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 this work included the development of an assay specific for active pararetroviruses in Florida citrus, and to use this assay to assess correlation between the active pararetrovirus and blight affected trees. An assay was initially developed using sequence inromation from the original work. Then leaves and roots were collected from over 50 trees from five geographically distinct locations. The majority of these trees were identified as being blight affected by water uptake testing, but putatively healthy trees were also sampled. In some cases, bark tissue from trunks was also collected for testing. RNA extractions were completed for all samples from all trees, and the presence of active pararetrovirus was assessed using the two primer sets selected in the optimization study from the previous quarter. Every tree that showed diminished water take up using the syringe injection test was positive for citrus blight associated pararetrovirus DNA. When the RNA extractions were treated with DNAse to eliminate potential genomic DNA sources of citrus blight associated pararetrovirus, all but 1 tree tested positive for pararetroviral RNA. However, no reverse transcription negative controls suggest that some samples still had low levels of genomic DNA, and these results need to be confirmed. Still, in initial analysis, there is a very strong correlation between the reduced water uptake via the syringe test and the presence of active citrus blight associated pararetrovirus. A third objective was to generate complete genome sequences for any and all active blight associated pararetroviruses and developing a active virus specific assay comprehensive enough to detect all blight associated pararetroviruses. As of the last quarter we had successfully generated a complete genome sequence for a blight associated active pararetrovirus, we are prepared to hand the sequence data to CRDF. In addition to the original genome sequence we have continued sequencing other pararetrovirus isolates from additional trees and geographic locations to determine levels of diversity. Six other complete genome sequences are underway. Also in our last quarter we had tested multiple assays for the active pararetrovirus on a large sample set to determine which of the assays was specific only to active pararetrovirus while still being inclusive enough to detect all active pararetroviruses. A single assay was developed that was very effective in detecting all active pararetrovirus samples in the 2015 sampling, this will meet the objective of the project. As a final check, an additional set of samples was taken in the summer of 2016 for testing and validating the new assay. This assay is ready for delivery to CRDF. .