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First Report of Phytoplasmas in Soybean, Alfalfa, and Lupinus sp. in Lithuania

Plants of cultivated soybean (Glycine max) and alfalfa (Medicago sativa) in Dotnuva and of wild Lupinus sp. in Ledakalnis, Lithuania, exhibited symptoms that suggested phytoplasmal infections. Soybean plants were of normal growth habit but exhibited veinal necrosis. Alfalfa and Lupinus plants exhibited stunting, abnormally small leaves, and witches'-broom symptoms. Diseases in the plants were termed soybean veinal necrosis (SVN), alfalfa stunt (AlfS), and Lupinus stunt (LupS), respectively. The presence of phytoplasmas in diseased plants was assessed using polymerase chain reaction (PCR) for amplification of phytoplasma-specific 16S rDNA. A phytoplasma-characteristic 1.2-kbp DNA fragment was amplified from all diseased plants but not from known healthy plants in nested PCRs in which the first DNA amplification was primed by primer pair P1/P7 and reamplification of DNA was primed by primer pair F2n/R2 (2,4). Products from the nested PCR primed by F2n/R2 were subjected to restriction fragment length polymorphism (RFLP) analysis, and the RFLP patterns obtained were compared with patterns previously published (1--4). On the basis of AluI, HaeIII, HhaI, HpaI, KpnI, MseI, and RsaI RFLP patterns, the SVN and LupS phytoplasmas were classified in group 16SrIII (peach X-disease phytoplasma group), subgroup B (III-B, type strain clover yellow edge phytoplasma), and the AlfS phytoplasma was classified in group 16SrI (aster yellows phytoplasma group), subgroup B (I-B, type strain aster yellows phytoplasma). Nucleotide sequences were determined for 16S rDNA fragments amplified from SVN and AlfS phytoplasmas in nested PCRs primed by F2n/R2. The sequences were deposited in GenBank under Accession nos. AF177383 for SVN and AF177384 for AlfS. Sequence similarity between the 16S rDNAs of SVN and Canadian clover yellow edge (strain CYE-C, GenBank Accession no. AF175304) phytoplasmas was 99.8%; sequence similarity between 16S rDNAs of AlfS and aster yellows (strain SAY, GenBank Accession no. M86340) phytoplasmas was 99.6%. The SVN phytoplasma 16S rDNA shared 100% sequence similarity with a 16S rDNA from the Lithuanian clover yellow edge (CYE-L, GenBank Accession no. AF173558) phytoplasma. The nucleotide sequence data supported the conclusion that the SVN and AlfS phytoplasmas were closely related to strains classified in subgroups III-B and I-B, respectively. Our findings extend the known geographic ranges of phytoplasma subgroups I-B and III-B to northern Europe, including Lithuania, and expand the known plant host ranges of these pathogens.

References: (1) R. E. Davis et al. Int. J. Syst. Bacteriol. 47:262, 1997. (2) R. Jomantiene et al. Int. J. Syst. Bacteriol. 48:269, 1998. (3) R. Jomantiene et al. HortScience 33:1069, 1998. (4) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998.