2006 Annual Report 4d.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 CRIS 5320-21000-011-00D "Molecular Resources for the Improvement of Tropical Crops." High Through-put PCR Assay for BBTV and CTV Resistant Mexican Lime A simple procedure has been optimized to prepare banana samples for real-time PCR system for detection of BBTV from a large number of banana samples. The real-time PCR system for detection of BBTV from a large number of banana samples from BBTV-infested banana fields. The assay has also been used to study the molecular interactions of BBTV with banana plants to develop control strategies. Transgenic Mexican lime plants have been produced. Banana Bunchy Top Virus: Aphid Vector Distribution and Imidacloprid Research Surveys were conducted in several banana orchards on Oahu to help develop a sampling plan for banana aphids and determine their within plant distribution. Banana orchards were sampled in Wailua, west and east Eva, Poamoho, Kunia, Kahuku and Waianae. A total of 2,011 banana plants were inspected during the survey period. Statistical analyses from these surveys have indicated that the most reliable site, of the banana plant to inspect for the presence of P. nigronervosa, is the area between the pseudostem and petiole of the lower leaf bract. Further analysis indicated that sampling for aphids in the middle & upper plant stratum increases the likelihood of receiving a false negative for the presence of aphids (e.g., concluding that aphids are absent when they are present). As such, sampling the lower plant stratum will significantly increase the chance of accurately identifying the presence of aphids compared with other areas of the banana plant. Additionally, through casual observation we have noted that the presence of ants may be a good indicator that aphids have colonized the plant. Young banana plants were treated with a foliar spray application of Provado 1.6F® at four concentrations of imidacloprid active ingredient: 120 ppm, 60 ppm, 24 ppm, and 12 ppm. Five plants were randomly chosen from each of the 4 spray treatments and control (H2O spray only) at 7 d intervals for 4 weeks. Leaf disc samples were obtained from the sprayed plants and used to determine levels of imidacloprid over a one month period. An ELISA plate reader was used to find out imidacloprid concentrations in the leaves. Analysis of leaf tissue for imidacloprid content showed that the concentration was consistently higher in older leaves than newly formed leaves. To determine the effect of Provado on P. nigronervosa mortality, survival of adult aphids was monitored on those plants treated with the four imidacloprid concentrations. Five aphids were placed on the sprayed banana leaves and leaves that formed after Provado application, respectively. After 72 h, these aphids were recorded as being alive and on the plant (AP), alive and off the plant (A) or dead (D). These aphid mortality tests were conducted weekly for one month. This insecticide remains quite effective, however, even after one month has passed. Mean loss of efficacy ranged from 20 to 38% at 120 and 12 ppm, respectively. This suggests that Provado may provide protection from P. nigronervosa for a minimum of one month following initial spray application. Results of the test also showed that Provado may be translocated through the banana plant. Banana leaves that appeared after foliar treatment showed mean aphid mortality rates ranging from 46 to 88% during the 4 week test period. These results indicate that newer developing banana leaves have moderate protection from P. nigronervosa, for several weeks. Banana Bunchy Top Virus Control and Education Program BBTV is still confined to the original geographic regions that existed at the start of this project. Work on BBTV video is nearing completion. BBTV field guides are being prepared for banana farmers who speak English and other languages. Field visits were made to farmers to advise them about BBTV identification and management. A field site on Lanai has been identified for a possible disease-free banana nursery to supply affected banana growers with disease-free planting material. BBTV extension publications were distributed at several client training sessions, including Master Gardeners. The Bunchy Top website is visited by a number of clients on a daily basis and serves as an excellent passive source of accurate information.