Studies on the Interaction of TSAMV and G. boliviana

1. Studies on the Interaction of TSAMV and G. boliviana

2. Outline • Brief background of the tobamovirus, Tropical soda apple mosaic virus (TSAMV) • Objectives of Study: 1. Determine whether G. boliviana transmits TSAMV from infected to uninfected plants 2. Determine whether G. boliviana adults discriminate between infected and uninfected plants for feeding and oviposition • Experiments 1. Virus transmission 2. Preference for feeding/oviposition

3. Background of TSAMV(Adkins et al 2007) • Mosaic symptoms suggestive of virus infection initially observed on foliage of TSA plants in pastures near Okeechobee, FL in fall of 2002; foliage exhibiting symptoms was taken back to the lab for isolation of the virus • Mechanical transmission of the virus from leaves of a symptomatic TSA plant to Nicotiana benthamiana was initial means used to isolate the virus • Laboratory isolation of novel tobamovirus from N. benthamiana, dubbed Tropical soda apple mosaic virus, was the end result

4. Background of TSAMV (cont’d) • To date, known means of transmission of TSAMV include: 1. Mechanical Transmission (wounding of plant’s epidermal cell layer and physical movement of virus into plant), 2. Seed Transmission (TSAMV contamination of seed from infected TSA—leading to infected seedlings— was found, suggesting that seed transmission may be important in TSAMV dissemination)

5. Background of TSAMV (cont’d) • Symptoms of TSAMV on TSA 1. Yellow-green mottling apparent in foliar mosaic 2. Reduced leaf size at points of expression • Effects on TSA 1. Reduced vigor 2. Eventual leaf drop/necrosis

6. Questions: • It is documented that TSAMV is transmitted mechanically and by contaminated seed, but can INSECTS SERVE AS VECTORS for spread of the virus? - While collecting data at Eave’s ranch in fall of 2005 and spring of 2006, we noticed several pockets of infected and uninfected plants with Gratiana feeding on the plants in each of the pockets. - Very limited research has been conducted in the area of insect transmission of the virus - Specifically, is G. boliviana a vector for TSAMV transmission from infected to uninfected plants? • Do G. boliviana adults discriminate between infected and uninfected plants for oviposition?

7. EXPERIMENTS • VIRUS TRANSMISSION • PREFERENCE FOR FEEDING/OVIPOSITION

8. EXPERIMENT 1

9. Virus Transmission • Objective -Determine whether G. boliviana transmits TSAMV from infected S. viarum to uninfected S. viarum

10. Materials and Methods • 6 S. viarum inoculated with TSAMV-infected N. benthamiana to serve as source plants for virus transmission to uninfected target plants • 6 Trials; 5 Reps. per Trial = 30 Reps.; 10 beetles per Rep. = 300 total • Beetles reared from egg stage to adults on uninfected plants (i.e., plants grown from seed sterilized in TSP solution) • 10 adults per source plant were released per rep. • Allowed to feed for 48h, removed and placed in cage with target plant and allowed to feed for 48h • Beetles removed from target plants and destroyed at the end of the last 48h • Target plants monitored daily for 50 days for visual signs of TSAMV • Samples taken and ELISA performed to determine presence of TSAMV in target plants

11. Materials and Methods*New gloves and forceps used between each rep. and trmt. to place beetles on plants.

12. Findings • None of the 30 replications resulted in transmission of TSAMV from the source plant to the target plant after 48h of feeding • ELISA (enzyme linked immunosorbent assay) testing and back inoculation of local lesion tobacco with leaf samples from the target plant from each rep. confirmed the same 2 findings each time: 1. No TSAMV lesions present 2. G. boliviana is not a vector for TSAMV transmission between TSA plants

13. EXPERIMENT 2

14. Preference for Oviposition • Objective -Determine whether G. boliviana adults discriminate between infected and uninfected S. viarum for feeding/oviposition

15. Materials and MethodsDual Choice Oviposition Host Preference • 12 S. viarum inoculated with TSAMV from Nicotiana spp. to serve as infected plants for test • 6 Replications: 6 TSAMV-infected plants, 6 uninfected plants, 30 newly mated females, 30 “virgin” males • Each rep. per individual bug dorm contained: 1 TSAMV-infected plant, 1 uninfected plant, 5 “virgin” males, 5 newly mated females • Trial run time: 52h (72h)* *(Test ended after 52h due to noticeable differences in plant ht. and overall vigor between infected and uninfected plants.) • Adults Counted for Plant Preference: 2, 4, 8, 24, and 52h • Eggs Counted: 24, 52h

16. Materials and Methods (cont’d) • Initial Test Parameters: -15 mature males from caged colony—all found to be copulating and with testis fully developed—placed with 30 “virgin” females for 48h -Separated after 48h, 30 newly mated females placed with 30 “virgin” males and divided into groups of 5 males/5 females per rep. • Final Measurements -Beetles and eggs destroyed after 52h -Plants from each trmt. cut at base of stem and fresh wt. taken -Plants dried @ 70C for 1wk and dry wt. taken

17. Material and Methods*Petri dish w/ adults placed between infected and uninfected plant

18. * P<0.05 Dual-choice oviposition Oviposition Host Preference (corrected for fresh weight w/ plant size as covariant) * * * * NS

19. Findings • Adults do discriminate between infected and uninfected plants • Eggs: Uninfected = 2 Infected = 0 • Mating Pairs Observed: Uninfected = 3 Infected = 1

20. Future Research • Effects of TSAMV-infected TSA on the Life History Traits of G. boliviana • Does feeding on infected plants have adverse effects on -developmental time -longevity -survival -fecundity of G. boliviana?

21. Acknowledgements Principal Researchers • Dr. Bill Overholt (UF, IRREC, BCRCL) • Dr. Scott Adkins, Dr. Erin Rosskopf (USDA, ARS) Support • Rodrigo Diaz, Jackie Markle, Douglas Gonzalez (Zamorano intern)

22. Finis