Association of a phytoplasma from ribosomal subgroup 16SrXII‐A with walnut witches’‐broom in Iran

Abstract

Walnut trees displaying symptoms similar to those caused by phytoplasmas, including foliar chlorosis, yellowing and shortening of internodes, were found in a fruit orchard in the north of Fars province, Iran in 2020 (Fig. 1). Samples were collected and analysed by PCR to detect and identify possible phytoplasma(s) associated with diseased plants. Four diseased and four asymptomatic walnut trees were sampled and analysed for the presence of phytoplasma. Total DNA was extracted from 0.1 g of midribs using the CTAB method. Nested PCR was performed using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) in the first round, and R16F2n/R16R2 (Smart et al., 1996) in the second. PCR products of the expected size, c. 1.2 kb, were obtained from all diseased plants but not from the symptomless ones. The nucleotide sequence was determined directly and submitted to GenBank (Accession No. MZ005313). A BLAST search showed that the phytoplasma associated with walnut witches’-broom had the highest sequence identity (99.9%) with Iranian alfalfa witches’-broom phytoplasma (KT763373), a member of the ribosomal subgroup 16SrXII-A (‘Candidatus Phytoplasma solani’). Virtual RFLP patterns obtained with iPhyclassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) supported the sequence analysis results. Walnut witches’-broom phytoplasma was first reported in Iowa, USA and was associated with the group 16SrIII (‘Ca. P. pruni’) (Yun & Harrington 2011). In Iran, the 16SrXII phytoplasma group has been reported in alfalfa, almond, aubergine, grapevine, peach, plum and tomato (Siampour et al., 2019). It has also been identified from Cannabis sativa (Sichani et al., 2011) and Salix babylonica (Zamharir & Taheri, 2017). In Fars province, walnut orchards are adjacent to stone fruit orchards and vineyards, so it is likely that this phytoplasma has been transferred from such crops to walnut trees. Since large areas of walnut are grown in Iran, this phytoplasma may be a serious threat to walnut cultivation. To the best of our knowledge, this is the first report of the association of a 16SrXII-A related phytoplasma with walnut witches’-broom globally. The authors would like to thank Dr. Sadegh Sadeghi and Mr. Mohsen Amiri for great help in the field.