Variation in mutations providing resistance to acetohydroxyacid synthase inhibitors in Cyperus difformis in China
Graphical abstract
Introduction
Acetohydroxyacid synthase (AHAS), also called acetolactate synthase (ALS), is a key enzyme in the biosynthesis of branched chain amino acids, valine, leucine and isoleucine, in plants and microorganisms (Durner et al., 1991; Devine and Shukla, 2000). AHAS catalyzes the reactions of both pyruvic acid to 2-acetolactate, and pyruvic acid and 2-ketobutyrate to 2-aceto-2-hydroxybutyrate (Schloss et al., 1988). AHAS inhibitors block the biosynthesis of the three branched chain amino acids, hindering protein synthesis during cell mitosis and making it difficult for plants to grow normally, thus leading to their consequent death. Since the introduction of AHAS inhibitors, they have been widely used in weed control due to their high control efficacy, wide herbicide spectrum and low environmental impact (Mazur and Falco, 1989). There are five chemical families of AHAS inhibitors: imidazolinones (IMI), sulfonylureas (SU), triazolopyrimidines (TP), pyrimidinylthio-benzoates (PTB) and sulfonylamino‑carbonyltriazolinones (SCT), and all act on the target enzyme of AHAS (Chaleff and Mauvais, 1984; Shaner and Studham, 1984; Gerwick et al., 1990; Stidham, 1991; Santel et al., 1999).
The extensive use of AHAS inhibitors has led to the rapid development of herbicide resistance. As early as 1982, Australia reported the first case of the evolved resistance of Lolium rigidum to AHAS inhibitors (Heap and Knight, 1986). Until now, 162 weed species have developed resistance to AHAS inhibitors globally, mostly due to point mutations in the AHAS gene that reduce the sensitivity of AHAS to herbicides (Heap, 2019). To date, 29 resistance-endowing amino acid substitutions were identified at 8 positions (Ala-122, Pro-197, Ala-205, Asp-376, Arg-377, Trp-574, Ser-653, and Gly-654) in the AHAS enzyme (Heap, 2019). Cross-resistance among AHAS-inhibiting herbicides can occur and is related to the mutation types of the target sites. For example, the Ala-122, Ser-653 and Gly-654 mutations confer resistance to IMI but not SU herbicides. The Pro-197 mutation confers resistance to SU and TP herbicides, while the Asp-376 or Trp-574 mutations provided resistance to all five families of AHAS-inhibiting herbicides (Pang et al., 2002; McCourt et al., 2005; Beckie and Tardif, 2012). However, the exact pattern of resistance due to specific amino acid substitutions can vary between species (Deng et al., 2016).
Cyperus difformis, also known as smallflower umbrella sedge, is an annual weed of the Cyperaceae family, a problematic weed in paddy fields with high levels of self-pollination (Merotto et al., 2009). C. difformis is widely distributed in central, east, north and northeast of China and has a serious impact on rice production. One hundred plants of C. difformis grown in 1 m2 can lead to a significant reduction in rice yield of 49–81% (Swain et al., 1975). AHAS inhibitors have been frequently applied to control C. difformis in paddy fields since the 1980s and there have been some reports of resistance of C. difformis. In some areas, C. difformis has developed cross-resistance to AHAS inhibitors, with reduced or no chemical control effects. A study in Korea found that C. difformis exhibited varying degrees of cross-resistance to imazosulfuron, bensulfuron-methyl, pyrazosulfuron-ethyl, bispyribac‑sodium, and imazapyr (Kuk et al., 2004). Furthermore, cross-resistance to AHAS inhibitors has also been found in Spain, America and Italy (Busi et al., 2006). Resistance mutations at Pro-197 (substituted by Ala, Ser or His) were also identified in C. difformis, conferring resistance to the AHAS inhibitors, azimsulfuron, halosulfuron-methyl, and bispyribace‑sodium (Ntoanidou et al., 2016; Tehranchian et al., 2015). The AHAS inhibitors used to control C. difformis in paddy fields in China are mainly pyrazosulfuron-ethyl, penoxsulam, bensulfuron-methyl, and halosulfuron-methyl. Pyrazosulfuron-ethyl and bensulfuron-methyl are very important herbicides to control C. difformis in paddy fields of China, and they have been commercialized since 1980s. However, the control effect of them is decreasing significantly recently. To date, there is limited information about resistance to AHAS inhibitors in C. difformis in China and the mutations conferring resistance.
The aims of this research were: 1) confirm the distribution of resistance to pyrazosulfuron-ethyl in C. difformis populations collected from paddy fields in 7 provinces of China; 2) sequence the AHAS gene and elucidate the mechanism of resistance to AHAS inhibitors; 3) detect the biochemical basis of resistant C. difformis based on the AHAS in vitro activity in the presence of different AHAS inhibitors; and 4) determine the cross-resistance patterns conferred by different AHAS mutations.
Section snippets
Plant material
A total of 38C. difformis populations were collected in paddy fields from 2011 to 2015 from the provinces of Heilongjiang, Liaoning, Hubei, Hunan, Anhui, Jiangsu and Jiangxi. YX11, YX12, YX13 and YX15 represent the populations of C. difformis collected in 2011, 2012, 2013 and 2015, respectively.
Soil from an area without an herbicide application history was sifted and mixed with nutrient soil in a 4:1 ratio (organic matter content >15% and total nitrogen, phosphorus and potassium contents >0.83%
Effect of pyrazosulfuron-ethyl on C. difformis populations
At 21 DAT with pyrazosulfuron-ethyl at the recommended dose of 30 g ha−1, the survival rates of all C. difformis populations were determined and are shown in Table 4. Among the 38 tested populations, 22 were dead and 16 still had surviving plants, 12 of which showed complete survival and 4 of which showed partial survival. The R populations were found in the provinces of Hunan, Jiangxi, Jiangsu and Anhui (Fig. 1).
Sequences comparison and identification of resistance mutation in AHAS
By comparing the differences between DNA and cDNA amplification products, we found
Discussion
Pyrazosulfuron-ethyl and bensulfuron-methyl were successfully developed in the late 1980s as highly efficient, broad-spectrum and low toxicity herbicides in paddy fields and have been popularly applied for weed control (Yamamoto et al., 1994). Previous studies have shown that AHAS inhibitors have a tendency to select for resistance rapidly (Powles and Yu, 2010). Until now, 26 species of weeds, such as Monochoria korsakowii, Lindernia micrantha, C. difformis and Sagittaria trifolia, have been
Acknowledgments
This work was supported by the National Natural Science Foundation of China (31972281) and the National Key Research and Development Program of China (2016YFD0200500). The authors thank all of the workers for their assistance in conducting this research. No conflicts of interest have been declared.
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