First Report of a 16SrI-B Group Phytoplasma Associated with a Yellows-Type Disease Affecting Tomato Plants in the Baja California Peninsula of Mexico.

Since 2000, a phytoplasma-like disease (locally known as "permanent yellowing") was observed on tomatoes (Lycopersicon esculentum Mill.) grown in the Valle de San Quintín in northern Baja California Peninsula. Affected plants showed general chlorosis, severe stunting, upwardly rolled leaves, bronzing of mature leaves, purple discoloration of veins, "little leaf", abnormal floral structures, and excessive branching of axillary shoots. Total DNA extracted from symptomatic and asymptomatic plants was used in nested (n)-PCR assays driven by phytoplasma-universal primer pair P1/P7 (3), followed by primer pair R16F2n/R16R2 (1) targeting the 16S ribosomal RNA gene of the putative phytoplasma. PCR conditions (direct and nested) were conducted as previously described (l,3). Restriction fragment length polymorphism (RFLP) patterns of nPCR-amplified products (≈ 1.25-kbp 16S rDNA fragments) digested with enzymes AluI, MseI, HhaI, and HpaII showed that 85% (17 of 20) of PCR-positive tomato samples had restriction patterns typical of phytoplasmas belonging to the aster yellows group, subgroup B (16SrI-B) "Candidatus Phytoplasma asteris" (2). Only 10% (2 of 20) of the samples were associated with a phytoplasma related to the 16SrXIII-A Mexican periwinkle virescence group (formerly group 16SrI, subgroup I). None of the symptomless plants tested positive. Subsequently, these results were confirmed by nPCR using 16SrI specific primer pair P1/Aint (4) and specific primers rp(I-B)F1/rp(I-B)R1 that amplify the ribosomal protein (rp) gene operon of aster yellows phytoplasma subgroup B (16SrI-B[rp-B]) (1). The presence of the phytoplasmas in symptomatic plants was confirmed by scanning electron microscopy. Characteristic yellow symptoms could be experimentally reproduced by graft inoculation of tomato seedlings (cv. Maya) with tissue of field-infected plants. Symptoms similar to those of field-grown diseased plants were observed consistently in most of the plants, and when graft transmitted from tomato to periwinkle (Catharantus roseus (L.) G. Don), symptoms of virescencent, small flowers were observed. In contrast, no symptoms were observed on plants grafted with tissues from healthy plants. In Baja California, it appears that at least two distinct phytoplasmas are involved in the disease complex. To our knowledge, this is the first molecular evidence of the presence of a phytoplasma associated with yellows-type diseases in the major tomato cultivation areas of the peninsula. References: (1) I.-M. Lee et al. Phytopathology 93:1368, 2003. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) B. Schneider et al. Page 369 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, San Diego, CA, 1995. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.