First report of a ‘Candidatus Phytoplasma solani’‐related strain (16SrXII‐A) associated with Althaea officinalis yellows disease in Iran

Abstract

Althaea officinalis (marsh mallow, Malvaceae) is a medicinal plant, the roots, leaves and flowers of which are usually used in traditional medicines in many countries, including Iran (Kianitalaei et al., 2019). During a survey in April 2022, a phytoplasma-type disease, tentatively named A. officinalis yellows (AOY), was observed in patches of A. officinalis plants naturally growing in Golestan, near Shiraz (Fars province, Iran). In a 2-ha surveyed area, the disease incidence was up to 5%. The characteristic symptoms were shoot proliferation from stems, little and deformed leaves, shortened internodes, yellowing, witches’ broom, stunting and plant sterility (Fig. 1 & 2). Total DNA was extracted from five diseased and five asymptomatic A. officinalis plants using a CTAB procedure (Zhang et al., 1998). To examine the presence of phytoplasma, extracted DNA samples were subjected to direct PCR using P1/P7 (Lee et al., 1998) and nested PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen and Lee, 1996). The expected amplicons of c. 1,800 and 1,250 bp (respectively) were obtained only in diseased plants. All five R16F2n/R16R2 amplicons were cloned into Escherichia coli DH5α cells using the InsT/A clone™ PCR product cloning kit (Sinnaclone, Iran) and three plasmids from recombinant colonies were sequenced. The obtained sequences were identical, and a consensus sequence was deposited in GenBank (Accession No. OP572231). A BLASTn search showed that the phytoplasma associated with AOY disease shared the highest sequence identity (100%) to those of Convolvulus arvensis witches’ broom phytoplasma strains, members of ‘Candidatus Phytoplasma solani’ (Salehi et al., 2019), which were detected in Iran in Fars, Lorestan and Yazd provinces in 2019. Phylogenetic analysis using the neighbour-joining method (MEGA7 program) showed that the phytoplasma strain associated with AOY disease clustered within the 16SrXII phytoplasma group closest to 16SrXII-A (‘Ca. P. solani’) subgroup (Fig. 3). The percentage sequence similarity of AOY phytoplasma to the reference strain for the species (AF248959) was 99.28%. In Iran, ‘Ca. P. solani’ (16SrXII-A) phytoplasma strains have been previously reported in several plants such as grapevine, hemp, alfalfa, tomato and bindweed (Siampour et al., 2019); however, to our knowledge this is the first report of ‘Ca. P. solani’ (16SrXII-A) associated with A. officinalis in Iran and 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.