D Subgroup Phytoplasma Associated with Parsley Witches’

J Phytopathol ORIGINAL ARTICLE Occurrence and Characterization of a 16SrII-D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran 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, Yazd 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 Yazd province (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: salehi_abarkoohi@yahoo.com 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 primer 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 province 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 inoculated 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 Â 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 transmission 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 collected 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

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