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4866

In this study, two polyclonal antibodies were produced against the Omp protein of ‘Candidatus Liberibacter asiaticus’. First, omp genes were sequenced to exhibit 99.9% identity among 137 isolates collected from different geographical origins. Then, two peptides containing the hydrophobic polypeptide-transport-associated (POTRA) domain and β-barrel domain, respectively, were identified on Omp protein. After that, these two peptides were overexpressed in Escherichia coli and purified by affinity chromatography to immunize the white rabbits. Finally, the antiserum was purified by affinity chromatography. The two Omp antibodies gave positive results (0.454 to 0.633, 1:1,600 dilution) in enzyme-linked immunosorbent assay against ‘Ca. L. asiaticus’-infected samples collected from different geographical origins but revealed negative results against other pathogen-infected, nutrient-deficient and healthy samples. The antibody against the POTRA domain of Omp protein could detect ‘Ca. L. asiaticus’ in 45.7% of the symptomatic samples compared with a 56.2% detection rate with a polymerase chain reaction assay. These new antibodies will provide a very useful supplement to the current approaches to ‘Ca. L. asiaticus’ detection and also provide powerful research tools for tracking distribution of this pathogen in vivo.

A novel halotolerant actinomycete, designated strain BNT52T, was isolated from soil collected from Cihanbeyli Salt Mine in the central Anatolia region of Turkey, and examined using a polyphasic taxonomic approach. The isolate was found to have chemical and morphological properties typical of the genus Amycolatopsis and formed a distinct phyletic line in the 16S rRNA gene tree. Strain BNT52T was most closely related to Amycolatopsis nigrescens CSC17Ta-90T (96.7 %), Amycolatopsis magusensis KT2025T (96.6 %), Amycolatopsis sulphurea DSM 46092T (96.6 %), Amycolatopsis dongchuanensis YIM 75904T (96.5 %), Amycolatopsis ultiminotia RP-AC36T (96.4 %) and Amycolatopsis sacchari DSM 44468T (96.4 %). Sequence similarities with other strains of species of the genus Amycolatopsis were lower than 96.2 %. The isolate grew at 20–37 °C, pH 6–12 and in the presence of 0–10 % (w/v) NaCl. The cell wall of the novel strain contained meso-diaminopimelic acid and arabinose and galactose as the diagnostic sugars. Major fatty acids were iso-C16 : 0 2-OH and iso-C16 : 0. The predominant menaquinone was MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylmethylethanolamine. The genomic DNA G+C content was 68.8 mol%. On the basis of the data from this polyphasic taxonomic study, strain BNT52T represents a novel species within the genus Amycolatopsis for which the name Amycolatopsis cihanbeyliensis sp. nov. is proposed (type strain BNT52T = KCTC 29065T = NRRL B-24886T = DSM 45679T).

In April of 2012, tobacco (Nicotiana tabacum) plants with symptoms resembling those caused by viral infection were observed in commercial fields in several departments in Nicaragua, including Esteli and Nueva Segovia. Heavy infestations of the psyllid Bactericera cockerelli, a major insect pest of potato and other solanaceous crops and vector of the bacterium “Candidatus Liberibacter solanacearum” (Lso) (2,3), were observed in the affected fields. All cultivars grown were affected and 5 to 100% of plants in each field were symptomatic. Symptoms on affected plants included apical leaf curling and stunting, overall chlorosis and plant stunting, young plant deformation with cabbage-like leaves, wilting, internal vascular necrosis of stems and leaf petioles, and overall poor leaf quality. Plant samples were collected from a total of three psyllid-infested fields in the municipalities of Esteli, Condega, and Jalapa (one field/municipality). The plant samples were first tested for viruses, including Potato virus Y, Tobacco mosaic virus, Cucumber mosaic virus, and Impatiens necrotic spot virus, using Immunostrips (Agdia, Elkhart, IN) and no virus was detected. Total DNA was extracted from leaf tissues of a total of 22 plants, including 17 symptomatic plants and five asymptomatic plants from two cultivars (Corojo and Habano) with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (2,4). The DNA samples were tested by PCR using specific primer pairs OA2/OI2c and OMB 1482f/2086r, to amplify a portion of 16S rDNA and the outer membrane protein (OMB) genes, respectively, of Lso (2). 16 rDNA and OMB gene-derived fragments of 1,168 and 605 bp, respectively, were amplified from the DNA of 13 of 17 (76.5%) symptomatic plants, indicating the presence of Lso. No Lso was detected in the five asymptomatic plants. DNA amplicons of three plant samples (one plant/field) with each primer pair were cloned and two to four clones of each of the six amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences was the same for all three locations (GenBank Accession Nos. KC768323, KC768324, and KC768325) and showed 100% identity to numerous 16 rDNA sequences of Lso in GenBank, including accessions HM245242, JF811596, and JX559779. Similarly, identical OMB consensus sequences were observed in all three locations (KC768331 and KC768332 for Jalapa and Condega, respectively) that are 97 to 100% identical to a number of Lso sequences in GenBank (e.g., CP002371, FJ914617, JN848754, and JN848752). A second OMB sequence was isolated from the Esteli sample (KC768333) that was 98% identical with the consensus sequences from this and other sites and 100% identical to an OMB sequence from Lso in GenBank (JN848754). To our knowledge, this is the first report of Lso associated with tobacco. Tobacco is an important crop in many parts of the world, including Central and South America. This bacterium has also caused millions of dollars in losses to potato and several other solanaceous crops in the Americas and New Zealand (3). In addition, this plant pathogen has been reported as serious pest of carrot in Europe, where it is associated with the psyllids Trioza apicalis and B. trigonica (1,4). References: (1) A. Alfaro-Fernandez et al. Plant Dis. 96:581, 2012. (2) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (3) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012. (4) J. E. Munyaneza et al. J. Econ. Entomol. 103:1060, 2010.