First Report of Candidatus Phytoplasma solani Infecting New Natural Host Plum (Prunus domestica L.) in Jordan.

Plum (Prunus domestica L., Rosaceae) is among the most important stone fruit species grown in Jordan, especially in the northeastern part of the country. In September 2017, 30 trees from a 2-ha orchard of cultivar ‘Early Queen’ showing possible phytoplasma symptoms of leaf yellowing and reddening, stunted growth, and witches’ broom were observed. Additionally, there were around 20 bindweed plants (Convolvulus arvensis L.) among the plum trees showing stunting and leaf reddening in the Mafraq region in northeast Jordan. Leaf samples were collected from 15 symptomatic and five asymptomatic plum trees and three symptomatic bindweed plants. Total DNA was extracted from all samples and analyzed by PCR using the phytoplasma universal primer pair P1/P7 (16S/23S RNA, Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR with primer pair R16F2n and R16R2 (16S rDNA, Gundersen and Lee 1996). The same samples were also tested by PCR using the 16SrXII-specific primers SecY1a and SecYR1 (secY, Lee et al. 2010) and by “stolbur” phytoplasma specific quantitative PCR (16S rDNA, Hren et al. 2007). DNA samples of five out of 15 P. domestica and all three bindweed samples tested positive for the presence of a phytoplasma by qPCR (Ct values of 19.5 to 26.2 and 33 to 34 for plum and bindweed, respectively) and nested PCR, yielding the expected PCR amplicons. Interestingly, no phytoplasma was detected in 10 samples from symptomatic plum trees as well as from all the asymptomatic trees. Bidirectional sequencing (Macrogen, Amsterdam, The Netherlands) was done for each PCR amplicon after gel purification. A BLASTn similarity analysis of the assembled consensus sequences derived from the plum and the bindweed host plants revealed that the sequences of phytoplasmas infecting P. domestica (accession nos. MH085227, MH085228, and MH085229) and C. arvensis (MH085225 and MH085226) in Jordan had identity of 99% to the deposited sequence of ‘Candidatus Phytoplasma solani’ from of Vitis vinifera L. in Jordan (KC835139) and the ‘Ca. P. solani’ strain sequence from Capsicum annuum L. from Serbia (AF248959). The same analysis based on the secY gene confirmed the highest identity (99%) of analyzed sequences of ‘Ca. P. solani’ (MK928324 to MK928326) with strains from Russia and Germany (GU004346 and FO393427, respectively). The identity of ‘Ca. P. solani’ was confirmed in plum and bindweed by virtual RFLP (iPhyClassifier, Zhao et al. 2009) and phylogenetic analyses (BioNumerics, Applied Math, Belgium). These results supported the first occurrence of ‘Ca. P. solani’ in plum in Jordan, where it may represent a potential threat to other already reported susceptible stone fruit hosts, such as peach, almond, and cherry. The reported presence of the bindweed in Jordan (Salem et al. 2013), a well-known phytoplasma natural reservoir (Mehle et al. 2011), and potential insect vectors (e.g., Hyalesthes obsoletus) (Quaglino et al. 2013) along with vegetatively propagated plant material (Bertaccini et al. 2014) may have contributed to the plum infection in Jordan.