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ORCHID VIRUSES
A Glimpse into the Current Status of the Common Viruses infecting Orchids*
For many years most amateur and professional orchid growers have been aware of orchid viruses, their means of transmission and methods of control. Although many surveys for orchid viruses have been conducted since the studies done by Zettler et al. (1978), Bodnaruk et al. (1979) and Wisler et al. (1979, 1986), no comparisons have been made to detect changes in the incidences of the two most common viruses, odontoglossum ringspot tobamovirus (ORSV) and cymbidium mosaic potexvirus (CymMV). Likewise, there are no reports of cymbidium ringspot tombusvirus (CymRSV) since the original report by Hollings and Stone, who first described this virus in 1963. Symptoms induced by both ORS\C a rigid rod- shaped virus, and CYIHMV a flexuous rod-shaped virus, include color break on flowers and occasionally necrotic lesions and mosaic patterns on leaves (Plates 29-32). Neither virus is known to have a natural vector and both are remarkably stable. This stability, along with inconsistent symptom expression, accounts for the prevalence of these viruses in orchids. Some of the viruses less commonly detected in orchids, such as cucumber mosaic cucumovirus (CMV), a spherical virus, also induce striking color break and distortion of flowers (Plate 33). Whereas the rhabdoviruses such as orchid fleck (OFV) cause chlorotic and necrotic streaking on leaves, dendrobium vein necrosis closterovirus (DVNV) causes veins on flowers to become brown and necrotic but does not cause obvious symptoms on leaves (Plate 34). Vanilla mosaic (Wisler et al., 1987) and vanilla necrosis (Pearson et al., 1993), members of the potyvirus group, can cause severe mosaic and distortion on leaves but are apparently only a concern in the vanilla- producing areas of the South Pacific (Plate 35). While tomato spotted wilt tospovirus (TS\/VV) induces chlorotic ringspots in infected leaves of certain orchids, the virus apparently remains localized and does not spread to new growth (Hu et al., 1992). Certain healthy, virus-free Phalaenopsis hybrids exhibit virus-like symptoms apparently based on the species involved in the crosses (Plates 36 & 37). Injury as well as fungal and bacterial pathogens can also cause virus- like symptoms in orchid leaves (Plate 38).
Odontoglossum ringspot virus and CymMV have no known natural vector aside from humans. These viruses are extremely stable and can persist on contaminated tools and pots (Lawson, 1995). Dendrobium vein necrosis virus and CMV are similar in that they can be transmitted by aphids (Zettler et al., 1990). Aphids also transmit potyviruses such as vanilla mosaic and vanilla necrosis viruses (Wisler et al., 1987; Pearson et al., 1993). Orchid fleck rhabdovirus can be mechanically transmitted (Brunt et al., 1997), but a natural vector remains unknown (Doi et al., 1977). Likewise, Hollings et al.(1977) reported that CymRSV could be mechanically transmitted with difficulty, but the natural vector was unknown. Tomato spotted wilt virus is transmitted by thrips and only became a concern with the outbreak of the western flower thrips (Frankliniella occidentalis) in Hawaii (Hu et al., 1992).
RESULTS
A total of 420 samples representing 35 genera of both terrestrial and epiphytic orchids were tested for CymRSV using DAS-ELISA. Because 40 samples from one source had already been determined to be infected with ORSV or CymMV by other workers prior to shipment to our lab, a total of 380 of the 420 samples were tested for ORSV and CymMV using I-ELISA. Either ORSV or CymMV (or both) were detected in all 18 sources sampled. Incidence of ORSV ranged from 0% to 67.5%, whereas the incidence of CymMV ranged from 0% to 70%. The percentage of virus- infected orchids (total of single and double infections) ranged from 1% to 47.5%. Of the 380 samples tested, only 42 were infected with both viruses.
Interestingly, of the 144 cymbidium plants tested, 31 were infected with ORSV, three were infected with CymMV and 21 were infected with both viruses. Cymbidium ringspot was not detected in any of the 420 orchids tested.
DISCUSSION
The incidence of CymMV compared to ORSV still remains much greater in most orchids with the exception of cymbidiums. Several studies of virus incidence in dendrobiums in Hawaii as well as other studies on multiple genera in Singapore and the United States also reported similar results (Hu et al., 1993; Wong et al., 1984; Okemura et al., 1984; Zettler et al., 1978). This difference is particularly obvious with Cattleya and Cattleya intergeneric hybrids in which 83 (42%) and 33 (17%) of the 197 plants tested were infected with CymMV and ORSV, respectively (Siegmann et al., 1999). The reverse is true, however, with regard to Cymbidium species and hybrids in which 31 (21.5%) and 3 (2%) of the 144 plants tested were infected with CymMV and ORSV, respectively (Siegmann ct al., 1999). This confirms the observations made by Francki (1966) more than 30 years ago in Australia.
The types of orchid collections sampled can greatly influence the ratio of healthy to diseased orchids as well as the ratio of ORSV to CymMV For example, our data confirm those of Preitas-Astua et al. (1999), who also reported that private and public orchid collections tend to have a higher incidence of infected plants whereas fewer infected plants can be found in the collections of commercial growers. Given the disparity in the incidence of ORSV and CymMV in cymbidiums, the number of CymMV-infected as well as the total number of virus-infected plants may be artificially low when the collection contains a greater number of cymbidiums as compared to other genera (Siegmann et al., 1999; Freitas-Astua et al., 1999).
Given the differences in nursery type, sampling procedures and orchid type, total incidence of virus appears to have peaked and then stabilized in the early to mid 1980s (Zettler et al., 1978; Bodnaruk et al., 1979; Wisler et al., 1979, 1986; and Okemura, 1984). For example, Zettler et al. (1978) reported a virus incidence ranging from 18% to 69% in nurseries in Florida. Wisler (1981) reported a 35% incidence in Florida while Wong et al. (1984) reported a virus incidence from 43% in a botanic garden to 73% in four commercial farms in Singapore. Siegman et al. (1999) reported a virus incidence ranging from 100% to 10% depending upon the nursery type. The overall percentage of infected orchids was approximately 38%. Virus incidence in older collections appears to have remained the same or decreased slightly. This decrease can be attributed to the gradual replacement of older plants with plants which have been indexed and determined to be virus free as well as increased emphasis on better horticultural practices (Freitas- Astua et al., 1999; Elliott et al., 1996; Hu et al., 1993). The longer a plant remains in cultivation, the greater the likelihood it may become virus infected (Hu et al., 1993; Okemura, 1984; Wisler, 1989). The more rapid decrease in virus incidence in commercial operations may be attributed to an increase in the use of seedling propagation and the use of more reliable virus indexing methods prior to mericloning.
Commercial nurseries rely heavily on seedling propagation and virus indexing prior to mericloning, and attempt to sell their retail stock as rapidly as possible. Thus, most of their orchids are virus free. These nurseries, however, usually keep plants used for breeding or show in another location. When possible, samples were collected from these older plants. Because this was not always possible and because some of the samples were collected from the plants intended to be sold, the number of virus-infected plants in commercial nurseries was lower than the number of infected plants collected from the established orchid collections. The percentage of infected plants in older collections, however, appears to remain about the same. When considering both commercial growers as well as private collections, the overall percentage of infected orchids appears to have decreased slightly, from an average of 43% to 38% over the last 20 years.
The improvement in virus control methods and horticultural practices over the last 20 years would most likely reduce the probability of detecting CymRSV in new collections or commercial orchid businesses, Likewise, the probability of plants becoming infected generally increases with respect to age of the plant and length of time a plant has been in cultivation (Hu et al., 1993: Freitas-Astua et al., 1999). Therefore, whenever possible, species and hybrids which have been in cultivation for 30 to 100 years were selected. While this virus was first detected in a few cymbidium plants during a routine survey of a commercial orchid nursery in Great Britain, the authors failed to indicate the species from which the virus was obtained. Therefore, cymbidium plants which may have been in cultivation since the time of the first report were requested from collections which may have existed at that time. Given these data as well as those of Freitas-Astua et al. (1999) and G. Martelli (pers. comm.), it is our opinion that cymbidium ringspot tombusvirus is either extremely rare or does not infect orchids.
While virus incidence in orchids appears to be declining, good control methods remain absolutely necessary. Good horticultural practices such as weed and insect control as well as sterilization of cutting tools and pots with household bleach or tri-sodium phosphate (TSP) are very important. All plant debris should be removed from cutting tools which should then be soaked for at least several minutes in a 10% solution of household bleach or a saturated solution of TSP to inactivate viruses (Lawson, 1995). Pots should be soaked in these solutions overnight. TSP may not be available in some areas due to environmental constraints. A product marketed as “'TSP” may actually be a tri-sodium phosphate substitute, but the efficacy as a virus control agent is uncertain at this time. Cutting tools can also be flamed in alcohol or placed in a 300 F oven for one hour. The use of milk for sterilizing tools has been discussed over the years, but according to Hu et al. (1994), it was not effective in prevention of virus transmission. Orchids known to be virus-infected do not necessarily need to be discarded but should be identified and moved to an isolated area of a greenhouse or to another greenhouse in which they will avoid contact with healthy plants. Perhaps the best control strategy is to consider and “treat all plants as if they are virus-infected" (Marv Ragan — pers. comm.,). Whereas the incidence of both ORSV and CymMV appears to be declining they remain the most common and problematic of the orchid viruses. Virus incidence appears to be declining more rapidly in the collections of commercial growers than in those of private collections. Cymbidium mosaic virus is apparently more easily transmitted and is more common than ORSV in most orchid genera. Odontoglossum ringspot virus, however, for reasons still unclear, is far more common than CymMV in cymbidiums. Many of the less commonly detected orchid viruses cause striking symptoms and are usually eliminated from a collection without having to resort to diagnostic testing. Because orchids infected with ORSV and CymMV may not have obvious symptoms, growers may wish to have particularly valuable plants tested for these viruses. Growers should also keep in mind that some plants with virus-like symptoms may not be infected with ORSV or CymMV. Should this be the case, they may wish to have these plants tested for other viruses which are known to infect orchids.
MATERIALS AND METHODS
Either 20 or 40 orchid leaf samples were collected from each of 18 sources throughout the United States (California, Florida, Georgia, Missouri, Pennsylvania, South Carolina, and Washington, DC). Symptomatic and asymptomatic leaves were selected from both epiphytic and terrestrial species and hybrids. Some collections known to contain a large number of cymbidium orchids were selected. Likewise, samples of species and hybrids known to have been in cultivation for many years were preferentially selected.
Orchid leaf samples were tested for ORSV and CymMV (Wisler et al., 1982) using antigen trapped indirect enzyme linked immunosorbent assay (I- ELISA). The procedure used was a modification of that reported by Elliott et al. (1996) and Yeh and Gonsalves (1984). Samples were triturated 1:10 (weight : volume) in extraction buffer (Na2SO3, polyvinyl pyrrolidone-40, bovine serum albumin, phosphate buffered saline plus Tween-20 (PBST), pH 9.6). Odontoglossum ringspot virus and CymMV antisera provided by Dr. F. W. Zettler (Wisler et al., 1982) were diluted 1:1,000 in conjugate buffer (bovine serum albumin, polyvinyl pyrrolidone-40, PBST, pH 7.4). Whole-molecule goat anti-rabbit alkaline phosphatase conjugate (Sigma: St. Louis, MO) was diluted 1:30,000 in conjugate buffer. Increased absorbance values due to non-specific reactions with cymbidium tissue were reduced or eliminated by cross-absorption with healthy cymbidium tissue ground in conjugate buffer in a ratio of 1 :5 (weight : volume). Orchid leaf samples were tested for CymRSV using double antibody sandwich ELISA (DAS-ELISA). CymRSV antiserum and CymRSV antiserum-enzyme conjugate (Agdia, Inc., Elkhart, IN) were diluted 1:300 and tissue samples were triturated 1:10 in extraction buffer (p1·l 7.4). CymRSV-infected Nicotiana benthamiana tissue was used for the positive control. Enzyme corijugate was detected in both I-ELISA and DAS-ELISA using p-nitrophenyl phosphate substrate diluted 1 mg/ml in substrate buffer (diethanolamine in water, pH 9.8). Although absorbance readings were taken at 15 minute intervals for one hour at a wavelength of 405 nm, only the 30—minute absorbance values (Am) were used for evaluation. I-ELISA and DAS-ELISA absorbance values representing the average of three wells per sample were considered positive if the absorbance values were equal to or greater than three times the healthy values (Sutula et al., 1986). Whenever possible, identical or closely related genera or hybrids were used as positive controls. Additional data on the incidence of ORSV, CymMV and CymRSV was obtained from surveys conducted by Elliott et al. (1996) and Freitas-Astua et al. (1999). Data were also provided by the Florida Division of Plant Industry Data from current studies were also compared with that from earlier surveys conducted by Wisler et al. (1979, 1982), Zettler et al. (1978), Bodnaruk et al. (1979) and Wong et al. (1984).
See also Orchids from seeds