PLANT PATHOLOGY HORTSCIENCE 37(3):543–546. 2002. Materials and Method Plant material. Double petunia samples Virus-associated Diseases of Double were collected from 15 greenhouses and 6 garden centers in Toledo, Cincinnati, Cleve- Petunia: Frequency and Distribution land, and Columbus, Ohio, in Spring 1997 and 1998. In 1997, growers and garden center managers were requested to send a sample of in Ohio Greenhouses 12 double petunia plants. Since the number of plants initially received was small, we col- M-C Sanchez-Cuevas and S.G.P. Nameth lected the material directly in greenhouses and Department of Plant Pathology, The Ohio Agricultural Research and garden centers. Only plants displaying virus- Development Center, The Ohio State University, Columbus, OH 43210 like symptoms were selected. We also in- cluded in the survey all double petunia samples Additional index words. ELISA, inclusion bodies, Petunia ×hybridia sent to the C.W. Ellett Plant and Pest Diagnos- Abstract. Double petunia plants expressing virus-like symptoms were collected in tic Clinic, The Ohio State Univ., Columbus. greenhouses and garden centers throughout Ohio in Spring 1997 and 1998 in an effort Plants were collected at different stages of to determine the frequency and distribution of petunia viruses present in the state. development, in plug trays, pots, and baskets. Direct antibody- sandwich and indirect enzyme-linked immunosorbent assay (ELISA) Plants were transplanted and maintained un- were conducted with commercial antisera made against 13 viruses, a potyvirus kit der insect-free greenhouse conditions, with capable of detecting 80 different potyviruses, and our antiserum raised against a ample distance between plants to minimize cross-contamination. Tissue (≈10 g) was har- tobamo-like virus inducing severe mosaic in double petunia. Viral-associated double- ° stranded ribonucleic acid (dsRNA) analysis and light microscopy for detection of vested from each plant and stored at –20 C. inclusion bodies were also carried out. ELISA, dsRNA analysis, and light microscopy Virus-like symptoms, such as mosaic, revealed the presence of tobacco mosaic tobamovirus, an unknown tobamo-like petunia ringspots, leaf deformation, leaf puckering, virus, tomato ringspot nepovirus, tobacco streak ilarvirus, and tobacco ringspot spots and/or stunting were recorded one week nepovirus. Tomato aspermy cucumovirus, tomato spotted wilt tospovirus, impatiens after transplanting or whenever they were even- necrotic spot tospovirus, alfalfa mosaic virus, cucumber mosaic cucumovirus, potato tually observed. virus X potexvirus, and chrysanthemum B carlavirus were not detected. No potyviruses Detection of viruses by ELISA. Leaves, were identified. A number of plants with virus-like symptoms tested negative for petals, and young stems from 544 symptom- all viruses. atic double petunia plants were tested by direct antibody-sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA) using a modi- Petunia ×hybridia Hort. is a one of six 1995). Symptoms induced by these viruses in fication (condensation of incubation time using members of the Solanaceae grown primarily infected petunia plants are variable, as they heat and shaking) of a previously described as an ornamental plant, and the most eco- are influenced by environmental conditions, procedure (Clark and Adams, 1977) for de- nomically important due to its horticultural strain of the virus, mixed infections with tecting alfalfa mosaic virus (AMV), CMV, value (Sink, 1984). It is a popular bedding other viruses, genetic diversity of petunia TAV, TSV, potato virus X potexvirus (PVX), plant, the third most valuable after geranium cultivars, and cultural conditions. Some in- TRSV, ToRSV, TMV, TSWV, INSV, and and impatiens (Hamrick, 1997) but it is also fected plants remain symptomless (Lesemann, CVB using commercial polyclonal antisera grown in pots, hanging baskets, and balcony/ 1996). According to Lesemann (1996), petu- produced in rabbits (Agdia, Elkhart, Ind.). window boxes (Lesemann, 1996). Petunia nia virus (PV)-associated diseases became DAS-ELISA was also conducted with plants propagate by seeds in nature, but new widespread after 1992, when asexually propa- polyclonal antibodies we produced against a species of vegetatively propagated petunia, gated hybrids were introduced from Japan possible new tobamo-like virus affecting such as “Supertunia’ and double petunias, without strict virus-indexing schemes. The double petunia tentatively named petunia virus, have been recently introduced in Europe and symptoms associated with virus diseases in and a commercial indirect ELISA screen which the United States as garden ornamentals petunias are stunting and deformation of the tests for 80 potyviruses (Agdia). During the (Lesemann, 1996; Sikron et al., 1995). Double foliage, light-green streaks, discolored and screening process with our antisera produced petunia plants provide the most impressive deformed flowers (Lindgren, 1993), irregu- against PV, it was determined that our antisera flowers of all bedding plants (Nau, 1991) and lar vein- yellowing, mottling, color breaking, did cross-react slightly with tomato mosaic are usually female sterile because of mal- and a reduction in the number and size of the virus and pepper mild mottle mosaic virus. formed or underdeveloped pistils (Goldsmith, flowers (Edwardson and Christie, 1997 a and Optical density at 405 nm was measured with 1968). b). Mixed virus infections apparently aggra- an MRX microplate reader (Dynex Technolo- Several viruses have been reported as vate symptoms (Leseman, 1996). gies, Chantilly, Va). Samples were considered naturally infecting petunias (Bellardi et al. The virus most often detected in petunias positive when the mean absorbance of dupli- 1996; Leseman, 1996; Mavric et al., 1996; in the United States is tobacco mosaic cate wells exceeded twice the mean absor- Pirone, 1978; Sikron et al., 1995), and the list tobamovirus [(TMV); Agdia, 1995; Lawson bance of appropriate healthy controls. Each of viruses capable of infecting petunias is and Hsu, 1994]. Other commonly detected plate included a healthy tissue sample and very long (Brunt et al., 1996), because petu- viruses are cucumber mosaic cucumovirus extraction buffer as negative controls. Tissue nias are often used as indicator hosts for virus (CMV), tobacco ringspot virus nepovirus known to be positive for a particular virus was identification assays (Lane, 1992). Virus dis- (TRSV), unknown potyviruses, tomato spot- used as the positive control. eases constitute a threat to the production and ted wilt tospovirus (TSWV), tomato ringspot Viral dsRNA extraction and analysis. Leaf, quality of the petunia crop (Sikron et al., nepovirus (ToRSV), occasionally tobacco flower petals, and young stem tissue (5 g) was streak ilarvirus (TSV), tomato aspermy used for analysis. Viral-associated double- Received for publication 5 Jan. 2001. Accepted for cucumovirus (TAV), and chrysanthemum vi- stranded ribonucleic acid (dsRNA) was ex- publication 28 Aug. 2001. This paper is a portion of rus B carlavirus (CVB) (Brunt et al., 1996) tracted and purified using a modification of a thesis submitted by M.-C. Sanchez-Cuevas. We The purpose of this study was to screen the CF-11 cellulose column chromatography thank greenhouses and garden centers in Ohio for providing the plants for this study. We also thank for the viruses infecting double petunia plants method described by Morris and Dodds, (1979) Mike Tiffany for information on important viral in commercial greenhouses and garden cen- and Jordan et al. (1983). diseases of petunias and for providing positive ters in Ohio and to determine their the fre- Light microscopy of inclusion bodies. Epi- samples for serology. quency and distribution. dermal strips from the underside of healthy HORTSCIENCE, VOL. 37(3), JUNE 2002 543 6762, p. 543-546 543 5/13/02, 1:14 PM PLANT PATHOLOGY and symptomatic double petunia and Turkish potyviruses detected by the potyvirus screen. infected plants identified by DAS-ELISA in tobacco plants were stained with a combina- In 1997, 8.5% of the plants collected (18/ 1998 were positive when tested with the com- tion of Calcomine Orange and Luxol Brilliant 212) were positive for virus infections. Of mercial antibodies against the common or Green, a general stain for proteins, and Azure these, 12 plants were positive for ToRSV, 5 for vulgare strain of TMV (TMV-c) and our own A (Aldrich Chemical Co., Milwaukee), a TSWV, and 1 for TSV. No mixed infections polyclonal antibodies raised against the un- nucleic acid-specific stain (Christie and were detected in 1997 (Table 1). The symp- known petunia virus (PV). All plants that tested Edwardson, 1994). The stained tissue was toms observed in TSWV positive samples positive for either or both of these viruses, observed with a Zeiss Axioscope microscope, varied from severe leaf puckering, leaf defor- expressed various degrees of mosaic, pucker- equipped with a Zeiss MC80 microscope mation and reduced leaf size, to inter-venial ing and leaf deformation. The absorbance val- camera (Carl Zeiss, Inc., Thornwood, N.Y.). chlorosis and small local lesions. Some of ues for the PV samples were usually lower in these symptoms (inter-veinal chlorosis and those samples that tested positive for both TMV Results local lesions) were also detected in plants and PV (mixed infection) (Table 1). infected with ToRSV. Those plants also ex- Some of the 1998 plants that expressed Plants collected in greenhouses and garden pressed ringspots