A virus causing a disease in mint (the aromatic and culinary plant) has recently become a problem in the Taurus Mountains, a mountain range in the Mediterranean region of Turkey. To detect the virus and investigate its distribution in the region, mint leaf samples were collected from the vicinity of spring areas in the plateaus of Antalya and Konya in 2009. It was found that Cucumber mosaic virus (CMV) was detected in 27.08% of symptomatic samples tested by DAS-ELISA. To the best of our knowledge, this is the first report of CMV on mint plants in this region of Turkey

A. Crescenzi

A. Hani

A. Samad

C. Ling

C. Varveri

D. O'Keefe

D. Sether

E. Chatzivassiliou

E. Johansen

F. Naghibi

G. Choi

G. Erdiller

H. Dorman

I. Tzanetakis

J. De Angelis

J. Duffus

J. Edwardson

J. Fletcher

J. Postman

M. Arli-Sokmen

M. Clark

M. Roossinck

M. Yilmaz

N. Kutluk-Yilmaz

N. Mimica-Dukic

P. Palukaitis

R. Dheepa

R. Grube

S. Flasinski

S. Unlu

V. Vicchi

Acta Botanica Croatica

Natural occurrence ofCucumber mosaic virusinfecting water mint (Mentha aquatica) in Antalya and Konya, TurkeyAvirus causing a disease in mint (the aromatic and culinary plant) has recently become a problem in the Taurus Mountains, a mountain range in the Mediterranean region of Turkey. To detect the virus and investigate its distribution in the region, mint leaf samples were collected from the vicinity of spring areas in the plateaus of Antalya and Konya in 2009. It was found that Cucumber mosaic virus (CMV) was detected in 27.08% of symptomatic samples tested by DAS-ELISA. To the best of our knowledge, this is the first report of CMV on mint plants in this region of Turkey.</jats:p

Mehmet Sevik

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Natural occurrence of Cucumber mosaic virus infecting water mint (Mentha aquatica) in Antalya and Konya, Turkey

Mehmet A. Sevik

HRČAK - Portal of Croatian Scientific and Professional Journals

Acta Bot. Croat. 71 (1), 187–193, 2012 CODEN: ABCRA25ISSN 0365–0588eISSN 1847-8476Short communicationNatural occurrence of Cucumber mosaic virusinfecting water mint (Mentha aquatica) in Antalyaand Konya, TurkeyMEHMET A. SEVIKDepartment of Plant Protection, Faculty of Agriculture, University of Ondokuz Mayis,Samsun, TurkeyAbstract – A virus causing a disease in mint (the aromatic and culinary plant) has recentlybecome a problem in the Taurus Mountains, a mountain range in the Mediterranean regionof Turkey. To detect the virus and investigate its distribution in the region, mint leaf sam-ples were collected from the vicinity of spring areas in the plateaus of Antalya and Konyain 2009. It was found that Cucumber mosaic virus (CMV) was detected in 27.08% ofsymptomatic samples tested by DAS-ELISA. To the best of our knowledge, this is the firstreport of CMV on mint plants in this region of Turkey.Key words: Disease, Cucumber mosaic virus, detection, Mentha aquaticaIntroductionMentha is a genus of flowering plants in the family Lamiaceae including about 30 spe-cies found in the temperate regions of the world (DORMAN et al. 2003). Species such as M.aquatica L. and M. longifolia (L.) are used as wild vegetables and culinary herbs (NAGHIBIet al. 2005). M. aquatica (water mint) is a perennial plant. As the name suggests, it occursin the shallow margins and channels of streams, rivers, pools, dikes, ditches, canals, wetmeadows, marshes and fens (LING 2011).Several viruses have been associated with disease symptoms in mint including Cucum-ber mosaic virus, Alfalfa mosaic virus, Tomato spotted wilt virus, Impatiens necrotic spotvirus, Arabis mosaic virus, Strawberry latent ringspot virus, Tobacco mosaic virus, To-bacco ringspot virus, Tomato leaf curl virus, Tomato aspermy virus, Cherry rasp leaf virus,Lychnis ringspot virus, Mint virus 1, Mint virus-2, Mint virus X, Mint vein banding associ-ated virus, Peppermint latent virus, and Peppermint stunt virus (VICCHI and BELLARDI1988, SETHER et al. 1991, DE ANGELIS et al. 1993, FLETCHER 2001, POSTMAN et al. 2004,TZANETAKIS et al. 2004, SAMAD et al. 2008, TZANETAKIS et al. 2010).ACTA BOT. CROAT. 71 (1), 2012 187* Corresponding author, e-mail: malis@omu.edu.trCopyright® 2012 by Acta Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:09Color profile: DisabledComposite  150 lpi at 45 degreesCucumber mosaic virus (CMV), the type member of the plant virus genus Cucumovirus(family Bromoviridae), has a wide host range and infects a great variety of important cropplants, making it one of the most economically significant plant viruses (PALUKAITIS et al.1992). CMV infects more than 1200 plant species (ROOSSINCK et al. 2001). Its ubiquitousnature may be attributed to its broad host range, non-persistent transmission by more than86 aphid species in the field (EDWARDSON and CHRISTIE 1991) and transmission throughseed in some hosts (O’KEEFE et al. 2007). Seed transmission plays a pivotal role in the sur-vival of the virus from season to season (JOHANSEN et al. 1994). Therefore, the managementof a CMV outbreak is difficult in the field (GRUBE et al. 2000). Ornamental plants, espe-cially those propagated vegetatively as bulbs or rhizomes may be virus reservoirs (FLA-SINSKI et al. 1995). Interestingly, water mint is a herbaceous rhizomatous perennial plamt.The rhizomes are wide-spreading, fleshy, and bear fibrous roots. Moreover, an importantnumber of plant species, including many weeds, have been reported to serve as reservoirsfor CMV during the intercropping season (CHATZIVASSILIOU et al. 2004).In previous studies, CMV infections have been frequently reported from Turkey on dif-ferent plant families showing mild to severe symptoms (ERDILLER and ERTUNÇ 1988,YILMAZ et al. 1995, ARLI-SOKMEN et al. 2005). In recent years, in mint-growing areasaround water springs of Antalya and Konya plateaus, a virus-like disease has become a ma-jor problem causing severe symptoms. The virus-like symptoms are exhibited by manymint plants, but little is known about the etiology of the disease. The objectives of the studywere to identify the virus and characterize the disease it caused.Materials and methodsSurveys and sample collectionIn 2009, surveys were performed in 14 natural spring water areas in some plateaus ofAntalya and Konya provinces containing mint plants. Leaf samples showing virus-likesymptoms were taken from mint plants exhibiting symptoms, placed in polythene bags andstored at –20 °C until use.Serological testingDAS-ELISA method was used to detect the virus in mint leaf samples and performedaccording to CLARK and ADAMS (1977) and the instructions of the CMV antiserum manu-facturer (Bioreba, Switzerland). Leaf samples with typical symptoms of virus infectionswere ground (1g leaf/5 mL buffer) in extraction buffer (PBS: 0.13 M NaCl, 0.014 MKH2PO4, 0.08 M Na2HPO4, 0.002 M KCl, pH: 7.4, containing 0.05% Tween-20), added tomicroplate wells (Nunc Microwell, Denmark) after coating with CMV-specific polyclonalantiserum diluted in carbonate buffer (pH: 9.6) and incubated at 4 °C overnight. Plates werewashed three times with PBS/Tween-20 buffer and coated with alkaline phosphatase-con-jugated antibody diluted in extraction buffer and incubated for 4h at 37 °C. After washing,p-nitrophenyl phosphate (Sigma) in diethanolamine substrate buffer (0.5 mg mL–1, pH:9.8) was added to the wells and incubated at room temperature for 30–180 min. Absor-bance values were read at 405 nm using a microplate reader (Tecan Spectra II). Extractionbuffer and healthy plants were used as negative controls. Samples were considered to be188 ACTA BOT. CROAT. 71 (1), 2012SEVIK M. A.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:09Color profile: DisabledComposite  150 lpi at 45 degreespositive when the absorbance values at 405 nm (A405) values exceeded the mean of the nega-tive controls by least a factor of three (KUTLUK-YILMAZ 2010).Biological testingNicotiana tabacum L. 'Samsun' was grown in a growth chamber at 23 °C with a 16 hlight/8 h dark photoperiod cycle. The samples of water mints collected (previously used forELISA) were used for preparation of inoculum in the buffer and all fully expanded leavesof plants at 2–4 leaves growth stage were inoculated.Results and discussionMost of the farmers and people of Antalya and Konya regions complained of an in-creased incidence of mosaic disease in mint plants over the past few years. Besides their cu-linary use, mint plants are also used during livestock grazing. It is well known that mint hasbeen used for centuries for its medicinal properties and in the food and fragrance industries(MIMICA-DUKIC et al. 2003).Visual surveys of the mint plants revealed a high incidence of leaf mosaic (Fig. 1), de-formation and severely affected apical plant growth. The newly growing apical leaves weresmall with very conspicuous symptoms. During the surveys, most spring areas containedplants with virus-like symptoms. Virus-like symptoms were observed in all 14 mints grownareas surveyed.CMV was detected by DAS-ELISA in the mint plants from all surveyed areas. The inci-dence of CMV in the symptomatic plants varied in the different springs. Serological assayresults revealed a variation in virus incidence among the different springs surveyed. The in-cidence of CMV was estimated as 27.08% in Antalya and Konya provinces, ranging from14.2% to 42.8% in the springs visited.ACTA BOT. CROAT. 71 (1), 2012 189DETECTION OF CUCUMBER MOSAIC VIRUS IN MINT PLANTSFig. 1. Symptoms associated with natural infections by CMV on water mint plants.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:10Color profile: DisabledComposite  150 lpi at 45 degreesIn the study, tests were conducted using several negative controls for CMV. Therefore,the range of absorbance values of negative controls varied from 0.066–0.118 at 405 nm.Positive samples gave absorbance values of 0.452– to 1.842 after 2 h substrate incubation(Table 1).The presence of CMV in water mint plant was verified in samples by transmission to in-dicator test plants, tobacco (N. tabacum). Tobacco plants mechanically inoculated with ex-tracts of ELISA-positive plants showed local necrotic spot on leaves 1 week after inocula-tion (Fig. 2). These symptoms were similar to those that were described previously for thevirus (CHOI et al. 2004).The cucumovirus CMV has been reported as the virus infecting different plants world-wide (VARVERI, and BOUTSIKA 1999) and different regions of Turkey (ERDILLER andERTUNÇ 1988, UNLU and GULDUR 2004), but infection of mint with CMV is reported for thefirst time in these regions. The report of CMV affecting Mentha was in 1966 in Germany,where it was found to be the cause of a mosaic disease of peppermint. CMV was found incultivated medicinal plants, including mint, in Hungary (TZANETAKIS et al. 2010). HANI(1971) studied the epidemiology of CMV in Switzerland and reported Mentha sp. as aweed host for the virus in the vicinity of tobacco fields where several CMV isolates werecausing problems. CMV was also isolated from Mentha sp. (VICCHI and BELLARDI 1988),M. piperita and M. palustris in Italy by CRESCENZI et al. (1993). M. pulegium for CMV wasfound infected for the first time by ELISA in Greece (CHATZIVASSILIOU et al. 2004).Weeds, native plants and seed transmission may have a significant effect on virus epi-demiology (DUFFUS 1971) and knowledge of weed reservoirs and vectors of viruses is es-sential for understanding the epidemiology. CMV-infected weeds may also play a signifi-cant role in its spread to crops. In 1998, 28 fields in Samsun, Turkey were surveyed and 222190 ACTA BOT. CROAT. 71 (1), 2012SEVIK M. A.Tab. 1. Range of absorbance values of positive and negative samples and incidence of CMV.No. of springssurveyedNo. ofsamples testedThe means DAS-ELISA absorbance valuesIncidence (%)Negative samples Positive samples1 12 0.084–0.215 0.767–1.231 16.62 4 0.090–0.193 0.829 25.03 8 0.059–0.330 0.452–0.669 37.54 10 0.104–0.364 0.860–1.076 20.05 6 0.093–0.316 1.290–1.842 33.36 5 0.110–0.299 1.082 20.07 7 0.075–0.317 0.773–0.803 42.88 7 0.060–0.262 1.318 14.29 9 0.124–0.355 0.793–0.916 22.210 8 0.078–0.326 0.510–0.599 37.511 7 0.066–0.303 0.685–0.697 28.512 6 0.088–0.249 0.860 16.613 3 0.115–0.354 0.965 33.314 4 0.128–0.338 0.495–0.852 28.5U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:10Color profile: DisabledComposite  150 lpi at 45 degreesweed samples were collected from pepper fields. Of the samples tested, 7.7% were found tobe infected with CMV. CMV was found to be the virus most frequently detected, in 13weed species out of 24 species (ARLI-SOKMEN et al. 2005). In experimental work, the virusis most frequently transmitted mechanically, and sap, purified virions, and viral RNA areall infectious via mechanical transmission (ROOSSINCK 2001). In addition, DHEEPA andPARANJOTHI (2010) reported that CMV from banana could be transmitted by rhizome inoc-ulation and mechanical methods. To help prevent CMV from spreading, farmers are rec-ommended to remove any mint plants infected with CMV near springs.The results obtained in this investigation clearly demonstrate that CMV is widely dis-tributed in different mint growing areas of Antalya and Konya, Turkey. This is the first re-port of CMV from water mint plants (M. aquatica) in Turkey.AcknowledgmentsI thank to D. Sevik for helping and assistance during surveys of these regions and sam-ple collections.ReferencesARLI-SOKMEN, M., MENNAN H, SEVIK M. A., ECEVIT O., 2005: Occurrence of viruses infield-grown pepper crops and some of their reservoir weed hosts in Samsun, Turkey.Phytoparasitica 33, 347–358.CHATZIVASSILIOU, E. K., EFTHIMIOU, K., DROSSOS, E., PAPADOPOULOU, A., POIMENIDIS, G.,KATIS, N. I., 2004: A survey of tobacco viruses in tobacco crops and native flora inGreece. European Journal of Plant Pathology 110, 1011–1023.CHOI, G. S., KIM, J. H., KIM, J. S., CHOI, J. K., 2004: Characterization of Cucumber mosaicvirus isolated from water chickweed (Stellaria aquatica). Plant Pathology Journal 20,131–134.ACTA BOT. CROAT. 71 (1), 2012 191DETECTION OF CUCUMBER MOSAIC VIRUS IN MINT PLANTSFig. 2. Virusinoculated tobacco (Nicotiana tabacum L. 'Samsun') showing local necrotic spots onleaves, characteristic of CMV infection.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:12Color profile: DisabledComposite  150 lpi at 45 degreesCLARK, M. R., ADAMS, A. M., 1977: Characteristics of the microplate method of enzymelinked immunosorbent assay for the detection of plant viruses. Journal of General Vi-rology 34, 475–483.CRESCENZI, A., BARBAROSSA, L., GALLITELLI, D., MARTELLI, G. P., 1993: Cucumber mosaiccucumovirus populations in Italy under natural epidemic conditions and after a satel-lite-mediated protection test. Plant Disease 77, 28–33.DE ANGELIS, J. D., SETHER, D. M., ROSSIGNOL, P. A., 1993: Survival, development, and re-production in western flower thrips (Thysanoptera, Thripidae) exposed to Impatiensnecrotic spot virus. Environmental Entomology 22, 1308–1312.DHEEPA, R., PARANJOTHI, S., 2010: Transmission of Cucumber mosaic virus (CMV) infect-ing banana by aphid and mechanical methods. Emirates Journal of Food and Agricul-ture 22, 117–129.DORMAN, H. J, KOSAR, M., KAHLOS, K., HOLM, Y., HILTUNEN, R., 2003: Antioxidant prop-erties and composition of aqueous extracts from mentha species, hybrids, varieties, andcultivars. Journal of Agricultural and Food Chemistry 51, 4563–4569.DUFFUS, J. E., 1971: Role of weeds in the incidence of virus diseases. Annual Review ofPhytopathology 9, 319–340.EDWARDSON, J. R., CHRISTIE, R. G., 1991: Cucumoviruses. In: CRC Handbook of virusesinfecting legumes, 293–319. CRC Press, Boca Raton.ERDILLER, G., ERTUNÇ, F., 1988: Identification of muskmelon viruses in Ankara province.Journal of Turkish Phytopathology 17, 47–56.FLASINSKI, S., SCOTT, S. W., BARNETT, O. W., SUN, C., 1995: Diseases of Peperomia,Impatiens and Hibbertia caused by Cucumber mosaic virus. Plant Disease 79, 843–848.FLETCHER, J. D. 2001: New hosts of Alfalfa mosaic virus, Cucumber mosaic virus, Potatovirus Y, Soybean dwarf virus, and Tomato spotted wilt virus in New Zealand. New Zea-land Journal of Crop and Horticultural Science 29, 213–217.GRUBE, R. C., ZHANG, Y., MURPHY, J. F., LOAIZA-FIGUEROA, F., LACKNEY, V. K., PROVVIDENTI,R., JAHN, M. K., 2000: New source of resistance to Cucumber mosaic virus in Capsi-cum frutescens. Plant Disease 84, 885–891.HANI, A. 1971: On the epidemiology of Cucumber mosaic virus in Tessin. Phytopatho-logische Zeitschrift 72, 115–144.JOHANSEN, E., EDWARDS, M. C., HAMPTON, R. O., 1994: Seed transmission of viruses: cur-rent perspectives. Annual Review of Phytopathology 32, 363–386.KUTLUK-YILMAZ, N.D. 2010: Interactions between beet necrotic yellow vein virus and beetsoilborne virus in different sugar beet cultivars. Anadolu Journal of Agricultural Sci-ences 25, 68–74.LING, C., 2011: Mentha aquatica. USGS Nonindigenous Aquatic Species Database, Gain-esville, FL.MIMICA-DUKIC, N., BOZIN, B., SOKOVIC, M., MIHAJLOVIC, B., MATAVULJ, M., 2003: Anti-microbial and antioxidant activities of three Mentha species essential oils. PlantaMedica 69, 413–419.192 ACTA BOT. CROAT. 71 (1), 2012SEVIK M. A.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:12Color profile: DisabledComposite  150 lpi at 45 degreesNAGHIBI, F., MOSADDEGH, M., MOTAMED, S. M., GHORBANI, A., 2005: Labiatae Family infolk Medicine in Iran: from Ethnobotany to Pharmacology. Iranian Journal of Pharma-ceutical Research 2, 63–79.O’KEEFE, D. C., BERRYMAN, D. I., COUTTS, B. A., JONES, R. A. C., 2007: Lack of seed coatcontamination with Cucumber mosaic virus in lupin permits reliable, large-scale detec-tion of seed transmission in seed samples. Plant Disease 91, 504–508.PALUKAITIS, P., ROOSSINCK, M. J., DIETZGEN, R. G., FRANCKI, R. I. B., 1992: Cucumber mo-saic virus. In: MARAMOROSCH, K., MURPHY, F. A. and SHATKIN, A. J. (eds.), Advances inVirus Research, 281–348. Academic Press, San Diego.POSTMAN, J. D., TZANETAKIS, I. E., MARTIN, R. R. 2004: First report of Strawberry latentringspot virus in Mentha sp. from North America. Plant Disease 88, 907.ROOSSINCK, M. J., 2001: Cucumber mosaic virus, a model for RNA virus evolution. Molec-ular Plant Pathology 2, 59–63.SAMAD, A., GUPTA, M. K., SHASANY, A. K., AJAYAKUMAR, P. V., ALAM, M., 2008: Bego-movirus related to Tomato leaf curl Pakistan virus newly reported in Mentha crops inIndia. New Disease Reports 18, 11.SETHER, D. M., DEANGELIS, J. D., ROSSIGNOL, P. A. 1991: First report of Tomato spottedwilt virus in peppermint (Mentha piperita). Plant Disease 75, 644.TZANETAKIS, I. E., POSTMAN, J. D., MARTIN, R. R., 2004: Identification, detection andphylogenetic analysis of new viruses infecting mint. Phytopathology 94, 104.TZANETAKIS, I. E., POSTMAN, J. D., SAMAD, A., MARTIN, R. R., 2010: Mint viruses: Beauty,stealth, and disease. Plant Disease 94, 4–12.UNLU, S., GULDUR, M. E., 2004: Determination of infection ratio of Cucumber mosaic vi-rus (CMV) on pepper in Sanliurfa. Journal of Faculty of Agriculture, Harran University8, 83–89.VARVERI, C., BOUTSIKA, K., 1999: Characterization of Cucumber mosaic cucumovirus iso-lates in Greece. Plant Pathology 48, 95–100.VICCHI, V., BELLARDI, M. G., 1988: The role of weeds in the epidemiology of Gladiolus vi-ruses and MLO. Acta Horticulture 234, 371–378.YILMAZ, M. A., BALOGLU, S., ÖZASLAN, M., GÜLDÜR, M. E., 1995: Plant viruses detected inhorticultural crops in the GAP region (in Turish). Proc Symposium of Plant ProtectionProblems and Prevention, Suggestions of Gap Region, Sanliurfa, Turkey, 241–250.ACTA BOT. CROAT. 71 (1), 2012 193DETECTION OF CUCUMBER MOSAIC VIRUS IN MINT PLANTSU:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:12Color profile: DisabledComposite  150 lpi at 45 degrees

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Acta Bot. Croat. 71 (1), 187–193, 2012 CODEN:ABCRA25ISSN 0365–0588eISSN 1847-8476Short communicationNatural occurrence of Cucumber mosaic virusinfecting water mint (Mentha aquatica) in Antalyaand Konya, TurkeyMEHMETA. SEVIKDepartment of Plant Protection, Faculty of Agriculture, University of Ondokuz Mayis,Samsun, TurkeyAbstract –Avirus causing a disease in mint (the aromatic and culinary plant) has recentlybecome a problem in the TaurusMountains, a mountain range in theMediterranean regionof Turkey. To detect the virus and investigate its distribution in the region, mint leaf sam-ples were collected from the vicinity of spring areas in the plateaus of Antalya and Konyain 2009. It was found that Cucumber mosaic virus (CMV) was detected in 27.08% ofsymptomatic samples tested byDAS-ELISA. To the best of our knowledge, this is the firstreport of CMV on mint plants in this region of Turkey.Key words: Disease, Cucumber mosaic virus, detection, Mentha aquaticaIntroductionMentha is a genus of flowering plants in the family Lamiaceae including about 30 spe-cies found in the temperate regions of the world (DORMAN et al. 2003). Species such as M.aquatica L. and M. longifolia (L.) are used as wild vegetables and culinary herbs (NAGHIBIet al. 2005). M. aquatica (water mint) is a perennial plant. As the name suggests, it occursin the shallow margins and channels of streams, rivers, pools, dikes, ditches, canals, wetmeadows, marshes and fens (LING 2011).Several viruses have been associated with disease symptoms in mint including Cucum-ber mosaic virus, Alfalfa mosaic virus, Tomato spotted wilt virus, Impatiens necrotic spotvirus, Arabis mosaic virus, Strawberry latent ringspot virus, Tobacco mosaic virus, To-bacco ringspot virus, Tomato leaf curl virus, Tomato aspermy virus, Cherry rasp leaf virus,Lychnis ringspot virus, Mint virus 1, Mint virus-2, Mint virus X,Mint vein banding associ-ated virus, Peppermint latent virus, and Peppermint stunt virus (VICCHI and BELLARDI1988, SETHER et al. 1991, DE ANGELIS et al. 1993, FLETCHER 2001, POSTMAN et al. 2004,TZANETAKIS et al. 2004, SAMAD et al. 2008, TZANETAKIS et al. 2010).ACTA BOT. CROAT. 71 (1), 2012 187* Corresponding author, e-mail: malis@omu.edu.trCopyright® 2012 byActa Botanica Croatica, the Faculty of Science, University of Zagreb. All rights reserved.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:09Color profile: DisabledComposite  150 lpi at 45 degreesCucumber mosaic virus (CMV), the typemember of the plant virus genus Cucumovirus(family Bromoviridae), has a wide host range and infects a great variety of important cropplants, making it one of the most economically significant plant viruses (PALUKAITIS et al.1992). CMV infects more than 1200 plant species (ROOSSINCK et al. 2001). Its ubiquitousnature may be attributed to its broad host range, non-persistent transmission by more than86 aphid species in the field (EDWARDSON and CHRISTIE 1991) and transmission throughseed in some hosts (O’KEEFE et al. 2007). Seed transmission plays a pivotal role in the sur-vival of the virus from season to season (JOHANSENet al. 1994). Therefore, the managementof a CMV outbreak is difficult in the field (GRUBE et al. 2000). Ornamental plants, espe-cially those propagated vegetatively as bulbs or rhizomes may be virus reservoirs (FLA-SINSKI et al. 1995). Interestingly, water mint is a herbaceous rhizomatous perennial plamt.The rhizomes are wide-spreading, fleshy, and bear fibrous roots. Moreover, an importantnumber of plant species, including many weeds, have been reported to serve as reservoirsfor CMV during the intercropping season (CHATZIVASSILIOU et al. 2004).In previous studies, CMV infections have been frequently reported from Turkey on dif-ferent plant families showing mild to severe symptoms (ERDILLER and ERTUNÇ 1988,YILMAZ et al. 1995, ARLI-SOKMEN et al. 2005). In recent years, in mint-growing areasaround water springs of Antalya and Konya plateaus, a virus-like disease has become ama-jor problem causing severe symptoms. The virus-like symptoms are exhibited by manymint plants, but little is known about the etiology of the disease. The objectives of the studywere to identify the virus and characterize the disease it caused.Materials and methodsSurveys and sample collectionIn 2009, surveys were performed in 14 natural spring water areas in some plateaus ofAntalya and Konya provinces containing mint plants. Leaf samples showing virus-likesymptoms were taken frommint plants exhibiting symptoms, placed in polythene bags andstored at –20 °C until use.Serological testingDAS-ELISA method was used to detect the virus in mint leaf samples and performedaccording to CLARK and ADAMS (1977) and the instructions of the CMV antiserum manu-facturer (Bioreba, Switzerland). Leaf samples with typical symptoms of virus infectionswere ground (1g leaf/5 mL buffer) in extraction buffer (PBS: 0.13 M NaCl, 0.014 MKH2PO4, 0.08 M Na2HPO4, 0.002 M KCl, pH: 7.4, containing 0.05% Tween-20), added tomicroplate wells (Nunc Microwell, Denmark) after coating with CMV-specific polyclonalantiserum diluted in carbonate buffer (pH: 9.6) and incubated at 4 °C overnight. Plates werewashed three times with PBS/Tween-20 buffer and coated with alkaline phosphatase-con-jugated antibody diluted in extraction buffer and incubated for 4h at 37 °C. After washing,p-nitrophenyl phosphate (Sigma) in diethanolamine substrate buffer (0.5 mg mL–1, pH:9.8) was added to the wells and incubated at room temperature for 30–180 min. Absor-bance values were read at 405 nm using a microplate reader (Tecan Spectra II). Extractionbuffer and healthy plants were used as negative controls. Samples were considered to be188 ACTA BOT. CROAT. 71 (1), 2012SEVIK M. A.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:09Color profile: DisabledComposite  150 lpi at 45 degreespositive when the absorbance values at 405 nm (A405) values exceeded the mean of the nega-tive controls by least a factor of three (KUTLUK-YILMAZ 2010).Biological testingNicotiana tabacum L. 'Samsun' was grown in a growth chamber at 23 °C with a 16 hlight/8 h dark photoperiod cycle. The samples of water mints collected (previously used forELISA) were used for preparation of inoculum in the buffer and all fully expanded leavesof plants at 2–4 leaves growth stage were inoculated.Results and discussionMost of the farmers and people of Antalya and Konya regions complained of an in-creased incidence ofmosaic disease inmint plants over the past few years. Besides their cu-linary use, mint plants are also used during livestock grazing. It is well known that mint hasbeen used for centuries for its medicinal properties and in the food and fragrance industries(MIMICA-DUKIC et al. 2003).Visual surveys of the mint plants revealed a high incidence of leaf mosaic (Fig. 1), de-formation and severely affected apical plant growth. The newly growing apical leaves weresmall with very conspicuous symptoms. During the surveys, most spring areas containedplants with virus-like symptoms. Virus-like symptomswere observed in all 14mints grownareas surveyed.CMV was detected by DAS-ELISA in the mint plants from all surveyed areas. The inci-dence of CMV in the symptomatic plants varied in the different springs. Serological assayresults revealed a variation in virus incidence among the different springs surveyed. The in-cidence of CMV was estimated as 27.08% in Antalya and Konya provinces, ranging from14.2% to 42.8% in the springs visited.ACTA BOT. CROAT. 71 (1), 2012 189DETECTION OF CUCUMBER MOSAIC VIRUS IN MINT PLANTSFig. 1. Symptoms associated with natural infections by CMV on water mint plants.U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:10Color profile: DisabledComposite  150 lpi at 45 degreesIn the study, tests were conducted using several negative controls for CMV. Therefore,the range of absorbance values of negative controls varied from 0.066–0.118 at 405 nm.Positive samples gave absorbance values of 0.452– to 1.842 after 2 h substrate incubation(Table 1).The presence of CMV inwater mint plant was verified in samples by transmission to in-dicator test plants, tobacco (N. tabacum). Tobacco plants mechanically inoculated with ex-tracts of ELISA-positive plants showed local necrotic spot on leaves 1 week after inocula-tion (Fig. 2). These symptoms were similar to those that were described previously for thevirus (CHOI et al. 2004).The cucumovirus CMV has been reported as the virus infecting different plants world-wide (VARVERI, and BOUTSIKA 1999) and different regions of Turkey (ERDILLER andERTUNÇ 1988, UNLU and GULDUR 2004), but infection of mint with CMV is reported for thefirst time in these regions. The report of CMV affecting Mentha was in 1966 in Germany,where it was found to be the cause of a mosaic disease of peppermint. CMV was found incultivated medicinal plants, including mint, in Hungary (TZANETAKIS et al. 2010). HANI(1971) studied the epidemiology of CMV in Switzerland and reported Mentha sp. as aweed host for the virus in the vicinity of tobacco fields where several CMV isolates werecausing problems. CMV was also isolated from Mentha sp. (VICCHI and BELLARDI 1988),M. piperita and M. palustris in Italy by CRESCENZI et al. (1993). M. pulegium for CMVwasfound infected for the first time by ELISA in Greece (CHATZIVASSILIOU et al. 2004).Weeds, native plants and seed transmission may have a significant effect on virus epi-demiology (DUFFUS 1971) and knowledge of weed reservoirs and vectors of viruses is es-sential for understanding the epidemiology. CMV-infected weeds may also play a signifi-cant role in its spread to crops. In 1998, 28 fields in Samsun, Turkey were surveyed and 222190 ACTA BOT. CROAT. 71 (1), 2012SEVIK M. A.Tab. 1. Range of absorbance values of positive and negative samples and incidence of CMV.No. of springssurveyedNo. ofsamples testedThe means DAS-ELISA absorbance valuesIncidence (%)Negative samples Positive samples1 12 0.084–0.215 0.767–1.231 16.62 4 0.090–0.193 0.829 25.03 8 0.059–0.330 0.452–0.669 37.54 10 0.104–0.364 0.860–1.076 20.05 6 0.093–0.316 1.290–1.842 33.36 5 0.110–0.299 1.082 20.07 7 0.075–0.317 0.773–0.803 42.88 7 0.060–0.262 1.318 14.29 9 0.124–0.355 0.793–0.916 22.210 8 0.078–0.326 0.510–0.599 37.511 7 0.066–0.303 0.685–0.697 28.512 6 0.088–0.249 0.860 16.613 3 0.115–0.354 0.965 33.314 4 0.128–0.338 0.495–0.852 28.5U:\ACTA BOTANICA\Acta-Botan 1-12\436 Sevik.vp26. o ujak 2012 14:49:10Color profile: DisabledComposite  150 lpi at 45 degreesweed samples were collected from pepper fields. Of the samples tested, 7.7%were found tobe infected with CMV. CMV was found to be the virus most frequently detected, in 13weed species out of 24 species (ARLI-SOKMEN et al. 2005). In experimental work, the virusis most frequently transmitted mechanically, and sap, purified virions, and viral RNA areall infectious via mechanical transmission (ROOSSINCK 2001). In addition, DHEEPA andPARANJOTHI (2010) reported that CMV from banana could be transmitted by rhizome inoc-ulation and mechanical methods. To help prevent CMV from spreading, farmers are rec-ommended to remove any mint plants infected with CMV near springs.The results obtained in this investigation clearly demonstrate that CMV is widely dis-tributed in different mint growing areas of Antalya and Konya, Turkey. This is the first re-port of CMV from water mint plants (M. aquatica) in Turkey.AcknowledgmentsI thank to D. Sevik for helping and assistance during surveys of these regions and sam-ple collections.ReferencesARLI-SOKMEN, M., MENNAN H, SEVIK M. 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Natural occurrence of Cucumber mosaic virus infecting water mint (Mentha aquatica) in Antalya and Konya, Turkey

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