Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran M. Salehi, S. A. Esmailzadeh Hosseini, E. Salehi & A. Bertaccini Folia Microbiologica Official Journal of the Institute of Microbiology, Academy of Sciences of the Czech Republic and Czechoslavak Society for Microbiology ISSN 0015-5632 Folia Microbiol DOI 10.1007/s12223-016-0476-5 1 23 Your article is protected by copyright and all rights are held exclusively by Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Folia Microbiol DOI 10.1007/s12223-016-0476-5 Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran M. Salehi1 & S. A. Esmailzadeh Hosseini2 & E. Salehi1 & A. Bertaccini 3 Received: 8 March 2016 /Accepted: 22 September 2016 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2016 Abstract During 2010–14 surveys in the major sesame Keywords 16SrII-D . 16SrVI-A . 16SrIX-C subgroups . growing areas of Fars, Yazd and Isfahan provinces (Iran), Circulifer haematoceps . Orosius albicinctus . Iran genetic diversity and vector transmission of phytoplasmas as- sociated with sesame phyllody were studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S Introduction ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas Phytoplasmas are members of the class Mollicutes, intracellu- referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, lar wall-less plant pathogens transmitted mainly by different and 16SrIX-C. The same analyses using 16S rDNA sequences leafhopper species, and inducing typical symptoms of from sesame phyllody-associated phytoplasmas retrieved yellowing, discoloration, witches’ broom, dwarfing, vires- from GenBank database showed the presence of cence, and phyllody in both wild and cultivated plants phytoplasmas clustering with strains in the same subgroups (Seemüller et al. 1998; Bertaccini et al. 2014). Phytoplasma in other Iranian provinces including Bushehr and Khorasan diseases cause significant yield losses worldwide in more than Razavi. Circulifer haematoceps and Orosius albicinctus, 1000 plant species from different plant families (Lee et al. known vectors of the disease in Iran, were tested for transmis- 2000; Bertaccini and Duduk 2009). sion of the strains identified in this study. C. haematoceps Sesame (Sesamum indicum L.) is an oilseed plant belong- transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, ing to family Pedaliaceae that is one of the most economically while O. albicinctus only transmitted 16SrII-D strains. Based important hosts of phytoplasmas. It has been described for the on the results of the present study and considering the reported first time in ancient Assyria and Babylon about four thousand presence of phytoplasmas belonging to the same ribosomal years ago. Due to high nutritional value, rich antioxidant com- subgroups in other crops, sesame fields probably play an im- pounds, tolerance to high temperatures, and short length of portant role in the epidemiology of other diseases associated plant growth, sesame is considered very important by farmers with these phytoplasmas in Iran. compared to the other oilseed plants. Its short growing cycle enables farmers to use it as an intermediate crop between harvesting and sowing of winter fall crops (Weiss 2000; Ashri 2007). Sesame phyllody is a very important disease * A. Bertaccini [email protected] especially in tropical and subtropical areas of the world where it causes significant economic losses (Sertkaya et al. 2007; 1 Plant Protection Research Department, Fars Agricultural and Natural Rao et al. 2015). Resources Research and Education Center, AREEO, Zarghan, Iran In recent years, phyllody has been produced great losses to 2 Plant Protection Research Department, Yazd Agricultural and the sesame farmers and it is one of the main reasons for the Natural Resources Research and Education Center, AREEO, reported reduction of its cultivated areas (Esmailzadeh Yazd, Iran Hosseini et al. 2015a). The disease was reported for the first 3 Department of Agricultural Sciences, Alma Mater Studiorum, time in Burma (Myanmar) (McGibbon 1924), and today, it has University of Bologna, Bologna, Italy been reported in many parts of the world. Phytoplasmas Author's personal copy Folia Microbiol infecting sesame plants were classified in 16SrI group sub- in Iran, little is known about the genetic diversity of the asso- group -B, 16SrII subgroups -A, -C, and -D, 16SrVI subgroup ciated phytoplasmas; therefore, the present work reports a -A and 16SrIX subgroup -C, and different leafhopper species study about genetic diversity and vector transmission of SP- were reported as vectors of the disease (Table 1). associated phytoplasmas in Iran. Sesame is one of the oldest plants grown in Iran, where its cultivation started around 2000 BC; currently, it is used as food and for medical purposes (Hue 1996). In Iran, total re- Materials and methods ported yield of sesame seeds is 28,000.00 t with 0.7 ton/ha (FAOSTAT 2013). Sesame phyllody (SP) disease was firstly Plant sampling observed in 1965 in Varamin (Mostafavi 1970), and subse- quently in most parts of the country, especially in tropical Sesame sampling areas were Fasa, Jahrom, Khafr, Mobarak and subtropical areas (Ibrahimi and Minasian 1975; Salehi Abad, Neyriz, Sarvestan and Zarghan (Fars province), Herat, and Izadpanah 1992; Dehgham et al. 2009; Esmailzadeh Yazd, Ashkezar (Yazd province), Ardestan, and Mahabad Hosseini et al. 2015b). Although the wide distribution of SP (Isfahan province). In each area, five sesame fields were Table 1 Sesame phyllody disease: associated phytoplasmas Phytoplasma Country Insect vector species Reference(s) and their distribution, taxonomy, group/ subgroup and insect vector 16SrI South Korea - Lee et al. 2004 16SrI India - Khan et al. 2007b 16SrI-B India Hishimonus phycitis Esmailzadeh Hosseini et al. 2015a, b Myanmar - Win et al. 2010 South Korea - Lee et al. 2004; Rao et al. 2015 16SrI-M India - Khan et al. 2007b; Manjunatha et al. 2012 16SrII Iran Orosius albicinctus Esmailzadeh Hoseini et al. 2007 16SrII Oman - Al-Sakeiti et al., 2005 16SrII-A Taiwan - Tseng et al. 2014 Thailand - Nakashima et al. 1995 16SrII-C India - Madhupriya et al. 2015;Nabietal.2015a 16SrII-D India - Madhupriya et al. 2015; Oman - Khan et al. 2007a Pakistan O. albicinctus Akhtar et al. 2008, 2009 Turkey O. albicinctus Ikten et al. 2014 16SrVI-A Turkey - Sertkaya et al. 2007 16SrIX Iran - Salehi et al. 2005 Turkey - Catal et al. 2013 16SrIX-C Turkey O. albicinctus Ikten et al. 2014 Unclassified Burkina Faso O. cellulosus Desmits and Laboucheix 1974 Etyopia - Kolte 1985 Iran Circulifer haematoceps Salehi and Izadpanah 1992 Iraq - Tamimi et al. 1989 Israel - Klein 1977 Mexico - Kolte 1985 Nigeria - Kolte 1985 Sudan C. haematoceps Choopanaya 1973;Kolte1985 Tanzania - Kolte 1985 Turkey - Kersting 1993 Uganda - Akhtar et al. 2009 Venezuela - Kolte 1985 -not determined Author's personal copy Folia Microbiol randomly selected for sampling both symptomatic plants and DNA extraction insects. During September, 300 sesame plants (5 plants per field) showing typical symptoms were selected for molecular Total DNAwas extracted from 0.2-g midrib tissue of naturally analyses and vector transmission studies. Plants used in the SP-infected sesame and symptomatic experimentally insect- latter study were grown from seed in an insect-proof green- inoculated sesame and periwinkle plants using Zhang et al. house, every 2 weeks sprayed with Metasystox insecticide. (1998) procedure. Total nucleic acids were extracted from Plastic cages were used for maintenance of leafhoppers on the field-collected leafhoppers by the Doyle and Doyle potted plants during the transmission trials. (1990) method. Total DNA extracted from symptomless ses- ame and periwinkle plants and insect samples from healthy colonies were used as negative controls. Positive control was a Insect transmission trials symptomatic periwinkle plant infected with Fars (Iran) alfalfa witches’ broom phytoplasmas (Salehi et al. 2005). From each sesame field, insects were collected two times (June and September) during the growing season by using a PCR amplification of 16S rRNA gene D-vac aspirator. Among collected leafhoppers, Circulifer haematoceps (Mulsant and Rey 1855) and Orosious Total DNA samples were tested for phytoplasma presence albicinctus Distant, 1918 Orosius x Orosius, two known SP using primer pair P1/P7 (Deng and Hiruki 1991;Schneider vectors in Iran, were separated to test their ability to transmit et al. 1995) followed by R16F2n/R16R2 (Gundersen and Lee SP phytoplasma strains. These specimens were identified by 1996). Primer pair P1/P7 amplifies a 1.8 kbp fragment of the comparison to the voucher specimen