First Report of a Natural Infection by Mexican Papita Viroid and Tomato Chlorotic Dwarf Viroid on Greenhouse Tomatoes in Mexico.

In early 2008, tomato plants (Solanum lycopersicum) grown in a large greenhouse facility located near Mexico City exhibited general stunting, leaf chlorosis at the top of the diseased plant that later turned bronze or purple, and reduced-sized fruits. Initially, diseased plants were confined to a 5-ha greenhouse, but the disease quickly spread to two additional 5-ha greenhouses in the summer of 2008. By the end of 2008, approximately 5% of tomato plants in 35-ha of greenhouse were infected. Sixteen diseased samples were collected, twelve in 2008 and four in 2009. Bioassays through mechanical inoculation with leaf extracts of diseased samples demonstrated the transmissibility of the causal agent to plants of tomato cvs. Horizon or Rutgers, which expressed symptoms that were similar to those on the source plants. Serological or PCR assays were negative for several commonly occurring greenhouse tomato viruses. However, an expected size product (~196 bp) was consistently detected by reverse transcription (RT)-PCR using pospiviroid-specific primers Pospil-RE and Pospil-FW (4) in all symptomatic samples or from the mechanically inoculated tomato plants. Preliminary analysis with sequences obtained from direct sequencing of amplicons revealed one dominant sequence with 94% identity to Mexican papita viroid (MPVd) (GenBank Accessions Nos. L78454 and L78456-L78463). However, further analysis of the cloned cDNAs indicated a mixed infection of two pospiviroids in two samples. Of 10 cDNA clones analyzed, 9 were MPVd-like sequences and one was sequence of Tomato chlorotic dwarf viroid (TCDVd). Further analysis using full genomic sequences obtained by RT-PCR with previously designed primers (2) or a new set of primers (MTTVd-F: 5' GGG GAA ACC TGG AGC GAA CTG G, and MTTVd-R: 5' GGG GAT CCC TGA AGC GCT CCT) revealed genetic diversity in this population. Eight of thirteen cloned cDNAs represented by the 359-nt sequence of isolate Mex8 (GenBank Accession No. GQ131572) had 93 to 94% nucleotide sequence identity to other MPVd isolates (L78454 and L78456-L78463). Five other cDNA clones represented by the 361-nt sequence of isolate HM2 (GenBank Accession No. GQ131573) were 99% identical to a TCDVd isolate recently identified in Arizona (GenBank Accession No. FJ822878) and 96 to 97% identical to TCDVd isolates from other areas (GenBank Accession Nos. AF162131 and AB329668). These results are the first evidence of a mixed infection of two viroids infecting tomatoes in Mexico. MPVd was first identified in Mexico on papita (S. cardiophyllum) in 1996 (1). The origin of TCDVd in this greenhouse was not determined, but TCDVd potentially can be seed transmitted in tomato (3). The close relationship between the Mexican and the U.S. isolates suggests that TCDVd in these two countries may share a common origin, likely from seed. To our knowledge, this is the first report of a natural infection of MPVd and TCDVd on tomatoes in Mexico. References: (1) J. P. Martinez-Soriano et al. Proc. Natl. Acad. Sci. U.S.A. 93:9397, 1996. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) R. P. Singh and A. D. Dilworth. Eur. J. Plant Pathol. 123:111, 2009. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.