2007 Annual Report 1a.Objectives (from AD-416) Determine the potential for increase and distance of spread of Leprosis (CiLV)in commercial and residential citrus in order to develop eradication/suppression/control strategies and evaluate their efficacy. Develop CiLV diagnosis/confirmation technology. 1b.Approach (from AD-416) Making disease assessments, collect data, monitoring vector populations, and collect meteriological variable information. Establish trap lines to determine long distance spread of leprosis. Correlate the prevalence of mite species with fluctuations in disease development. Determine the long distance spread of the virus emanating from an infested grove. Determine the temporal increase of Leprosis. Model the distance of disease spread within groves. Develop a temporal model to estimate disease increase for predictive purposes and economic/crop loss estimates. Correlate the prevalence of mite species with fluctuations in disease development. Evaluate the effect of mite vector control on epidemic increase and spread. 3.Progress Report This report serves to document research conducted under a reimbursable agreement between ARS and APHIS, with additional details of research found in the report for the parent CRIS 6618-22000-034-00D, Domestic, Exotic and Emerging Diseases of Citrus, Vegetables and Ornamentals (DEED). The spatial and temporal progress of disease and its vector were compared as function of phenology stage of citrus, weather conditions, and vector control. Six field plots were established near Matão, São Paulo State, Brazil. The central tree of each block was inoculated with virus infected and mite infested branches and fruits. First mites were observed in Novr/04 and mite populations and mite-infested trees started to increase in Mar/05. There was a strong relation between number of mites observed and number of infested trees. Peaks of mite population were coincident wit peaks of infested trees incidence. Mite population is very well established and distributed in all plots. Of ten weed species inoculated with CiLV, only Commelina benghalensis is a host for CiLV and is one of the most important weed species in citrus orchards in São Paulo State. In addition windbreak/shrub trees grown near citrus orchards were tested and Malvaviscus arboreus, Hibiscus rosa-sinensis, Bixa orellana and Grevillea robusta were confirmed as CiLV hosts. Confirmation was by symptom analyses, back inoculation to Citrus sinensis, transmission electron microscopy, RT-PCR, and sequence analyses of the amplicons. The prevalence fo the endosymbiont Brevipalpus phoenicis populations was studied. The Cardinium bacterium was found in 42 out of the 44 samples tested. A sequenced region within the 16S rDNA of 37 mite populations from all the major geographic regions of Brazil was examined and similarity ranged from 89 to 100%. Since this region is known to be very conserved, other regions were sequenced as well, such as the ITS and the gene that codes for gyrase B. The best RNA extraction method was (a CTAB protocol), the best storage condition (up to 120 days, at -20oC or 4oC), and minimal number of individuals (3) for the detection of Brevipalpus mites’ endosymbionts. The results obtained were submitted to Experimental and Applied Acarology and accepted for publication on March 27, 2007. We were able to eliminate the endosymbiont from the mites using the antibiotic tetracycline, high temperature, and cobalt-60 irradiation. Non-cured females and cured females (with and without endosymbiont) and males (without endosymbiont) were placed onto the CiLV inoculum source for 72 hours. 10 mites from each of the three groups were collected and tested for the presence of CiLV by RT-PCR to determine if they had acquired the virus. The results showed that, regardless of the presence of the bacterium, B. phoenicis mites are able to acquire CiLV. All three groups of mites were transferred to healthy citrus plants. Results suggest that non-treated female mites are more efficient in transmitting the virus, but all groups were able to transmit CiLV.