Publications

4428

Abstract: We collected 209 Ixodes persulcatus ticks in various regions of Russia, including the southern Urals and western and eastern Siberia. Using PCR amplification and sequencing of the citrate synthase‐encoding gene (gltA), we detected a new rickettsial genotype, which we named ‘Candidatus Rickettsia tarasevichiae.’ This bacterium was found in 9.27%, 10.0%, and 20.5% of the ticks collected in western Siberia, eastern Siberia, and the southern Urals, respectively. ‘Candidatus Rickettsia tarasevichiae’ exhibited a 98% and 96% nucleotide sequence homology, with the 16S rDNA and gltA sequence, respectively, of R. canadensis, a rickettsia previously only found in Haemaphysalis leporispalustris ticks in North America. The phylogenetic analysis of ‘Candidatus Rickettsia tarasevichiae’ and other Rickettsia species allowed the creation of a new cluster with high bootstrap values within the Rickettsia genus involving this rickettsia, R. canadensis, and three uncultured rickettsiae from plant insects.

During July 2002, surveys of mandarin orchards were conducted in Punakha Valley and Wangdue districts of Bhutan. Symptoms of the greening disease were observed in most of the orchard. The incidence of disease was recorded up to 30% in 24 private orchards with more than 5,000 trees total. Affected trees were generally stunted with leaves showing symptoms of mottling. Sometimes, symptoms were seen only on one part of the canopy. The greening disease is caused by a fastidious phloem restricted bacterium, “Candidatus Liberibacter asiaticus” in Asian countries and “Candidatus Liberibacter africanus” in African countries. To confirm the presence of this bacterium causing greening disease in Bhutan, 33 leaf samples were collected from seven locations in Bhutan and stored at -80°C. Petioles and midribs were used for extraction of DNA using DNeasy Plant Mini Kit (Qiagen Gmbh, Hilden, Germany). Polymerase chain reaction (PCR) was initially performed with a sample from Rimchu, Bhutan using primer pair 5′TATAAAGGTTGACCTTTCGAGTTT/5′ACAAAAGCAGAAATAGCACGAACAA previously designed for amplification of ribosomal protein genes of β-operon of two liberibacter species (1). An amplicon of approximately 700 bp was obtained. The size of the PCR product is similar to that amplified from “Candidatus Liberibacter asiaticus”. The amplicon was cloned in pGEM-T easy vector and sequenced. The clone was 703 nt long and showed 100% sequence homology with the corresponding sequence of “Candidatus Liberibacter asiaticus” confirming that “Candidatus Liberibacter asiaticus” is the cause of greening disease in Bhutan. Later, one sample from each location was analyzed and found to be positive to greening. To our knowledge, this is the first report of this bacterium and greening disease in Bhutan, and citrus greening appears to be the main cause of declining citrus in the Punakha Region of Bhutan. Reference: (1) A. Jocquellet et al. Page 363 in: Proc. Conf. Int. Organ. Citrus Virol. 14th. IOCV, Riverside, CA, 2000.