FIRST REPORT OF A 16Sr IV GROUP PHYTOPLASMA ASSOCIATED WITH LETHAL YELLOWING IN AGAVE TEQUILANA
B. E. González-Pacheco, R. I. Rojas-Martínez, D.L. Ochoa-Martínez, L. Silva-Rosales
Mexico, which is considered the origin and the biodiversity center of the family Agavaceae, hosts 117 of the 155 known species. Agave tequilana Weber cv. Azul is a national icon and the most widely cultivated species for the production of tequila, a widely known alcoholic beverage. In spring 2013, a disease of A. tequilana was observed in Tala, (Jalisco, Mexico) which greatly reduced its availability for tequila production. Affected plants showed extensive chlorosis, followed by yellowing and necrosis of stem and leaf tissues. As the disease progressed, leaves collapsed and hung downwards around the central head. To investigate the possibility of a phytoplasma infection, DNA was extracted according to Lee et al. (1993) from 25 symptomatic and 25 symptomless plants. A nested PCR was performed using two universal primer sets specific for the phytoplasma 16S rRNA gene, i.e. R16mF2/R16mR1 followed by R16F2n/R16R2 (Gundersen and Lee, 1996). The expected 1,200 bp product was obtained from 88% of the symptomatic plants. The PCR products were cleaned with a Wizard kit (Promega, USA) and cloned in Escherichia coli using a TOPO-TA cloning kit (Invitrogen, USA) in accordance with manufacturer’s instructions. When nucleotide sequences of the amplified products (accession Nos KJ156364, KJ156365) were compared with those available in GenBank, it was found that the agave phytoplasma was most similar (99.98%) to the Texas phoenix palm phytoplasma (USA, JF791816) and Sabal mexicana decline phytoplasma (Mexico, GU473588). Phylogenetic and putative restriction site analysis of 16Sr DNA indicated that the phytoplasma associated with A. tequilana is closely related to the lethal yellows 16SrIV group. To our knowledge, this is the first report of a 16SrIV group phytoplasma associated with a disease of A. tequilana.