Abstract

1. Changes in Symptom Phenotype Resulting From Mixed Virus Infection of Melon (Cucumis melo) Joseph HanDepartment of Biology, Hartnell College, Salinas, CA 93908 William M. Wintermantel, Ph.D. Dept. of Virology USDA-ARS Abstract A field of canteloupe (Cucumis melo) in the San Joaquin Valley of California, exhibiting severe symptoms of necrosis and mosaic were found to be infected by multiple viruses in the fall of 2011. Nucleic acid extraction followed by reverse transcription-polymerase chain reaction (RT-PCR) testing identified three distinct viruses, Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV) and Lettuce necrotic stunt virus (LNSV). Finding multiple viral infections is not unusual, but discovering LNSV whose normal host range was not known to include systemic (whole plant) infection of melon is more significant. Mechanical inoculations were performed on young cantaloupe plants to attempt toreplicate the symptoms from the field incident and determine which virus combinations producedthe severe symptoms seen in the field. Inoculations included individual viruses (one virus per plant), all combinations of two viruses, and inoculation with all three suspected viruses. The results should clarify how multiple infections alter symptom developmenton this important crop and may determine the cause of the systemic necrosis symptom. Figure 2. Procedure of infecting plants with source viruses in preparation for ELISA technique Results Figure 4. Photographs portraying the symptoms resulting from a variety of inoculation combinations. Observations were noted one week after the first set of inoculations and continued thereafter. LNSV- Necrotic local legions on inoculated leaf, chloratic spots CMV- Dark/light alternating mosaic patterns, most likely systemic WMV- Slight discoloration becoming apparent, most likely systemic CMV+LNSV- Necrotic legions on inoculated leaves, newer leaves conveying mosaic discoloration (2/2 plants) WMV+LNSV- Mosaic patterns appear systemic, necrotic spots on inoculated leaves (½) CMV+WMV- New leaves developing misshapen, seeing systemic mosaic symptoms, no necrotic symptoms (2/2) CMV+WMV+LNSV- Necrotic spots on inoculated leaves, new leaves misshapen, mosaic patterns arising on newer leaves (2/3). Plant appears to have ceased growing, death ensuing (1/3) As shown in figure 4 the symptoms of the melon tested do not resemble the strange symptoms found in the San Joaquin Valley (Figure 1.). The degree of necrosis along the veins of the tested plants were cut short due to the death and collapse of the leaves. The charts in Figure 5. portray the quantitative results collected by analyzing the ELISA. Each plant was sampled by an inoculated leaf and a newly formed leaf to test if the virus went systemic, referred to as top/bottom simplification. The LNSV chart clearly represents an isolated form of infection, with the antiserum not interacting with new leafs. The ELISA data conveyed is an averaged summary between the data of two trials to show consistency. Conclusions Thus far the test results have yet to replicate the exact symptoms of the infections that occurred in the San Joaquin Valley. The plants inoculated with all three viruses did portray more severe symptoms than the other combinations of infections, but the symptoms did not convey a systemic infection of the LNSV. It appears that the plants were able to isolate the LNSV in necrotic legions. The results did however convey interesting results regarding the relationships between the three tested viruses. Alongside visual symptoms, the ELISA procedure further detailed the dominance of CMV in the Cucumis Melo variety. In mixed infections, the CMV readings were usually around 3x that of any other virus present in the testings. The WMV was able to coexist with the CMV but struggled with the LNSV. The LNSV existed only on inoculated leaves. The underlining effects of the original infectious symptoms are still yet to be fully understood. There may be variables yet accounted for that will require further research. 1. Create Virus preparation, using Cellulite (diatomaceous earth), phosphate buffer (ph 7.4) and a fresh source of the virus in symptomatic plants leaves. Use a mortar and pestle to grind the plant leaves, Celluliate and phosphate buffer together into a juice. 2. Mechanical Inoculation: Wearing disposable gloves, rub the virus preparation on the surface of the host plant leaves in such a way to break surface cells without causing too much direct damage. Note younger leaves need only to be grazed. 3. Allow plants to stand for 5 minutes 4. Spray gently with tap water to remove inoculum residue. 5. Allow plants to stand for approximately 30 minutes to recover prior to placing plants in greenhouse or growth chamber. 6. Water plants daily and maintain temperatures averaging 25oC 7. Symptoms should begin to appear in roughly a week in which careful scoring of the plants conditions should begin. Include Details of plants health. 8. At the 2-3 week mark, each plant should be sampled. Take .5 grams of one of the original inoculated leaves and .5grams of a new leaf. 9. Complete the Enzyme-linked Immunoabsorbent Assay and record results. 10. Two trials have been analyzed with the third needing more time for completion,. Figure 5. Enzyme-linked Immunosorbent Assay Results Figure 1. Photograph of a Melon plant from the San Joaquin Valley exhibiting symptoms of necroses along the veins Figure 1. Photograph of a Melon plant from the San Joaquin Valley exhibiting symptoms of necroses along the veins Figure 1. Photograph of a Melon plant from the San Joaquin Valley exhibiting symptoms of necroses along the veins Future Research Further research might involve utilizing different temperatures and inoculation periods for the various combinations of viruses which may result in symptoms closer to those found in the original infected plants. Materials and methods The following materials, equipment and facilities were provided by Dr. Wintermantel, USDA-ARS, Salinas: Balance for weighing samples, ELISA grinding bags and hand roller, Nunc Maxisorp 96-well plate, Bio-tek 96-well reader and software, Micropipettor (single and 8-channel) and sterile tips, incubator set at 37oC, Ice and bucket, paper towels, weigh boats, small flasks and stir bars, Sigma phosphatase tablets, melon plants (cultivar Topmark) Virus sources (WMV, LNSV, CMV) a green house and growth chamber. Different combinations of viruses inoculated to melons. Figure 3 . Visual description of generic Enzyme-linked immunosorbent assay (ELISA) technique. Source thefreedictionary.com.Note: CMV, WMV and LNSV are not rotaviruses Acknowledgments A special thanks my mentor Dr. William M. Wintermantel for providing his time and expertise, and to Laura Hladky and Art Cortez in the Wintermantel Lab for training and assistance with this project. A deep gratitude also goes to the Tanimura Family Foundation for the grant which funded this research project. Laboratory The Wintermantel lab is located in the Virology Department of the United States Department of Agriculture – Agricultural Research Service Station 1636 East Alisal Street Salinas, California 93905. The lab is a part of the Crop Improvement and Protection Research Unit.