Characterisation of a phytoplasma associated with sunflower phyllody in Fars, Isfahan and Yazd provinces of Iran

Abstract

Sunflower (Helianthus annuus) is an important oil crop in Iran. During 2010-2012 surveys, symptoms of sunflower phyllody were observed in sunflower fields of Abarkooh, Yazd (Yazd province), Fasa (Fars province) and Nain (Isfahan province). Affected plants showed proliferation of abnormal heads, virescence, phyllody of main flowers and axillary buds along the stem (Fig. 1). Total DNA was extracted from 0.3 g of fresh suspected phyllody flowers of affected sunflower plants and also from healthy plants using the procedure of Zhang et al. (6). Two universal primer pairs, P1/P7 (Deng & Hiruki, 1; Schneider et al., 4) and R16F2n/R2 (Gundesen & Lee 2), were used to target the phytoplasma 16S rRNA gene. Phytoplasma DNA fragments of ~1.8 and ~1.250 kbp were amplified, respectively, from direct and nested PCR assays from eight symptom-bearing sunflower plants (two samples per location) but not from symptomless plants. Eight P1/P7 DNA fragments amplified from Abarkooh, Yazd, Fasa and Nain phyllody-affected sunflower plants were separately cloned and sequenced. The 16S rDNA sequences shared 100% sequence identity with each other, with that of the Abarkooh isolate (ASP) being deposited in GenBank (Accession No. KJ016231). BLAST search showed the highest sequence identity with phytoplasma sequences of phytoplasma members of the 16SrII group (‘Candidatus Phytoplasma aurantifolia’). Phylogenetic analysis of the ASP 16S rDNA sequence and those of reference from other phytoplasma groups (MEGA version 5.0) showed that the ASP phytoplasma clustered with the 16SrII phytoplasma, closely related to ‘Ca. P. australasia’ of subgroup 16SrII-D (Fig. 2). Computer-simulated restriction analysis with AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI, and TaqI enzymes using pDRAW32 software (http://www.acaclone.com) revealed that the RFLP patterns of the ASP phytoplasma (Fig. 3) were identical to those of ‘Ca P. australasia’. To our knowledge this is the first report of the association of a 16SrII phytoplasma with a sunflower phyllody disease in Iran. Mixed infections of phytoplasma groups 16SrII and 16SrVI (‘Ca. P. ulmi’) in a witches'-broom affected sunflower were previously reported from the Kerman province of Iran (Tazehkand et al., 5). Since alfalfa and sugar beet witches'-broom caused by 16SrII related phytoplasmas are economically important diseases in the Abarkooh region (Salehi et al., 3), the identification of a 16SrII phytoplasma affecting sunflower suggests that the 16SrII phytoplasma isolates limited to the same geographical location of Abarkooh may share similar epidemiological constraints.