Plant material

Thirty-one field populations were collected from winter wheat fields rotated with rice in Anhui, Jiangsu and Shandong provinces from June 2011 to June 2012 and are coded as A××, J×× and S××, respectively. All the fields have been sprayed by fenoxaprop-p-ethyl in the last 5 yr. A known susceptible population, TS was collected from the Mountain Taishan scenic spot, Taishan District, Shandong Province. Seeds from at least 20 mature plants were randomly collected by hand and bulked. The seeds were air-dried and stored in paper bags at room temperature until use. The collecting position is shown in Table 1.

Table 1: Information on the collection location and herbicide history. 

Fenoxaprop- p -ethyl single-rate test

The experiment was conducted from September 2013 to December 2013. The herbicide sensitivity assessment procedure was the same for every sample. Seeds were germinated and planted in plastic pots (d = 12 cm) as previously described (^{Li et al., 2014}). Next, the seedlings were transferred to a greenhouse (temperature was maintained at approximately 15 to 25 °C with 75% RH and natural sunlight) and watered as needed. The plants were thinned to 10 evenly sized plants per pot at the two-leaf stage. The plants were sprayed with 62.1 g ai ha^-1 (1×) fenoxaprop-p-ethyl (69 g L^-1 EW, Bayer, Hangzhou, China) at the three- to four-leaved-stage using a compressed air, moving-nozzle cabinet sprayer equipped with one flat fan nozzle (9503EVS, Teejet Technologies, Springfield, Illinois, USA) and calibrated to deliver 450 L ha^-1 at 0.28 MPa. The plants treated with water were selected as a control. Every treatment contained one pot and all treatments were replicated three times. All plants were returned to the greenhouse after the herbicide treatment. Plant survival was assessed visually 21 d after the herbicide was applied. Severely injured plants were classified as sensitive, while surviving plants that expanded new green leaves were classified as resistant.

Fenoxaprop- p -ethyl dose-response experiment

To determine the level of resistance in resistant populations, dose-response experiments were performed using 11 populations from December 2013 to May 2014. The plant and spray procedure were as described above except each pot only contained five seedlings. The rates of herbicide application are described in Table 2. The plants were cut at the soil surface, and the fresh weight were recorded 21 d after treatment (DAT) after the plant survival was assessed. The treatments were replicated three times, and the experiments were performed twice.

Table 2: Herbicide dose used to test the resistance index. 

Note: The recommended dose of fenoxaprop-p-ethyl for use in fields is 62.1 g ai ha^-1.

Statistics

All regression analyses were performed using Sigma Plot 12.0 (Systat Software, Chicago, Illinois, USA). Fresh-weight data were subjected to a nonlinear regression analysis using the log-logistic equation (^{Seefeldt et al., 1995}):

where C is the lower limit, D is the upper limit, b is the relative slope around the herbicide dose that yields 50% growth inhibition (GR₅₀ ), x is the herbicide rate, and y is the growth response (percentage of the untreated control). The significances of the regression parameters were determined using the t-test method (P = 0.05). The fitted equations were used to estimate the GR₅₀ value. The resistance index (RI) was calculated as the GR₅₀ of the resistant population divided by the GR₅₀ of the susceptible population to indicate the level of resistance for the resistant population. Data sets from repeated experiments were analyzed by ANOVA (IBM SPSS Statistics for Windows, Version 20.0; IBM Corp., Armonk, New York, USA). The data were pooled for subsequent analyses as the variance between repeated experiments was nonsignificant. Similarly, the median lethal concentration (LC₅₀) was attained according to the plant survival rate.

Identification of resistant amino acid substitution

The seeds were germinated and planted as described above except each pot only included one seedling. The pots were placed in the same greenhouse and watered as needed. The seedlings were managed as described above. Approximately 50 mg shoot tissue from each individual plant at the three- to four-leaf stage was cut and immediately frozen in liquid nitrogen for further analyses. The total DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method (^{Doyle and Doyle, 1990}). The forward primer 5'--TTTCCCAGCGGCAGACAGAT-3', and reverse primer 5'--TCCCTGGAGTCTTGCTTTCA-3' were used to amplify a 1437-bp fragment that coded 479 amino acids containing all the seven known substitutions that confer resistance to ACCase-inhibiting herbicides (^{Bi et al., 2015}). The PCR experiments were performed in a final volume of 25 μL, containing 1 μL genomic DNA (approximately 30 ng), 1 μL each primer (10 μM), 2.5 μL 10× Trans EasyTaq buffer (Mg2+ Plus, TransGen Biotech, Beijing, China), 2 μL deoxynucleotide triphosphate mixture (2.5 mM, TransGen Biotech), 0.25 μL Trans EasyTaq DNA polymerase (2.5 units), and 17.25 μL distilled deionized H₂O. The PCR experiments were performed using a thermal cycler (T100, Bio-Rad Laboratories, Hercules, California, USA) programmed for an initial denaturation step of 94 °C for 5 min followed by 35 cycles of 50 s at 94 °C, 50 s at 58 °C, and 100 s at 72 °C. A final extension cycle for 10 min at 72 °C was also included.

The PCR products were visualized by electrophoresis on 1.0% agarose gel running in 1× Tris-acetate-ethylenediaminetetraacetic acid buffer. The intended bands were extracted from 1.0% agarose gel using the EasyPure Quick Gel Extraction kit (TransGen Biotech) and then cloned using pEASY-T1 vector (TransGen Biotech). The recombinant plasmids were introduced into competent Escherichia coli (Trans1-T1 Phage Resistant Chemically Competent Cell, TransGen Biotech) in accordance with the manufacturer's instructions. The positive clones were sequenced on an ABI PRISM 3730 DNA sequencer (Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China). In total, 10 individual plants from each population were sequenced. At least five clones for each biological replicate were sequenced. The sequence data for the resistant and susceptible populations were compared to determine whether a nucleotide substitution was associated with resistance. Sequence data for each population were aligned and compared using DNAMAN version 5.2.2 software (Lynnon Biosoft, Quebec, Canada).

RESULTS AND DISCUSSION

Fenoxaprop- p -ethyl single-rate test

For the single-rate test, 21 populations were severely injured, and no new leaves expanded after treatment with fenoxaprop-p-ethyl at the recommended dose (62.1 g ai ha^-1) compared with the untreated control. Certain populations, such as TS and A02, were completely killed. Eight of nine populations from Anhui province were efficiently controlled by 1× fenoxaprop-p-ethyl, and the number from Jiangsu province and Shandong province were 7/15 and 6/7 respectively. Fenoxaprop-p-ethyl did not efficiently control 10 populations (A01, J01, J02, J03, J05, J09, J11, J13, J14 and S03) at the same dose, even completely failed to control several populations, such as J11, J13, J14 and S03.

Fenoxaprop- p -ethyl dose-response experiments

The two whole-plant experiments did not show different results. Therefore, data were combined for the subsequent analyses. In the dose-response experiment, all field populations tested showed high-level resistance to fenoxaprop-p-ethyl. The LC₅₀ value for J03 was 91.28 g ai ha^-1, which was the lowest in all resistant populations, but still was much higher than the recommended rate 62.1 g ai ha^-1. The GR₅₀ values varied from 91.55 to 2798.87 g ai ha^-1 with RI values from 14.6 to 445.0, which were compared to the susceptible TS population (Table 3).

Table 3: The level of resistance to fenoxaprop-p-ethyl in 10 Beckmannia syzigachne populations. 

GR₅₀: Herbicide dose required to decrease plant fresh weight by 50% compared with the untreated control. Each value represents the mean ± standard error (SE).

RI: Resistance index calculated by dividing the GR₅₀ value of the resistant population by the susceptible population.

Fenoxaprop-p-ethyl is one of the main herbicides that have been used in wheat fields to selectively control grass weed in China since the 1990s. Widespread and continuous use of fenoxaprop-p-ethyl has produced resistant weeds. Beckmannia syzigachne field populations resistant to fenoxaprop-p-ethyl were first identified in 2008 in Danyang county, Jiangsu province (^{Liu and Zhang, 2008}). Next, B. syzigachne field populations that are resistant to fenoxaprop-p-ethyl were successively identified in Jurong county Jiangsu province and Lujiang county Anhui province (^{Lv et al., 2012}; ^{Li et al., 2014}). In this research, 31 B. syzigachne field populations were treated with fenoxaprop-p-ethyl to investigate their resistance to fenoxaprop-p-ethyl. The results show that 10 of the 31 field populations have evolved a high level of resistance to fenoxaprop-p-ethyl (Table 3). Eight of the 10 resistant populations were collected in Jiangsu province. The data indicates that agriculture in the Jiangsu province has been more severely disturbed by fenoxaprop-p-ethyl resistant B. syzigachne.

Identification of resistant amino acid substitution

A portion of the CT gene was amplified and sequenced to compare the sequence and determine the resistance mechanisms (the gene information is deposited in National Center for Biotechnology Information (NCBI( with the accession number KT291176-KT291186). The sequence comparison showed several key amino acid substitutions in the CT region of the resistant populations (Table 4). Ile-1781-Leu, Ile-1781-Val, Ile-2041-Asn, Asp-2078-Gly and Gly-2096-Arg substitutions were identified in the J09, J11, J13, J14 and S03 populations, respectively. The key substitution is the target-site-resistant mechanism to fenoxaprop-p-ethyl in the corresponding populations.

Table 4: Key amino acids in the carboxyltransferase domain of fenoxaprop-p-ethyl-susceptible Alopecurus myosuroides and fenoxaprop-p-ethyl-resistant and -susceptible Beckmannia syzigachne. 

Note: The known resistance amino acid substitution is in bold for the resistant populations.

Moreover, no known substitutions conferring resistance to ACCase-inhibiting herbicides were identified in the A01, J01, J02, J03 and J04 populations. Resistance to fenoxaprop-p-ethyl in these populations is highly suspected to be conferred by non-target-site resistance mechanism.

To date, 12 substitutions at seven positions in the CT domain have been identified as the basis for resistance to ACCase-inhibiting herbicides in grass weed (^{Beckie and Tardif, 2012}; ^{Kaundun et al., 2013 b}). Five substitutions at four positions (Table 4) have been identified in this research, which contain the less reported Ile-1781-Val and Gly-2906-Ala. The Trp-2027-Cys substitution was also identified in a fenoxaprop-p-ethyl-resistant B. syzigachne population in our previous research (^{Li et al., 2014}). These results indicate that B. syzigachne exhibits rich diversity target-site resistance mechanisms for fenoxaprop-p-ethyl resistance even though it is a diploid, self-pollinated grass weed. Due to the different cross-resistance patterns associated with different ACCase substitutions, this diversity will make it more difficult to control fenoxaprop-p-ethyl resistant B. syzigachne.

Other mechanisms except the resistant substitution are also likely involved in fenoxaprop-p-ethyl-resistant B. syzigachne field populations. Currently, little is known about other resistant mechanisms except the insensitive target enzyme in B. syzigachne, especially the NTSR mechanism. Compared to the single-gene encoded TSR mechanism, NTSR mechanism likely encoded by multiple genes has unpredictable cross-resistance patterns and more complex genetic patterns (^{Délye et al., 2011}; ²⁰¹³).