A phytoplasma associated with ‘amachamiento’ disease of dry common bean in Costa Rica

Dry common bean plants (Phaseolus vulgaris) from the main productionregionsof Costa Rica have been affected by a disease, locally called ‘amachamiento’. Main symptoms are a severe loss of bean pods due to flowering reduction or abortion, interveinal chlorosis, deformed leaves with corrugated midrib (Fig. 1); with diseased plants remaining green at harvest. Morales et al. (1999) associated ‘amachamiento’ to Cowpea chlorotic mottle virus (CCMV), but CCMV was shown not to induce such symptoms in bean (Gámez, 1976). A total of 104 plants with symptoms were collected and analyzed by DAS-ELISA for CCMV, and also three comoviruses and five potyviruses commonly infecting P. vulgaris. Negative DAS-ELISA results were shown by 57% of plants for all viruses studied. Nested PCR with universal 16S rRNA phytoplasma primers (P1/P7 and R16R2-R16F2n) was used to search for phytoplasma in those symptom-bearing plants negative for DAS-ELISA, and three symptomless plants. R16R2-R16F2n PCR products of 1.2-kb were amplified from more than 60 plants with symptoms, but not from the symptomless plants. RFLP patterns with RsaI, HhaI, KpnI, BfaI, HaeIII, HpaII, AluI, and MseI characterized the ‘amachamiento’ phytoplasma as a member of group 16SrI, ‘Candidatus Phytoplasma asteris’, subgroup 16SrI-B. BLAST comparisons of the 16S rRNA sequence of the ‘amachamiento’ phytoplasma (GenBank Accession No. FJ603645)from two cloned PCR amplicons (TA Cloning kit, Invitrogen), showed a 99% of identity with those of 16SrI phytoplasmas (Fig. 2). However, they were distantly related to the dry bean phyllody phytoplasma from Washington (AY496003), and those 16SrI phytoplasmas recorded in chayote (Sechium edule) and Erythrina poeppigiana in Costa Rica (Villalobos et al., 2002; Saborío-R et al., 2007). This is the first report of a phytoplasma associated with ‘amachamiento’ disease in Costa Rica.