Target Site Resistance Mechanism of Protoporphyrinogen Oxidase Inhibiting Herbicides in Eleusine indica
Type of DegreePhD Dissertation
Crop Soils and Environmental Sciences
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Goosegrass is considered as one of the five most troublesome weeds in the world. Protoporphyrinogen oxidase (PPO) with two isoforms, chloroplast targeted (PPO1) and mitochondrial targeted (PPO2), is a step in the biosynthesis of chlorophyll and heme. PPO1 and PPO2 are herbicide target sites of PPO inhibitors. Target-site mutations reported to date conferring resistance to PPO inhibitors have all been in PPO2. Oxadiazon is a unique PPO inhibitor utilized for goosegrass control in field courses and crop management. Studies were conducted to better understand target site resistance mechanism of PPO inhibitors. First, two previously confirmed oxadiazon preemergence resistant and susceptible goosegrass biotypes were evaluated to different PPO inhibitors. Two goosegrass biotypes were confirmed to be resistant to oxadiazon, but not to other structurally unrelated PPO inhibitors, such as lactofen, flumioxazin and sulfentrazone. A novel mutation A212T was identified in PPO1, conferring resistance to oxadiazon in in vitro enzyme and Escherichia coli functional expression system. Computational structural modeling provided a mechanistic explanation for reduced herbicide binding to the variant protein: the presence of a methyl group of Threonine 212 changes PPO1 active site and produces repulsive electrostatic interactions that repel oxadiazon from the binding pocket. Secondly, A binary goosegrass populations collected from different locations were submitted to the Herbicide Resistance Diagnostic Lab at Auburn University to evaluate for resistance to oxadiazon at 1.12 kg ha-1. Eleven of the 21 suspected populations were diagnosed as R based on postemergence screen. PCR sequencing results showed that there are 9 new resistant populations encoded a nonsynonymous SNP resulting in A212T amino acid substitution in PPO1, except for one population. A dCAPs primer and leaf chlorophyll fluorescense can be effectively utilized as a combination assay to detect the A212T mutation of all populations containing the mutation without sequencing. There is no other reported mutations occurred either in PPO1 or PPO2 of any populations. The target-site mutation A212T in PPO1 have been further substantiated as the primary mechanism of oxadiazon resistance. However, this does not preclude other non-target site mechanisms conferring resistance to oxadiazon in Sandestin or in future populations. At last, three different PPO isoforms from susceptible goosegrass plants (S-PPO1, S-PPO2) and resistant goosegrass plants (R-PPO1) were extracted and transformed to hemG mutant E. coli strain to evaluate the sensitivity response with three PPO inhibitors: oxadiazon, lactofen and sulfentrazone. The results showed that oxadiazon inhibits the PPO1 isoform greater than PPO2, lactofen inhibits the PPO2 isoform greater than PPO1, and sulfentrazone inhibits both PPO1 and PPO2 similarly, indicating that oxadiazon may prefer to binding chloroplast PPO1 isoform.