2007 Annual Report 1a.Objectives (from AD-416) The overall objective of this cooperative research project is to minimize the impact of two economically important plant viral diseases, Banana Bunchy Top and Citrus Tristeza. The objectives are as follows: . 1)determine BBTV aphid vector distribution within plantations, both spatially and temporarily.. 2)Determine the effect of imadocloprid (Provado ®, Bayer) on banana aphid mortality, reproductive rate and transmission of BBTV to bananas.. 3)Optimize PCR procedures to test large numbers of banana samples for BBTV.. 4)Develop an island-wide plan for management of BBTV for Hawaii Island.. 5) Coordinate and focus the efforts of groups and agencies involved in determining the current distribution, local management and suppression, and education about BBTV on an island-wide basis. 6)Create and distribute multi-faceted education efforts to teach growers and the general public on how to recognize and what to and what not to do if they find BBTV.. 7)Develop a high throughput RT-PCR assay for CTV while monitoring CTV resistance in field trials.. 8)Develop CTV resistant Mexican lime through genetic engineering. 1b.Approach (from AD-416) For objective. 1)collect spatial and aphid distribution from field sites and map BBTV distribution using ARC-GIS and update on a CTAHR web site for BBTV.. 2)Analyze imadocloprid (Provado®, Bayer) concentrations and latency after treatment to determine the effects of treatment using a microwell plate assay. Objective. 3)Optimization of PCR BBTV assay for a 96 well reaction plate format by testing different DNA purification methods. For objectives. 4)and. 5)form a "Banana Action Group" to provide a coordinated effective response to BBTV on Hawaii Island to minimize the movement of BBTV into unaffected areas, through the development of educational materials and timely coordinated regulatory actions. For objective. 6)create educational materials about BBTV including but not limited to, news releases, BBTV website, educational leaflets, Banana IPM guide and others. For objective. 7)optimize existing RT-PCR protocol for CTV to adapt to a high throughput assay. For objective. 8)through development and incorporation of CTV resistant gene constructs, fully transformed lime plants will be developed. Documents SCA with U-HI-Manoa. Formerly 5320-21000-010-10S (06/06). 3.Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Hawaii at Manoa. Additional details of research can be found in the report for the parent project 5320-21000-011-00D Molecular Resources for the Improvement of Tropical Crops. Banana Bunchy Top Virus: Aphid Vector Distribution and Imidacloprid Research Results suggest that banana aphids are preferentially found on banana suckers below heights of 1.5 meters and are less likely to be found on larger plants especially banana plants in the flowering or fruiting stage. Those found on nearly mature or fruiting banana plants are frequently at low densities (less than 10 individuals/plant). Additionally, we found that spraying in the plant canopy is not needed because aphids are rarely found in that area of the plant. This information will allow growers to spray selectively resulting in a reduction in insecticide use. Additionally, we found that ants may not be used as an indicator for the presence of aphids which was believed by many. This is tentatively because ants found on banana plants may be tending other insects such as mealybugs and scales. High Through-put PCR Assay for BBTV and CTV Resistant Mexican Lime The high-throughput real-time PCR (TaqMan) method was optimized and used for detecting Banana bunchy top virus (BBTV) in asymptomatic banana plants surrounding symptomatic plants in a banana farm in Hawaii. We found that BBTV could be detected from about 20% of the asymptomatic banana plants from 100 ‘Dwarf Brazilian’ samples that were sampled. Embryogenic cell suspensions (ECS) of banana cultivar ‘Dwarf Brazilian’ have been transformed using Agrobacterium tumefaciens with four constructs derived from the Rep gene of the Hawaiian isolate of BBTV. The protocol for regeneration of plants from embryogenic cell suspensions of ‘Dwarf Brazilian’ has been optimized. Putatively transgenic lines that have survived in vitro culture on media containing selective antibiotics have been regenerated into plantlets and acclimated in soil under greenhouse conditions. These plants are currently being challenged in bioassays with aphids reared on BBTV-infected banana plants, and will be evaluated for expression of bunchy top symptoms and BBTV resistance. Any plants that show BBTV resistance will be further characterized by molecular techniques including Real-time PCR and Southern blot hybridizations. Banana Bunchy Top Virus Control and Education Program Assisted and consulted with the Maui Invasive Species Committee to provide educational materials to a new outbreak of BBTV on two farms on Maui. Developed and coordinated with Hawaii Banana Industry Association to develop a new policy for distribution of disease-free tissue cultured bananas produced by CTAHR. Presented plan for approval by Hawaii Department of Agriculture for a certified seed program for bananas in Hawaii. Conducted banana disease workshops for organic farmers on Molokai, Hawaii and Maui. Conducted separate banana workshops for farmers on Honokaa and Keaau, Hawaii. This project is monitored through meetings, email and telephone communications, and on-site visit.