Key West Nightshade, a New Experimental Host for Plant Viruses Scott Adkins and Erin N. Rosskopf, U.S. Department of Agriculture, Agricultural Research Service, United States Horticultural Research Laboratory, 2001 South Rock Road, Fort Pierce, FL 34945 mosaic virus (TMV), and Pepper mild ABSTRACT mottle virus (PMMoV), viruses generally Adkins, S., and Rosskopf, E. N. 2002. Key West nightshade, a new experimental host for plant confined to short-lived herbaceous plants, viruses. Plant Dis. 86:1310-1314. because it continues to grow for many months following inoculation. Key West nightshade (Solanum bahamense) is a perennial solanaceous weed found in the ex- treme southern portion of Florida. It can be propagated by seed and cuttings and is absent from MATERIALS AND METHODS the noxious weed lists of all U.S. states. Its susceptibility to five viruses common to Florida was Field collection of S. bahamense. evaluated by mechanical inoculation of leaves with Tomato spotted wilt virus (TSWV), Tobacco While hiking on Bahia Honda in the Flor- mosaic virus (TMV), Pepper mild mottle virus (PMMoV), Cucumber mosaic virus (CMV), and a putative tobamovirus recently isolated from hibiscus in Florida (HV). TSWV induced ida Keys, we observed a single shrub-like chlorotic rings on inoculated leaves and mosaic and malformation of uninoculated leaves. CMV tree with red-orange fruit (Fig. 1) that were induced necrotic local lesions on inoculated leaves. No symptoms were observed following morphologically similar to those of S. inoculation with TMV, PMMoV, or HV. TSWV, TMV, and PMMoV systemically infected S. americanum. As part of our continuing bahamense as determined by the use of enzyme-linked immunosorbent assay, reverse transcrip- search for a long-term TSWV host suited tion-polymerase chain reaction, viral-associated double-stranded RNA analysis, and/or indicator to our needs, we collected fruit and ex- hosts. Active growth of infected plants continued for 7 months following inoculation, making S. tracted seeds, which we subsequently bahamense suitable for long-term maintenance of viruses in planta. We suggest that S. ba- planted in the greenhouse. Three seeds hamense may be a useful host for virus culture collections and for studies involving large num- germinated and grew into plants identified bers of virus isolates where fresh, infected tissue is continuously required. as Key West nightshade, S. bahamense. These three plants were maintained as stock plants for propagation. Inoculation of S. bahamense. In addi- Although plant virologists generally fo- experimental hosts are annuals. A perennial tion to TSWV, four other viruses com- cus their research on economically impor- plant species easily manipulated under monly found in Florida were used to make tant crops, there are several instances experimental conditions and susceptible to an initial determination of the “virus where noncrop plants merit consideration. commonly studied plant viruses may find range” of S. bahamense and to evaluate its Such plants, frequently weeds, are impor- use in virus culture collections and re- suitability as a host for use in virology tant (i) reservoirs for viruses causing eco- search with viruses that lose infectivity experiments. Viruses tested were: TMV nomic losses in crop plants, (ii) experimen- upon storage. strain U1 (kindly provided by Dennis tal hosts for detection, identification, As part of our research on Tomato spot- Lewandowski), Florida isolates of maintenance, or easier manipulation of ted wilt virus (TSWV) diversity, we have PMMoV (2), a putative tobamovirus re- such viruses, and (iii) targets for biocontrol tested many solanaceous plants for their cently detected in hibiscus (HV; 1), Cu- by viruses (8,18). Weeds have long been response to infection. American black cumber mosaic virus (CMV; kindly pro- known to harbor plant viruses and the vec- nightshade, Solanum americanum, is regu- vided by Mark Gooch), and TSWV. tors that transmit them (6,14). Since they larly employed as an indicator host in our Inocula were prepared from virus-infected can serve as an important source of inocu- studies. However, S. americanum, like all leaf tissue of D. stramonium (TSWV), lum for crop plants, numerous weed spe- other plants tested to date, has a very short tobacco (Nicotiana tabacum cv. Xanthi; cies have been explored (naturally infected useful life following TSWV infection. TMV, PMMoV, and CMV), and Cheno- and/or experimentally inoculated) as reser- Short host life represents a limitation for podium quinoa (HV). Inoculum for TSWV voir hosts. A great many of these are in the our research because we have determined was prepared by homogenization of in- Solanaceae (3,4,7,15,20,27), and some that fresh leaf tissue is a better source of fected leaf tissue in 0.5% (wt/vol) sodium weed species, e.g., Datura stramonium, inoculum, viral RNA, and viral protein sulfite containing 1% (wt/vol) Celite as an have proven to be useful experimental than frozen leaf tissue. We therefore have a abrasive using a mortar and pestle. Inocula hosts (6). Additional experimental and/or need for a TSWV host that will continue for TMV, PMMoV, HV, and CMV were indicator hosts from multiple plant genera active growth following infection. prepared by homogenization of the in- are available to virologists. Many of these In this report, we examined the “virus fected leaf tissue in 20 mM sodium phos- are also in the Solanaceae (5,19), espe- range” of a previously unstudied perennial phate buffer (pH 7.0) containing 1% cially the genus Nicotiana (25,26). Al- member of the Solanaceae, Key West (wt/vol) Celite. Each of five independent though a few of these species are perenni- nightshade (Solanum bahamense L.). The groups of S. bahamense plants (two or als (16,17), the vast majority of term “virus range,” coined by Christie and three plants each) was inoculated with one Crawford (9), is an assessment of the vi- of the five viruses. Cheesecloth was used ruses to which a particular host plant is to apply inocula to several marked leaves Corresponding author: Scott Adkins susceptible. Representatives of three dif- per plant. A sixth group of plants was E-mail: [email protected] ferent virus genera were assayed. While mock-inoculated with phosphate buffer. Accepted for publication 11 July 2002. the virus range reported was not developed Determination of “virus range.” In- by testing all possible viruses that may oculated plants were monitored weekly for infect S. bahamense, the results with the symptom development. Following the first Publication no. D-2002-0926-01R five viruses assessed here demonstrate the appearance of symptoms on TSWV- This article is in the public domain and not copy- utility of this plant as a new experimental inoculated (marked) leaves 1 month post- rightable. It may be freely reprinted with custom- ary crediting of the source. The American Phyto- host. We show that S. bahamense is a use- inoculation, uninoculated leaves were col- pathological Society, 2002. ful long-term host for TSWV, Tobacco lected from all plants and tested for the 1310 Plant Disease / Vol. 86 No. 12 presence of the input virus by at least two from 7-g samples of uninoculated S. ba- 1C). Vegetative propagation via cuttings of the following techniques: enzyme-linked hamense leaf tissue following a protocol from the original three plants was found to immunosorbent assay (ELISA), reverse previously published (24), although only a be a much easier and more expedient transcription-polymerase chain reaction (RT- single cycle of cellulose chromatography means of plant production, especially with PCR), viral-associated double-stranded (ds) was used. DsRNA was analyzed by elec- the use of commercially available auxin RNA analysis, and/or indicator host inocu- trophoresis on native 5% polyacrylamide (Rootone, Green Light Co., San Antonio, lation. A commercially available ELISA kit gels and detected by silver staining using a TX). Cuttings rooted and were ready for (Agdia, Elkhart, IN) routinely employed in commercially available kit (Bio-Rad, Her- inoculation in 2 weeks, while 2 to 3 our laboratory was used to test for TSWV. cules, CA). Upper noninoculated leaf tis- months were required for seed to germi- RT-PCR was used for detection of TSWV, sue from inoculated S. bahamense plants nate and produce plants suitable for propa- PMMoV, and TMV according to standard was homogenized and used to inoculate gation by cuttings. protocols (22,23) with virus-specific prim- appropriate indicator hosts for TMV, “Virus range” of S. bahamense. ers (Table 1). Briefly, first strand cDNA PMMoV, HV, and CMV based on the lit- Chlorotic rings and ring patterns developed was synthesized by Moloney murine leu- erature (1,10,28,29). on S. bahamense leaves inoculated with kemia virus reverse transcriptase (Promega, TSWV by 4 weeks postinoculation (Fig. Madison, WI) at 50°C for 45 min. This RESULTS 2A) on two of three inoculated plants. was followed by 30 cycles of PCR ampli- Culture of S. bahamense. Although the Symptoms of systemic infection, including fication with Taq polymerase at 94°C for three plants started from field-collected malformation of leaves (Fig. 2B), a gener- 45 s, 55°C for 45 s, and 72°C for 1 min. seed grew vigorously and flowered pro- alized mosaic, and localized necrosis, were Products were analyzed by electrophoresis fusely (Fig. 1A and B) in our greenhouse, readily apparent several weeks later on on native 2% agarose gels and detected by no fruit were produced. This was in strik- these plants, an observation confirmed by ethidium bromide staining. DsRNA analy- ing contrast to S. americanum, which pro- the use of ELISA and RT-PCR (Table 2). sis was selected for examination of infec- duces abundant fruit in our greenhouse. RT-PCR with TSWV-specific primers tion by TMV, PMMoV, HV, and CMV, as Hand pollination of S. bahamense flowers TSWV723 and TSWV722 (Table 1) ampli- these four viruses are amenable to detec- was necessary for fruit production, but fied the expected 620-bp product from total tion by this method.