First report of a 16SrII‐C phytoplasma associated with Artemisia sieberi witches’‐broom disease in Iran

Abstract

Artemisia sieberi (Asteraceae) is an important perennial plant that is common in the pastures, mountains and deserts of Iran. In 2019, A. sieberi showing little leaf, yellowing, internode shortening and witches’-broom symptoms (Fig. 1) were observed around Abarkooh (Yazd province, Iran). The disease incidence was up to 5%. Total DNA was extracted from 0.2 g of fresh midrib tissue from 10 diseased and five symptomless plants and was tested for the presence of phytoplasma DNA by direct PCR using the P1/P7 primers and nested PCR using the P1/P7 and the R16F2n/R16R2 primers (Lee et al., 1998). Expected amplicons of 1.8 and 1.25 kb were obtained from all symptomatic but not from asymptomatic plants. R16F2n/R16R2 amplicons yielded the same RFLP patterns as those obtained from the 16SrII-C subgroup (Lee et al., 1998) after restriction with AluI, HhaI, HinfI, HpaII, MseI and RsaI enzymes. Three nested PCR products were cloned and sequenced. The sequences were identical and a consensus sequence was deposited in GenBank (Accession No. MZ151480). Sequence comparison by BLAST analysis showed the highest identity with members of ‘Candidatus Phytoplasma aurantifolia’ species (e.g. MH363612 and KJ442846). Phylogenetic analysis using the neighbour-joining method (MEGA software version 6.0) confirmed that the A. sieberi witches’-broom phytoplasma clustered within the 16SrII phytoplasma group clade, closest to strains affiliated to the 16SrII-C subgroup (Fig. 2). Computer-simulated analysis with 17 restriction endonucleases using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) showed that the RFLP pattern derived from the A. sieberi witches’-broom phytoplasma 16S rRNA gene resembled the reference pattern of the 16Sr group II, subgroup C (AJ293216), with a pattern similarity coefficient of 0.99. The occurrence of 16SrII-C phytoplasma strains was previously reported from Iran on Daucus carota, Medicago sativa, Prunus dulcis, Vicia faba (Siampour et al., 2019), Alhagi maurorum (Esmaeilzadeh-Hosseini & Salehi, 2021), P. armeniaca (Rasoulpour et al., 2019) and P. persica (Salehi et al., 2020). Previous studies in Iran have shown that Circulifer haematoceps can transmit 16SrII group phytoplasmas; this leafhopper is polyphagous and could feed on A. sieberi, which may act as a reservoir host for 16SrII-C phytoplasma strains. Association of a 16SrII-D phytoplasma strain with A. sieberi has been previously reported from the Hormozgan province, Iran (Hemmati & Nekoohi, 2019). To our knowledge this is the first report of A. sieberi as a new host of the 16SrII-C phytoplasma subgroup in Iran. This paper is part of the results of project no. 100-11-81-108 approved and supported by Agricultural Research, Education and Extension Organization (AREEO), Ministry of Agriculture, Iran.