Publications

4937

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.

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).