Citrus blight continues to be a major economic problem in citrus groves in Florida. Thousands of trees each year succumb to citrus blight, with estimated losses at over $60 million per year. The disease can occur on all common citrus cultivars, and Carrizo citrange are especially susceptible. Early symptoms are zinc deficiency in the leaves which may disappear, zinc accumulation in the phloem and eventually high zinc levels in the xylem. Blockage of xylem tissues with amorphous plugs follows with reduced water uptake. The causal agent of citrus blight is unknown. However, symptoms and all of the characteristics associated with citrus blight can be reproduced by root graft inoculations. Therefore in a project previously funded by CRDF we used NGS RNA sequencing protocols to look for novel viruses in roots of sweet orange with blight, but not present in roots of healthy trees, or trees affected by HLB. We identified several related endogenous pararetroviruses related to Petunia Vein Clearing Virus (PVCV) using a collection of 10 RNA libraries prepared from 10 different root samples collected from healthy trees or those with blight or HLB. The objectives of the proposed work are the following: 1. Generate a complete genome sequence for CBAPRV. This has been completed. We would like to transmit the complete sequence to CRDF. 2. Develop a highly specific RT-PCR assay that can determine when CBAPRV is active. This has been completed. The primers are ready to be transferred to CRDF. 3. Use this assay to screen a large number of trees from blight affected areas in Florida. This has been completed. The correlation between presence of the active viral RNA and blight is very high (96%). In addition, active viral RNA has never been found in healthy trees. 4. Transmission tests to determine if CBAPRV is the causal agent of citrus blight. This has been attempted, without success using multiple approaches. In the quarter just ending we have focused our efforts on the final objective. Previous sampling of 100 blight affected trees and 20 healthy trees from 5 blight affected areas (two sequential years) in Florida demonstrated a strong but not 100% correlation between the presence of CBAPRV RNA (the hallmark of active exogenous virus) and the onset of blight. 96% of the trees that were identified as affected by citrus blight have active viral CBAPRV RNA in leaves and roots, and none of the non-blighted trees have evidence of CBAPRV RNA. This is a strong correlation, but the 4% of trees that have been identified as blighted without the presence of CBAPRV suggests two possibilities: 1) the presence of the active virus is associated with the stresses of blighted trees, but the virus is not the causal agent of the disease, or 2) the method of sampling for CBAPRV RNA is not 100% effective. The final objective attempts to address which of these possible explanations is correct. Attempts to transmit the virus by aphids and psyllids were unsuccessful, in agreement with previous litereature. We have grafted CBAPRV onto a variety of rootstocks and scions, and these trees tested positive for viral DNA but not RNA. We will continue to monitor these trees, as well as applying a number of stresses (drought, cold, heat, etc…) in an attempt to induce blight. This work will continue beyond the scope of the project. In addition, attempts at purification of viral particles identified some likely particles, but these particles were only found in roots of blighted trees. Thus the efficiency of purification was very low. We may attempt to do further purifications and subsequent mechanical inoculations if enough particles can be generated, but that will occur beyond the scope of this project.