First report of 'Candidatus Phytoplasma solani' associated with grapevine Bois noir disease in Jordan

Background and objectives Bois noir (BN) is a disease of the grapevine yellows (GY) complex associated with phytoplasmas of the species 'Candidatus Phytoplasma solani', taxonomic subgroup 16SrXII-A, mainly transmitted by the polyphagous Cixiidae Hyalesthes obsoletus Signoret (Alma et al., 1987; Quaglino et al., 2009, 2013). BN is largely spread in Europe and in Countries of the Mediterranean Basin, but in the last years its presence was reported also in South America, in South Africa, and in China (Botti and Bertaccini, 2006; Gajardo et al., 2009; Duduk et al., 2010). In the present work, we communicate the first report of BN in Jordan. Materials and Methods A survey was carried out in Jordan vineyards in August and October 2012, grapevine plants showing typical grapevine yellows (GY) disease symptoms were observed. In the same vineyards, bindweed plants showing stunting and leaf chromatic alteration were found suggesting the involvement of phytoplasmas in the disease etiology. Total DNA was extracted from leaf veins of 25 symptomatic and two asymptomatic grapevines, and from five symptomatic and two asymptomatic bindweeds. Phytoplasma detection by nested PCRs carried out using universal primer pairs P1/P7 followed by R16F2n/R16R2 (F2n/R2) (Lee et al., 1998). DNAs from periwinkle plants infected by ‘Ca. P. asteris’ strain SAY (group 16SrI), ‘Ca. P. solani’ strain STOL (group 16SrXII), and 'Ca. P. ulmi’ strain EY1 (group 16SrV), were used as positive controls. DNAs from healthy periwinkle and reactions without template DNA were employed as negative controls. F2n/R2 PCR products amplified from grapevines and bindweeds were sequenced and analysed through the software BlastN (sequence identity), BioEdit (alignment), iPhyClassifier (group/subgroup affiliation), and MEGA 5 (phylogeny). Results and Discussion Amplification of a band of the expected size (1250 nt) in three grapevines and in five bindweeds, and in the positive controls through 16S rDNA nested PCRs was observed. No amplification with DNA from 22 symptomatic grapevines, probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform (Constable et al., 2003), nor from asymptomatic plants and negative controls. Amplicons were not produced with DNA from 22 symptomatic grapevines (probably because samples were collected late in the growing season and phytoplasma distribution in plants was non-uniform (Constable et al., 2003), nor from asymptomatic plants and negative controls. PCR products were sequenced by commercial services in Italy (Primm, Milan) and Korea (Macrogen Inc., Soul). Representative 16S rDNA nucleotide sequences were deposited in NCBI GenBank with accession 30 nos. KC835139 (from grapevine) and KC835140 (from bindweed). The 16S rDNA nucleotide sequences of phytoplasmas identified in grapevine and bindweed in Jordan shared >99.5% sequence identity with 'Ca. Phytoplasma solani' reference strain STOL (AF248959), and carried identical STOL-unique signature sequences and distinguishing sequence blocks (Quaglino et al., 2013). Phylogenetic and in silico RFLP (Figure 2) analyses confirmed the affiliation of phytoplasma strains identified in grapevine and bindweed in Jordan to the species 'Ca. Phytoplasma solani' (subgroup 16SrXII-A), opening an avenue to future studies on the dissemination and impact of Bois noir (BN) in Jordan. These studies may add new information about BN, previously reported in neighboring countries (Davis, et al., 1997; Choueiri et al., 2002). Further studies will investigate the role of Hyalesthes obsoletus Signoret, a polyphagous Cixiidae responsible for the BN phytoplasma transmission in Europe, and other possible insect vector(s) in the BN spread in Jordan.