Bulletin of Insectology 68 (2): 311-319, 2015 ISSN 1721-8861 Characterization and vector identification of phytoplasmas associated with cucumber and squash phyllody in Iran 1 2 3 4 Mohammad SALEHI , Majid SIAMPOUR , Seyyed Alireza ESMAILZADEH HOSSEINI , Assunta BERTACCINI 1Plant Protection Research Department, Fars Agricultural and Natural Resources Research and Education Center, AREEO, Zarghan, Iran 2Department of Plant Protection, College of Agriculture, Shahrekord University, Shahrekord, Iran 3Plant Protection Research Department, Yazd Agricultural and Natural Resources Research and Education Center, AREEO, Yazd, Iran 4Department of Agricultural Sciences, Alma Mater Studiorum University of Bologna, Italy Abstract Phytoplasmas associated with cucumber phyllody (CuP) and squash phyllody (SqP) in Yazd province of Iran were characterized by molecular analyses and biological studies. Orosius albicinctus leafhoppers testing positive for phytoplasma presence by polymerase chain reaction (PCR) successfully transmitted CuP and SqP phytoplasmas to healthy cucumber and squash plants. The phytoplas- mas were also transmitted by O. albicinctus from cucumber and squash to periwinkle, alfalfa, cucumber, carrot, sesame, sunflower, pot marigold, eggplant, squash, tomato and parsley. Both phytoplasmas induced similar symptoms in the post-inoculated plants. Restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA nested PCR products identified the CuP, SqP and O. albicinctus phytoplasmas as members of the 16SrII group. Sequence identity and phylogenetic analysis confirmed the placement of these phytoplasmas in the same clade of other phytoplasmas belonging to 16SrII group. Virtual RFLP analyses on 16S rDNA sequences allowed the affiliation of SqP phytoplasma to subgroup 16SrII-D, while the CuP phytoplasma was identified as represen- tative of a new subgroup 16SrII-M. This is the first report on molecular characterization of the CuP and SqP phyllody phytoplas- mas, including subgroup affiliation, identification of their leafhopper vector and determination of their plant host range in Iran. Key words: cucumber, squash, phyllody, 16SrII phytoplasma, characterization, leafhopper, Orosius albicinctus, Iran. Introduction Iran is among the top ten producers of cucumber after China with field cultivation of 8,700 ha and a year pro- Phytoplasmas are cell-wall less bacteria that have very duction of 1,811,630 tonnes (FAOSTAT, 2013). During small genome sizes and are amongst the smallest self- field surveys in 2004, cucumber (C. sativus) and squash replicating living organisms (Bertaccini et al., 2014). (C. pepo) plants showing phyllody symptoms were ob- They infect numerous important food, fibre, fodder and served in Abarkooh and Yazd areas (Yazd province, timber crops, causing significant crop losses, and are Iran). The present work reports biological and molecular transmitted by sap-feeding insects. Phytoplasma dis- characterization of phytoplasmas associated with cu- eases of the Cucurbitaceae family have been reported in cumber (CuP) and squash phyllody (SqP), and the Cucumis sativus L., Cucurbita maxima Duchesne, Cu- transmission experiments to identify insect vector(s) of curbita mixta Pangalo, Cucurbita pepo L., Lagenaria both SqP and CuP. leucantha Rusby, Lagenaria siceraria (Molina) Stand- ley (McCoy et al., 1989; Seemüller et al., 1998), Cu- curbita moschata Duchesne (Montano et al., 2006), Materials and methods Luffa cylindrica L. (McCoy et al., 1989; Lee et al., 1993; Gundersen et al., 1994; Montano et al., 2007a), Disease incidence Momordica charantia L. (McCoy et al., 1989; Montano In cucumber cultivations of Chahgeer five fields per et al., 2000), Sechium edule (Jacquin) Swartz (McCoy crop were selected randomly and sampling was carried et al., 1989; Montano et al., 2000; Villalobos et al., out randomly at five points in 1,000 m2 fields within a 2002), and Sicana odorifera (Vellozo) Naudin 1 m2 on a diagonal transect across each of the five (Montano et al., 2007b). Phytoplasmas associated with fields. The percentage of CuP disease incidence was diseases in cucurbitaceae plants were molecularly char- calculated by counting number of plants with symptoms acterized in C. pepo from Italy (Seemüller et al., 1998) out of total number of plants observed using the formula and in S. edule from Costa Rica (Villalobos et al., 2002) given below. where 16SrI group (‘Ca. P. asteris’) was detected. In C. No. of symptomatic plants % disease incidence = × 100 pepo from Australia and Egypt (Davis et al., 1997; No. of plants observed Omar and Foissac, 2012) and in S. edule, M. charantia, S. odorifera, C. moschata, and L. cylindrica, from Bra- Source of phytoplasmas zil, 16SrII group phytoplasmas were identified (Montano Cucumber and squash plants with typical symptoms of et al., 2000; 2006; 2007a; 2007b). In L. cylindrica, phyllody were selected in fields located in Chahgeer lo- 16SrVIII group phytoplasmas were detected in Taiwan cation in Abarkooh area (140 Km west of Yazd), trans- (Lee et al., 1993; Gundersen et al., 1994). ferred to a greenhouse located in Zarghan (Fars prov- ince, Iran) and used as sources for biological and mo- Host range studies lecular studies of the associated phytoplasmas. Leaf Followed taxonomical identification, non-inoculative midribs (0.3 g) from symptomatic cucumber and squash colonies of O. albicinctus were developed by transfer- plants collected in the fields and from the experimen- ring single fertilized females to a healthy sugar beet tally post-inoculated plants were subjected to total DNA plants for egg deposition and subsequent hatching. Non- extraction. A periwinkle [Catharanthus roseus (L.) inoculative colonies were frequently monitored for SqP G. Don] plant infected with a ‘Ca. P. aurantifolia’ and CuP phytoplasma presence by nested PCR. Highly (Salehi et al., 2002) was used as positive control. inoculative O. albicinctus colonies were developed by Healthy C. sativus, C. pepo, C. roseus, Medicago sativa transferring adult O. albicinctus from non-inoculative L. and Solanum lycopersicum L. grown from seeds col- colonies to infected cucumber and squash plants, and lected in Zarghan fields were used as negative controls. the resulting young adults used to inoculate cucumber, squash, periwinkle, sunflower, sesame, alfalfa, carrot, DNA extraction and PCR detection of phytoplasmas sugar beet, arugula, parsley, rapeseed, onion, pot mari- Total DNA was extracted from plant (Zhang et al., gold, eggplant and tomato plants (table 1). The inocula- 1998) and insect samples following the protocol of tion test for each plant species consisted of caging Doyle and Doyle (1990). The DNA quality and concen- twenty five inoculative leafhoppers of each species on tration was estimated by spectrophotometer and agarose five plants in a pot. Fifteen plants (in 3 pots) for each gel electrophoresis (Sambrook et al., 1989). For PCR, species were inoculated. The inoculation feeding time 100 ng of total DNA extract was used. The universal on each plant species was three weeks. After the acqui- primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et sition access period (AAP), plants were sprayed with al., 1995) was used to amplify the 16S rRNA operon insecticide and transferred to a separate insect-proof comprising the 16S rRNA gene, 16S-23S rRNA genes greenhouse for the monitoring of disease symptom ap- spacer region and the 5' end of the 23S rRNA gene. The pearance and PCR testing. Twenty five non-inoculative amplification products were diluted 1: 29 with sterile O. albicinctus were fed on five plants of each plant spe- deionized water and 1 µL was amplified in a nested cies used as negative controls. Cucumber, squash and PCR with the primer pair R16F2n/R2 (Gundersen and test plants used in host range studies were grown from Lee, 1996). The PCR reaction was performed in 50 µL seed in a greenhouse sprayed with insecticide every two reaction mixtures containing 0.4 µM of each primer, weeks. Six months post-inoculation, plants were tested 0.2 mM of each dNTP, 1.25 U Taq DNA polymerase for phytoplasma presence detection by nested-PCR as- (CinnaGen, Iran) and 5 µL 1X Taq polymerase buffer. says. The reaction cycled 35 times in a Bio-Rad (USA) ther- mal cycler with the following parameters: denaturing RFLP analyses for 1 min at 94 °C (2 min of initial denaturation), an- Products from R16F2n/R2 nested PCR were digested nealing for 2 min at 55 °C and primer extension for with restriction endonucleases AluI, HhaI, HinfI, HpaII, 3 min at 72 °C (10 min of final extension). PCR condi- MseI, RsaI and TaqI (Fermentas, Vilnius, Lithuania). tions for the nested PCR were the same except that the RFLP profiles were analyzed on 2% agarose electropho- annealing temperature was 58 °C. Following PCR, 5 µL resis gels followed by staining with ethidium bromide of each PCR product were electrophoresed in a 1% and visualization under a UV transilluminator. The 16S (w/v) agarose gel containing 0.3 µg/mL ethidium rDNA virtual RFLP patterns of CuP and SqP phyto- bromide in 0.5 X TBE buffer (22.5 mM Tris-borate, plasmas were analyzed and compared to that of other 1 mM EDTA, pH 8.0) to verify amplification of target phytoplasmas using iPhyClassifier (Zhao et al., 2009). DNA. Each 16S rDNA fragment was digested in silico with 17 distinct restriction enzymes [AluI, BamHI, BfaI, BstUI Vector identification, PCR examination and phyto- (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, plasma transmission KpnI, MboI (Sau3AI), MseI, RsaI, SspI and TaqI].