Control citrus Huanglongbing (HLB) by counteracting the SA hydroxylase of Candidatus Liberibacter asiaticus

Control citrus Huanglongbing (HLB) by counteracting the SA hydroxylase of Candidatus Liberibacter asiaticus

Report Date: 09/12/2016
Project: 15-028   Year: 2016
Category:
Author: Nian Wang
Sponsor: Citrus Research and Development Foundation

The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). Our previous study indicate that Las contains a functional SA hydroxylase that degrades SA and its derivatives. SA and its derivatives play important roles in plant defenses. Las employs SA hydroxylase to suppress plant defenses. Our central hypothesis is that we can improve HLB management by counteracting SA hydroxylase. We will focus on counteracting SA hydroxylase using inhibitors based on structure based design. This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush and March 2016 during spring flush. In addition, three field trials for different compounds including SA are being arranged. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush. Trunk injection in August and September, 2016 during summer and late summer-fall flush. HLB disease severity and disease incidence surveys were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016; Las titers in the leaf midrib and roots of selected spray-treated and control trees were determined right before treatment and at 6 months after first treatment using qPCR. HLB disease severity and Las titer in the leaf midrib and roots of selected soil drench �treated trees were determined before treatment in April, 2016 and at 6 months after the 1st application will be done in October, 2016. HLB disease severity and Las titer in the leaf midrib and roots of selected trunk injection �treated trees were determined before treatment in June, 2016. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We are continuing to identify SA hydroxylase inhibitors. The SA hydroxylase expressing tobacco plants could be used to test the inhibitors. The


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