Franco-Lara, Liliana

AbstractPhytoplasmas are bacteria transmitted by insects that can cause plant diseases. In Bogotá 'Candidatus Phytoplasma asteris' and ' Candidatus Phytoplasma fraxini', infect 11 species of urban trees, weeds, grass, potato and strawberry. A set of primers, that amplify both phytoplasmas species were designed and used for absolute and relative qPCR quantification of the 16SrRNA gene. The primers AJ-16Sr-F/AJ-16Sr-R allowed the amplification of ‘Ca. P. asteris’, ‘Candidatus Phytoplasma palmae’, ‘Ca. P. fraxini’ and ‘Candidatus Phytoplasma phoenicium’, not of ‘Candidatus Phytoplasma pruni’. Absolute qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Two species-specific hydrolysis probes, AJ-16SrI-Cy5.5 and AJ-16SrVII-TexRed, were designed to detect 'Ca. P. asteris' and 'Ca. P. fraxini' respectively, using the AJ-16Sr-F/AJ-16Sr-R primers. For relative quantification, the 18SrRNA gene was used as normalizer. Relative qPCR detected phytoplasmas between 1 × 109 and 1 × 103 copies/μL DNA extract. Multiplex reactions allowed the specific quantification of 'Ca. P. asteris', 'Ca. P. fraxini' in comparison to the normalizer. qPCR methods were validated on natural hosts Andean oak trees and leafhoppers. The relative quantification values were higher for 'Ca. P. fraxini' (x̅ RQ = 3203.1 ± 2622,9 n = 14) compared with 'Ca. P. asteris' (x̅ RQ = 14.9 ± 24,5 n = 6) in oak tree samples. In the leafhoppers, the relative quantification values ranged between RQ = 26.5 and RQ = 294,927.3 for 'Ca. P. fraxini’ and RQ = 34.8 and RQ = 1722.2 for 'Ca. P. asteris'. In conclusion, although absolute qPCR allowed the quantification of phytoplasmas by comparing Cq (quantification cycle) values of samples with a standard curve, it did not allow to differentiate between 'Ca. P. asteris' and 'Ca. P. fraxini'. In contrast, relative qPCR assays using specific hydrolysis probes allowed the specific detection and quantification of each phytoplasma, in individual and mixed infections in insect vectors and plant hosts.

Phytoplasmas have been associated with a disease that affects trees of at least 11 species from different botanic families in Bogotá, Colombia. ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma fraxini’ are the major groups of phytoplasma in the area of Bogotá. In this study, the genetic diversity within ‘Ca. P. asteris’ and ‘Ca. P. fraxini’ was studied in five urban tree species: Croton species (Euphorbiaceae), Fraxinus uhdei (Oleaceae), Magnolia grandiflora (Magnoliaceae), Populus nigra (Salicaceae) and Quercus humboldtii (Fagaceae). Analyses of the 16S rRNA gene using nested PCR, RFLP and sequencing showed that phytoplasmas of ‘Ca. P. asteris’ could be assigned to: subgroup 16SrI-B; a new subgroup named 16SrI-AF, with a restriction pattern similar to that of 16SrI-B; and a new subgroup named 16SrI-AG, with a restriction pattern similar to that of 16SrI-K and 16SrI-AH with a restriction pattern similar to that of 16SrI-AC. ‘Ca. P. fraxini’ isolates belonged to a new subgroup named 16SrVII-G, with a restriction pattern similar to that of 16SrVII-A. To complement the identification of the phytoplasma strains, we amplified nonribosomal genes such as leuS and secA. Unexpectedly, it was observed that in 16 trees in which 16S rRNA gene analysis showed the presence of ‘Ca. P. fraxini’ only, the leuS or secA primers amplified sequences exclusively affiliated to ‘Ca. P. asteris. In those plants, sequences belonging to ‘Ca. P. fraxini’ leuS or secA genes were not amplified. The present work contributes to the identification of novel strains of both species in Colombia, and supports previous suggestions that phytoplasmas in South America are highly variable.