Publications

4483

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.

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.

An obligate anaerobic, Gram-staining-negative, mesophilic, cellulolytic bacterium, strain H1T, was isolated from the rumen content of yak. Cells were straight to slightly curved rods, 0.8–1.0×3.0–4.0 μm in size, non-motile and encapsulated with mucous materials. Elliptical and terminal spores that swelled the cells were produced occasionally. The strain grew at 25–45 °C (optimum, 38 °C) and pH 6.0–7.8 (optimum, pH 6.7). Cellulose, cellobiose, xylan, xylose and maltose were used as carbon and energy sources, but not glucose. Products from cellulose and cellobiose fermentation were formic acid, acetic acid, carbon dioxide and trace amounts of ethanol, lactic acid and succinic acid. The genomic DNA G+C content was 33.7±1.2 mol%. The predominant fatty acids were C16 : 0(27.1 %), C14 : 0(9.2 %) and iso-C16 : 0(6.4%). Based on the 16S rRNA gene sequence analysis, strain H1Twas affiliated to the clostridial rRNA cluster XIVb and showed the highest 16S rRNA gene sequence similarity toClostridium lentocellumDSM 5427T(96.0 %). These two strains formed a distinct lineage of the family ‘Lachnospiraceae’. Based on data from this polyphasic taxonomic study, a new genus,Cellulosilyticumgen. nov., is proposed.Cellulosilyticum ruminicolasp. nov. is proposed for strain H1T. The type strain ofCellulosilyticum ruminicolasp. nov. is strain H1T(=CGMCC 1.5065T=JCM 14822T).Clostridium lentocellumwas reclassified in the new genus asCellulosilyticum lentocellumcomb. nov. (type strain RHM5T=ATCC 49066T=DSM 5427T=NCIMB 11756T).

From 2006 to 2009, all commercial potato fields in Azacualpa F.M. Honduras were heavily infested with the potato psyllid Bactericera cockerelli (Sulc.). Plants exhibited interveinal chlorosis, vein-greening, downward curling, stunting, above ground tuber formation, and brownish flecks in some tubers. Disease incidence ranged from 50 to 95%. Leaf samples and psyllids were collected from seven fields in two potato-growing regions of Honduras. Total DNA was purified from the leaves of 30 symptomatic and three asymptomatic plants. DNA was extracted from 20 adult and 10 immature (4th to 5th instar) psyllids according to Frohlich et al (1). PCR primers, PSY680F 5′-GTTCGGAATAACTGGGCGTA-3′ and PSY1R 5′-CCCATAAGGGCCATGAGGACT-3′, were used to amplify a 680-bp fragment of the 16S rDNA for the recently described “Candidatus Liberibacter physallaurous” (2) and “Ca. L. solanacearum” (3). PSY1R/PSY680F primer design was based on the association of a previously undescribed liberibacter with vein-greening symptoms in greenhouse tomato plants in Arizona from 2006 to 2007 (GenBank Accession No. GQ926918) that lead to the hypothesis that a similar bacterium could be associated with symptomatic potato plants in Honduras. PCR amplification, cloning, and sequencing of the resultant 16S rDNA amplicons indicated that 17 of 30 potato plants, 8 of 20 adult and 7 of 10 third to fourth instar psyllids, respectively, were positive for liberibacter based on 99 to 100% shared nucleotide sequence (nt) identity with the analogous sequence from liberibacter (EU812558 [2]). To substantiate these results, a second molecular marker was targeted using the 1611F and 480R primers (~980 bp) that amplify the 16S-23S-ITSrDNA of liberibacter (2) for selected liberibacter-positive samples (above). Amplicons of the expected size were obtained from 12 of 17 potato and 7 of 10 immature psyllids. No PCR product of the expected size was obtained from asymptomatic potato samples or the PCR negative (water) control. The resultant PCR amplicons were cloned and 12 to 15 clones per amplicon were sequenced. The sequences were aligned and the percentage pair wise nt identity was calculated by Clustal W revealing that the 16S rDNA and 16S-23S-ITS sequences, respectively, shared 99 to 100% nt identity with each other. BLAST analysis against the NCBI database indicated that the 16S rRNA sequences from potato plants (GQ926922) and immature psyllids (GQ926923), and the 16S-ITS-23S sequence from potato plants (GQ926924) and immature psyllids (GQ926925), shared 98.5 to 100% nt identity with ‘Ca. Liberibacter’ reported from potato (EU812556; [2,4]) and tomato (EU812558, EU812559, EU935005; [2,3]). Evidence for the widespread presence of liberibacter and the potato psyllid in potato fields in Honduras, together with foliar and tuber symptoms that are reminiscent of those recently described in potato plants in the United States affected with ‘zebra chip’ disease (4), suggest that a similar or identical disease of the potato also occurs in Honduras. This emergent disease poses a serious threat to potato production in Honduras and elsewhere in Central America. References: (1) D. R. Frohlich et al., Mol. Ecol. 8:1683, 1999. (2) A. K. Hansen et al. Appl. Environ. Microbiol. 78:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007.