First report of a ‘Candidatus Phytoplasma aurantifolia’ related strain (16SrII‐C) associated with Convolvulus virgatus witches'‐broom disease in Iran

Abstract

Convolvulus virgatus (Convolvulaceae) is a perennial shrub which grows mainly in desert and shrubland biomes in Iran, the Arabian Peninsula and Pakistan (Wood et al., 2015). In a 2020 survey for phytoplasma diseases, witches'-broom disease of C. virgatus (WBDCV) was observed in the Roodan area (Hormozgan province, Iran). The main symptoms of WBDCV were flower and stem proliferation, shortened internodes, little leaf, severe witches'-broom, yellowing, reddening, plant sterility and decline (Figs. 1, 2). Total DNA was extracted from leaves and leaf petioles of five symptom-bearing and five symptomless C. virgatus plants using a CTAB-based method. DNA samples were tested for the presence of phytoplasmas by direct-PCR using primer pair P1/P7 (Lee et al., 1998) and nested PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). The expected amplicons of c. 1,800 bp and 1,250 bp were obtained in direct and nested-PCR respectively, from all diseased plants but not symptomless ones. Two R16F2n/R16R2 amplicons were randomly selected and cloned into Escherichia coli DH5α cells using the PCR TA Cloning Kit (SinaClon, Cat. No.:CL5841, Iran) and three plasmid DNAs from recombinant colonies were sequenced. The obtained sequences were identical, and a consensus sequence was deposited in GenBank (Accession No. ON361565). BLASTn analysis of the obtained sequence showed the highest sequence identity (98.7-99.9 %) with phytoplasmas related to ‘Ca. P. aurantifolia’ (16SrII group). Phylogenetic analysis with MEGA11 software using the neighbour-joining method Fig. 3 revealed that the WBDCV phytoplasma clustered within the ‘Ca. P. aurantifolia’ (16SrII) group clade, closest to the strains of subgroup 16SrII-C. In Iran, phytoplasma strains belonging to 16SrII-C subgroup have been reported previously in Bushehr, Fars (adjacent to Hormozgan province) and Yazd provinces in several plant species including Daucus carota, Medicago sativa, Prunus dulcis, Vicia faba (Siampour et al., 2019), Alhagi maurorum and Artemisia sieberi (Esmaeilzadeh-Hosseini & Salehi, 2021); however, this is the first report of a ‘Ca. P. aurantifolia’-related strain in C. virgatus associated with WBDCV in Iran and most probably, in the world. The authors would like to thank the head of the Fars Agricultural and Natural Resources Research and Education Centre, Shiraz, Iran, for support and encouragement.