Determining the role of a novel virus in Citrus Blight

Determining the role of a novel virus in Citrus Blight

Report Date: 04/06/2015
Project: 921   Year: 2015
Category: CLas Bacteria
Author: William Schneider
Sponsor: Citrus Research and Development Foundation

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. In the quarter just ending we have expanded the correlation studies for the blight related pararetrovirus. Eight sets of primers for reverse transcription-PCR detection of viral RNA (an indicator of virus replication and biological activity) were tested on the original set of blight affected, HLB infected and healthy citrus trees. Two sets of primers were identified that were the most specific to the blight related pararetrovirus. These were applied to an additional 8 samples from two different Florida locations with blight problems. As with the original testing, the blight associated pararetrovirus RNA was only found in blight affected trees, and was present in all trees identified as blight affected via water uptake testing. More significantly, it was determined that the RNA from the blight associated pararetrovirus was present in both the roots and canopy of affected trees, indicating that blight affected trees may be identifiable using a leaf based test. Based on these results, an additional sampling was conducted to complete the correlation objective. 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 are underway as a first step to completing the first objective. In addition, progress is being made towards generation of a complete genome sequence for the blight associated pararetrovirus. A primer walking strategy is being implemented to extend known sequences in both directions to generate a full genome.


Your browser does not support pdfs, click here to download the file.