Using Improved Diagnostics to Determine Citrus Blight Prevalence

Using Improved Diagnostics to Determine Citrus Blight Prevalence

Report Date: 04/15/2024
Project: 23-021   Year: 2024
Category: Other
Author: Ronald Brlansky
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them:

Objective 1: The development of diagnostic assays. The antibody has been successfully used to identify potential Citrus Blight (CB) infected trees. We are continuing to work on an ELISA protocol. The beginning of the quarter we overcame challenges with some non-specific binding issues. We were successful overcoming those challenges by making modifications to our protocol resulting in clearly labeling of the MP antigen positive control wells that the while all other wells remained clear as expected. We then tested protein extracted from different trees and were not able to detect the CB movement (CBMP) protein, despite positive results with dot blots. This leads us to consider that the protein extraction samples may be below the limit of detection for ELISA. We are currently concentrating those samples to re-test them during the third quarter. Additional optimization considerations will be made to continue to develop an ELISA method. We are also exploring the use of a different membrane for dot blots. Polyvinylidene fluoride (PVDF) is a hydrophobic membrane that has been shown to increase the surface binding area for protein binding assays such as western blots and dot blots. We are currently modifying the dot blot method to see if we can increase the amount of bound protein on the Nitrocellulose of PVDF membrane which could increase our ability to detect CBMP in protein samples.

Objective 2: The goal of this objective is to determine how prevalent inserted copies of viral DNA are in commercial citrus. The virus inserts a copy Citrus Blight associated Pararetrovirus (CBaPRV) into the host DNA as a regular part of the viral life cycle. For objective 2 we are using PCR to screen DNA from 10 citrus varieties (a minimum of 3 trees per variety) for the presence of inserted viral DNA. By the end of the second quarter, we have surpassed the number of varieties by a large number, 36 in total, including 6 different species (orange, lemon, lime, grapefruit, mandarin, tangerine and tangelo) including 11 different varieties of rootstock. All tested citrus varieties are positive indicating that CBaPRV DNA has been inserted in their genome. This means that each tree is capable of being infected if the proper triggering conditions occur. We have tested some citrus relatives and found that Murraya species do not have the presence of the inserted virus.

2. Please state what work is anticipated for next quarter:

Objective 1: Next quarter we plan to continue develop an ELISA protocol and work though current challenges. We will continue optimizing the dot blot using a PVDF membrane. We will repeat the previous 30 samples tested using a PVDF membrane instead of a nitrocellulose membrane. We anticipate adding 10-20 more samples for a total of 40-50 samples. We will continue to generate data to assess the diagnostic capacity of this method compared to other existing methods (i.e. water uptake and PCR).

Objective 2: We need to test at least three trees for many of the varieties, to determine if the virus is present in all members of the variety. For several varieties, we have only tested a single tree, which is insufficient to determine prevalence.

3. Please state budget status (underspend or overspend, and why):

We are on track with our budget for this quarter and have purchased supplies necessary to carry out the objectives of the grant. We are on track for salaries this quarter.

4. Please show all potential commercialization products resulting from this research, and the status of each:

At this time there is no potential commercialization of any products associated with this research.

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