First Report of "Candidatus Liberibacter solanacearum" in Tomato Plants in México.

Tomato (Solanum lycopersicum) plants exhibiting symptoms resembling those of permanent yellowing disease (known in Mexico as "permanente del tomate") that is commonly associated with phytoplasmas (1) were observed in tomato fields in Sinaloa, México in March 2009. Plant symptoms also resembled those caused by "Candidatus Liberibacter solanacearum" infection (2). Affected plants showed an overall chlorosis, severe stunting, leaf cupping, purple discoloration of veins, excessive branching of axillary shoots, and leaf scorching (1,2). Symptom incidence ranged from 18 to 40%. To investigate whether liberibacter is associated with permanent yellowing disease of tomato in México, eight symptomatic and five asymptomatic tomato plants were collected from two fields in La Cruz de Elota and Culiacán, Sinaloa. Total DNA was extracted from the top whole leaf tissue of symptomatic and asymptomatic plants with cetyltrimethylammoniumbromide (CTAB) buffer (3,4). DNA samples were tested by PCR using primer pairs OA2/OI2c and CL514F/CL514R, which amplify a sequence from the 16S rDNA and rplJ and rplL ribosomal protein genes, respectively, of "Ca. L. solanacearum" (2,4). The DNA samples were also tested for phytoplasmas with nested PCR using universal primer pairs P1/P7 and fU5/rU3 (3). DNA from five and four symptomatic plants yielded the expected 1,168-bp 16S rDNA and 669-bp rplJ/rplL amplicons, respectively, indicating the presence of liberibacter. Extracts from asymptomatic plants yielded no products with these primers. Amplicons generated from three symptomatic plants with each primer pair were cloned into pCRII-TOPO plasmid vectors (Invitrogen, Carlsbad, CA) and three clones of each of these amplicons were subsequently sequenced in both directions (ACGT, Inc., Wheeling, IL). BLAST analysis of the 16S rDNA consensus sequence (GenBank Accession No. FJ957897) showed 100% identity to 16S rDNA sequences of "Ca. L. solanacearum" amplified from S. betaceum (EU935004) and S. lycopersicum (EU834130) from New Zealand (2), and "Ca. L. psyllaurous" from potato psyllids (EU812559). The rplJ/rplL consensus sequence (GenBank Accession No. FJ957895) was 100% identical to the analogous rplJ and rplL "Ca. L. solanacearum" ribosomal protein gene sequence amplified from S. lycopersicum (EU834131) from New Zealand (2) and 'Ca. Liberibacter' sp. sequence amplified from zebra chip-infected potatoes from Lancaster, CA (FJ498803). No phytoplasmas were detected in the symptomatic tomato plants. To our knowledge, this is the first report of "Ca. L. solanacearum" associated with tomatoes in México. In 2008, this bacterium was detected in glasshouse tomatoes in New Zealand and caused millions of dollars in losses to the commercial glasshouse tomato industry (2). References: (1) R. L. Holguín-Peña et al. Plant Dis. 91:328, 2007. (2) L. W. Liefting et al. Plant Dis. 93:208, 2009. (3) J. E. Munyaneza et al. J. Econ. Entomol. 100:656, 2007. (4) J. E. Munyaneza et al. Plant Dis. 93:552, 2009.