested PCR (P1/P7 then fPD/rPDS), one selection had 39 of 79 nursery trees (49%) that were PD positive, while the other selection had 27 of 96 trees (28%) testing as PD positive. PCR amplification of DNA isolated from leaves of six of the propagated trees, with primer pair fPD/rPDS, yielded an ~1,400-bp product that was sequenced. A consensus sequence of 1,313 bp (GenBank Accession No. GU565959) was subjected to a nucleotide BLAST search of the NCBI database and showed 100% nt identity with sequences of phytoplasmas PD1 (AJ542543) and PYLR (Y16394). Subsequently, the PD-positive results from leaf, dormant shoot, and root tissues from the original seedling trees were confirmed by PD-specific nested PCR. On the original seedling trees, visible symptoms typical of PD, especially premature leaf coloration, were observed in late summer 2008 and samples taken of green and red leaves were subjected to PD-specific PCR. Red leaves were PD-positive, while green leaves were mostly PD-negative. Pear leaves, dormant shoots, and roots collected from research and commercial orchards in southern Ontario in 2007 and 2008 were subjected to PD-specific nested PCR (P1/P7 then fPD/rPDS), AP-specific nested PCR (P1/P7 then fO1/rO1) (2), as well as the universal phytoplasma nested PCR (P1/P7 then fU5/rU3), resulting in the identification of PD-positive trees of several cultivars. The sequence of the 1,057-bp amplicon from accession PYR0190 (selection HW615), with AP-specific primers fO1/rO1, was deposited in GenBank (GU475131). Although there have been no previous reports of PD in Ontario, Canada, it would appear that PD has been present for some time based on the number and distribution (both geographic and cultivar) of positive samples. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (3) E. Seemüller et al. J. Plant Pathol. 80:3, 1998. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.