Identification of biochemical and molecular mechanisms of resistance to glufosinate and glyphosate in Eleusine indica

Abstract

Herbicides are important tools in agriculture. They allow for a simple and effective method of weed control for growers in order to meet the global food demand. Unfortunately, intensive herbicide usage with diminishing diversity of weed control methods has resulted in weeds evolving resistance to herbicides. Herbicide resistance is now a major issue and challenge for growers globally. One of the problematic weed species, especially in the tropical and warm climate regions is Eleusine indica. Eleusine indica is a pernicious weed that is prone to evolve resistance to herbicides. Currently, global incidences of evolved resistance in E. indica include eight different herbicide sites of action, including glufosinate.

Working with a glufosinate-resistant E. indica population from Malaysia, the resistance profile was further characterised and assessed for multiple resistance. Glufosinate resistance was confirmed in the E. indica population, with the GR50 (rate required to reduce the growth by 50%) and LD50 (rate required to kill 50% of the population) R/S ratios being 5- and 14-fold, respectively. More importantly, multiple resistance was observed, with the selected glufosinate-resistant subpopulation (R*) exhibiting a very high level of glyphosate resistance. The GR50 and LD50 R/S ratios obtained were 12- and 144-fold, respectively for glyphosate. This population had also evolved resistance to paraquat, albeit at a low level (GR50 and LD50 R/S ratios 2 to 3-fold, respectively). This species is the first to be reported to have evolved resistance to all three non-selective herbicides. Additionally, resistance to several ACCase-inhibiting herbicides, namely fluazifop-p-butyl, haloxyfop-p-methyl and butroxydim, was caused by a Trp-2027-Cys substitution in the ACCase protein sequence.

In order to investigate the glufosinate resistance mechanism(s) in the R* population, activity of glutamine synthetase (GS) (the target -site of glufosinate) was compared in the S and R* populations. No difference in enzyme sensit ivity towards glufosinate was observed. Specific GS activity was also similar between S and R*. Differences in foliar uptake and translocation of [14C]-glufosinate were not significant between the two populations. HPLC analysis of glufosinate metabolism did not detect any metabolites in S or R* plants. Consequently, the resistance mechanism to glufosinate is not due to an insensitive target-site, target-site over production, differential glufosinate uptake and translocation, nor enhanced glufosinate metabolism, and remains to be determined.

Sequencing of the glyphosate target gene, EPSPS, in the highly glyphosate resistant E. indica population revealed that a double mutation in the EPSPS gene, i.e. Thr-102-Ile and Pro-106-Ser (TIPS), was responsible for the high level glyphosate resistance. Importantly, this double mutation is similar to the first commercialised transgenic, glyphosate-tolerant EPSPS in maize, but has never been reported to occur naturally. Dose-response experiments showed that the naturally evolved TIPS mutants are 180-fold (LD50 based) more resistant to glyphosate than the wild type (WT) E. indica plants, and 32-fold more resistant than the Pro-106-Ser (P106S) (LD50 based) mutants. EPSPS inhibition assays also revealed similar results, with the TIPS EPSPS enzyme activity showing very high glyphosate resistance relative to wild type (WT) EPSPS (2600-fold) and P106S EPSPS (600-fold). Interestingly, the highly resistant TIPS mutant exhibited a resistance cost in terms of vegetative growth and seed production, while no resistance cost was observed for plants with the P106S mutation. Plants with the TIPS mutation had a higher basal shikimic acid (the substrate for EPSPS) level and lower tryptophan (a downstream product) levels than WT and P106S plants. The evolution of the TIPS double mutation is likely a sequential event, with the P106S mutation being selected first, followed by the T102I mutation, creating the highly glyphosate resistant TIPS EPSPS.
Working with a glufosinate-resistant E. indica population from Malaysia, the resistance profile was further characterised and assessed for multiple resistance. Glufosinate resistance was confirmed in the E. indica population, with the GR50 (rate required to reduce the growth by 50%) and LD50 (rate required to kill 50% of the population) R/S ratios being 5- and 14-fold, respectively. More importantly, multiple resistance was observed, with the selected glufosinate-resistant subpopulation (R*) exhibiting a very high level of glyphosate resistance. The GR50 and LD50 R/S ratios obtained were 12- and 144-fold, respectively for glyphosate. This population had also evolved resistance to paraquat, albeit at a low level (GR50 and LD50 R/S ratios 2 to 3-fold, respectively). This species is the first to be reported to have evolved resistance to all three non-selective herbicides. Additionally, resistance to several ACCase-inhibiting herbicides, namely fluazifop-p-butyl, haloxyfop-p-methyl and butroxydim, was caused by a Trp-2027-Cys substitution in the ACCase protein sequence.

In order to investigate the glufosinate resistance mechanism(s) in the R* population, activity of glutamine synthetase (GS) (the target -site of glufosinate) was compared in the S and R* populations. No difference in enzyme sensit ivity towards glufosinate was observed. Specific GS activity was also similar between S and R*. Differences in foliar uptake and translocation of [14C]-glufosinate were not significant between the two populations. HPLC analysis of glufosinate metabolism did not detect any metabolites in S or R* plants. Consequently, the resistance mechanism to glufosinate is not due to an insensitive target-site, target-site over production, differential glufosinate uptake and translocation, nor enhanced glufosinate metabolism, and remains to be determined.

Sequencing of the glyphosate target gene, EPSPS, in the highly glyphosate resistant E. indica population revealed that a double mutation in the EPSPS gene, i.e. Thr-102-Ile and Pro-106-Ser (TIPS), was responsible for the high level glyphosate resistance. Importantly, this double mutation is similar to the first commercialised transgenic, glyphosate-tolerant EPSPS in maize, but has never been reported to occur naturally. Dose-response experiments showed that the naturally evolved TIPS mutants are 180-fold (LD50 based) more resistant to glyphosate than the wild type (WT) E. indica plants, and 32-fold more resistant than the Pro-106-Ser (P106S) (LD50 based) mutants. EPSPS inhibition assays also revealed similar results, with the TIPS EPSPS enzyme activity showing very high glyphosate resistance relative to wild type (WT) EPSPS (2600-fold) and P106S EPSPS (600-fold). Interestingly, the highly resistant TIPS mutant exhibited a resistance cost in terms of vegetative growth and seed production, while no resistance cost was observed for plants with the P106S mutation. Plants with the TIPS mutation had a higher basal shikimic acid (the substrate for EPSPS) level and lower tryptophan (a downstream product) levels than WT and P106S plants. The evolution of the TIPS double mutation is likely a sequential event, with the P106S mutation being selected first, followed by the T102I mutation, creating the highly glyphosate resistant TIPS EPSPS.