Publications

3806

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.

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 H1T was affiliated to the clostridial rRNA cluster XIVb and showed the highest 16S rRNA gene sequence similarity to Clostridium lentocellum DSM 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, Cellulosilyticum gen. nov., is proposed. Cellulosilyticum ruminicola sp. nov. is proposed for strain H1T. The type strain of Cellulosilyticum ruminicola sp. nov. is strain H1T (=CGMCC 1.5065T=JCM 14822T). Clostridium lentocellum was reclassified in the new genus as Cellulosilyticum lentocellum comb. nov. (type strain RHM5T=ATCC 49066T=DSM 5427T=NCIMB 11756T).

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.