J Phytopathol

ORIGINAL ARTICLE Occurrence and Characterization of a 16SrII-D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Mohammad Salehi1, Seyyed Alireza Esmailzadeh Hosseini2, Elham Salehi1 and Assunta Bertaccini3

1 Plant Protection Research Department, Fars Agricultural and Natural Resources Research and Education Center, AREEO, Shiraz, Iran 2 Plant Protection Research Department, Agricultural and Natural Resources Research and Education Center, AREEO, Yazd, Iran 3 Department of Agricultural Sciences, Plant Pathology, Alma Mater Studiorum - University of Bologna, Bologna, Italy

Keywords Abstract 16S rRNA, ‘Candidatus Phytoplasma australasia’, dodder and graft transmission, During 2010–2013 surveys for the presence of phytoplasma diseases in molecular analysis, parsley diseases (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short Correspondence spindly shoots from crown buds, little leaf, yellowing, witches’ broom, M. Salehi, Plant Protection Research stunting, flower virescence and phyllody. The disease causative agent was Department, Fars Agricultural and Natural dodder transmitted from symptomatic parsley to periwinkle and from Resources Research and Education Center, AREEO, Shiraz, Iran. periwinkle to periwinkle by grafting inducing phytoplasma-type symp- E-mail: [email protected] toms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally Received: June 12, 2016; accepted: August 9, inoculated periwinkle plants in direct polymerase chain reaction (PCR) 2016. using phytoplasma primer pair P1/P7 or nested PCR using the same pri- mer pair followed by R16F2n/R16R2 primers. Restriction fragment length doi: 10.1111/jph.12520 polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd pro- vince belong to 16SrII-D phytoplasma subgroup. This is the first report of association of a 16SrII-related phytoplasma with PrWB disease in Iran.

European, Middle East and American cuisine (Athar Introduction et al. 1999; Mozafarian 2007a,b). The parsley impor- Phytoplasmas are cell wall-less phloem inhabiting tance is attributed to its high vitamin content (mainly bacteria affecting more than 1000 plant species vitamin C), antioxidants and some mineral elements including many economically important fruits, veg- such as iron and volatile oils that play an important etables, cereals and trees. Characteristic symptoms role in the pharmaceutical and food industries (Lopez associated with phytoplasma infection include yellow- et al. 1999; Sidra et al. 2014). The Iranian production ing, little leaf, flower virescence, phyllody, prolifera- of fresh vegetables including parsley is 3 646 570 tons tion and sterility, witches’ broom and dwarfing (Lee (FAOSTAT 2013). et al. 2000; Bertaccini et al. 2014). Phytoplasmas are There is only one report available of aster yellows vectored by leafhoppers, planthoppers and psyllids phytoplasmas in parsley with yellows symptoms in (Weintraub and Beanland 2006); however, they can Canada (Khadhair et al. 1998) and one report on the also be transmitted by dodder, grafting and vegetative presence of Spiroplasma citri in asymptomatic parsley propagation (Lee and Davis 1992). (Gera et al. 2011). During 2010–2013 surveys for the Vegetables are infected by phytoplasmas belonging presence of phytoplasma diseases in Yazd province to different 16S ribosomal RNA groups worldwide (Iran) parsley plants showing excessive development (Lee et al. 2003, 2004). Parsley [Petroselinum crispum of short spindly shoots from crown buds, little leaf, (Mill.) Fuss, 1866, Apiaceae] is a biennial herb native yellowing, stunting, flower virescence and phyllody to the Mediterranean region and is widely used in were observed, the main common symptom was the

Ó 2016 Blackwell Verlag GmbH 1 Parsley witches’ broom phytoplasma M. Salehi et al. witches’ broom, and therefore, the disease was named of disease symptoms and polymerase chain reaction parsley witches’ broom (PrWB). The main objective of (PCR) assays. the present work was to identify and characterize the For long-term maintenance, the PrWB agent was phytoplasmas detected in the symptomatic parsley then graft transmitted to periwinkle plants. Small plants. axillary shoots from a newly infected dodder inocu- lated periwinkle plant from each area were used as scions and side grafted on five 12-week-old seed- Materials and Methods grown periwinkle plants. Each donor plant received two scions. Grafted areas were wrapped with parafilm Source of the disease and plants covered with plastic bags for a week to Sampling of symptomatic parsley was carried out in maintain humidity. Abarkooh, Khatam, Herat-Marvast, Mehreez and Yazd areas, Iran. From each area, two parsley fields DNA extraction and polymerase chain reaction assays were surveyed and two parsley plants (one plant per field) with typical symptoms of witches’ broom Total DNA was extracted from 0.2 g midrib tissue of (PrWB) were selected, potted and transferred to a 10 naturally witches’ broom affected parsley plants greenhouse. They were biweekly sprayed with Meta- from Abarkooh, Khatam, Herat-Marvast, Mehreez systox-R insecticide and used as the sources of the and Yazd areas (two symptomatic plants per area) and PrWB disease in biological and molecular studies. graft and dodder inoculated periwinkle plants (Zhang Twelve-week-old seed-grown periwinkle [Catharan- et al. 1998). Total DNA was extracted from symptom- thus roseus (L.) G. Don] plants were used in dodder less parsley and periwinkle plants and a symptomatic and graft transmission for in vivo maintenance of the periwinkle plant infected with Fars alfalfa witches’ PrWB agent. broom phytoplasma (Salehi et al. 2005), a 16SrII-C strain [(Acc. No. DQ233) (Salehi et al. 2014)]; they were used as negative and positive controls, respec- Disease incidence tively. DNA samples were tested for phytoplasma In each area, four fields were selected randomly and presence using universal phytoplasma primer pair P1/ sampling was carried out at five points in 1000 m2 P7 (Deng and Hiruki 1991; Schneider et al. 1995) in fields within a 1 m2 on a diagonal transect across each direct PCR and followed by R16F2n/R16R2 (Gunder- of the five fields. The percentage of PrWB disease inci- sen and Lee 1996) primer pairs in nested PCR assays. dence was calculated by number of plants with symp- Polymerase chain reaction conditions and reagents toms out of total number of plants observed using the were as reported (Salehi et al. 2014). Polymerase formula given below. chain reaction products were separated in 1% agarose Per cent disease incidence gels in 1X TBE buffer [108 g Tris-HCl, 55 g boric acid, 40 ml EDTA (0.5 M), pH 8.0]. DNA bands were No. of plants infected ¼ 100 stained with ethidium bromide and visualized with a Total no. of plants observed UV transilluminator. The molecular weight of the PCR products was estimated by comparison with 100- Dodder and graft transmission bp DNA ladder (Fermentas, Vilnius, Lithuania). Dodder (Cuscuta campestris Yunk.) was used for trans- mission of the PrWB agent from 10 symptomatic pars- Restriction fragment length polymorphism (RFLP) ley plants from Abarkooh, Khatam, Herat-Marvast, analysis Mehreez and Yazd areas (two symptomatic plant per area) to periwinkle plants. For this purpose, seeds of Restriction fragment length polymorphism analysis of dodder were collected in a phytoplasma-free sugar nested PCR products was used for preliminary identi- beet field, germinated on moist filter paper, and seed- fication of the phytoplasma associated with PrWB dis- lings were transferred to 10 witches’ broom affected ease. The R16F2n/R16R2 amplicons were digested parsley plants (each plant in one pot) maintained separately with AluI, HaeIII, HhaI, HpaII, MseI, RsaI under an insect-proof greenhouse. Four weeks later, and TaqI restriction enzymes according to the instruc- each dodder-infested plant was placed adjacent to tions of the manufacturer (Fermentas) at 37°C (65°C three healthy seed-grown periwinkle plants. After for TaqI) overnight. The restriction products were 5 weeks, periwinkle plants were freed of dodder and then separated by a 6.7% polyacrylamide gel elec- kept in the insect-free greenhouse for the observation trophoresis and stained with ethidium bromide. DNA

2 Ó 2016 Blackwell Verlag GmbH M. Salehi et al. Parsley witches’ broom phytoplasma

bands were visualized with a UV transilluminator. MEGA6 (Tamura et al. 2013). Acholeplasma laidlawii The resulting RFLP patterns were compared with was used as an out-group to root the trees. Bootstrap- those previously published for 16S rDNA from other ping was performed 1000 times to estimate the stabil- phytoplasma strains (Lee et al. 1998). ity and support for the branches. The 16S rDNA sequence identity between strains was evaluated after alignments generated using homology matrix distance Cloning and sequencing of PCR products option of DNAMAN version 4.02 (Lynnon Biosoft, Que- Ten P1/P7 primed PCR products from the 10 bec, Canada). witches’ broom affected parsley plant samples col- lected from five fields (one field per area and two Virtual RFLP analysis samples per field) in the surveyed areas were ligated onto pTZ57R/T vector and cloned into Virtual RFLP analysis using the iPhyClassifier tool Escherichia coli DH5a cells using InsT/A cloneTM (Zhao et al. 2009) was determined for subgroup affili- PCR Product Cloning Kit (Fermentas) according to ation of PrWB phytoplasma strains under study. RFLP the manufacturer’s instructions. The presence of the profiles of the PrWB phytoplasma strains under study correct insert was confirmed by restriction endonu- were compared on the 1248-bp fragment (R16F2n/R2 clease analysis using EcoR1 and Pst1 enzymes. Plas- region of 16S rRNA gene) to those of 16SrII subgroups mid DNA from three recombinant colonies was -A to -L. Each aligned DNA fragment was digested purified using High Pure Isolation kit (Roche, Man- in silico with 17 distinct restriction enzymes: AluI, nheim, Germany). Sequencing was performed by BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, Macrogen (South Korea) on both strands using HinfI, HpaI, HpaII, KpnI, MboI(Sau3AI), MseI, RsaI, SspI M13 (-21) forward (50-TGTAAAACGACGGCCAGT- and TaqI and restriction profiles were compared 30) and M13 (-29) reverse (50-CAGGAAACAGCTAT- to those of the reported phytoplasma groups and GACC-30) primers (Macrogen, DNA sequencing ser- subgroups. vice, South Korea). A database search of homologous sequences was performed by BLAST Results analyses to determine the closest phytoplasma rela- tives of the PrWB phytoplasma. Disease symptoms and incidence Occurrence of witches’ broom symptoms was Sequence homology and phylogenetic analysis observed in both one- and two-year-old parsley plants R16F2n/R16R2 primed sequences of 26 phytoplasma in all surveyed areas. Symptoms were excessive devel- strains including PrWB phytoplasma were separately opment of short spindly shoots from crown buds, little aligned, and a phylogenetic tree was generated using leaf, yellowing, witches’ broom, stunting (Fig. 1a),

Fig. 1 (a) Yellowing, little leaf, internode shortening, witches’ broom and stunting in a parsley plant in a Chahgeer (Yazd province, Iran) field compared with asymptomatic pars- ley plants (behind). (b) Flower proliferation, phyllody, and witches’ broom in inflorescence of a parsley plant.

Ó 2016 Blackwell Verlag GmbH 3 Parsley witches’ broom phytoplasma M. Salehi et al.

flower virescence and phyllody (Fig. 1b). Disease inci- (Fig. 2). The minimum time elapsing between inocu- dence was variable, and maximum infection rates in lation and symptom expression in dodder and graft Abarkooh, Khatam, Herat-Marvast, Mehreez and inoculated periwinkle plants was 51 and 28 days, Yazd were 6.7, 5, 4.8, 4 and 3.2%, respectively. respectively.

Graft and dodder transmission PCR amplification and RFLP analysis Thirty of 30 dodder inoculated and 25 of 25 graft inoc- In direct PCR using P1/P7 primer pair and nested PCR ulated periwinkle plants developed phytoplasma-type using P1/P7 followed by R16F2n/R16R2 primer pair, symptoms including flower virescence and phyllody all symptomatic parsley and experimentally infected periwinkle plants and positive control were positive, and DNA fragments of approximately 1800 and 1200 bp, respectively, were amplified (not shown). No amplifications were observed in DNA samples from symptomless plants in both direct and nested PCR assays. The RFLP patterns from the R16F2n/R16R2 ampli- cons of the 10 symptomatic parsley plants (two plants per area) and five dodder inoculated periwinkle plants (one plant per area) were identical (Fig. 3a) and refer- able to those previously published for members of the peanut witches’ broom group (16SrII) (Lee et al. 1998).

Nucleotide sequence analyses P1/P7 PCR products of 10 PrWB phytoplasma strains were successfully cloned and sequenced: obtained sequences showed 100% identity among each others. Full length 16S rRNA gene sequence of a representa- tive of these sequences, Chahgeer PrWB (C-PrWB) phytoplasma, was submitted to GenBank database under the accession number KU501295. BLAST search showed that C-PrWB strain had maximum identity (99%) with members of peanut witches’ broom (16srII) phytoplasma group. Phylogenetic analysis (Fig. 4) showed that the C-PrWB strain clusters in the branch that encloses the Fig. 2 Virescence and phyllody in a periwinkle plant dodder inoculated phytoplasma strains from group 16SrII, and was most with parsley witches’ broom agent.

Fig. 3 (a) Polyacrylamide gel showing RFLP profiles of a 16S rDNA amplified by nested PCR using P1/P7 followed by R16F2n/R16R2 primer pairs from parsley witches’ broom phyto- plasma in Chahgeer of Abarkooh (Yazd pro- vince, Iran). PCR products were digested by the enzymes shown in the picture. (b) Virtual RFLP of Chahgeer parsley witches’ broom phy- toplasma (GenBank accession no. KU501295) generated with iPhyClassifier (Zhao et al. (a) (b) 2009).

4 Ó 2016 Blackwell Verlag GmbH M. Salehi et al. Parsley witches’ broom phytoplasma closely related to the ‘Candidatus Phytoplasma aus- Virtual RFLP analysis tralasia’ strains (GenBank accession nos. JQ868448 Virtual RFLP analysis (Fig. 3b) confirmed that the pat- and Y10096). The homology percentages between tern of PrWB phytoplasma was identical (similarity 16S rDNA sequences (Table 1) showed that among coefficient 1.00) to the reference pattern of 16Sr 16SrII subgroups (A-L), C-PrWB strain had maximum group II, subgroup D (GenBank accession no. identity (100%) with ‘Ca. P. australasia’ (GenBank Y10097). accession no. Y10096).

Fig. 4 Phylogenetic tree constructed by the Neighbor-joining method of 16S rRNA gene sequences from 26 phytoplasmas and A. laidlawii, as the out-group. The position of Chahgeer parsley witches’ broom phytoplasmas is bolded. Numbers at the nodes are bootstrap (confidence) values based on 1000 repetitions.Ca. P.’, ‘Candidatus Phytoplasma’; CWB, cactus witches’ broom. GenBank accession numbers for sequences are in parenthesis, and ribosomal subgroups are on the left. Bar, 1 nucleotide substitution per 100 nucleotides.

Table 1 Pairwise homology (%) among Chahgeer parsley witches’ broom phytoplasma and selected phytoplasmas in group 16SrII using 16S rRNA gene sequences a ABCDEF GHI J KL

CPrWB 99.8 98.7 98.5 100 98.6 98.6 98.4 98.2 98.0 98.1 98.2 98.0 aA, peanut witches’ broom (16SrII-A, GenBank accession no. L33765); B, ‘Ca. P. aurantifolia’ (16SrII-B, GenBank accession no. U15442); C, faba bean phyllody (16SrII-C, GenBank accession no. X83432); D, ‘Ca. P. australasia’ (16SrII-D, GenBank accession no. Y10096); E, Pichris echioides phyllody (16SrII-E, GenBank accession no. Y16393); F, cotton phyllody (16SrII-F, GenBank accession no. EF186827); G, cactus witches’ broom strain YN23 (16SrII-G, GenBank accession no. EU099568); H, cactus witches’ broom strain YN24 (16SrII-H, GenBank accession no. EU099569); I, cactus witches’ broom strain YN06 (16SrII-I, GenBank accession no. EU099550); J, cactus witches’ broom strain YN07 (16SrII-J, GenBank accession no. EU099552); K, cactus witches’ broom strain YN28 (16SrII-K, GenBank accession no. EU099572); L, cactus witches’ broom strain YN01 (16SrII-L, GenBank accession no. EU099546); C-PrWB, Chahgeer parsley witches’ broom phytoplasma (GenBank accession no. KU501295).

Ó 2016 Blackwell Verlag GmbH 5 Parsley witches’ broom phytoplasma M. Salehi et al.

Discussions Esmailzadeh Hosseini SA, Khodakaramian G, Salehi M, Fani SR, Mirchenari SM, Salehi E, Bertaccini A. (2015b) On the basis of disease symptoms, dodder transmis- Incidence, distribution and economic importance of sion and PCR amplification, PrWB in Yazd province alfalfa witches’ broom disease in Sistan-Baluchestan was associated to phytoplasma presence. Restriction (Iran) and characterization of associated phytoplasma. fragment length polymorphism analyses indicated Phytopathogenic Mollicutes 5:32–38. that the associated phytoplasma is a member of FAOSTAT. (2013). Internet Resource: http://faostat3. 16SrII-D subgroup. The sequence homology and fao.org (verified May, 2016). phylogenetic analyses further confirmed this classifi- Gera A, Maslenin L, Weintraub P, Mawassi M. (2011) cation. In our knowledge, this is the first report of Phytoplasma and spiroplasma diseases in open-field association of a 16SrII-related phytoplasma with crops in Israel. Bull Insectol 64(Suppl):S53–S54. PrWB disease. Reported phytoplasmas associated with Gundersen DE, Lee I-M. (1996) Ultrasensitive detection of parsley diseases belong to 16SrI phytoplasma group phytoplasmas by nested-PCR assays using two universal (Khadhair et al. 1998). As the 16SrII-D subgroup primer pairs. Phytopath Medit 35:144–151. phytoplasmas have been previously detected in sun- Khadhair AH, Kawchuk LM, Taillon RC, Botar G. (1998) flower (Salehi et al. 2015a) and squash (Salehi et al. Detection and molecular characterization of an aster 2015b) in Yazd province, it is suggested that in these yellows phytoplasma in parsley. Can J Plant Path areas parsley, as a biennial plant, can serve as 16SrII- 20:55–61. D phytoplasma reservoir for infection of these and Lee I-M, Davis RE. (1992). Mycoplasmas which infect other susceptible plant hosts. Further investigations plants and insects. In: Maniloff J, McElhaney RN, Finch are needed to determine the transmission by insect LR, Baseman JB. (eds) Mycoplasmas: Molecular Biology and Pathogenesis. Washington, D.C, American Society vectors of PrWB phytoplasma to other plant species of Microbiology, pp 379–390. present in these areas. In Abarkooh (Yazd province) Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. area, Circulifer haematoceps leafhopper have been (1998) Revised classification scheme of phytoplasmas reported as vector of the alfalfa witches’ broom dis- based on RFLP analyses of 16SrRNA and ribosomal pro- ease that is associated with a 16SrII-C phytoplasma tein gene sequences. Int J Syst Bacteriol 48:1153–1169. (Esmailzadeh Hosseini et al. 2015a,b). Evaluation of Lee I-M, Davis RE, Gundersen-Rindal DE. (2000) Phyto- vectoring ability of C-PrWB phytoplasma with plasma: phytopathogenic mollicutes. Annu Rev Micro- C. haematoceps remains to be determined. biol 54:221–255. Lee I-M, Martini M, Bottner KD, Dane RA, Black MC, Acknowledgements Troxclair N. (2003) Ecological implications from a molecular analysis of phytoplasmas involved in an aster This research was supported by a grant from Fars yellows epidemic in various crops in Texas. Phytopathol- Agricultural and Natural Resources Research and ogy 93:1368–1377. Education Center, AREEO, Shiraz, Iran. Lee I-M, Gundersen-Rindal DE, Davis RE, Bottner KD, Marcone C, Seemuller€ E. (2004) ‘Candidatus Phyto- plasma asteris’, a novel taxon associated with aster References yellows and related diseases. Int J Syst Bacteriol Athar N, Spriggs TW, Liu P. (1999) The Concise New Zeal- 54:1037–1048. and Food Composition Tables, 4th edn. Wellington, New Lopez MG, Sancheze-Medoza IR, Ochoa-Alejo N. (1999) Zealand, New Zealand Institute for Crop & Food Comparative study of volatile components and fatty Research Ltd, Palmerston North, New Zealand and acids of plants and in vitro cultures of parsley (Pet- Ministry of Health, 1999. roselinum crispum) (Mill) Nym ex Hill. J Agric Food Chem Bertaccini A, Duduk B, Paltrinieri S, Contaldo N. (2014) 47:3292–3296. Phytoplasmas and phytoplasma diseases: a severe threat Mozafarian V. (2007a) A Dictionary of Iranian Plant to agriculture. Am J Plant Sci 5:1763–1788. Names. Tehran, Farhang Moaser press 671p. Deng SJ, Hiruki C. (1991) Amplification of 16S ribosomal- Mozafarian V. (2007b) Umbelliferae. In: Assadi M, RNA genes from culturable and nonculturable molli- Khatamsaz M, Maasoumi AA. (eds) Flora of Iran, No. cutes. J Microbiol Meth 14:53–61. 54. Tehran, Research Institute of Forests and Range- Esmailzadeh Hosseini SA, Khodakaramian G, Salehi M, lands, pp 596. Fani SR, Bolok Yazdi HR, Raoufi D, Jadidi O, Bertaccini Salehi M, Heydarnejad J, Izadpanah K. (2005) Molecular A. (2015a) Status of alfalfa witches’ broom phytoplasma characterization and grouping of 35 phytoplasmas from disease in Iran. Phytopathogenic Mollicutes 5(1-Suppl): central and southern . Iran J Plant S65–S66. Pathol 41:62–64.

6 Ó 2016 Blackwell Verlag GmbH M. Salehi et al. Parsley witches’ broom phytoplasma

Salehi E, Salehi M, Taghavi SM, Izadpanah K. (2014) A a culinary herb of Mediterranean region. Pak J Pharm 16SrII-D Phytoplasma strain associated with tomato Sci 27:193–202. witches’ broom in Bushehr province. Iran J Crop Prot Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 3:377–388. (2013) MEGA6: Molecular Evolutionary Genetics Salehi M, Esmailzadeh Hosseini SA, Salehi E. (2015a) Analysis Version 6.0. Mol Biol Evol 30: Characterisation of a phytoplasma associated with sun- 2725–2729. flower phyllody in Fars, Isfahan and Yazd provinces of Weintraub P, Beanland L. (2006) Insect vectors of phyto- Iran. New Dis Rept 3:6. plasmas. Ann Rev Entomol 51:91–111. Salehi M, Siampour M, Esmailzadeh Hosseini SA, Bertac- Zhang YP, Uyemoto JK, Kirkpatrick BC. (1998) A small- cini A. (2015b) Characterization and vector identifica- scale procedure for extracting nucleic acids from woody tion of phytoplasmas associated with cucumber and plants infected with various phytoplasmas for PCR assay. squash phyllody in Iran. Bull Insectol 68:311–319. J Virol Methods 71:45–50. Schneider B, Seemuller€ E, Smart CD, Kirkpatrick BC. Zhao Y, Wei W, Lee M, Shao J, Suo X, Davis RE. (1995) Phylogenetic classification of plant pathogenetic (2009) Construction of an interactive online phyto- mycoplasmalike organ-isms or phytoplasmas. In: Razin plasma classification tool, iPhyClassifier, and its appli- S, Tulley JG. (eds) Molecular and Diagnostic Procedures cation in analysis of the peach X-disease phytoplasma in Mycoplasmology, Vol. I. S. New York, Aca-demic group (16SrIII). Int J Syst Evol Microbiol 59:2582– Press, pp 369–380. 2593. Sidra M, Shahzad H, Farnaz M. (2014) Critique of medici- nal conspicuousness of parsley (Petroselinum crispum):

Ó 2016 Blackwell Verlag GmbH 7