Publications

4364

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.

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.

Carrot (Daucus carota) plants with symptoms resembling those of carrot psyllid (Trioza apicalis) damage (3,4) were observed in 14 commercial fields in southern Finland in August 2008; all cultivars grown were affected at approximately 5 to 35% symptomatic plants per field. T. apicalis, a pest of carrots in northern and central Europe, can cause up to 100% crop loss (3,4). Symptoms on affected plants included leaf curling, yellow and purple discoloration of leaves, stunted growth of shoots and roots, and proliferation of secondary roots (3,4). Given recent association of liberibacter with several annual crops affected by psyllids (1,2), an investigation on whether this bacterium is associated with symptoms of psyllid damage on carrots was conducted. Total DNA was extracted from petiole tissue of 20 symptomatic and 18 asymptomatic plants (cv. Maestro, Nanda, Nipomo, Nerac, and Fontana) sampled from 10 psyllid-infested fields in southern Finland, as well as 15 plants (cv. Primecut, Cheyenne, and Triple Play) grown from seed in an insect-free greenhouse, with the cetyltrimethylammoniumbromide (CTAB) method (2). DNA was also extracted from 10 carrot roots (cv. Nantura) of plants continuously exposed to field-collected carrot psyllid colonies in the laboratory. DNA samples were tested by PCR using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rplJ/rplL ribosomal protein genes, respectively, of “Candidatus Liberibacter solanacearum” (1,2). A 1,168 bp 16S rDNA fragment was detected in DNA from 1 asymptomatic and 16 symptomatic plants and a 669 bp rplJ/rplL fragment was amplified from DNA from 19 symptomatic and 6 asymptomatic plants, indicating presence of liberibacter. DNA from all 10 root samples yielded similar amplicons with both primer pairs. DNA from all the greenhouse carrot plants yielded no amplicon. Amplicons from DNA from three petioles and three roots with each primer pair were cloned (pCR2.1-TOPO; Invitrogen, Carlsbad, CA) and three clones of each of the 12 amplicons were sequenced (MCLAB, San Francisco, CA). BLAST analysis of the 16S rDNA consensus sequences from petiole and root tissues (GenBank Accession Nos. GU373049 and GU373048, respectively) showed 99.9% identity to those of “Ca. L. solanacearum” amplified from Capsicum annuum (FJ957896) and Solanum lycopersicum (FJ957897) from Mexico, and “Ca. L. psyllaurous” from potato psyllids (EU812559). The rplJ/rplL consensus sequences from petioles and roots (GenBank Accession Nos. GU373051 and GU373050, respectively) were 97.9% identical to the analogous rplJ/rplL “Ca. L. solanacearum” ribosomal protein gene sequence from solanaceous crops in New Zealand (EU834131) and to “Ca. Liberibacter” sp. sequence from zebra chip-affected potatoes in California (FJ498803). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with a nonsolanaceous species and the first report of this pathogen outside of North and Central America and New Zealand (1,2). References: (1) L. W. Liefting et al. Plant Dis. 93:208, 2009. (2) J. E. Munyaneza et al. Plant Dis. 93:552, 2009. (3) G. Nehlin et al. J. Chem. Ecol. 20:771, 1994. (4) A. Nissinen et al. Entomol. Exp. Appl. 125:277, 2007.

Abstract Analyses of archaeal membrane lipids are increasingly being included in ecological studies as a comparatively unbiased complement to gene-based microbiological approaches. For example, crenarchaeol, a glycerol dialkyl glycerol tetraether (GDGT) with a unique cyclohexane moiety, has been postulated as biomarker for ammonia-oxidizing Archaea (AOA). Crenarchaeol has been detected in Nitrosopumilus maritimus and ‘Candidatus Nitrosocaldus yellowstonii’ representing two of the three lineages within the Crenarchaeota containing described AOA. In this paper we present the membrane GDGT composition of ‘Candidatus Nitrososphaera gargensis’, a moderately thermophilic AOA, and the only cultivated Group I.1b Crenarchaeon. At a cultivation temperature of 46 °C, GDGTs of this organism consisted primarily of crenarchaeol, its regioisomer, and a novel GDGT. Intriguingly, ‘Ca. N. gargensis’ is the first cultivated archaeon to synthesize substantial amounts of the crenarchaeol regioisomer, a compound found in large relative abundances in tropical ocean water and some soils, and an important component of the TEX86 paleothermometer. Intact polar lipid (IPL) analysis revealed that ‘Ca. N. gargensis’ synthesizes IPLs similar to those reported for the Goup I.1a AOA, Nitrosopumilus maritimus SCMI, in addition to IPLs containing uncharacterized headgroups. Overall, the unique GDGT composition of ‘Ca. N. gargensis’ extends the known taxonomic distribution of crenarchaeol synthesis to the Group I.1b Crenarchaeota, implicating this clade as a potentially important source of crenarchaeol in soils and moderately high temperature environments. Moreover, this work supports the hypothesis that crenarchaeol is specific to all AOA and highlights specific lipids, which may prove useful as biomarkers for ‘Ca. N. gargensis’-like AOA.