Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations

    Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations Long Du, Weitang Liu, Guohui Yuan, Wenlei Guo, Qi Li, Jinxin Wang PII: DOI: Reference:

S0048-3575(15)30014-6 doi: 10.1016/j.pestbp.2015.07.005 YPEST 3846

To appear in: Received date: Revised date: Accepted date:

26 December 2014 21 July 2015 23 July 2015

Please cite this article as: Long Du, Weitang Liu, Guohui Yuan, Wenlei Guo, Qi Li, Jinxin Wang, Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations, (2015), doi: 10.1016/j.pestbp.2015.07.005

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ACCEPTED MANUSCRIPT Cross-resistance patterns to ACCase-inhibitors in American sloughgrass (Beckmannia syzigachne Steud.) homozygous for specific ACCase mutations

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Long Dua, Weitang Liua, Guohui Yuana, Wenlei Guoa, Qi Lia, Jinxin Wanga*

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a Key Laboratory of Pesticide Toxicology and Application Technique, College of Plant Protection, Shandong Agricultural University, Shandong Tai’an 271018, PR China *Corresponding author.

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Fax: +86 538 8241114. E-mail address: [email protected]

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Abstract:

American sloughgrass is a troublesome annual grass weed in winter wheat field rotated with

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rice in China. The overreliance on acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides

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has resulted in resistance evolution in this weed. In this study, the cross-resistance patterns to

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fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl, haloxyfop-p-methyl, sethoxydim, clethodim and pinoxaden were established using purified plants individually homozygous for

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specific mutant ACCase alleles. Results indicated that 1781Leu allele endows high-level resistance

to

APPs,

CHDs

and

pinoxaden

while

confers

moderate

resistance

to

haloxyfop-p-methyl. The 2027Cys and 2041Asn alleles endow high-level resistance to APPs and pinoxaden and lower level resistance to CHDs. The 2078Gly allele confers high-level resistance to all herbicides tested in this study, however, moderate resistance to sethoxydim. The 2096Ala very likely endows high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim. In addition, one undefined resistance mechanism was involved in population SD-04. Key words: Beckmannia syzigachne; ACCase gene; cross-resistance; mutation; homozygote

ACCEPTED MANUSCRIPT 1. Introduction Commercial

ACCase-inhibiting

herbicides

are

grouped

into

three

classes:

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aryloxyphenoxypropionates (APPs), cyclohexanediones (CHDs), and phenylpyrazolin class

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herbicide pinoxaden. Repeated use of ACCase-inhibiting herbicides in different cropping systems has selected 46 ACCse-resistant species [1], including American sloughgrass (Beckmannia syzigachne Steud.). The ACCase-inhibiting herbicide resistance is mainly caused by alterations of

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the ACCase enzyme (target-site resistance, TSR). To date, 13 distinct amino acid substitutions

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located in CT domain of the plastidic ACCase gene have been reported: Ile1781Leu [2], Ile1781Val [3], Ile1781Thr [4], Trp1999Cys [5], Trp1999Leu [6], Trp1999Ser [7], Trp2027Cys [8,

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9], Ile2041Asn [10, 11], Ile2041Val [10], Asp2078Gly [8-10, 12], Cys2088Arg [6, 9, 13],

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Gly2096Ala [14] and Gly2096Ser [15]. In contrast, non-target-site resistance (NTSR) is generally

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considered to be an intricate issue that can be due to enhanced metabolic degration of the herbicides. NTSR is relatively difficult to study than TSR, and NTSR is consindered to be an

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destructive threating for weed management [16-21]. For TSR, different ACCase mutations may confer distinct cross-resistance patterns to ACCase-inhibitors [8, 14, 22, 23], and the homo/heterozygous status of the plants for the mutation can also influence the resistance levels [6, 14]. In general, among the common mutant ACCase alleles, 1781Leu, 2078Gly and 2088Arg mutant alleles can confer resistance to APPs, CHDs and pinoxaden [2,24,25]; 2027Cys and 2041Asn alleles can cause resistance to APPs and pinoxaden, but not to CHDs [6, 10, 24]; 2096Ala allele confers resistance mainly to APPs [14]. American sloughgrass is a troublesome weed mainly infesting winter wheat rotated with rice in the Yangtze River delta and southwest region of China [25]. American sloughgrass is an annual or

ACCEPTED MANUSCRIPT shortlived perennial bunchgrass with stout, leafy stems (culms) that are 60 to 100 cm tall. The flowerhead (inflorescence) is a very narrow, upright spike 20 to 30 cm long. It has a double row of

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densely compacted, single-flowered spikelets on one side of the panicle branches [26]. Most

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American sloughgrass are diploid plants (2n=14), while the others are polyploidy [27]. Since 2008, farmers in Jiangsu province of China have observed that fenoxaprop-p-ethyl at recommended field rate failed to control this weed after several years of successful control. Previous studies

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documented that isoforms 1781Leu and 2027Cys conferred highlevel resistance to

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fenoxaprop-p-ethyl in American sloughgrass populations[28, 29]. So far, Ile2041Asn, Asp2078Gly, Gly2096Ala have been observed in American sloughgrass populations. Although related studies

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established that Ile2041Asn, Asp2078Gly, Gly2096Ala mutations resulted in resistance to

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fenoxaprop-p-ethyl, the resistance patterens to other ACCase inhibitors attributed to above

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mentioned mutated alleles have not been described. In addition, the resistance level to fenoxaprop-p-ethyl characterized in previous study was estimated at population level without

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excluding the presence of NTSR. Thus, the objective of this research was to obtain dereived wild-type and homozygous mutant segregating plants sharing homogenized genetic backgrounds and establish more accurate cross-resistance patterns associated with specific ACCase mutations at plant level using homozygous plants. 2. Materials and methods 2.1 Plant materials Four American sloughgrass populations, previously analysed by ACCase molecular analysis, collected in 2011 and 2012 were used to produce nine subpopulations. The plants from each subpopulation were homozygous for specific ACCase mutant allele or wild-type allele.

ACCEPTED MANUSCRIPT Furthurmore, the genetic background of homozygous wild-type (W/W) and homozygous mutant plants (M/M) were minimised as much as possible. One susceptible population SD-12 collected

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from an uncultivated land in Tai’an, Shandong province of China where had no herbicide

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application history was used as control. This methodological approach enables the independent comparison of derived segregating plants versus ACCase-susceptible plants. Seeds from each population were stored at -20 °C for 7 days and then were deposited in Petri

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dishes containing two layers of filter paper soaked with 5 mL distilled water and placed in

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chamber at 25/15 °C (12h day/12h night temperatures), and 75% relative humidity. After germination, seeds were sown in plastic pots and transferred to a glasshouse (temperature

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maintained at approximately 15-25 °C, 75% humidity, and natural sunlight). In order to obtain

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segregating wild-type (W/W) and homozygous mutant (M/M) plants sharing homogenized genetic

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backgrounds from each population, at the 3-leaf stage, the presence of all known mutations at ACCase was investigated in seedings by genotyping. SNPs markers used for genotyping were

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listed in Table 3. One mother plant heterozygous (W/M) for one specific mutant ACCase allele and contained no other known mutant ACCase alleles was used to generate segregating plants. Each plant was isolated within a pollen-proof enclosure. All plants were watered and fertilized as needed. Seeds from per plant were collected as a bulk sample and stored at room temperature for 2 months to enable after-ripening, then germinated and grown to the 3-leaf stage, then genotyped as above described. Ten Wild-type (W/W) and ten homozygous mutant (M/M) ACCase plants from the same bulk sample were selected as mother plants to produce seeds used for subsequent experiments. Each group of 10 plants was cultured as above described. Ripe seeds were harvested as derived segregating subpopulation from each group of 10 plants and labelled as described in

ACCEPTED MANUSCRIPT Table 1. Ten progeny plants were randomly selected from each subpopulation for ACCase sequencing as described by Li et al. [28], and all plants were confirmed to be homozygous for a

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wild-type allele or specific ACCase mutant allele.

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2.2 Herbicides and chemicals

The herbicides used for dose–response tests were as follows: fenoxaprop-p-ethyl (69 g L-1 EW, Bayer CropScience), clodinafop-propargyl (15% WP, Syngenta), fluazifop-p-butyl (15% EC,

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Ishihara Sangyo Kaisha), haloxyfop-p-methyl (108 g L-1 Dow AgroSciences), sethoxydim (12.5%

Pinoxaden (5% EC, Syngenta).

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2.3 Whole-plant dose-response study

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EC, Zhongnong Zhushang Agrochemical), clethodim (240 g L-1 EC, Mindleader Agroscience),

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Nine groups of derived segregating plants, along with control plants were used in a whole-plant

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experiment in a glasshouse to determine cross-resistance to ACCase-inhibitors. Seeds were germinated and sown in 12-cm diameter plastic pots filled with moist loam soils and cultured as

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described above. Seedings were thinned to 5 plants per pot before herbicide treatment. Herbicide treatments were applied to the plants at three or four-leaf stage using a compressed air, moving nozzle cabinet sprayer equipped with one Teejet 9503 EVS flat fan nozzle. The sprayer was calibrated to deliver 450 L ha-1 water at 0.28 MPa. All pots were randomly placed in glasshouse. Herbicide rates used in Whole-plant dose-response were listed in Table 2. At the 21 days after treatment (DAT), the plants were cut at the soil surface and oven-dried for 72 h at 80 oC. Then the dry weight data were recorded. All treatments were replicated three times and the experiments were conducted twice. 2.4 Statistical analysis

ACCEPTED MANUSCRIPT Differences among treatment means were compared using least significant difference (LSD) test at the 5% level of significance. The ANOVA (SPSS Version 20.0, SPSS Inc.) showed no

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significant difference between the two run experiments, and the data were pooled. Dose-response

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assay was obtained by nonlinear regression using four parameters logistic response equation (1) proposed by Seefeldt et al. [30].

(1)

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Where C is the lower limit, D is the upper limit, b is the slope at herbicides dose resulting in 50%

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growth inhibition (GR50) relative to the no treated control. In the regression equation, the herbicides dose was the independent variable (x) (g a.i. ha−1 ) and the growth response (percentage

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of the control) was the dependent variable (y).

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The resistance factor (R/S ratio, Rf) was determined using GR50 value of derived wild-type

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segregating population compared with that of susceptible control population or determined using GR50 value of derived mutant segregating population compared with that of corresponding derived

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wild-type segregating population. 3. Results

3.1 Genotype frequency screen Just as shown in Table 1, populations JS-04 and AH-12 had similar frequencies of wild-type, heterozygous and homozygous plants for mutant 1781Leu (W/W: W/M: M/M = 25: 6.6: 68.4) and 2027Cys (W/W: W/M: M/M = 20.8: 8.3: 70.8), respectively. Two types of ACCase mutations 2041Asn and 2078Gly were detected in population JS-32. The proportion of each genotype was 79.1: 2.8: 11.2: 1.1: 5.6 corresponding to wild-type: heterozygous 2041Asn mutation: homozygous 2041Asn mutation: heterozygous 2078Gly mutation: homozygous 2078Gly mutation.

ACCEPTED MANUSCRIPT In population JS-32, out of 535 genotyping plants, only one individual plant contained both isoform 2041Asn and 2078Gly, moreover, the two mutations were all heterozygous. Heterozygous

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2096Ala plants showed a large proportion (63.4%) in population SD-04, the proportions of

plants of the control population SD-12 were wild-type. 3.2 Sensitivity screen of derived wild-type plants derived

there

plants

were

no

I/I1781-JS-04,

W/W2027-AH-12,

known ACCase

mutations

I/I2041-JS-32

conferring

resistance

and to

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D/D2078-JS-32,

wild-type

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For

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wild-type and homozygous 2096Ala plants were 10.4% and 26.2%, respectively. As expected, all

ACCase-inhibitors in those plants, and the GR50 values were no significantly different with

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susceptible plants SD-12 to fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl,

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haloxyfop-p-methyl, sethoxydim, clethodim, pinoxaden, respectively (Fig 1, Table 4). It was very

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likely that contribution of NTSR was little or no in plants L/L1781-JS-04, C/C2027-AH-12, N/N2041-JS-32, G/G2078-JS-32. As for derived wild-type plants G/G2096-SD-04, resistance to

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fenoxaprop-p-ethyl, clodinafop-propargyl and pinoxaden was observed, and the resistance factors were 63.5, 4.6 and 3.3 respectively (Fig 1 a, b, g, Table 4). The presence of NTSR or unknown mutant ACCase isoforms might be confirmed in population SD-04 and derived segregating plants from population SD-04. 3.3 Cross-resistance patters to ACCase inhibitors Purified plants homozygous for 1781Leu, 2027Cys, 2041Asn, 2078Gly alleles respectively all showed high-level resistance (Rf>10) to APPs fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl and haloxyfop-p-methyl except plants L/L1781-JS-04 which showed a moderate resistance (Rf 5~10) to haloxyfop-p-methyl, with a Rf of 5.4 (Table 5).

ACCEPTED MANUSCRIPT Plants L/L1781-JS-04 had high-level resistance to sethoxydim and clethodim, with Rf of 14.2 and 14.0, respectively (Table 5).Derived resistant plants C/C2027-AH-12 and N/N2041-JS-32

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showed lower cross resistance (Rf 2~5) to sethoxydim and clethodim. Plants G/G2078-JS-32 had

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moderate resistance to sethoxydim and high-level resistance to clethodim.

Plants L/L1781-JS-04, C/C2027-AH-12, N/N2041-JS-32 and G/G2078-JS-32 were considered to be uniformly high resistant to pinoxaden (Table 4, 5), a newly marketed ACCase-inhibiting

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herbicide in China.

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Purified plants A/A2096-SD-04 conferred high resistance to APPs fenoxaprop-p-ethyl, clodinafop-propargyl, fluazifop-p-butyl and haloxyfop-p-methyl, but moderate resistance to

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sethoxydim, clethodim and pinoxaden (Compared with control SD-12).

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4. Discussion

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4.1 Sensitivity screen of derived wild-type plants In this study, five derived wild-type segregating subpopulations were obtained from original

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populations. The sensitivity of derived wild-type plants was estimated, and the results showed that there was no significant difference compared with control plants except derived wild-type G/G2096-SD-04 plants. As a result, it indicated that the contribution of NTSR was little or negligible in derived homozygous mutant ACCase plants L/L1781-JS-04, W/W2027-AH-12, I/I2041-JS-32, D/D2078-JS-32 and original populations JS-04, AH-12, JS-32. It is interesting that the resistance to fenoxaprop-p-ethyl, clodinafop-propargyl and pinoxaden observed in derived wild-type plants G/G2096-SD-04 which did not possess any known ACCase mutations. This may attributed to NTSR or novel SNPs. Study on the potential resistant mechanism for herbicide resistance in population G/G2096-SD-04 is currently on the way in authors’ laboratory.

ACCEPTED MANUSCRIPT 4.2 Cross-resistance patterns to ACCase-inhibitors endowed by homozygous mutant ACCase alleles

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Different mutant ACCase isoforms may cause different spectrum of resistance. This study

alleles

(1781Leu,

2027Cys,

2078Gly,

2096Ala,

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illustrated the cross-resistance patterns associated with five different homozygous mutant ACCase respectively)

to

three

families

of

ACCase-inhibitors in American sloughgrass at plant level.

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The cross-resistance patterns attributed to TSR had been documented in previous studies. In

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general, the 1781Leu mutation can confer high-level resistance to almost all three classes of ACCase-inhibitors in Alopecurus myosuroides Huds. [34], Avena fatua L.[15], Lolium rigidum

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Gaudin.[35]. Crystal structure analysis, using yeast ACCase, showed that 2027Cys and 2041Asn

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were located near the first aryl ring of haloxyfop, a unique feature of APPs [36, 37]. As a result,

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the 2027Cys and 2041Asn ACCase are correlated with resistance to APPs but not CHDs. This theory is supported by bioassay in A. myosuroides [8, 10, 24], Phalaris paradoxa [38], L. rigidum

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[9, 10] and Avena sterilis [22], A. fatua [15, 39, 40]. Previous reports indicated that isoform 2027Cys could confer high-level resistance to pinoxaden [24], and isoform 2041Asn could confer moderate or no resistance to pinoxaden [6, 37, 39]. High-level resistance to APPs, CHDs and pinoxaden endowed by 2078Gly has been reported in A. myosuroides [24], P. paradoxa [38], A.fatua [15, 29, 39], L. rigidum [11]. The 2096Ala mutation can confer resistance to APPs and pinoxaden, but generally not to CHDs [6, 8, 23, 24, 40]. In this study, whole-plant bioassays showed that homozygous 1781Leu ACCase presented a similar resistance spectrum compared with previous studies. The 1781Leu can confer high-level resistance to all herbicides used except haloxyfop-p-methyl (moderate resistance). Crystal

ACCEPTED MANUSCRIPT structures of the CT domain of yeast ACCase in complex with ACCase-inhibitors shows that the 1781Leu mutation is located in a binding pocket that is occupied by a methyl or ethyl group in all

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three groups of ACCase-inhibitors, as a result, the 1781Leu mutation can conferred

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cross-resistance to all three classes of herbicides [34]. So far, isoform 1781Leu has been known as the most common mutant resistant ACCase isoform [29], and isoform 1781Leu usually causes high-level resistance to all three groups of ACCase-inhibitors. The pervasive resistance to

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ACCase-inhibitors endowed by 1781Leu mutation may explain the prevalence of this state.

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In this paper, the mutations 2027Cys and 2041Asn conferred resistance to APPs and pinoxaden, and this result was accorded those previous reports mentioned above. It is interesting that the

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cross-resistance patterns for CHDs endowing by 2027Cys and 2041Asn were different from the

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previous studies. In this study, homozygous 2027Cys and 2041Asn mutations both exhibited lower

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level resistance to CHDs. This phenomenon may due to the sensitivity reduction of ACCase to CHDs resulted from homozygous 2027Cys and 2041Asn mutations, and this assumption should

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be confirmed by further analysis of ACCase enzyme activity. For mutation 2078Gly, compared with previous studies, the similar cross-resistance pattern was observed in American sloughgrass. However, this study found that the 2078Gly allele confers moderate resistance to sethoxydim rather than high-level resistance outlined by previous reports. The different cross-resistance pattern maybe attributed to specific weed species and/or other factors such as assumption discussed earlier in regard to the 2027Cys and 2041Asn mutations. Although CHDs and pinoxaden can be able to control some specific APPs-resistant weeds, they may be not as sustainable ACCase resistance management tool in ACCase-based resistance American sloughgrass. This finding will be helpful for further herbicides resistance investigation in weeds.

ACCEPTED MANUSCRIPT Resistance to fenoxaprop-p-ethyl, clodinafop-propargyl and pinoxaden was confirmed in derive wild-type plants G/G2096-SD-04. Because there was no known resistance-endowing ACCase

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mutation in those plants, we speculated this might result from NTSR and/or unknown mutant

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ACCase isoforms. It is reasonable to believe that the same unknown resistance mechanism might exist in original population SD-04 and derived homozygous mutant ACCase plants A/A2096-SD-04, and this inconclusive resistance mechanism may confer resistance to

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fenoxaprop-p-ethyl, clodinafop-propargyl and pinoxaden in American sloughgrass. In spite of this,

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it was very likely to confirm that homozygous 2096Ala ACCase could confer high-level resistance to fluazifop-p-butyl, haloxyfop-p-methyl and moderate resistance to sethoxydim because derived

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wild-type population G/G2096-SD-04 was susceptible to those herbicides. The level of resistance

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to fenoxaprop-p-ethyl, clodinafop-propargyl, clethodim and pinoxaden endowed by homozygous

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2096Ala ACCase can not be determined exactly because the presence of inconclusive resistance mechanism can also lead to resistance to those herbicides in plants G/G2096-SD-04.

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In conclusion, cross-resistance to ACCase inhibitors was characterized in American sloughgrass by using homozygous 1781Leu, 2027Cys, 2041Asn, 2078Gly and 2096Ala ACCase mutation plants, respectively. In addition, one unknown mechanism responsible for ACCase-inhibitors resistance was observed in population SD-04. This unknown mechanism may be challenging the management of American sloughgrass escape.

Acknowledgements This research was supported by the National Natural Science Foundation of China (31471787, 31171866) and the Special Fund for Agroscientific Research in the Public Interest (201303031).

ACCEPTED MANUSCRIPT The authors thank all the workers for assistance in conducting this research. Reference

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Beckmannia syzigachne, M.A. Thesis, Shanghai, China, Shanghai Jiaotong Univ. (2008)

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[27] Darris, D., A. Bartow, and R. Wynia. 2004. Plant fact sheet for American sloughgrass

Corvallis, OR.

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(Beckmannia syzigachne). USDA-Natural Resources Conservation Service, Plant Materials Center,

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[28] L. Li, Y. Bi, W. Liu, G. Yuan, J. Wang, Molecular basis for resistance to fenoxaprop-p-ethyl in American sloughgrass (Beckmannia syzigachne Steud.), Pestic. Biochem. Physiol. 105 (2013) 118–121.

[29] Q. Yu, M.S. Ahmad-Hamdani, H. Han, M.J. Christoffers, S.B. Powles, Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat ( Avena fatua): insights into resistance evolution in a hexaploid species, Heredity 110 (2013) 220–231. [30] S.S. Seefeld, J.E. Jensen, E.P. Fuerst, Log-logistic analysis of herbicides dose response relationships, Weed Technol. 9 (1995) 218–227. [31] L. Li, L. Du, W. Liu, G. Yuan, J. Wang, Target-site mechanism of ACCase-inhibitors

ACCEPTED MANUSCRIPT resistance in American sloughgrass (Beckmannia syzigachne Steud.) from China, Pestic. Biochem. Physiol. 110 (2014) 57–62.

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[32] L. Li, Resistance of American sloughgrass(Beckmannia syzigachne Steud.) to

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fenoxaprop-p-ethyl, M.A. Thesis, Tai’an, China, Shandong Agric. Univ. (2014) [33] W. Guo, G. Yuan, W Liu, Y. Bi, L Du, C. Zhang, Q. Li, J. Wang, Multiple resistance to ACCase and AHAS-inhibiting herbicides in shortawn foxtail (Alopecurus aequalis Sobol.) from

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China, Pestic. Biochem. Physiol, doi:10.1016/j.pestbp.2015.04.006.

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[34] A.C. Brown, S.R. Moss, Z.A. Wlilson, L.M. Field, An isoleucine to leucine substitution in the ACCase of Alopecurus myosuroides (black-grass) is associated with resistance to the herbicide

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sethoxydim, Pestic. Biochem. Physiol. 72 (2002) 160–168.

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[35] A. Tal, B. Rubin, Molecular characterization and inheritance of resistance to

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ACCase-inhibiting herbicides in Lolium rigidum, Pest Manag. Sci. 60 (2004)1013–1018 [36] L.P.C. Yu, Y.S. Kim, L. Tong, Mechanism for the inhibition of the carboxyltransferase

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domain of acetyl-coenzyme A carboxylase by pinoxaden, Proc. Natl. Acad. Sci. USA.107 (2010) 22072–22077.

[37] H. Zhang, B. Tweel, L. Tong, Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfopand diclofop, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 5910 – 5915. [38] O. Hochberg, M. Sibony, B. Rubin, The response of ACCase-resistant Phalaris paradoxa populations involves two different target site mutations, Weed Res. 49 (2009) 37 –46. [39] H. Cruz-Hipolito, M.D. Osuna, J.A. Dominguez-Valenzuela, N. Espinoza, R.D. Prado, Mechanism of resistance to ACCase-inhibiting herbicides in wild oat ( Avena fatua) from Latin

ACCEPTED MANUSCRIPT America, Agric. Food Chem.59 (2011) 7261–7267.

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[40] H.J. Beckie, F.J. Tardif, Herbicide cross resistance in weeds, Crop Prot. 35 (2012) 15–28.

ACCEPTED MANUSCRIPT Table 1 American sloughgrass populations used to produce the segregating plants.

2011

mutational pattern

Jiangsu Danyang

Ile1781Leu

W/Wa

W/Mb

M/Mc

Total

25

6.6

68.4

228

2012

Anhui Lujiang

Trp2027Cys

20.8

JS-32

2012

Jiangsu Jintan

Ile2041Asn

79.1

Asp2078Gly

79.1

MA

Shandong Yutai

a

216

W/W2027-AH-12e C/C2027-AH-12f

2.8

11.2

535

I/I2041-JS-32e N/N2041-JS-32f

1.1

5.6

0

0.19

0

Gly2096Ala

10.4

63.4

26.2

D

2011

L/L1781-JS-04f

70.8

Ile2041Asn/ Asp2078Glyd

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SD-04

I/I1781-JS-04e

8.3

NU

AH-12

Derived segregating plants

T

JS-04

Location

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Year

SC R

Populations

Plant frequency（%）of each genotype

D/D2078-JS-32e G/G2078-JS-32f

164

G/G2096-SD-04e A/A2096-SD-04f

W/W refers to wild-type ACCase plants. W/M refers to heterozygous mutant ACCase plants. c M/M refers to homozygous mutant ACCase plants. d Plant containing heterozygous 2041Asn ACCase and heterozygous 2078Gly ACCase. e Derived wide-tpye segregating plants. f Derived segregating plants homozygous for specific ACCase mutation.

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b

ACCEPTED MANUSCRIPT Table 2 Herbicide treatments applied for dose-response tests. Dosage (g a.i. ha-1) Herbicides

Derived wild-type plants

Fenoxapropp-ethyl

62.1, 186.3, 558.9, 1676.7, 5030.1, 15090.3

2.3, 6.9, 20.7, 62.1, 186.3, 558.9, 1676.7, 5030.1

1.9, 3.9, 7.8, 15.5, 31.05, 62.1a

Clodinafoppropargyl

67.5, 135, 270, 540, 1080, 2160

2.5, 7.5, 22.5, 67.5, 135, 270

2.1, 4.2, 8.4, 16.9, 33.8, 67.5

fluazifop-pbutyl

135, 405, 1215, 3645, 10935, 32805

5, 15, 45, 135, 405, 1215

1.67, 5, 15, 45, 135, 405

Haloxyfop-pmethyl

6.3, 18.9, 56.7, 170.1, 510.3, 1530.9, 4592.7, 13778.1

Sethoxydim

6.9, 20.8, 62.5, 187.5, 562.5, 1687.5, 5062.5

Clethodim

10, 30, 90, 270, 810, 2430

Pinoxaden

5.6, 16.7, 50, 150, 450, 1350

IP

SC R

NU

0.7, 2.1, 6.3, 18.9, 56.7, 170.1

2.3, 6.9, 20.8, 62.5, 187.5, 562.5

2.3, 6.9, 20.8, 62.5, 187.5, 562.5

1.1, 3.3, 10, 30, 90, 270

1.1, 3.3, 10, 30, 90, 270

0.62, 1.86, 5.6, 16.7, 50, 150

0.62, 1.86, 16.7, 50, 150

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D

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2.1, 6.3, 18.9, 56.7, 170.1, 510.3

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The recommend field rate is indicated by an underscore.

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a

Control

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Derived resistant plants

5.6,

ACCEPTED MANUSCRIPT

Primers

Sequence (5’-3’)

F-2027 R-2027

CGCGAAGGATTGCCTCTGTTCATCCTTGCTAACTA AATTCTGGATCAAGCCTACC

F-2041 R-2041

AAATCTTGCTTCCGTGTTGG TCTTTGAGTTCCTCTGACCTG

F-2078 R-2078

CAPS(dCAPS)patterns (fragment sizes, bp) Wide type

Mutant type

References

62

EcoT22 I

120, 35

165

Yu et al. [29]

Trp2027 TGg

56

Mae I

294, 35

329

Li et al. [31]

Ile2041 AtT

55

ECoR I

228, 974

1202

Li [32]

ATTGCCTCTGCTCATCCTTGCTAACTGG CATAGCACTCAATGCGATCTGGGTTTATCTTGATA

Asp2078 GaT

60

EcoRⅤ

181, 35

216

Guo [33]

F-2096

GAGGGGCTTGGGTTGTGATT

R-2096

CCCTCCAGGCAACAAAAGCA

Ala2096 GcC

65

HaeIII

561

486, 75

Li [32]

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Primer name starting with F, forward primer; primer name stating with R, reverse primer. Mismatched base is underlined in dCAPS primer. c Mutant nucleotide of the target codon is indicated in lowercase. b

Restriction enzyme

Ile1781 aTA

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CTGACTGACGAAGACCATGATCG AGAATACGCACTGGCAATAGCAGCACTTCCATGCA

Tm used in PCR

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F-1781 R-1781

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a

Target codonc

b

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a

CR

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Table 3 CAPS( or dCAPS) makers used for genotyping.

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Table 4 Estimated GR50 values for herbicides for derived segregating plants

18.3±0.82 694.2±171.3 19.3±4.10 1587.3±378.2 21.3±2.4 1332.0±293.2 17.8±1.4 2357.8±540.7 649.1±64.0 991.4±94.8 10.2±1.9

10.0±0.94 224.6±27.0 12.8±0.133 193.2±23.3 12.6±2.9 626.6±137.2 13.4±2.7 191.2±10.8 41.6±14.4 229.6±20.7 9.13±0.72

21.4±0.51 1505.2±99.9 27.3±6.89 708.8±46.2 26.7±2.2 1529.4±46.8 28.8±0.13 606.4±22.0 40.3±6.3 1737.2±153.0 22.6±0.85

Haloxyfop-pmethyl

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Fluazifop-p-butyl

10.0±2.22 54.1±18.6 5.92±1.13 63.4±7.2 8.9±0.3 1151.4±188.5 10.2±2.6 123.0±27.6 6.6±0.47 160.6±35.2 6.20±0.26

Sethoxydim

Clethodim

Pinoxaden

29.6±5.45 420.3±76.3 50.9±8.9 146.2±28.8 23.8±1.2 65.2±10.3 26.5±2.1 208.3±13.7 29.1±4.9 181.4±40.2 30.6±0.44

11.36±3.42 159.4±29.7 16.7±0.6 61.9±3.3 13.6±2.0 41.6±26.1 11.1±2.0 142.1±54.8 29.04±4.9 90.1±10.0 16.6±0.81

6.67±0.21 121.0±18.9 5.94±0.33 62.3±4.7 6.19±1.17 94.2±7.2 7.5±0.47 96.0±18.2 21.9±1.1 65.0±2.5 6.68±0.16

GR50 refers to the herbicide ratio required to decrease plant fresh weight by 50% compared to the untreated control. Each value represents the mean ± standard error.

AC

a

Clodinafoppropargyl

CE P

I/I1781-JS-04 L/L1781-JS-04 W/W2027-AH-12 C/C2027-AH-12 I/I2041-JS-32 N/N2041-JS-32 D/D2078-JS-32 G/G2078-JS-32 G/G2096-SD-04 A/A2096-SD-04 Susceptible SD-12

Fenoxaprop-p-ethyl

US

Population

CR

GR50(g a.i. ha-1)a

T

ACCEPTED MANUSCRIPT

W/W2027-AH-12 C/C2027-AH-12

1.89 155.4

I/I2041-JS-32 N/N2041-JS-32

2.09 130.4

D/D2078-JS-32 G/G2078-JS-32

1.7 230.8

G/G2096-SD-04

63.5

A/A2096-SD-04

97.0

a

82.2

1.41 21.2

62.5

1.4 68.7

132.5

1.5 21.0 4.6

1.5

25.1

Rfa

22.5

0.95 66.6

15.1

1.21 31.4

49.7

1.18 67.7

14.3

Rfb

1.3 26.8

Rfa

70.3

76.9

1.62 8.7

26.0

0.96 10.2

57.3

1.44 185.8

21.0

1.6 19.8

1.8

5.4

CR

37.9

1.10 24.6

Rfb

Haloxyfop-p-methyl

US

1.79 63.5

Rfa

MA N

I/I1781-JS-04 L/L1781-JS-04

Rfb

Fluazifop-p-butyl

TE D

Rfa

Clodinafop-propargyl

CE P

Fenoxaprop-p-ethyl

AC

Population

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Table 5 Estimated resistance factors for hebicides for derived segregating plants.

Rfb

Rfa

5.4

0.97 13.7

10.7

1.67 4.8

129.4

0.78 2.1

12.0

0.86 6.8

1.06 42.7

25.9

Sethoxydim Rfb

Rfa

14.2

0.682 9.6

2.8

1.0 3.7

2.6

0.82 2.5

7.9

0.70 8.5

0.95 24.4

5.9

Clethodim Rfb

Rfa

Rfb

14.0

1.00 18.1

18.1

3.7

0.89 9.3

10.5

3.0

0.93 14.1

15.2

12.8

1.1 15.5

12.8

1.7 6.2

5.4

Pinoxaden

3.3 3.2

9.7

2.9

Rf refers to resistance factor and was calculated using GR50 value of the derived segregating plants compared with that of the susceptible control plants. Rf was calculated using GR50 value of the derived segregating resistant plants compared with that of corresponding derived wild-type segregating plants, respectively. b

ACCEPTED MANUSCRIPT

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Fig.1. Dose-response curve for above ground dry weights of derived plants treated with increasing rates of seven ACCase inhibitors. Each point represents the mean of two experiments, each containing three replicates. Dry weight is expressed as a percentage of the untreated check. Error bars represent the ± standard error based on P = 0.05.

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ACCEPTED MANUSCRIPT

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Graphical asbtract

ACCEPTED MANUSCRIPT Highlights



Obtain wild-type (W/W) and homozygous mutant plants (M/M) from one heterozygous

IP

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mother pant (W/M).

The cross-resistance patterns to ACCase-inhibitors were established using purified plants.



One undefined resistance mechanism was involved in population SD-04.

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