Glyphosate resistance in Eleusine indica: EPSPS overexpression and P106A mutation evolved in the same individuals

Highlights

• Four goosegrass populations from tea plantations in Zhejiang Province were evolved resistance to glyphosate.

• EPSPS amplification in SH and SY populations resulted in moderate resistance (RI = 7.2 and 7.9, respectively).

• P106S mutation (6.7% of individuals) and EPSPS amplification (another 80.0%) in CX lead to low-level resistance (RI = 4.9).

• P106A mutation and EPSPS amplification in the same individuals evolved in CA confer high-level resistance (RI = 13.4).

Abstract

Goosegrass is one of the most widespread weeds in orchards and tea plantations in China, and glyphosate is a popular herbicide used to control it. However, high glyphosate selection pressure has led to some populations becoming resistant. The objectives of this research were to determine resistance levels and possible resistance mechanisms of goosegrass populations from several tea plantations in Zhejiang Province in China. The resistance indexes in four goosegrass populations (SH, SY, CA and CX) ranged from 4.9 to 13.4, and lower shikimate accumulation in these populations compared with a glyphosate-susceptible (GS) population confirmed their resistance to glyphosate. No mutations in the target gene EPSPS were found in populations SH and SY, however, the expression of EPSPS in these two populations was 9.3 and 29.7 times higher than that in the GS population, respectively. In the CX population, a P106S mutation in EPSPS was found in 6.7% of the individuals and another 80.0% of individuals had EPSPS amplification. In population CA, all the individuals had a P106A mutation and 86.7% of them had amplification in EPSPS. The EPSPS copy numbers ranged from 5.2 to 62.3 in these four resistant populations. There was a positive correlation between signal intensities of primary anti-EPSPS antibody and the copy number of the EPSPS protein, as indicated by immunoblot analysis. In population CA, with high-level resistance to glyphosate, both P106A mutation and amplification in EPSPS evolved in the same individuals in this population.

Introduction

Goosegrass (Eleusine indica (L.) Gaertn.) is a widespread annual grass in temperate and tropical regions and is considered as one of the world's worst weeds (Holm et al., 1977). In China, this weed is widespread in orchards, vegetable gardens, tea plantations and general field crops, such as corn (Zea mays L.), soybean (Glycine max (Linn.) Merr.) and cotton (Gossypium spp.) (Zhang, 2003; Yang et al., 2012). The presence of goosegrass leads to a reduction of yield in infested crops (Holm et al., 1977; Ma et al., 2015). This weed species had been efficiently controlled by different herbicides up to now. However, herbicide resistance has also evolved in goosegrass in China, especially to the herbicide glyphosate (Zhu et al., 2018). Studies have reported that glyphosate-resistant (GR) goosegrass populations occur in orchards throughout South China (Zhang et al., 2015; Chen et al., 2015a). In addition, some populations have evolved multiple resistance to paraquat, glyphosate and glufosinate (Hu et al., 2018).

Glyphosate is one of the most popular nonselective herbicides worldwide, especially in regions where GR transgenic crops are cultivated (Duke and Powles, 2008). Glyphosate competitively inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; E.C. 2.5.1.19) in the shikimate pathway, preventing the synthesis of the aromatic amino acids Tyr, Phe and Trp, leading to plant death (Steinrücken and Amrhein, 1980). Due to a widespread reliance on glyphosate for weed control for decades, 47 weed species, including goosegrass, have evolved glyphosate resistance (Heap, 2019). Many studies have focused on clarifying the resistance mechanism of GR weeds and selecting the best strategy to manage them (Heap and Duke, 2018). Mutations involving amino acid substitutions at conserved region and the amplification of EPSPS are target-site mechanisms conferring glyphosate resistance in weeds (Baerson et al., 2002; Yu et al., 2015; Gaines et al., 2010; Perotti et al., 2019; Li et al., 2018; Ngo et al., 2018). Transposable elements were reported to be related to EPSPS amplification in Amaranthus palmeri (Gaines et al., 2013). In addition, tandem amplification of EPSPS was found in the GR weed K. scoparia (Jugulam et al., 2014). Some reports have found that amplified copies of EPSPS in the GR weed A. palmeri are present in the form of extrachromosomal circular DNA molecules with various conformations that can be transmitted to the next generation by tethering to mitotic and meiotic chromosomes (Koo et al., 2018). Research has also suggested that an altered EPSPS gene promoter is related to its overexpression (Zhang et al., 2019). Strong glyphosate selection pressure resulted in amino acid substitutions evolved in EPSPS, in addition, mutation and overexpression of EPSPS were found in the same population (Perotti et al., 2019; de Castro Grossi Brunharo et al., 2019). Many weed species have evolved non-target-site mechanisms of glyphosate resistance, and this resistance can be conferred by increased metabolism, reduced translocation or uptake, or enhanced vacuolar sequestration of glyphosate, respectively (Heap and Duke, 2018; Van Horn et al., 2018; Moretti et al., 2018). GR goosegrass was shown to contain target site mutations at amino acid residues 106 or double mutations at 102 and 106 (Takano et al., 2018; Chen et al., 2015b). The first double mutation found in EPSPS was in the goosegrass population in Malaysia (Yu et al., 2015).

In China, some goosegrass populations are hard to control with glyphosate following many years of selection pressure. In this research, we measured the glyphosate resistance levels of goosegrass populations collected from tea plantations in Zhejiang Province of China and examined possible mechanisms of glyphosate resistance.