First Report of Association of a ‘Candidatus Phytoplasma aurantifolia’ 16SrII-D Related Phytoplasma with Geranium (Pelargonium hortorum) Little Leaf Disease in Iran

Geranium, an important ornamental plant belong to the family Geraniaceae, is widely cultivated in urban landscapes and private gardens all over the world. During a survey in spring 2019, a few seed-propagated geranium plants exhibiting typical symptoms induced by phytoplasmas, including pale green and little leaves, phyllody, virescence, and witches’-broom symptoms, were observed in Jiroft, Kerman province of Iran. Total genomic DNA extracted from leaf midribs and petioles of the three symptomatic and two asymptomatic plants was used as a template in a nested polymerase chain reaction (PCR) assay using universal phytoplasma primer pairs P1/P7 followed by R16F2n/ R16R2 (Gundersen and Lee 1996) to amplify a part of the 16S rDNA phytoplasma genome as previously described by Azadvar et al. (2011). The generated 1.8- and 1.25-kb amplicons in the first and second round of the nested PCR assay, respectively, from each of the three symptomatic plant samples revealed phytoplasma infection of the plants. No amplicon was generated in PCR assays by using the DNA templates from symptomless plants. The purified PCR products were directly sequenced (Bioneer, South Korea) in both forward and reverse directions. The consensus sequences were analyzed by the iPhyClassifier tool (Zhao et al. 2009) and pDRAW32 software (AcaClone software, http://www.acaclone.com) for 16Sr group/subgroup phytoplasma identification. The 16S rDNA nucleotide sequences from the three diseased plants were identical (GenBank accession no. MT187639). iPhyClassifier analysis showed that the F2nR2 16S rRNA encoding gene sequence of the phytoplasma associated with geranium little leaf (GrLL) disease in Iran was identical (similarity coefficient 1.00) to those of the reference pattern of 16Sr group II subgroup D (GenBank accession no. Y10097) and had 98.7% similarity with the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession U15442). Virtual restriction fragment length polymorphism (in silico RFLP) analysis of the 16S rDNA F2nR2 sequence with the pDRAW32 program using AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, RsaI, Sau3AI, SspI, and TaqI endonucleases further confirmed that the GrLL phytoplasma belong to subgroup II-D. The 16SrII-D subgroup phytoplasma has been previously reported in association with Calendula officinalis (Esmailzadeh Hosseini et al. 2016), Helianthus annuus (Salehi et al. (2015), and Petunia hybrida (Faghihi et al. 2014) ornamental plants in Iran. We also found many petunia (P. hybrida) plants showing little leaf, yellowing, phyllody, and witches’-broom symptoms next to the GrLL-infected plants, which hosted the same phytoplasma (GenBank accession no. MT187637). A 16SrII group-related phytoplasma has previously been reported associated with Pelargonium capitatum weed plant exhibiting severe stunting and necrosis in Australia (Lee et al. 2010). To our knowledge, this is the first report of a ‘Ca. P. aurantifolia’ 16SrII-D subgroup-related phytoplasma associated with geranium (Pelargonium hortorum) little leaf disease in the world.