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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.

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.

Huanglongbing (HLB) is a serious disease of citrus worldwide. Three different ‘Candidatus Liberibacter’ species are associated with HLB: ‘Ca. Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ (1). ‘Ca. L. africanus’ and its vector, Trioza erytreae, are both heat sensitive, and when present, occur in citrus when temperatures remain below 30 to 32°C. In Africa, ‘Ca. L. africanus’ and T. erytreae have been reported in South Africa, Zimbabwe, Malawi, Burundi, Kenya, Somalia, Ethiopia, Cameroon, and Madagascar (1). Inspection of citrus trees in orchards and budwood sources in nurseries located in the warmer citrus-growing areas of Tigray and North Wollo in northern Ethiopia revealed nearly 100 trees with symptoms of leaf yellowing with a blotchy mottle pattern, dead branches, and decreased fruit quality and yield. Two symptomatic sweet orange budwood trees and three symptomatic orchard plants were sampled in April 2009, along with three healthy-looking sweet orange plants. DNA was extracted from 200 mg of desiccated leaf midribs using the CTAB method (4) and subjected to conventional PCR using the primer pairs A2/J5 (2) and OI2/23S1 (3) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon and the 16S/23S ribosomal intergenic regions, respectively, of ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’. Positive PCR reactions were obtained for all five symptomatic samples with both primer pairs. PCR amplicons of 703 bp (A2/J5) and 892 bp (OI2/23S) recovered from two of these samples were purified, cloned, and sequenced. BLAST analysis revealed that the nucleotide sequences we obtained for the ribosomal protein (GenBank Accessions Nos. GQ890155 and GQ890156) shared 100% identity with each other and 99% identity with sequences of ‘Ca. L. asiaticus’ from Brazil (DQ471904), Indonesia (AB480161), China (DQ157277), and Florida (CP001677). Similarly, the 16S/23S ribosomal intergenic sequences (GU296538 and GU296539) shared 100% identity with each other and 99% identity with homologous ‘Ca. L. asiaticus’ sequences from Brazil (DQ471903), Indonesia (AB480102), China (DQ778016), and Florida (CP001677) and contained two tRNA genes as occurs in ‘Ca. L. asiaticus’ but not in ‘Ca. L. africanus’ (3). To our knowledge, this is the first report of ‘Ca. L. asiaticus’ in Africa. The presence of ‘Ca. L. asiaticus’ is a threat for warmer citrus-growing areas of Africa that are less favorable for ‘Ca. L. africanus’ and T. erytreae. In areas where ‘Ca. L. asiaticus’ was confirmed, symptomatic trees must be promptly eradicated and surveys to determine spread of the disease and its vectors are necessary. References: (1) J. M. Bove. J. Plant Pathol. 88:7, 2006. (2) A. Hocquellet et al. Mol. Cell. Probes 13:373, 1999. (3) S. Jagoueix et al. Int. J. Syst. Bacteriol. 47:224, 1997. (4) M. G. Murray and W. F Thompson. Nucleic Acids Res. 8:4321, 1980.

In August 2008, 30% of tomato (Solanum lycopersicum) plants in plots in Lubbock County, Texas showed yellowing, lateral stem dieback, upward leaf curling, enlargement of stems, adventitious roots, and swollen nodes. Yellowing in leaves was similar to that seen with zebra chip disease (ZC) of potato that was confirmed in a potato field 112 km away in July 2008 and was associated with a ‘Candidatus Liberibacter’ species (1), similar to findings earlier in 2008 in New Zealand and California (2,3). Tissue from four symptomatic plants of cv. Spitfire and two of cv. Celebrity were collected and DNA was extracted from midribs and petioles with a FastDNA Spin Kit (Qbiogene, Inc., Carlsbad, CA,). PCR amplification was done with 16S rRNA gene primers OA2 and OI2c, which are specific for “Ca. Liberibacter solanacearum” from potato and tomato and amplify a 1.1-kb fragment of the 16S rRNA gene of this new species (1,3). Amplicons of 1.1 kb were obtained from all samples and these were sequenced in both orientations (McLab, San Francisco, CA). Sequences of the 16S rRNA gene were identical for both Spitfire and Celebrity and were submitted to the NCBI as GenBank Accession Nos. FJ939136 and FJ939137, respectively. On the basis of a BLAST search, sequence alignments revealed 99.9% identity with a new species of ‘Ca. Liberibacter’ from potato (EU884128 and EU884129) in Texas (1); 99.7% identity with the new species “Ca. Liberibacter solanacearum” described from potato and tomato (3) in New Zealand (EU849020 and EU834130, respectively) and from the potato psyllid Bactericera cockerelli in California (2) (EU812559, EU812556); 97% identity with ‘Ca L. asiaticus’ from citrus in Malaysia (EU224393) and 94% identity with both ‘Ca. L. africanus’ and ‘Ca. L. americanus’ from citrus (EU921620 and AY742824, respectively). A neighbor-joining cladogram constructed using the 16S rRNA gene fragments delineated four clusters corresponding to each species, and these sequences clustered with “Ca. L. solanacearum”. A second PCR analysis was conducted with the CL514F/CL514R primer pair, which amplifies a sequence from the rplJ and rplL ribosomal protein genes of “Ca. L. solanacearum”. The resulting 669-bp products were 100% identical to a sequence reported from tomato in Mexico (FJ498807). This sequence was submitted to NCBI (GU169328). ZC, a disease causing losses to the potato industry, is associated with a ‘Candidatus Liberibacter’ species (1–3) and was reported in Central America and Mexico in the 1990s, in Texas in 2000, and more recently in other states in the United States (4). In 2008, a “Ca. Liberibacter solanacearum” was detected on Capsicum annuum, S. betaceum, and Physalis peruviana in New Zealand (3). Several studies have shown that the potato psyllid, B. cockerelli, is a potential vector for this pathogen (2,4). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” in field tomatoes showing ZC-like foliar disease symptoms in the United States. 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. 93:208, 2009. (4) G. A. Secor et al. Plant Dis. 93:574, 2009.

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.