Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in

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

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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 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 (). 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 identified previously by ribosomal operon which encloses the 16S rRNA gene, the the British Museum (UK). Specimens of C. haematoceps and 16S–23S intergenic spacer region (SR) and a portion of the O. albicinctus randomly selected from each area (10 speci- 5′ region of the 23S rRNA gene and R16F2n/R16R2 primer mens of each species per area) were separately tested for phy- pair that amplifies 1.25 kbp of 16S rRNA gene. PCR condi- toplasma presence using nested PCR assay. For transmission tions were as described previously (Salehi et al. 2011). The trials, non-inoculative and inoculative colonies of C. amplifications were carried out in a programmable haematoceps and O. albicinctus leafhoppers were allowed to thermocycler (Bio-Rad, USA); PCR products were electro- grow in an insect-proof greenhouse (no. 1), while healthy test phoresed in 1 % agarose gels in TAE buffer and visualized plants (sesame, sugar beet, and periwinkle) were grown in a with a UV transilluminator following ethidium bromide stain- separate insect-proof greenhouse (no. 2). Non-inoculative ing. The molecular weight of the PCR products was estimated colonies of C. haematoceps and O. albicinctus were sepa- by comparison with 100-bp DNA ladder (Fermentas, Vilnius, rately established by transferring single-fertilized females Lithuania). on healthy sugar beet plants for egg deposition and subse- quent hatching. Non-inoculative colonies and sugar beet plants were frequently monitored by nested PCR for phy- Nucleotide sequencing and analysis toplasma presence. Inoculative colonies were developed by transferring adult C. haematoceps and O. albicinctus from Five nested PCR products of samples from each surveyed non-inoculative colonies to sesame plants (infected by dif- areas in Fars province were ligated onto pTZ57R⁄T vector ferent SP phytoplasma strains and maintained in green- and cloned into Escherichia coli DH5a cells using InsT ⁄ A house no.2). In the time of inoculation, caged healthy ses- cloneM PCR Product Cloning Kit (Fermentas, Vilnius, ame and periwinkle plants (free of insects) were brought to Lithuania) according to manufacturer instructions. The pres- greenhouse no. 1 and inoculative leafhoppers (200 insects ence of the correct insert was confirmed by restriction endo- per species) were transferred on healthy plants (10 insects nuclease analysis using EcoR1 and Pst1 enzymes. Plasmid per plant) under the cages. The inoculation feeding time on DNA from cultures of recombinant colonies was purified each plant species was 10 days. After the inoculation ac- using GF-1 PCR Clean-Up Kit (Vivantis, Malysia, HQ). cess period (IAP), the insects under the cages were killed Sequencing was performed by Macrogen (South Korea) on using Metasystox insecticide, cages were removed, and both strands by using M13 (−21) forward (5′-TGTA test plants (in pots) were transferred to greenhouse no. 2 AAACGACGGCCAGT-3′)andM13(−29) reverse (5′- (which was insect-free and insect-proof) for monitoring CAGGAAACAGCTATGACC-3′) primers (BioNeer, DNA disease symptoms development and sampling for PCR as- sequencing service, S. Korea). The Yazd and Isfahan SP says. Fifty non-inoculative specimens of each leafhopper strains were directly sequenced using nested PCR products species fed on five plants of each species were used as obtained with P1/P7 and R16mF2/R16mR2, and after assem- negative controls. Three months post-inoculation, plants bling, the resulting sequences were trimmed to the R16F2n/ were molecularly tested for phytoplasma presence. R16R2 fragment (1250 bp). A database search of homologous Author's personal copy

Folia Microbiol sequences was then performed by Blast analyses at the Results National Center for Biotechnology Information (NCBI). Symptomatology Virtual RFLP analysis Occurrence of SP disease was observed in the all surveyed areas including Fasa, Jahrom, Khafr, Mobarak Abad, Neyriz, Virtual RFLP analysis using iPhyClassifier (Zhao et al. 2009) Sarvestan, Zarghan (Fars province), Herat, Yazd, Ashkezar was used to determine subgroup affiliation of SP (Yazd province), Ardestan and Mahabad (Isfahan province). phytoplasmas. Each aligned DNA fragment was digested in Disease incidence rate in some sesame fields in Zarghan and silico with 17 distinct restriction enzymes: AluI, BamHI, BfaI, Ashkezar approached 100 %. The prevalent symptoms of dis- BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, ease were severe little leaf, internode shortening, witches’ KpnI, MboI(Sau3AI), MseI, RsaI, SspI, and TaqI that have broom (Fig. 1a), flower virescence, phyllody, proliferation been used for the phytoplasma 16S rDNA RFLP analysis (Lee and sterility (Fig. 1c), cracking of seed capsules (Fig. 1d), et al. 1998). germination of seeds in capsules, dwarfing, and yellowing.

Sequence homology and phylogenetic analysis DNA amplification Partial 16S rDNA sequences of 12 SP phytoplasma strains from Fasa, Jahrom, Khafr, Mobarak Abad, Neyriz, After 35 cycles, DNA fragments of approximately 1.8 and Sarvestan, Zarghan (Fars province), Herat, Yazd, Ashkezar 1.2 kbp were amplified in direct and nested PCR, respectively, (Yazd province), Ardestan and Mahabad (Isfahan province) from 300 naturally SP-affected sesame plants (25 samples per (present study) and seven SP phytoplasma sequences from area) from 12 areas in Fars, Yazd, and Isfahan provinces and Iranian Fars, Bushehr and Khorasan Razavi provinces, re- from all the experimentally inoculated symptomatic sesame trieved from GenBank (Table 2) were separately aligned and and periwinkle plants. Neither control plants nor leafhopper related phylogenetic trees and sequence homologies generated samples tested positive in direct PCR. Forty-three out of 120 using MEGA6 software (Tamura et al. 2013). Acholeplasma samples of C. haematoceps from all surveyed areas and 29 out laidlawii wasusedasanoutgrouptorootthetree. of 120 samples of O. albicinctus from Mobarak Abad, Fasa, Bootstrapping was performed 1000 times to estimate the sta- Jahrom, Khafr, Yazd, Ashkezar, Mahabad, and Ardestan were bility and support for the tree branches. positive in nested PCR assay.

Table 2 Sesame phyllody phytoplasma strains characterized Sesame phyllody strain Location (province) GenBank Acc. no. Ribosomal group in this study Bushehr2 Bushehr KC429654 16SrII Jahrom Fars KF607109 16SrII Khafr1 Fars KT265706 16SrII Mobarak Abad Fars KT265705 16SrII Mahabad Isfahan KU297203 16SrII Yazd Yazd KT923671 16SrII Bushehr1 Bushehr KC429656 16SrVI Fasa3 Fars KT265697 16SrVI Jahrom1 Fars KT265698 16SrVI Jahrom2 Fars KT265699 16SrVI Jahrom4 Fars KT265700 16SrVI Neyriz Fars KT265699 16SrVI Khorasan Razavi1 Khorasan Razavi JF508514 16SrVI Darab Fars KF607108 16SrIX Fasa1 Fars KT265703 16SrIX Sarvestan Fars KT265704 16SrIX Zarghan Fars KT265702 16SrIX Sabzevar Khorasan Razavi KF774193 16SrIX Khorasan Razavi2 Khorasan Razavi JF508515 16SrIX Author's personal copy

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ABJahrom4, Neyriz, Khorasan Razavi1, Bushehr1, and Fasa3 were referable to those of ‘Candidatus Phytoplasma trifolii’ (Genbank Acc. no. AY390261), rep- resentative of 16SrVI-A subgroup; however, while the sequences of strains Khorasan Razavi1 and Bushehr1 were identical to the one of ‘Ca. P. trifolii,’ sequences of Jahrom1, Jahrom2, Jahrom4, Neyriz, and Fasa3 were dif- ferentiable from it in DraI, EcoRI, MseI, and RsaI restric- tion profiles. Based on the same enzymes, the sequence of CD Fasa 3 also was differentiable from the others and from ‘Ca. P. trifolii’. Zarghan, Fasa1, Darab, Sarvestan, Khorasan Razavi2, and Sabzevar strains produced profiles identical to the one of Knautia arvensis phyllody phyto- plasma (GenBank Acc. no. EF186823), classified in 16SrIX-C subgroup (Lee et al. 2012). Sequences of strains Khafr, Yazd, Mahabad, Bushehr2, and Jahrom showed identical patterns to the one of ‘Ca. P. australasia’ (GenBank Acc. no. Y10096), a 16SrII-D phytoplasma (Lee et al. 2010), while the sequence of Mobarakabad strain was distinguishable from the one of Fig. 1 Symptoms of sesame phyllody disease. a Severe little leaf, ‘Ca. P. austaliense’ by HinfI virtual restriction profile ’ internode shortening, and witches broom symptoms in phyllody- (Fig. 2). affected sesame field in Zarghan area (Fars province). b Healthy sesame plant. c Flower virescence and phyllody. d Cracking of seed capsules Phylogenetic analyses Nucleotide sequence analyses The phylogenetic trees generated by the analysis of Sixty R16F2n/R16R2 amplified fragments of SP 1.2 kbp 16S rRNA genes belonging to representatives or phytoplasmas from 12 areas in three provinces were se- subgroups of 16SrI, 16SrII, 16SrIII, 16SrIV, 16SrVI, quenced. In each area, one example of identical sequences 16SrIX, and 16SrXII phytoplasma groups, and SP phyto- was submitted to the GenBank; totally, 12 sequences from plasma strains from Iran showed that Jahrom1, Jahrom2, nine areas including Fasa (two sequences), Jahrom (three se- Jahrom4, Neyrizi, and Fasa3 (Fars province), Bushehr1 quences), Khafr, Mobarak Abad, Neyriz, Sarvestan, Zarghan, (Bushehr province), and Khorasan Razavi1 (Khorasan Yazd, and Mahabad (one sequence per area) were submitted Razavi province) clustered with 16SrVI; Zarghan, Fasa1, under accession numbers (Acc. nos.): KT265703, KT265697, Sarvestan, Darab (Fars province), Khorasan Razavi2, and KT265698, KT265699, KT265700, KT265706, KT265705, Sabzevar (Khorasan Rasavi province) with 16SrIX while KT265701, KT265704, KT265702, KT923671, and Mobarak Abad, Khafr, Jahrom (Fars province), Yazd KU297203, respectively. BLAST search using these se- (Yazd province), Mahabad (Isfahan province), and quences and similar sequences (1.2 kbp of ribosomal Bushehr2 (Bushehr province) clustered with16SrII phyto- RNA operon) retrieved from GenBank showed that three plasma group (Fig. 3). Phylogenetic trees and percent se- SP phytoplasma strains from Jahrom (Jahrom1, Jahrom2, quence homology (Table 3) confirmed the results of vir- Jahrom4) in addition to one strain from each of Fasa tual RFLP analyses. In these analyses, Jahrom1, Jahrom2, (Fasa3), Neyriz, Bushehr1, and Khorasan Rasavi1 blast Jahrom4, Neyrizi, Fasa3, Bushehr, and Khorasan Razavi1 with 16SrVI group phytoplasmas, while Darab, Fasa1, strains were closer to vinca virescence phytoplasma Sarvestan, Zarghan, Khorasan Razavi2, and Sabzevar blast (Genbank Acc. no. AY500817), enclosed in the 16SrVI- with 16SrIX group phytoplasmas, Bushehr2, Jahrom, A subgroup (Lee et al. 2010) than other subgroups of Mobarak Abad, Khafr, Yazd, and Mahabad strains with 16SrVI group. These strains had maximum homology – BCa P. trifolii’ 16SrII group phytoplasmas. (99.2 99.8 %) with . (GenBank Acc. no. AY390261), representative of 16SrVI-A subgroup. Zarghan, Fasa1, Sarvestan, Darab, Khorasan Razavi2, Virtual RFLP analysis and Sabzevar SP strains clustered with Knautia arvensis phyllody phytoplasma (Genbank Acc. no. EF18682) rep- Results of virtual RFLP analyses of the 1.2 kbp 16S resentative of 16SrIX-C subgroup (Lee et al. 2012)show- rDNA of sequences from strains Jahrom1, Jahrom2, ing maximum homology (99.8–99.9 %) with this strain. Author's personal copy

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Fig. 2 Virtual RFLP profiles generated with program iPhyClassifier from in silico digestion of the R16F2n/R16R2 DNA fragments of the sesame phyllody phytoplasmas and of selected representatives of subgroups of 16SrVI-A, -B, and - C, 16SrIX-D and -E and 16SrII-F and -G. Profiles are in a, ‘Ca. P. tr if oli i’ (Acc. no. AY39026) (16SrVI-A); in b,Jahrom1, Jahrom2, Jahrom4, and Neyriz1, Khorasan Razavi1; in c,Fasa3;in d, Knautia arvensis phyllody phytoplasma strain KAP (Acc. no. EF186823) (16SrIX-C); in e, profiles of Darab, Fasa1, Sarvestan, Zarghan, Khorasan Razavi2, Sabzevar; in f; ‘Ca.P. austaliense’ (Acc. no. Y1009) (16SrII-D) and profiles of Bushehr, Fasa1, Jahrom5, Esfahan, Yazd strains; in g, Mobarak Abad strain. Boxes show the patterns of differentiating enzymes; MW, virtual ØX174 HaeIII digested size marker

Mobarak Abad, Khafr, Jahrom, Bushehr2, Yazd, and the only species reared in caged sesame plants. Out of 120 Mahabad clustered with ‘Ca. P. australiense’,arepresen- DNA samples of C. haematoceps 42 specimens from tative of 16SrII-D subgroup with sequence homology of Fasa, Jahrom, Khafr, Mobar Ababad, Neyriz, Sarvestan, 99.9–100 %. Based on these results, 16SrVI-A strains Zarghan, Yazd and Isfahan were positive in nested PCR were identified in Fars, Bushehr, and Korasan Razavi assay. In the case of O. albicinctus,32samplesfrom provinces, extending from south to north Iran; 16SrII-D Jahrom, Fasa, Kafr, Mobarak, Darab, Yazd, and Isfahan strains were present in Fars, Bushehr, Isfahan, and Yazd were PCR positive. provinces, central and southern regions, and 16SrIX in In the trials for the transmission of SP strains identified Fars, and Khorasane Razavi, central and northern regions. in this study (Table 4), C. haematoceps leafhopper trans- It appears that 16SrII-D is present only in the central mitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas regions of Iran. from affected sesame plants to healthy sesame and peri- winkle plants. Twenty-one out of 30 sesame seedlings and Vector transmission 19 out of 30 periwinkle plants inoculated with each of these phytoplasma strains showed symptoms and were C. haematoceps and O. albicinctus (Fig. 4)werethemost positive in PCR assay. O. albicinctus only transmitted abundant leafhoppers collected in the sesame fields and 16SrII-D phytoplasma and seven of 10 inoculated sesame Author's personal copy

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Fig. 3 Phylogenetic tree constructed by the Neighbor- Joining method using partial 16S rRNA gene sequences (1250 bp) of 48 phytoplasmas and A. laidlawii as the outgroup; ‘Ca. P.’: BCandidatus phytoplasma^; CWB cactus witches’ broom; numbers at the nodes are bootstrap (confidence) values are based on 1000 repetitions; only bootstrap values higher than 70 are reported; GenBank accession numbers for sequences are given in parentheses after the phytoplasma names, Iranian sesame phyllody phytoplasmas are in bold; bar 1 nucleotide substitution per 100 nucleotides Author's personal copy

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Table 3 Pairwise homology (%) among 19 Iranian sesame phyllody phytoplasma strains and selected phytoplasma representatives of 16SrII, 16SrVI, and 16SrIX subgroups using partial 16S rRNA gene sequences

Ribosomal subgroups Iranian SP strains and related 16Sr groups

16SrII * A B C D E F G H I J K L Mobarak Abad 99.8 98.9 98.6 99.9 98.5 98.7 98.5 98.5 98.1 98.2 98.3 98.1 Khafr 99.9 99.0 98.7 100 98.6 98.8 98.6 98.5 98.2 98.3 98.4 98.2 Yazd 99.9 99.0 98.7 100 98.6 98.8 98.6 98.5 98.2 98.3 98.4 98.2 Jahrom 99.9 99.0 98.7 100 98.6 98.8 98.6 98.5 98.2 98.3 98.4 98.2 Bushehr2 99.9 99.0 98.7 100 98.6 98.8 98.6 98.5 98.2 98.3 98.4 98.2 Mahabad 99.7 98.7 98.5 99.7 98.4 98.5 98.4 98.3 98.0 98.0 98.1 98.0 16SrVI ** ABCDEF HI Jahrom1 99.2 98.9 98.9 99.0 98.6 99.0 97.9 96.9 Jahrom2 99.4 99.1 99.1 99.2 98.7 99.2 98.1 97.1 Jahrom4 99.4 99.1 99.1 99.2 98.7 99.2 98.1 97.1 Neyriz 99.4 99.1 99.1 99.2 98.7 99.2 98.1 97.1 Fasa3 99.7 99.3 99.3 99.4 99 99.4 98.3 97.3 Bushehr1 99.8 99.5 99.5 99.6 99.2 99.6 98.5 97.5 Khorasan Razavi1 99.8 99.5 99.5 99.6 99.2 99.6 98.5 97.5 16SrIX *** ABCDEF Khorasan Razavi2 99.4 99.0 99.8 99.5 99.4 99.0 Darab2 99.4 99.0 99.8 99.5 99.4 99.0 Sabzevar 99.4 99.1 99.9 99.6 99.5 99.1 Fasa1 99.4 99.1 99.9 99.6 99.5 99.1 Zarghan1 99.4 99.2 99.8 99.7 99.4 99.0 Sarvestan1 99.4 99.2 99.8 99.7 99.4 99.0

*A, peanut witches’ broom (16SrII-A, Acc. no. L33765); B, ‘Ca. P. aurantifolia’ (16SrII-B, Acc. no. U15442); C, faba bean phyllody (16SrII-C, Acc. no. X83432); D, ‘Ca.P.australasia’ (16SrII-D, Acc. no. Y10096); E, Pichris echioides phyllody (16SrII-E, Acc. no. Y16393); F, cotton phyllody (16SrII-F, Acc. no. EF186827); G, cactus witches’ broom strain YN23 (16SrII-G, Acc. no. EU099568); H, cactus witches’ broom strainYN24 (16SrII-H, Acc. no. EU099569); I, cactus witches’ broom strain YN06 (16SrII-I, Acc. no. EU099550); J, cactus witches’ broom strain YN07 (16SrII-J, Acc. no. EU099552); K, cactus witches’ broom strain YN28 (16SrII-K, Acc. no. EU099572); L, cactus witches’ broom strainYN01 (16SrII-L, Acc. no. EU099546); Mobarak Abad (Acc. no. KT265705), Khafr (Acc. no. KT265706), Yazd (Acc. no. KT923671), Jahrom (Acc. no. KF607109), Bushehr2 (Acc. no. KC429654) and Mahabad (Acc. no. KU297203), Iranian sesame phyllody phytoplasma strains **A, ‘Ca.P.trifolii’ (16SrVI-A, Acc. no. AY390261); B, strawberry multiplier disease (16SrVI-B, Acc. no. AF190224); C, Illinois elm yellows (16SrVI- C, Acc. no. AF409070); D, periwinkle little leaf (16SrVI-D, Acc. no. AF228053); E, Centaurea solstitialis virescence (16SrVI-E, Acc. no. AY270156); F, Catharanthus phyllody phytoplasma (16SrVI-F, Acc. no. EF186819); H, portulaca little leaf phytoplasma (16SrVI-H, Acc. no. EF651786); I, ‘Ca.. P. sudamericanum’ (16SrVI-I, Acc. no. GU292081). Jahrom1 (Acc. no. KT265698), Jahrom2 (Acc. no. KT265699), Jahrom4 (Acc. no. KT265700), Neyriz (Acc. no. KT265701), Fasa3 (Acc. no. KT265697), Bushehr1 (Acc. no. KC429656) and Khorasan Razavi1 (Acc. no. JF508514), Iranian sesame phyllody strains ***A, pigeon pea witches’ broom (16SrIX-A, Acc. no. AF248957); B, ‘Ca.. P. phoenicium’ (16SrIX-B, Acc. no. AF515636); C, Knautia arvensis phyllody phytoplasma (16SrIX-C, Acc. no. EF186823); D, Lactuca sativa phyllody (16SrIX-D, Acc. no. DQ889748); E, Juniperus witches’ broom (16SrIX-E, Acc. no. GQ925918); F, Honduran Gliricidia little leaf (16SrIX-F, Acc. no. AF361017); Khorasan Razavi2 (Acc. no. JF508515), Darab (Acc. no. KF607108), Sabzevar (Acc. no. KF774193), Fasa1 (Acc. no. KT265703), Zarghan (Acc. no. KT265702) and Sarvestan (Acc. no. KT265704), Iranian sesame phyllody strains and nine of 10 periwinkle plants were symptomatic and strains were severe, moderate, and almost absent, respectively. PCR positive. Neither C. haematoceps nor O. albicinctus More yellowing was seen in 16SrVI-A strain inoculated per- from non-inoculative colonies were able to transmit SP iwinkles than in those inoculated with 16SrIX-C and 16SrII-D phytoplasma strains. strains (Fig. 5). Disease symptoms in vector inoculated sesame plants were similar to those of naturally diseased plants, while same age periwinkle plants inoculated at the same time with different SP Discussion phytoplasma strains show differentiable symptomatology. Little leaf, internode shortening, and stunting in periwinkle Occurrence of sesame phyllody phytoplasma disease was re- plants inoculated with 16SrIX-C, 16SrVI-A, and 16SrII-D ported from different sesame growing areas in Iran (Salehi and Author's personal copy

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Fig. 4 Circulifer haematoceps (a)andOrosious albicinctus (b)vectors of all the identified strains and of 16SrII strain, respectively, of sesame phyllody phytoplasma in Iran

Izadpanah 1992; Dehghan et al. 2009;EsmailzadehHosseini et al. 2015b), but a comprehensive studies on diversity and classification of sesame phyllody strains was never per- formed. In the present work, molecular identification of phytoplasmas associated with sesame phyllody disease and ability of C. haematoceps and O. albicinctus to vector differ- ent sesame phyllody phytoplasma strains is reported. Based on Blast search using partial 16S rRNA gene, SP phytoplasma strains from 21 areas in five provinces are shown to belong to Fig. 5 Reaction of periwinkle plants 2 months old to the insect vector inoculation carried out in the same time with different SP phytoplasma three 16Sr phytoplasma groups including 16SrII, 16SrVI, and strains, 130 days after the beginning of transmission trials. a Severe little 16SrIX. Collectively, virtual RFLP, phylogenetic analyses, leaf, internode shortening and stunting in a periwinkle plant inoculated and percent sequence homology showed that all 16SrIX phy- with a 16SrIX phytoplasmas. b Yellowing moderate little leaf, internode toplasma strains were related to -C subgroup, while those shortening and stunting in a periwinkle plant inoculated with a 16SrVI-A phytoplasmas. c Virescence, phyllody, and nearly normal leaves and in- classified in the 16SrVI and 16SrII groups were identical or ternodes in a periwinkle plant inoculated with 16SrII-D phytoplasmas. d very similar to phytoplasmas in 16SrVI-A and 16SrII-D sub- Two healthy seed grown periwinkle plants groups, respectively. Different reaction of periwinkle plants to SP phytoplasma strains confirmed the genetic diversity of SP disease agents in Iran. lettuce (Salehi et al. 2007a; Lee et al. 2012) and -E in tomato The 16SrIX-C subgroup phytoplasmas have been previ- (Jamshidi et al. 2014) were also reported. In Iran, in addition ously detected in almond (Salehi et al. 2006) and eggplant to sesame, tomato (Jamshidi et al. 2014) was reported as host (Tohidi et al. 2015) in Fars and Hormozgan provinces of of 16SrVI-A phytoplasmas. Phytoplasmas associated with Iran; however, other subgroups of 16SrIX group including - safflower phyllody (Salehi et al. 2009), cabbage yellows B in almond and GF-677 trees (Motamedi et al. 2016), -D in (Salehi et al. 2007b), greenhouse cucumber phyllody

Table 4 Transmission of sesame phyllody strains identified in this study by Circulifer haematoceps and Orosius albicinctus leafhoppers

Total samples PCR positive Transmission of sesame phyllody phytoplasmas to experimental hostsa collected samples 16SrII-D 16SrVI-A 16SrIX-C

Sesame Periwinkle Sesame Periwinkle Sesame Periwinkle

C. haematoceps 120 42 9/10 6/10 7/10 4/10 5/10 9/10 O. albicinctus 120 30 10/10 9/10 0/10 0/10 0/10 0/10 a Number of infected plants/number of inoculated plants Author's personal copy

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(Esmailzadeh Hosseini et al. 2015c), and alfalfa witches’ Bertaccini A, Duduk B, Paltrinieri S, Contaldo N (2014) Phytoplasmas broom (Esmailzadeh Hosseini et al. 2015b) are other three and phytoplasma diseases: a severe threat to agriculture. Am J Pl Sci 5:1763–1788 16SrVI group phytoplasma reported in the country, but their Catal M, Ikten C, Yol E, Ustun R, Uzun B (2013) First report of 16SrIX subgroup affiliation was not determined. Other reported hosts group phytoplasma associated with sesame phyllody in Turkey. of 16SrII-D phytoplasmas in Iran are squash (Salehi et al. Plant Dis 97:835 2015b), tomato (Salehi et al. 2014), sunflower (Salehi et al. Choopanya D (1973) Mycoplasma like bodies associated with sesamum phyllody in Thailand. Phytopathology 63:1536–1537 2015a), and alfalfa (Esmailzadeh Hosseini et al. 2015a). 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Focus – related to 16SrI and 16SrVI groups with C. haematoceps 12:13 15 Esmailzadeh Hosseini SA, Khodakaramian G, Salehi M, Fani SR, was reported from Iran (Salehi et al. 2007b, 2011), to our Mirchenari SR, Elham S (2015a) Incidence, distribution and eco- knowledge, this is the first report of transmission of 16SrII nomic importance of alfalfa witches’ broom disease in Sistan- and 16SrIX group phytoplasmas by this insect vector. Baluchestan (Iran) and characterization of associated phytoplasma. In all surveyed areas also O. albicinctus was collected but Phytopath Moll 5:1–7 Esmailzadeh Hosseini SA, Salehi M, Khodakaramian G, Bolok Yazdi only 16SrII-D phytoplasmas associated with sesame phyllody HR, Salehi M, Jafari Nodooshan A, Jadidi O, Bertaccini A were transmitted by this species. This is in agreement with (2015b) Status of sesame phyllody and its control methods in reports from Turkey demonstrating the transmission of only Yazd, Iran. 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