First Report of a ‘Candidatus Phytoplasma fraxini’-Related Strain Associated with Potato in Colombia

Potato is one of the most important crops in Colombia, with a total production of 2,819,020 tons in 149,060 ha in 2017 (FAOSTAT 2018). More than 10 varieties of Solanum tuberosum and S. phureja are grown in Colombia; among them, variety Superior covers 20% of the cultivated area. In the last 5 years, potato growers have reported a new disease in Cundinamarca state, and our objective was to test for the presence of phytoplasmas in affected plants. Mejia et al. (2011) associated phytoplasmas of the groups 16SrV and 16SrXII with potatoes in central Colombia. In May 2015, we sampled a crop of the variety Superior from Tausa, Cundinamarca (5°10′53.2″N, 73°51′26.1″W). More than 70% of the plants showed symptoms such as curly and yellowing leaves with purple margins, and abnormally short or long internodes. Five symptomatic and five nonsymptomatic plants were sampled. Total DNA was extracted from the leaves by a modified cetyltrimethylammonium bromide protocol method (Prince et al. 1993), and the extracts were tested by nested polymerase chain reaction (PCR) with phytoplasma 16SrDNA universal primers P1A/P7A followed by R16mR1/R16mF2 for sequencing, and P1A/P7A followed by R16F2n/R16R2 for restriction fragment length polymorphism (RFLP) analysis with restriction enzymes AluI, MseI, and RsaI (Gundersen and Lee 1996; Lee et al. 2004). Amplicons were obtained for all symptomatic plants, although in one sample the band was too faint for analysis. The other four amplicons produced RFLP patterns consistent with group 16SrVII with all the restriction enzymes. These amplicons were sequenced by Macrogen, Korea. Good quality sequences were obtained from two samples (GenBank nos. MK956091 and MK956092). These sequences were analyzed with the iPhyClassifier suite (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), and both had 99.8% similarity with the reference strain ‘Candidatus Phytoplasma fraxini’ (GenBank no. AF092209). The sequence MK956091 had a similarity coefficient of 0.97, suggesting that it belongs to a new subgroup, with the most similar reference pattern that of 16SrVII-A (GenBank no. AF092209). The other sequence was not analyzed because it was too short. A phylogenetic tree was built by the neighbor-joining method with 1,000 bootstrap replicates, including representative phytoplasmas sequences, and the two sequences clustered with ‘Ca. P. fraxini’ isolates from Colombia and the United States. Phytoplasmas were not detected in the nonsymptomatic plants. Finally, leaf petioles of symptomatic plants that were positive by nested PCR were studied by transmission electron microscopy (Devonshire 2013). Translucent cells that resemble phytoplasmas were observed in the sieve elements of the phloem tissue. Although the prevalence of this disease has not been estimated, potato growers report these symptoms increasingly. ‘Ca. P. fraxini’ is known to infect urban tree species in Bogota such as Fraxinus uhdei, Liquidambar styraciflua, Populus nigra, and Quercus humboldtii, among others (Franco-Lara et al. 2014; unpublished results). Interestingly, the new 16SrVII subgroup detected in potatoes is also present in urban trees such as P. nigra (GenBank nos. MH795203 to MH795207) and in Q. humboldtii (MH795215 and MH795216). Bogotá city is located in Cundinamarca state; these results suggest that the pathogen is moving from the trees to the crops.