Publications

4729

ABSTRACT An immunocompromised patient presented with febrile episodes, an erysipelas-like rash, and thromboembolic complications. Amplification of 16S rRNA gene sequences from blood and sequence analysis revealed “ Candidatus Neoehrlichia mikurensis.” We report the first case of human disease caused by “ Ca . Neoehrlichia mikurensis.”

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.

A Gram-negative, aerobic, rod-shaped bacterium, designated strain CL-33T, was isolated from the encrusting pore coral Montipora aequituberculata collected from seawater off the coast of southern Taiwan. 16S rRNA gene sequence analysis showed that the strain clustered closely with Endozoicomonas elysicola MKT110T (96.7 % similarity). The novel strain required NaCl for growth and exhibited optimal growth at 25 °C and in the presence of 2–3 % NaCl. Predominant cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 39.6 %), summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c; 32.8 %) and C16 : 0 (12.0 %). The DNA G+C content of strain CL-33T was 50.0 mol%. The results of physiological and biochemical tests allowed the clear phenotypic differentiation of this isolate from E. elysicola. It is evident from the genotypic, phenotypic and chemotaxonomic data presented that strain CL-33T represents a novel species of the genus Endozoicomonas, for which the name Endozoicomonas montiporae sp. nov. is proposed. The type strain is CL-33T (=LMG 24815T =BCRC 17933T).

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.