Mutation at residue 376 of ALS confers tribenuron-methyl resistance in flixweed (Descurainia sophia) populations from Hebei Province, China

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Highlights

  • Two ALS genes were found in flixweed and only one of them had mutations related to resistance to tribenuron.

  • There were two mutation sites Pro197Ser and Asp376Glu of ALS conferring the resistance and the latter mutation was firstly found in flixweed.

  • Mutation Asp376Glu of ALS induced slight cross resistance to florasulam, but mutation Pro197Ser did not.

Abstract

The acetolactate synthase (ALS) inhibitor tribenuron has been used continuously for approximately twenty years as an herbicide in winter wheat fields in China. Flixweed (Descurainia sophia) has evolved resistance to tribenuron, due to multiple amino acid mutations at the 197th residue of ALS. In this study, the molecular basis of tribenuron resistance was investigated using two resistant populations, Xingtai (XT) and Shijiazhuang (SJ), and two susceptible populations, Cangzhou (CZ) and Handan (HD). Whole-plant tests and ALS activity assays showed that the two resistant populations were highly resistant to tribenuron. Targeted amplification of ALS genes from the four populations showed that there were two ALS genes in each population, and both of them were expressed in flixweed; the full coding lengths of the two ALS genes were 1998 bp and 2004 bp. Mutations related to tribenuron resistance in flixweed were located in only the 1998 bp paralog. An ALS activity assay showed that the resistant population SJ displayed slight cross-resistance to florasulam, with a resistance factor of 4.81, but the resistant population XT did not have cross-resistance to florasulam. The resistant population XT was found to carry the previously reported mutation Pro197Ser, but the resistant population SJ carried a different mutation, Asp376Glu, known from other weeds but novel in flixweed. Our results demonstrated that multiple versions of ALS genes exist in flixweed and that mutations at multiple sites may result in ALS-inhibitor resistance in this weed.

Introduction

Flixweed (Descurainia sophia), a broadleaf weed, is widely distributed in winter crops, forage, and rangelands throughout the world [1], [2], [3], [4]. It is the most important weed in winter wheat and causes yield losses of up to 20% in northeastern and northwestern China [5].

Tribenuron, a sulfonylurea (SU) acetolactate synthase (ALS) inhibitor, was produced by Dupont Company in the early 1980s and introduced to China in 1988 for the selective control of flixweed and other broad-leaved weeds, such as Capsella bursa-pastoris and Silene conoidea [4]. This herbicide has been extensively used in the last two decades due to its high efficacy at low dosage, low impact on non-target organisms and high selectivity. Currently, its use accounts for over half of the total herbicide usage area for broadleaf weed control in wheat fields in China. However, flixweed has evolved strong resistance to this herbicide due to poor usage. Florasulam, a triazolopyrimidine (TP) ALS inhibitor, was produced by Dow Agroscience in the mid-1990s [6]. Due to the development of evolved resistance to tribenuron in flixweed, florasulam is replacing tribenuron for broadleaf weed control in wheat fields in China.

ALS is the only target enzyme of ALS inhibitors, so weeds are prone to evolve resistance to these herbicides. At present, 151 weed species have evolved resistance to this mode of action worldwide [7]. Their resistance mechanisms are based on both target-site mutations and non-target-site mutations. However, the most common resistance mechanism is reduced target-site sensitivity to herbicide due to single-nucleotide polymorphisms in the ALS gene [8]. Ten sites of amino acid substitutions conferring resistance to ALS inhibitors have been reported (numbering standardized in accordance with the Arabidopsis thaliana sequence): Ala122, Pro197, Ala205, Asp376, Arg377, Trp572, Trp574, Phe578, Ser653 and Gly654 [9], [10], [11]. Of these, mutation at the Pro197 site is the most common, followed by mutation at Trp574, and then at Ser653.

Some papers have reported that multiple ALS mutations conferring inhibitor resistance have been found in populations of single weed species, such as Raphanus raphanistrum with the Pro197Ala, Pro197Thr, or Trp574Leu mutations [12]; Sisymbrium orientale with the Pro197Ile or Trp574Leu mutations [13]; Sinapis arvensis with the Trp574Leu or Pro197Ser mutations [13]; Amaranthus tuberculatus with the Trp574Leu, Ser653Asp, or Ser653Thr mutations [14]; Apera spica-venti with the Pro197Thr, Pro197Asn, Trp574Leu, or Arg377His mutations [15]; and Kochia scoparia with the Pro197Gln (Ser, Lys, Thr, Ala, Leu, Met, Arg, and Asp), Asp376Glu, or Trp574Leu mutations [16]. However, until now, only mutation at Pro197 had been reported in tribenuron-resistant flixweed populations [17], [18], [19], [20], [21]. It was unknown whether there were any other mutations affording tribenuron resistance in populations of this weed. The objectives of this study were to investigate other potential mutations of tribenuron-resistant flixweed populations and to evaluate their cross-resistance to florasulam using an ALS activity assay. The findings are useful for the design of an ALS-inhibitor rotation strategy for controlling resistant flixweed populations.

Section snippets

Seed collection

Seed samples of the four flixweed populations used for this study were collected from wheat fields in Hebei province, China. The seeds of two resistant populations were collected from the wheat fields of Xingtai (population XT) and Shijiazhuang (population SJ), where tribenuron has been applied for over fifteen years, and the seeds of two susceptible populations were collected from the wheat fields of Handan (population HD) and Cangzhou (population CZ). No tribenuron or other herbicides had

Whole-plant bioassay

The results of the whole-plant bioassay showed that the flixweed populations collected from Handan and Cangzhou, were highly susceptible to tribenuron, and their ED50 values were less than 0.1 g a.i. ha 1 (Table 1). Flixweed populations collected from Xingtai and Shijiazhuang, where tribenuron had been continually applied for more than 15 years, were highly resistant to this herbicide, and their ED50 values were 224.9 and 336.0 g a.i. ha 1, respectively.

In vitro ALS assay

The tribenuron I50 values for the susceptible

Discussion

Continuous application of ALS-inhibiting herbicides easily induces resistance evolution in weeds [24]. Tribenuron was extensively used to control flixweed for approximately two decades in China. The long-term use of this herbicide has resulted in herbicide resistance in this weed. Several resistant populations have been reported in wheat-growing regions throughout China [17], [18], [19], [20], [21]. As far as we know, this study is the first to have documented tribenuron resistance in flixweed

Conclusions

In this study, two new populations, XT and SJ, of flixweed were found to be resistant to tribenuron in China; the population SJ had slight cross-resistance to florasulam, but the population XT did not. Two ALS genes, of 1998 bp and 2004 bp in length, are transcribed in flixweed. In the population XT, the evolved tribenuron resistance was due to a mutation in ALS of Pro197 to Ser as previously reported, but in the population SJ, the resistance was due to another mutation, Asp376 to Glu.

Acknowledgments

The authors gratefully acknowledge all the workers for assistance in conducting this research. This research was funded by National Natural Science Foundation of China (31301679) and the Natural Science Foundation of Hebei Province (C2014301006).

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    • Florasulam resistance status of flixweed (Descurainia sophia L.) and alternative herbicides for its chemical control in the North China plain

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      Citation Excerpt :

      Tribenuron-methyl, a sulfonylurea (SU) ALS-inhibiting herbicide, was introduced in China in 1988 to control dicot weeds in wheat fields. Up to now, however, 7 kinds of amino acid substitutions endowing target-site resistance (TSR) to tribenuron-methyl, have been reported in flixweed in China at Pro197, Asp 376 and Trp 574, including P197S, P197L, P197T, P197H, P197Y, D376E and W574L (Deng et al. 2015; Deng et al. 2017; Xu et al. 2015). In addition, non-target-site resistance (NTSR) to tribenuron-methyl, which may endow wider and unpredictable cross-resistance to other herbicides with or without the same mode of action was also confirmed in flixweed in China (Yang et al. 2016).

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