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. 2. Develop a highly specific RT-PCR assay that can determine when CBAPRV is active. This has been completed. 3. Use this assay to screen a large number of trees from blight affected areas in Florida. This is largely completed. 4. Transmission tests to determine if CBAPRV is the causal agent of citrus blight. This is underway. In the quarter just ending we have focused our efforts on the remaining objectives. During this quarter we have been validating the CBAPRV assay that was developed, and further sampling blight affected areas in Florida to assess the correlation between CBAPRV and blight affected trees. At this point, there is a strong but not 100% correlation between the presence of CBAPRV RNA (the hallmark of active exogenous virus) and the onset of blight. 94% 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 6% 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. To assess which of these possible explanations is correct we are moving on to attempted transmissions of the virus. There is no evidence that the virus is insect transmitted, so we have begun to work in two directions: 1) grafting of virus present tissue onto a variety of rootstocks and scions, and 2) attempts at identification and purification of viral particles that could be used to establish modified Koch’s postulates. The final quarter of work will focus exclusively on these efforts. Some additional testing of field materials collected in August of 2016 will also be completed.