First Report of 'Candidatus Phytoplasma pyri' Causing Peach Yellow Leaf Roll (PYLR) in Spain

'Candidatus Phytoplasma prunorum,' which causes European stone fruit yellows (ESFY), is the prevalent phytoplasma affecting Prunus spp. in Europe. It is closely related to 'Ca. P. pyri,' which causes pear decline (PD) in pear trees. Both phytoplasma belong to the ribosomal group 16Sr-X and are naturally transmitted by different species of Cacopsylla spp. (4). In North America, 'Ca. P. pyri' is responsible for peach yellow leaf roll (PYLR), transmitted by Cacopsylla pyricola from pear to peach trees (1). In Spain, 'Ca. P. prunorum' is widespread on Prunus spp., but its occurrence on Prunus persicae is very low and 'Ca. P. pyri' is present in every pear orchard (3). During 2012, a previously unreported syndrome including early reddening, leaf curling, decline, abnormal fruits, and in some cases chlorosis and death of peach trees was reported on peach in Lleida, northern Spain. Symptoms were different to ESFY and PYLR, in that flowering disorders such as ESFY or yellows were not apparent, and reddening and decline were the most common symptoms. The disease was present in a wide range of varieties and rootstocks, suggesting insect transmission in an area where C. pruni, vector of 'Ca. P. prunorum,' was not previously reported, but C. pyri was abundant in pear orchards. Shoot samples from 20 symptomatic peach trees were collected in seven orchards within a 2 km² area with an estimated incidence of 40%, which was higher in the borders. DNA was extracted from 1 g of leaf midribs and phloem tissue and amplified with ribosomal universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 and specific primers fO1/rO1 that target the 16Sr-X group (3). The final PCR products were digested with RsaI enzyme. Amplifications with non-ribosomal specific primers, Imp ESFY, Imp PD A and Imp PD B that amplify sequences of gene Imp, that encode a phytoplasma membrane protein, were also carried out (2). Tissue samples with ESFY and PD and peach seedlings were used as positive and negative controls, respectively. Amplified PCR products were sequenced and compared to sequences deposited in GenBank. Phytoplasmas were detected in 18 of the 20 samples analyzed. No phytoplasmas were detected in negative peach controls. All digestions of fO1/rO1 PCR products from peach samples showed a PD profile, while no ESFY profile was detected. All samples were positive with specific primers Imp PD A and B. None of the peach samples were positive with the specific Imp-ESFY primers. Sequencing of R16 and Imp PDA and B amplicons revealed the presence of a stable isolate. The sequences were submitted to the European nucleotide archive (ENA) with the accession nos. HG737345 and HG737344. Based on the 16S rDNA sequence, this strain is 100% homologous to the reference strain PD1 (GenBank Accession No. AJ542543) and 99.55% homologous to strain PD 33 Lib (GenBank FN600725) based on the Imp gene sequence. This is the first report of PD phytoplasma in peach trees in Spain, and the first report in Europe of PD phytoplasma causing economically important outbreaks in peach orchards, following a pattern that could be similar to PYLR in North America. This strain is genetically closer to some European or Middle Eastern PDs than to North American PYLR. References: (1) C. L. Blomquist et al. Plant Dis. 86:759, 2002. (2) J. L. Danet et al. Microbiology 157:438, 2011. (3) M. Garcia-Chapa et al. J. Phytopathol. 151:584, 2003. (4) E. Seemüller et al. Int. J. Syst. Evol. Microbiol. 54:1217, 2004.