Plant Pathol. J. 27(1) : 44-52 (2011) DOI: 10.5423/PPJ.2011.27.1.044 The Plant Pathology Journal pISSN 1598-2254 eISSN 2093-9280 © The Korean Society of Plant Pathology Detection of Viruses Infecting Stone Fruits in Western Mediterranean Region of Turkey Bayram Çevik Nejla Yardimci and Handan Çulal-Klllç Department of Plant Protection, Faculty of Agriculture, Suleyman Demirel University 32260 Isparta, Turkey (Received on October 2, 2010; Accepted on February 8, 2011) Field surveys were conducted in 45 stone fruit orchards biologically and taxonomically different and belong to in seven districts of Isparta Province located in western different genera of single strand positive sense RNA Mediterranean region of Turkey important for stone viruses. The most common RNA viruses found in stone fruit production. Leaf samples were collected from 175 fruits include Apple mosaic ilarvirus (ApMV), Prunus trees showing virus-like symptoms. These samples were necrotic ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus first tested by ELISA for five different RNA viruses (PDV), Plum pox potyvirus (PPV), and Apple chlorotic including Apple mosaic ilarvirus (ApMV), Prunus necrotic leafspot trichovirus (ACLSV). These viruses are widely ringspot ilarvirus (PNRSV), Prune dwarf ilarvirus (PDV), Plum pox potyvirus (PPV), Apple chlorotic leafspot tri- distributed in stone fruit production areas of the world and chovirus (ACLSV). While no ApMV and PPV infection some of them found with infection rate of 25-100% in was found, 46, 24 and 16 samples were tested positive certain regions of the Mediterranean basin (Myrta et al., for PDV, ACLSV and PNRSV, respectively, in ELISA 2003). Frequent mixed infections with two or more of these showing about 45% of symptomatic trees in the region viruses ranging from 8 to 22% were observed in some were infected with at least one of these viruses. In addi- orchards. Combination of high infection rate and frequent tion, it was found that nine sweet cherry trees were mixed mixed infections causes significant yield reduction up to infected with two or three of these viruses and PDV with 57% and economical losses in some region (Dominguez et an infection rate of 26.3% was the most widespread virus al., 1998; Myrta et al., 2003; Varveri and Bem, 1995). in symptomatic trees in western Mediterranean region. Stone fruit trees were mainly tested for virus infection by Thirty samples were selected and tested by a multiplex serological methods, especially by ELISA. Even though RT-PCR (mRT-PCR) for simultaneous detection of these viruses. While PPV was not detected, more than ELISA is a reliable test appropriate for routine diagnostic half of the tested 20 samples were individually or mixed screening, it has low sensitivity and pathogens present at infected with ApMV, ACLSV, PNRSV and PDV. The very low titer may not be detected. Therefore, nucleic acid mRT-PCR results were confirmed by detection of these methods such as RT-PCR, hybridization and combination viruses individually in some of the field samples using of RT-PCR and ELISA were developed (Herranz et al., RT-PCR with primes specific to each virus. Com- 2005; Pallas et al., 1998; Sanchez-Navaro et al., 1998; Saade parison of ELSA and mRT-PCR results of 30 samples et al., 2000). More recently multiplex RT-PCR (mRT-PCR) showed that numbers of infected and mixed infected method enabling simultaneous detection of multiple viruses samples as well as infection and mixed infection rates have been developed and become a desirable assay for were significantly higher in RT-PCR (20 and 66.7%) routine diagnostic tests because it is more sensitive and than in ELISA (14 and 46.7%). The results confirm that require less time, labor and cost (Hassan et al., 2006; Menzel mRT-PCR is more sensitive than ELISA. et al., 2002; Sanchez-Navaro et al., 2005). A mRT-PCR Keywords : ELISA, multiplex RT-PCR, RNA viruses, stone assay was developed for simultaneous detection of eight fruits, virus detection different stone fruit viruses including ApMV, ACLSV, PNRSV, PDV, PPV, ApLV, APLPV and PBNSPaV. The method was successfully used for detection of these viruses A large number of RNA viruses cause economically on stone fruit trees in the field (Sanchez-Navarro et al., important diseases on stone fruits worldwide (Dunez, 1988; 2005). Myrta et al., 2003; Nemat, 1986). Most of these viruses are Presence of stone fruit viruses has previously been report- ed in Turkey. ApMV, ACLSV, PNRSV, PDV and PPV commonly found in commercial orchards in different stone *Corresponding author. fruit growing regions including Aegean (Azeri, unpublished Phone) +902462114737, FAX) +902462371693 E-mail) [email protected] data), eastern Mediterranean (Gazel and Çaglayan, unpublished Detection of Stone Fruits Viruses in Turkey 45 data; Yildizgördü Çali, unpublished data), eastern Anatolian phosphatase. Absorbance values of alkaline phosphatase (Elibüyük and Erdiler, 1998; Sipahioglu et al., 1999; were measured at 405 nm with VersaMax microplate reader Sipahioglu and Baloglu, unpublished data), central Anatolian (Molecular Devices, Sunnyvale, CA). Samples with absor- (Elibüyük, 2003) regions. Some of these viruses were also bance values greater than twice the mean absorbance read- found nurseries in certain regions (Sertkaya et al., 2004; ing of healthy controls obtained from Agdia (Elkhart, USA) Ulubaçs and Ertunç, 2004). In addition, mixed infection of were considered positive for each virus. two or more of these viruses were detected in some regions by ELISA. Nucleic acid isolation. Different nucleic acid isolation Isparta Province located in the western Mediterranean methods were used for isolation of total nucleic acid and region of Turkey is an important fruit production region. dsRNA. Total nucleic acids were isolated from stone fruit However, presence of viruses on stone fruits is largely un- leaf samples using previously published methods (Astruc et known in the region. So far only one study was conducted el., 1996) with minor modifications. About 100 mg leaf for attempting to determine presence of stone fruit viruses tissue was first grounded with liquid nitrogen in 2 ml tube in Isparta. As part of a national survey 16 stone fruit with plastic passel attached to hand held drill and homo- samples from a collection orchard in Isparta were tested for genized in 1 ml of extraction buffer (100 mM Tris-HCl, 50 ACLSV and PNRSV. Only one sample was tested positive mM EDTA, pH 8.0, 500 mM NaCl, 10 mM β-mercapto- for ACLSV by ELISA (Ulubaçs and Ertunç, 2005) and one ethanol). After the addition of 50 ml of 20% SDS the peach and plum tree were found to be infected with homogenate was incubated at 65 oC for 20 min. Then, 250 PNRSV by RT-PCR in the collection orchard (Ulubaçs and ml of 5 M KOAc were added and the mixture was incubat- Ertunç, 2004). In a regional survey, ApMV was detected on ed on ice for 20 min. After the removal of unwanted plant apple orchards in Isparta (Yardimci and Eryigit, 2005). debris by centrifugation at 10,000 rpm for 15 min at 4 oC, However, no comprehensive surveys and tests were con- nucleic acids were recovered from the supernatant by ducted in the region to detect stone fruit viruses in com- ethanol precipitation. It was washed with 70% cold ethanol, mercial orchards. In this study, stone fruit orchards were air dried, resuspended in 50 ml of sterile water and stored at screened for five different RNA viruses including ACLSV, −80 oC. ApMV, PDV, PNRSV and PPV by serological and mole- A previously reported dsRNA isolation method (Morris cular assays. Infection rate of these viruses in symptomatic and Dodds, 1979) was modified and used for dsRNA trees in different locations and different Prunus hosts was isolation from some samples from which RNA was not determined. In addition, the sensitivity of ELISA and mRT- recovered by total nucleic isolation protocol described PCR was compared for evaluation of different detection above. Briefly, about 5 g leave tissue were first ground in a assays for virus screening on stone fruits in Isparta. mortar and homogenized in 10 ml of STE buffer (0.1 M NaCl, 0.05 M Tris -HCl, 0.1 mM EDTA, pH 6.8), then Materials and Methods mixed with 10 ml phenol, chloroform-pentanol (25:1 v:v), with 2% bentonit and 10% SDS. The mixture was centri- Field surveys. Surveys were conducted in spring of 2005 in fuged at 10,000 rpm for 20 min, 95% ethanol was added a number of commercial and one collection stone fruit into the supernatant and after overnight incubation at 4 oC it orchards in nine different districts of Isparta Province was fractionated on Whatman CF-11 cellulose columns in located in south-western Turkey. Leaf samples were collect- the presence of ethanol to select dsRNA. Then, dsRNAs ed from a total of 175 different stone fruit trees including bound cellulose columns were eluted from the cellulose in sweet cherry, sour cherry, peach, apricot and plum showing ethanol free STE buffer, concentrated by ethanol precipita- virus or virus-like symptoms. Locations and the hosts of tion, resuspended in 50 µl sterile water and stored at −80 oC. 175 samples collected during field surveys are shown in Table 2. Multiplex reverse transcription polymerase chain reac- tion (mRT-PCR). The mRT-PCR method previously report- Double antibody sandwich enzyme linked immuno- ed for simultaneous detection of eight different stone fruit sorbent assay (DAS-ELISA). All leaf samples were test- viruses was modified and used for detection of five viruses ed for the presence of five different RNA viruses infecting including ACLSV, ApMV, PDV, PPV and PNRSV in this stone fruits including ACLSV, ApMV, PNRSV, PDV and study using primers listed in Table 1. About 1-5 µl of total PPP by enzyme linked immunosorbent assay (ELISA) nucleic acids or dsRNA from the different extraction pro- using specific ELISA detection kits for respective viruses cedures were denatured by incubation at 70 oC or 95 oC for 5 (Agdia, Elkhart, USA) based on previously reported DAS- min and used to perform a 25 µl mRT-PCR reaction using ELISA method (Clark and Adams, 1977) with alkaline MMuLV reverse transcriptase (Biobasic, Canada) and Taq 46 Bayram Çevik Nejla Yardimci and Handan Çulal-Klllç Table 1.