First Report of ‘Candidatus Phytoplasma trifolii’-Related Strain Associated With a New Disease in Tomato Plants in Zacatecas, Mexico

Abstract

Tomatillo (Physalis ixocarpa) is an important crop in Mexico with a total of 698,016 tons on 42,882 ha in 2016. In July 2015, symptoms of yellowing, stunted growth, foliar deformation, and phyllody were observed in approximately 12% of tomatillo plants from 25 ha of commercial crops in Zacatecas, Mexico (22°43′17.1″ N, 102°41′06.5″ W). Total DNA was extracted from nine symptomatic and four symptomless tomatillo plants. Direct and nested PCR assays targeting the 16S rRNA gene were used to confirm the association of phytoplasma with the symptomatology. The primers used for direct PCR were P1 5′-AAGAGTTTGATCCTGGCTCAGGATT-3′ and Tint 5′-TCAGGCGTGTGCTCTAACCAGC-3′ (Smart et al. 1996), and for nested PCR, R16F2n 5′-GAAACGACTGCTAAGACTGG-3′ and R16R2 5′-TGACGGGCGGTGTGTACAAACCCCG-3′ (Gundersen and Lee 1996). No PCR products were obtained from the symptomless plants. The nested PCR amplicons (1.2 kb) amplified from all symptomatic plants were cloned separately and directly sequenced. BLAST analysis of the 16S rDNA sequences revealed that they shared 100% sequence identity to each other and 99.0% sequence identity with those of the 16SrVI group, ‘Candidatus Phytoplasma trifolii’ strains. Computer simulated RFLP analysis of the Zacatecas tomatillo phytoplasma sequence (GenBank accession no. MG775048) was performed using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) and RFLP profiles were compared with each phytoplasma group and subgroup (Zhao et al. 2013) confirming that the analyzed sequence shared 98.9% identity with the reference strain (AY390261). Finally, virtual RFLP patterns were identical to those of the group 16SrVI, subgroup A. Although some reports associate phytoplasmas of the group 16SrVI with diseases in other solanaceous crops such as chili pepper and tomato in Mexico (Mauricio-Castillo et al. 2015; Salas-Munoz et al. 2016), there are no reports about the presence of Ca. P. trifolii related to tomatillo diseases. To our knowledge, this is the first report of Ca. P. trifolii associated with symptomatic tomatillo plants. The presence of phytoplasmas belonging to 16SrVI group on tomatillo could therefore represent a serious threat for this valuable crop in Mexico.