ESTABLISHMENT OF SOME VIRUSES POLYPHAGUES ON ECONOMICALLY IMPORTANT ESSENTIAL OIL BEARING AND MEDICINAL PLANTS IN BULGARIA

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1 ESTABLISHMENT OF SOME VIRUSES POLYPHAGUES ON ECONOMICALLY IMPORTANT ESSENTIAL OIL BEARING AND MEDICINAL PLANTS IN BULGARIA B. Dikova Plant Protection Institute, 2230, Kostinbrod, Bulgaria Correspondence to: Bistra Dikova ABSTRACT Viruses polyphagues such as cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), broad bean wilt virus I (BBWV I) and potato virus Y (PVY) were established on some of the following cultures: coriander (Coriandrum sativum L.), fennel (Foeniculum vulgare L.), valerian (Valeriana officinalis L.), purple coneflower (Echinacea purpurea L. Moench), sage (Salvia sclarea L.), catmint or catnip (Nepeta cataria L.), basil (Ocimum basilicum L.), wormwood (Artemisia absinthium L.) and lovage (Levisticum officinalis L.). The studies were carried out by serological ELISA method (double antibody sandwich ELISA DAS ELISA). The indicator method was used for identification of isolates belonging to cucumber mosaic virus (CMV) for future investigations. A comparison between CMV isolates from essential oil-bearing and medicinal cultures and an isolate of CMV from cucumber was done on indicators plants. Keywords: essential oil bearing and medicinal plants, viruses-polyphagues Introduction Cucumber mosaic virus (CMV) was isolated from coriander (Coriandrum sativum L.) in Bulgaria (7). A reduction in the size of ill vs. healthy plants was observed. The ill plants resembled the typical dwarfed appearance of coriander because of internodes shortening and concentration of young leaves, lower leaves became entirely chlorotic with green stripes only along the main veins. Lower leaves were reddish along the periphery (7). Another virus polyphague on coriander - alfalfa mosaic virus (AMV) - was found in Yemen (1). The same virus was proven in coriander plants with mosaic and chloroses in Oman (9). AMV and CMV were established on valerian (Valeriana officinalis), the first virus caused bright yellow spots on a large part of valerian leaf s laminas and the second one pale green mosaic spotting (4 and 7). CMV infection was responsible for significant variations in the relative compositions of the major constituents in root extracts of purple coneflower (Echinacea purpurea L.Moench), (6). The amount of essential oil obtained from sage plants (Salvia sclarea L.) infected with broad bean wilt virus I (BBWV I) was only 2/3 of that from healthy material (5). According to Kovachevsky et al., 1995, AMV caused bright yellow mosaic especially on young leaves of Salvia sclarea (7). CMV produced yellow spotting with oak leaf outline along the leaf s veins of the secondary branches of basil (Ocimum basilicum L.) (7). The objective of the study was the establishment of some wide spread viruses-polyphagues on economically important essential oil-bearing and medicinal plants and to identify some CMV isolates from these cultures for comparison with a vegetable CMV isolate. Materials and methods Plantations in the trial fields of the Institute of Roses and Essential Oil-Bearing Plants (IREOBP) in Kazanlak were investigated and samples of essential oil-bearing and medicinal plants were collected in the summer and autumn of Average samples from leaves with symptoms of virus diseases were prepared with parts of stems and roots of smaller tufts or single plants included, whenever possible. An isolate of CMV, originating from spotted and defective fruit of long-fruited cucumber was used as a positive control for this virus and for comparison with CMV isolates from 80

2 essential oil-bearing cultures on indicator plants. The positive controls for other viruses are given in Table 1. The samples were analyzed by ELISA method (DAS ELISA), (2), with antisera produced by the German company LOEWE, Biochemica, to cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV), broad bean wilt virus I (BBWV I) and Potato virus Y (PVY). The indicator plant samples were tested by DAS ELISA for the establishment of CMV presence. The extinction values were measured by spectrophotometer SUMAL PE, Carl Zeiss, Jena. All samples from essential oil-bearing and medicinal plants with values three times higher than negative controls were assumed virus positive, because essential oil-bearing and medicinal plants were in advanced age, phases of flowering and seed production. The extinction values for the indicator plants were assumed virus positive when the samples had values twice higher than negative controls, because these indicator plants were young. Some of essential oil-bearing and medicinal plants assumed as positive for CMV were analyzed by indicator method on the plant from the following species: Cucumis sativus (cucumber) cv. Levina, Cucurbita pepo var. giramonia (zucchini marrow) cv. Isobilna, Nicotiana tabacum cv. Samsun NN and Nicotiana tabacum cv Results and Discussion The average values from the measured positive extinction values for each virus are presented in Table 1. One, two or three viruses were found in 35 of a total of 102 samples of coriander, fennel, valerian, purple coneflower, sage, catmint, basil, wormwood and lovage. They were 34 % of the tested plants. AMV and BBWV I in mixed infection were established in yellow sprig of fennel from a plant with chlorotic leaflets, different from ordinary green sprigs of other plants. The extinctions measured for each of the viruses were and optical density (OD), respectively. CMV was diagnosed on five fennel sprigs from different plants, collected in the trial field of IREOBP with the following extinction values: 2.030, 0.491, 0.733, and OD. Establishment of viruses on essential oil bearing and medicinal plants Plant species Total number of samples Number of samples with Average extinction values for viruses Optical density virus CMV AMV BBWV I PVY Coriander Coriandrum sativum L Fennel Foeniculum vulgare L Valerian Valeriana officinalis L Purple coneflower Echinacea purpurea L Moench Sage Salvia sclarea L Catmint Nepeta cataria L Basil Ocimum basilicum L Wormwood Artemisia absinthium L Lovage Levisticum officinalis L K+ cucumber fruit K+ alfalfa sprig with mosaic K+ sugar beet isolate of BBWV K+ potato cv.nadejda with PVY K- coriander K- fennel K- valerian K- purple coneflower K- sage K- catmint K- basil K- wormwood K- lovage PBS-T puffer Legend: K+ and K- positive and negative controls; PBS-T phosphate buffered solution, contained Tween 20 TABLE 1 The same viruses AMV, CMV and BBWV I were established in coriander, although limited number of samples were analyzed due to advanced age of the plants (Table 1). CMV was proven in valerian leaves with mosaic spotting SPECIAL EDITION/ON-LINE YEARS OF ACADEMIC EDUCATION IN BIOLOGY

3 from light green and dark green areas of leaf s laminas. CMV was also isolated from old valerian leaves with yellow spotting. The measured extinction values for CMV were: 1.589, 0.926, 2.012, 2.055, and OD. In addition to CMV, AMV was isolated in valerian in previous studies (4). The symptoms caused by AMV and CMV were very different. AMV caused bright yellow elongated spots (4, 7). CMV was proven in purple coneflower leaves and the following extinction values were measured: 2.030, and OD. The symptoms were pale green spotting on these leaves with curly laminas due to the different growth of pale green and dark green areas of the leaves. CMV, AMV and PVY were established in a spotted and much smaller dwarfed purple coneflower leaf and the extinction values were: 0.532, and OD, respectively. CMV was isolated in catmint plants and the following extinction values were obtained: 0.522, and OD. Typical symptoms of CMV were mosaic of light green and dark green spots as opposed to a normal green healthy leaf. AMV and BBWV I in mixed infection were proven in one catmint plant and the extinction values were: and OD, respectively. AMV was established in one bright yellow catmint plant and high extinction was measured OD. CMV, AMV and BBWV I were found in different sage plants, showing various symptoms. So CMV was proven in sage plant with light green spots on the leaves and extinction value for this virus was OD. CMV and AMV in mixed infection were established in bright yellow spotted leaves of other sage plant with extinction values of and OD. AMV and BBWV I in mixed infection were established in a third sage plant with spotted leaves and extinction values were and OD, respectively. CMV and AMV were found in different mosaic spotted basil plants and the extinction for the viruses were: and OD. CMV was only proven in young pale green spotted leaf of one lovage plant with crinkled deformation with extinction value of OD. Our present studies confirmed the established viruses by other authors: on coriander CMV and AMV; on valerian CMV; on purple coneflower CMV and AMV; on sage AMV and BBWV I and on basil CMV (1, 4, 5, 6, 7, 9). Economically important plants such as coriander and fennel were cultivated on large areas in Bulgaria during the last ten years and widespread viruses-polyphagues were found in them CMV, AMV and BBWV I. There were some reports regarding the antiviral effect of essential oils on plant viruses and this aspect theoretically presumed their use in control of virus diseases. Essential oil, isolated from fennel in concentration 3000 ppm totally inhibited the formation of local lesions on the leaves of plants, infected with potato virus X (PVX), tobacco mosaic virus (TMV) and tobacco ring spot virus (TRSV), (8). In our study viruses were proven in 7 from 18 fennel plants and extinction values for these viruses were triple the negative controls and even higher. Fennel plants that were viruscarriers represented less than half of all analyzed plants from this species. CMV was established in 5 fennel plants of the virus-carriers and AMV and BBWV I in 1 plant for each of them. From 8 analyzed samples of different lovage tufts only 1 sample was a virus-carrier, although a lot of tufts manifested symptoms of viral diseases. Similar conclusions can be drawn for the other essential oil-bearing and medicinal species as well (Table 1). Diagnosis of virus diseases in them was probably inhibited to a certain degree by the antiviral effect of essential oils. In our study, the real number of positive virus-carrier samples was probably bigger than reported. Identification of CMV isolates from essential oil-bearing and medicinal cultures is a continuous process, obstructed by mixed virus infection and essential oil inhibiting effect. We succeeded to obtain three CMV isolates from valerian, catmint and basil and compare them with CMV isolate of cucumber fruit. The reaction of these CMV isolates on indicator plants and established virus concentration by measured extinction value for each of them are presented in Table 2. CMV isolates of catmint and basil caused identical symptoms on cucumber, zucchini squash and tobacco plants. The same CMV isolates of catmint and basil manifested on Nicotiana tabacum cvs. Samsun NN and 1146 light mosaic, oak leaf figures without deformation of leaf s laminas, but CMV isolate of cucumber fruit caused severe mosaic, stunting and deformation of leaf s laminas such as narrow leaves (Table 2). Cucumber and zucchini marrow plants, infected by CMV isolates of catmint, basil, valerian and cucumber fruit manifested similar symptoms, such as local lesions on cotyledons and systemic spotting and mosaic on the next leaves, smaller or bigger degree of stunting and deformations. Tobacco cultivars Nicotiana tabacum cv. Samsun NN and cv. 1146, infected by the same isolates, caused different symptoms. CMV isolate of valerian manifested on Samsun NN and 1146 mosaic and light deformation very different from stunting and narrow leaves, caused by CMV isolate of cucumber fruit, but also different from the symptoms, caused by CMV isolates of catmint and basil. Similar differences in reaction of CMV isolates on 82

4 Nicotiana tabacum var. Xanthii and cv. Samsun were established in previous studies (3). It is possible that phenotype differences in reaction of isolates are related to genotype (strain) differences in CMV isolates. Reaction of indicator plants to cucumber mosaic virus (CMV) Kind of isolate Indicator plant Description of symptoms Extinction values for CMV Optical density Catmint Cucumber, cv. Levina LLc / M, Mo Catmint Zucchini marrow, cv. Isobilna LLc, LLn /M, Mo, D 1.761, 2.091* Catmint Tobacco, cv. Samsun NN L / Mo, OL 0.250, Catmint Tobacco, cv L / Mo, OL Valerian Cucumber, cv. Levina LLc / M, Mo Valerian Zucchini marrow, cv. Isobilna LLc, LLn, / M, Mo, D 1.522, * Valerian Tobacco, cv. Samsun NN L / M Valerian Tobacco, cv L / M Basil Cucumber, cv. Levina LLc / M, Mo Basil Zucchini marrow, cv. Isobilna LLc, LLn / M, Mo, D Basil Tobacco, cv. Samsun NN L / Mo, OL Basil Tobacco, cv L / Mo, OL Fennel Zucchini marrow, cv. Isobilna L / Mo Purple coneflower Zucchini marrow, cv. Isobilna L / Mo Cucumber Cucumber, cv. Levina LLc / M, Mo, D Cucumber Zucchini marrow cv. Isobilna LLc, LLn / M, Mo, D Cucumber Tobacco, cv. Samsun NN L / M, Mo, D, NL 0.589, 0.519* Cucumber Tobacco, cv L / M, Mo, D, NL Cucumber K+ Spotted fruit M, Mo, D K- Symptomless cotyledons PBS-T** puffer Legend: * - more than one test LLc, LLn local chlorotic and local necrotic lesions L local latent infection M mosaic, systemic reaction Mo systemic mottling D systemic deformation of leaf s lamina OL systemic reaction with necrotic figures Oak Leaf on leaf s lamina NL systemic reaction, presenting deformation as narrow leaves ** - phosphate buffered solution, contained Tween 20 0 no symptoms, symptomless leaves Conclusions The present results lead to the following more important conclusions: One to three viruses polyphagues were established on nine economically important essential oil bearing and medicinal plants. Cucumber mosaic virus (CMV), alfalfa mosaic virus (AMV) and broad bean wilt virus I (BBWV I) were proven in essential oil-bearing cultures coriander and fennel, widely spread on large areas in Bulgaria. Cucumber mosaic virus (CMV) was identified in the following cultures: coriander, fennel, valerian, purple coneflower, sage, catmint, basil, wormwood and lovage. TABLE 2 Differences were found in reaction of CMV isolates, originating from catmint, basil and valerian on Nicotiana tabacum cv. Samsun NN and cv.1146 in comparison to the reaction of one isolate from vegetable culture cucumber. Acknowledgment We would like to thank the Management of the Institute of Roses and Essential Oil Bearing Plants in Kazanlak as well as my colleague researchers for the possibility to investigate trial fields and collect plant samples. REFERENCES 1. Alhubaishi A. A., Walkey D. G. A., Webb M. J. W., SPECIAL EDITION/ON-LINE YEARS OF ACADEMIC EDUCATION IN BIOLOGY

5 Bolland C. J., Cook A. A. (1987) FAO Plant Protection Bulletin, 35, Clark M. F., Adams A. N. (1977) J. Gen. Virol., 34, Dikova, B. (1984) Ph. D. Thesis. Plant Protection Institute, Kostinbrod, Bulgaria. 4. Dikova B. (2006c) Plant Science, 43, Hudaib M., Bellardi M. G., Rubies- Autonell C., Flori J., Cavrini V. (2001) Farmaco, 56, Hudaib M., Flori J., Bellardi M. G., Rubies-Autonell C., Cavrini V. (2002) J Pharma Biomed Anal., 29, Kovachevsky I., Markov M., Yankulova M., Trifonov D., Stoyanov D., Kacharmazov V. (1995) Virus and virus-like diseases of cultivated plants, Sofia, p Shukla H. S., Dubey P., Chatuvedi R. V. (1988) Agronomy, 9, Zadjali A. D., Matrooshi A. R., Moghal S. M. (2002) Agricultural Sciences, 7,

Science & Technologies CELERY MOSAIC VIRUS ON FOENICULUM VULGARE IN BULGARIA. 7 Shosse Bankya Str., 1080 Sofia, Bulgaria, E mail:

Science & Technologies CELERY MOSAIC VIRUS ON FOENICULUM VULGARE IN BULGARIA. 7 Shosse Bankya Str., 1080 Sofia, Bulgaria, E mail: CELERY MOSAIC VIRUS ON FOENICULUM VULGARE IN BULGARIA Bistra Dikova 1 and Hristo Lambev 2 1 Nikola Poushkarov Institute for Soil Science, Agrotechnologies and Plant Protection 7 Shosse Bankya Str., 1080