First Report of a New Phytoplasma Subgroup, 16SrIII-T, Associated with Decline Disease Affecting Sweet and Sour Cherry Trees in Lithuania.

During July 2007, sweet (Prunus avium) and sour cherry (P. cerasus) trees exhibiting disease symptoms suggestive of possible phytoplasma infection were observed in a large orchard in the Kaunas Region of Lithuania. Samples of leaf tissue were collected from 13 sweet cherry trees that were affected by a decline disease (designated cherry decline, ChD) characterized by symptoms that included leaf reddening and premature leaf drop and two sour cherry trees exhibiting proliferation of branches and nonseasonal flowering. To assess the diseased trees for phytoplasma infection, DNA was extracted with a Genomic DNA Purification Kit (Fermentas, Vilnius, Lithuania) and used as template in nested PCRs, primed by phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 for amplification of 16S ribosomal (r) DNA sequences (1,2). The 1.2-kbp DNA sequences amplified from all 15 trees were subjected to restriction fragment length polymorphism (RFLP) analyses with AluI, MseI, KpnI, HhaI, HaeIII, HpaII, RsaI, HinfI, TaqI, Sau3AI, and BfaI. The collective profiles indicated that DNAs were derived from two different phytoplasmas. One of them, designated ChD phytoplasma, was found in 11 sweet cherry trees and two sour cherry trees and tentatively classified as a member of new subgroup designated 16SrIII-T in 16S rDNA RFLP group 16SrIII (X-disease phytoplasma group). It was observed that the ChD phytoplasma caused different symptoms in sweet and sour cherry. The amplified ChD phytoplasma 16S rDNA was cloned in Escherichia coli, sequenced, and the sequence deposited in the GenBank database (Accession No. FJ231728). The ChD phytoplasma 16S rDNA shared 98.4 and 98.6% sequence identity with the 16S rDNAs from stone fruit-infecting phytoplasmas associated with western X-disease (GenBank Accession No. L04682) and Canada X-disease (GenBank Accession No. L33733), respectively, indicating that the three strains are closely related. Interestingly, the ChD phytoplasma 16S rDNA shared 99.8% sequence identity with 16S rDNA from one operon (rrnB, GenBank Accession No. AF370120) from a phytoplasma previously found to be associated with dandelion virescence (DanVir) disease in Lithuania. The operon rrnA (GenBank Accession No. AF370119) shared 99.3% sequence identity (2). The high similarity of the ChD 16S rRNA gene sequence to that of DanVir rrnB suggests the possibility that ChD and DanVir may belong to a single phytoplasma species and that dandelion is possibly an alternate host of ChD phytoplasma. The other phytoplasma, found in two sweet cherry trees, was classified in subgroup 16SrI-B of 16S rDNA RFLP group 16SrI ('Candidatus Phytoplasma asteris' and related strains) and was designated cherry proliferation phytoplasma (GenBank Accession No. FJ231729). Thus, in Europe, cherry may be affected by diseases associated with phytoplasmas belonging to groups 16SrI, 16SrIII, 16SrX, and 16SrXII (3,4). The infections by diverse phytoplasma strains and species underscore the need for production of phytoplasma-free planting stock and for intensified research to reduce ecological and economic impacts of these phytoplasmas. References: (1) D. E. Gunderson and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) R. Jomantiene et al. Eur. J. Plant Pathol. 108:507, 2002. (3) S. Paltrinieri et al. Acta Hortic. 550:365, 2001. (4) D. Valiunas et al. J. Plant Pathol. 91:71. 2009.