Publications

4369

Based on an earlier survey of putative psyllid vectors of apple proliferation (AP), carried out in 2009 and 2010, Cacopsylla picta (Förster) populations infected with ‘Candidatus Phytoplasma mali’ were detected in at least two commercial apple (Malus domestica Borkh.) orchards in southern Finland (1). To establish the presence of ‘Ca. P. mali’ in apple trees, a survey was conducted in 17 commercial apple orchards in August 2012. Phytosanitary inspectors tracked the source of the ‘Ca. P. mali’ by collecting 33 leaf samples from trees showing probable symptoms. Typical symptoms, including elongated stipules and witches' broom, were rare. Total DNA was extracted from leaves using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and screened for ‘Ca. P. mali’ with real-time PCR (2) and the commercial Apple Proliferation Group – complete PCR reaction kit (Loewe Biochemica GmbH, Sauerlach, Germany). Two samples tested positive and results were confirmed with TaqMan PCR and conventional PCR assays and DNA sequencing in the Food and Environment Research Agency (Fera), in the United Kingdom. One positive sample was taken from an orchard in Lohja, southern Finland, where high ‘Ca. P. mali’ incidence in overwintered C. picta was observed in 2010 (1). ‘Ca. P. mali’ was found in a >40-year-old ‘Red Melba’ tree with witches' broom but without elongated stipule symptoms. The other positive sample was collected from an orchard in the Aland Islands, where the infected ‘Lobo’ tree showed symptoms of elongated stipules. This orchard was not monitored for AP vectors. No small fruit symptoms were noted by inspectors or growers in either of the orchards. The positive samples were further analyzed for subtypes using PCR/RFLP and primers AP13/AP10 (3). The amplicons (776 bp) were sequenced and digested with HincII and BspHI (New England BioLabs Inc., Ipswich, MA) following manufacturer's instructions. Both samples proved to be apple proliferation subtypes AT-1 on the basis of RFLP and the sequenced 776-bp region. Sequences of the 776-bp amplicon of the Lohja and Aland isolates showed 100% and 99% identity, respectively, with sequences of apple proliferation isolates (accession nos. L22217.1 and CU469464.1) in GenBank. Both suspected psyllid vectors of ‘Ca. P. mali’ C. picta and C. melanoneura (Förster) occur in Finland, but their distribution, abundance, and transmission specificity is inadequately documented. The next step to evaluate the risk of spread of apple proliferation in commercial orchards is an extensive survey of the occurrence of Cacopsylla species infected with ‘Ca. P. mali’. References: (1) A. Lemmetty et al. B. Insectol. 64:257, 2011. (2) P. Nikolić et al. Mol. Cell. Probes. 24:303, 2010. (3) W. Jarausch et al. Mol. Cell. Probes 14:17, 2000.

A novel anaerobic bacterium that could ferment amino acids and organic acids was isolated from an anaerobic, propionate-oxidizing enrichment culture originating from soil of a rice field in Japan. Cells of the isolate, designated strain 4F6ET, were Gram-staining-negative, oxidase- and catalase-negative, non-spore-forming, vibrio-shaped, motile rods (0.8×2.0–2.5 µm) with two or three lateral flagella. Growth occurred at 20–42 °C (optimum at 37–40 °C), at pH 6.4–8.4 (optimum at pH 7.3) and at 0–1.5 % (w/v) NaCl (optimum at 0–0.5 %). Good growth occurred on glycine, serine, cysteine, pyruvate and citrate, whereas poor growth was observed on threonine, glutamine, l-malate, α-ketoglutarate, peptone and Casamino acids. In co-culture with the hydrogen-utilizing methanogen Methanobacterium formicicum JCM 10132T, strain 4F6ET oxidized alanine, valine, leucine, isoleucine, methionine, aspartate, glutamate, histidine, asparagine and fumarate. Yeast extract was required for growth. The G+C content of genomic DNA was 61.9 mol%. A phylogenetic analysis based on comparison of the 16S rRNA gene sequence showed that the type strains of Fretibacterium fastidiosum , Aminobacterium colombiense and Aminobacterium mobile , members of the family Synergistaceae , were the closest relatives of strain 4F6ET, with low sequence similarities (89.3, 89.5 and 86.2 %, respectively). Strain 4F6ET contained iso-C13 : 0 (24.43 %), iso-C15 : 0 (16.47 %) and C19 : 1ω11c/C19 : 1ω9c (16.32 %) as the major fatty acids, which differed from those of F. fastidiosum , Aminobacterium colombiense and Aminobacterium mobile . On the basis of phenotypic, chemotaxonomic and phylogenetic differences between strain 4F6ET and the type strains of F. fastidiosum and Aminobacterium species, we propose that strain 4F6ET represents a novel genus and species, Aminivibrio pyruvatiphilus gen. nov., sp. nov. The type strain of Aminivibrio pyruvatiphilus is strain 4F6ET ( = JCM 18417T = DSM 25964T).

Tomato (Lycopersicum esculentum) crops grown in several departments of Honduras and heavily infested with the psyllid Bactericera cockerelli were observed in April of 2012 with plant symptoms suggestive of “Candidatus Liberibacter solanacearum” infection. B. cockerelli is a serious pest of potato, tomato, and other solanaceous plants and a vector of “Ca. L. solanacearum” (1,2,3,4). The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (2,3). Disease incidence ranged from 5 to 50% symptomatic plants per field. Tomato (cv. Pony) plant samples were collected from two psyllid-infested commercial fields in the municipalities of Danli and Comayagua in the departments of El-Paraiso and Comayagua, respectively. Total DNA was extracted from leaf tissues of 50 and 20 symptomatic and asymptomatic plants, respectively, with the cetyltrimethylammonium bromide (CTAB) buffer extraction method (1,3). The DNA samples were tested for “Ca. L. solanacearum” by PCR with primer pairs specific for 16S rDNA (OA2 and OI2c) and the outer membrane protein gene (OMB 1482f and 2086r) of the bacterium (1,2). Ten (20%) of the 50 symptomatic tomato samples were positive for “Ca. L. solanacearum” using both primer pairs and the remaining samples were negative for the bacterium with both primer sets. None of the 20 asymptomatic plants tested positive for “Ca. L. solanacearum”. Amplicons from DNA of two plant samples (one plant/municipality) with each primer pair were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences from the clones (deposited in GenBank as Accession Nos. KC768321 and KC768322) were identical for both locations and showed 99 to 100% identity to several “Ca. L. solanacearum” sequences in GenBank (e.g., JN848753, JN84856, and HM246509). The OMB consensus sequences from the two tomato plants (deposited in GenBank as KC768329 and KC768330) were 100% identical to OMB sequences of Lso in GenBank (CP002371 and JN48754, respectively). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” associated with tomato crops in Honduras. This bacterium has caused millions of dollars in losses to the tomato industry in the United States, Mexico, and New Zealand (2,3,4). Serious damages to tomato crops due to “Ca. L. solanacearum” could expand throughout Central America, especially in those countries where B. cockerelli occurs. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009. (4) J. E. Munyaneza. Am. J. Pot. Res. 89:329, 2012.

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, tomato plants (Solanum lycopersicum) grown near the town of Yuroconte in the municipality of La Palma, Chalatenango, El Salvador, were observed with symptoms resembling those of “Candidatus Liberibacter solanacearum” infection. The symptoms included overall chlorosis, severe stunting, leaf cupping, excessive branching of axillary shoots, and leaf purpling and scorching (1,2,3). Disease incidence in several fields in the area ranged from 40 to 60%. Heavy infestations of the potato/tomato psyllid, Bactericera cockerelli, were observed in the affected fields and this insect has been shown to transmit “Ca. L. solanacearum” to tomato and other solanaceous species (1,2,3). Leaf samples and psyllids were collected from one of the fields and total DNA was purified from the leaves of 8 and 10 symptomatic and asymptomatic plants, respectively (2,3). DNA was also extracted from the psyllids and the samples were tested by PCR for species confirmation. PCR oligonucleotide primers specific for both 16S rDNA (OA2 and OI2c) and a gene for a surface antigen for the outer membrane protein (OMB) (OMB 1482f and 2086r) of “Ca. L. solanacearum” were used to confirm the presence of the bacterium in infected tomatoes (1). Four of the eight symptomatic tomatoes (50%) tested positive for “Ca. L. solanacearum” using both primer pairs and all asymptomatic plants were negative for the bacterium. The collected psyllids were first identified through a morphological key, then verified using species-specific PCR primers (CO1 F3 and CO1 meltR) that generated a 94-bp fragment that was consistent with DNA from B. cockerelli (4). Amplicons generated with DNA from two plant samples with each primer pair were cloned and four clones of each of the four amplicons were sequenced. BLASTn analysis of the 16S rDNA consensus sequences from the clones (1,168 bp; deposited in GenBank as Accession Nos. KC768318 and KC768319) showed 100% identity to “Ca. L. solanacearum” sequences in GenBank (HM246509 and HM245242, respectively). Two OMB consensus sequences were 98% identical (deposited in GenBank as KC768326 and KC768327) and both sequences were 97 to 100% identical to a number of “Ca. L. solanacearum” sequences in GenBank (e.g., CP002371, FJ914617, JN848754, and JN848752). To our knowledge, this is the first report of “Ca. L. solanacearum” associated with tomato in El Salvador and the first formal report of the bacterium in the country. This bacterium has caused millions of dollars in losses to the tomato industry in New Zealand, Mexico and the United States (2,3). Tomatoes are an economically important commodity in Central America and are severely damaged by “Ca. L. solanacearum” infection. The confirmation of “Ca. L. solanacearum” infections in El Salvador alerts the agricultural sector to the presence of this serious pathogen. References: (1) J. M. Crosslin. Southwest. Entomol. 36:125, 2011. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. Plant Dis. 93:1076, 2009. (4) K. D. Swisher et al. Environ. Entomol. 41:1019, 2012.

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.