Glyphosate is the most important and widely used herbicide in world agriculture. Intensive glyphosate selection has resulted in the widespread evolution of glyphosate-resistant weed populations, threatening the sustainability of this valuable once-in-acentury agrochemical. Field-evolved glyphosate resistance due to known resistance mechanisms is generally low to modest. Here, working with a highly glyphosate-resistant Eleusine indica population, we identified a double amino acid substitution (T102I + P106S [TIPS]) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant individuals. This TIPS mutation recreates the biotechnology-engineered commercial first generation glyphosate-tolerant EPSPS in corn (Zea mays) and now in other crops. In E. indica, the naturally evolved TIPS mutants are highly (more than 180-fold) resistant to glyphosate compared with the wild type and more resistant (more than 32-fold) than the previously known P106S mutants. The E. indica TIPS EPSPS showed very high-level (2,647-fold) in vitro resistance to glyphosate relative to the wild type and is more resistant (600-fold) than the P106S variant. The evolution of the TIPS mutation in crop fields under glyphosate selection is likely a sequential event, with the P106S mutation being selected first and fixed, followed by the T102I mutation to create the highly resistant TIPS EPSPS. The sequential evolution of the TIPS mutation endowing high-level glyphosate resistance is an important mechanism by which plants adapt to intense herbicide selection and a dramatic example of evolution in action.

A novel glyphosate resistance double point mutation (T102I/P106S, TIPS) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene has been recently identified for the first time only in the weed species Eleusine indica. Quantification of plant resistance cost associated with the TIPS and the often reported glyphosate resistance single P106S mutation was performed. A significant resistance cost (50% in seed number currency) associated with the homozygous TIPS but not the homozygous P106S EPSPS variant was identified in E. indica plants. The resistance cost associated with the TIPS mutation escalated to 85% in plants under resource competition with rice crops. The resistance cost was not detected in nonhomozygous TIPS plants denoting the recessive nature of the cost associated with the TIPS allele. An excess of 11-fold more shikimate and sixfold more quinate in the shikimate pathway was detected in TIPS plants in the absence of glyphosate treatment compared to wild type, whereas no changes in these compounds were observed in P106S plants when compared to wild type. TIPS plants show altered metabolite levels in several other metabolic pathways that may account for the expression of the observed resistance cost.

An Eleusine indica population was previously reported as the first global case of field-evolved glufosinate resistance. This study re-examines glufosinate resistance and investigates multiple resistance to other herbicides in the population. Dose-response experiments with glufosinate showed that the resistant population is 5-fold and 14-fold resistant relative to the susceptible population, based on GR 50 and LD 50 R/S ratio respectively. The selected glufosinate-resistant subpopulation also displayed a high-level resistance to glyphosate, with the respective GR 50 and LD 50 R/S ratios being 12-and 144-fold. In addition, the subpopulation also displayed a level of resistance to paraquat and ACCase-inhibiting herbicides fluazifop-P-butyl, haloxyfop-P-methyl and butroxydim. ACCase gene sequencing revealed that the Trp-2027-Cys mutation is likely responsible for resistance to the ACCase inhibitors examined. Here, we confirm glufosinate resistance and importantly, we find very high-level glyphosate resistance, as well as resistance to paraquat and ACCase-inhibiting herbicides. This is the first confirmed report of a weed species that evolved multiple resistance across all the three non-selective global herbicides, glufosinate, glyphosate and paraquat.

Goosegrass (Eleusine indica), regarded as one of the world's worst weeds, is highly pernicious to cash crop-growers in Malaysia. Following reports in 2009 that glufosinate-ammonium failed to adequately control goosegrass populations in Kesang, Malacca, and Jerantut, Pahang, Malaysia, on-site field trials were conducted to assess the efficacy of glufosinate-ammonium towards goosegrass in both places. Preliminary screenings with glufosinate-ammonium at the recommended rate of 495 g ai ha -1 provided 82% control of the weed at a vegetable farm in Kesang, while the same rate failed to control goosegrass at an oil palm nursery in Jerantut.The ensuing greenhouse evaluations indicated that the "Kesang" biotype exhibited twofold resistance, while the "Jerantut" biotype exhibited eightfold resistance towards glufosinate-ammonium, compared to susceptible goosegrass populations. The occurrence of glufosinate resistance in goosegrass calls for more integrated management of the weed to prevent escalating resistance and further proliferation of the weed in Malaysia.

Glufosinate resistance in this resistant population is not due to an insensitive GS, or increased activity, or altered glufosinate uptake and translocation, or enhanced glufosinate metabolism. Thus, target-site resistance is likely excluded and the exact resistance mechanism(s) remain to be determined. © 2017 Society of Chemical Industry.