Greenhouse and field evaluation of a novel HPPD-inhibiting herbicide, QYM201, for weed control in wheat

QYM201, 1-(2-chloro-3-(3-cyclopropyl-5-hydroxy-1-methyl-1H-pyrazole-4-carbonyl)-6-(trifluoromethyl)phenyl)piperidin-2-one), is a newly developed HPPD- (4-hydroxyphenylpyruvate dioxygenase; EC 1.13.11.27) inhibiting herbicide for weed control. Experiments were carried out to determine the effect of QYM201 on weeds and its safety for wheat in the glasshouse and field. The results indicated that at doses of 90 and 135 g active ingredient (a.i.) ha−1 QYM201 was highly effective against both grass and broadleaf weeds, such as Alopecurus aequalis Sobol., Alopecurus japonicus Steud, and Capsella bursa-pastoris Medic. In a wheat hybrid tolerance experiment, QYM201 showed a high level of safety for most of the 17 tested wheat hybrids, and the SI values reached ≥5.7 in the selectivity index study. To determine application rules for QYM201, field experiments were conducted in 2016 and 2017. During this time, 90 to 270 g a.i. ha−1 post-emergence herbicide application (POST) was sufficient to supply satisfactory all-season control of Alopecurus aequalis Sobol., Descurainia sophia [L.] Schur., and Malachium aquaticum (L.) Fires. No damage to wheat plants was observed. In order to increase wheat yield and deliver effective weed control, a dosage of 90 to 180 g a.i. ha−1 is suggested. In conclusion, the herbicide QYM201 is safe to use in wheat fields to control winter weeds.

In order to determine the spectrum of weed control, the safety to different wheat hybrids, and the selectivity of QYM201 among 3 commonly planted wheat hybrids and 4 common weeds, experiments were carried out in the greenhouse. In addition, field experiments were conducted to determine the effect of QYM201 on weed control in winter wheat fields with different rates of 6% QYM201 oil dispersion (OD) during the 2015-2016 and 2016-2017 growing seasons in Shandong province.

Results
Greenhouse experiments. Effectiveness of weed control. At all rates of application, QYM201 was effective on many of the tested weed species including grass weeds and broadleaf weeds. Treated weeds exhibited symptoms of bleach injury at 5 days after treatment (DAT), eventually undergoing necrosis and death at 20 DAT. At the dosage of 90 g a.i. ha −1 , QYM201 was highly effective against 3 of the treated weeds, and dry weight inhibition of A. aequalis, M. aquaticum, and D. sophia. were up to 93%, 91%, and 92%, respectively. Meanwhile dry weight inhibition of A. japonicus, and L. arvense were 86% and 87%, respectively. Weed injury increased according to application rate -higher rates leading to greater injury. At a dosage of 135 g a.i. ha −1 , 8 of the treated weeds were controlled by QYM201, with dry weight reductions ranging from 91% to 95% these weeds included A. aequalis, A. japonicus, C. bursa-pastoris, M. aquaticum, V. didyma, P. kengiana, L. arvense, and D. sophia (Table 1). However, even at high doses some weed species, such as L. multiflorum, B. japonicus, A. squarrosa, and E. helioscopia, showed only slight sensitivity to QYM201 with dry weight reductions of 21%, 23%, 20%, and 19%, respectively.
Wheat hybrid tolerance. QYM201 was safe for most of the treated wheat in the greenhouse experiment. When treated at 360 g a.i. ha −1 , most of the tested wheat hybrids were tolerant to QYM201 with reductions of <7% and herbicide damage <20% (Table 2). However, Huamai 5, Yangfumai 4, and Yangmai 158 were sensitive to QYM201, showing dry weight reductions of 13%, 12%, and 17%, respectively. The damage caused by QYM201 to these 3 hybrids ranging from 12% to 17% (Table 2). Wheat hybrids Zhengmai 10, Hengguan 35, Haomai 1, and Xinong 979 became sensitive to QYM201 with dry weights inhibited by beyond 10%, while crop injury caused by the herbicide was up to 28% at a dose of 540 g a.i. ha −1 (Table 2) Table 3). The high GR 50 values clearly indicated that QYM201 was safe for the 3 tested wheat hybrids and that the 4 weed species were also effectively controlled (Table 3). In addition, experimental results showed that A. aequalis and M. aquaticum   Table 5). The grain yield of wheat increased with increasing rates of QYM201 from 90 to 270 g a.i. ha −1 (Table 5). Specifically, in the field experiments of 2017, a 19% yield increase was achieved by increasing the rate from 0 to 270 g a.i. ha −1 . The yield in hand-weeded plots could be reduced by about 20% in two years, and none of the herbicide treatments led to superior grain yields compared with hand-weeded plots ( Table 5).

Discussion
The chemical structure of the herbicide QYM201 ( Fig. 1), is similar to that of topramezone, which is a typical HPPD-inhibitor 22 . Furthermore, weeds or crops that have been treated with QYM201 present with typical HPPD inhibitor injury characteristics. Thus QYM201 is suggested to be a novel member of the group of chemicals that inhibits 4-hydroxyphenylpyruvate dioxygenase 15 . The greenhouse bioassay results indicated that the weed control spectrum of QYM201 was broader than that of most commonly used herbicides in wheat fields; at all rates of application, QYM201 was able to control both grass weeds and broadleaf weeds. Post emergence applications of herbicides such as fluroxypyr and tribenuron-methyl are highly efficient against a large number of broadleaf weeds but provide only limited control for grass weeds 23,24 . Whereas fenoxaprop-P-ethyl and mesosulfuron-methyl are sufficiently effective against many grass weeds, they are not effective for broadleaf weeds 25 . The ability of QYM201 to control both grass and broadleaf weeds may make it a preferred choice over any other ordinary herbicides for weed control in wheat fields. Importantly, we found that QYM201 was highly effective in its control of A. aequalis and A. japonicus, which are the most harmful weeds to wheat yield worldwide. The widespread application of herbicides has led to the rapid evolution of A. aequalis and A. japonicus herbicide tolerance throughout the world. In some areas of China, A. aequalis has developed resistance to ALS inhibitors 26,27 , and/or ACCase inhibitors 27,28 , while A. japonicus has developed resistance to chlorsulfuron 28,29 , to fenoxaprop-P-ethyl [30][31][32] , to isoproturon 30 , and/or to pinoxaden 31 . Therefore, QYM201 will be helpful in controlling these resistant weeds. However, more attention should be given to preventing the  32 . However, the efficacy of QYM201 on a greater number of weed species that occur in wheat fields needs to be tested before recommendation of its widespread application.
In crop safety experiments, under all tested application rates, QYM201 was safe for most of the 17 tested hybrid wheat varieties. These results strongly suggested that QYM201 is an excellent alternative herbicide for controlling weeds in wheat fields. Moreover, the SI values were identified for JM22, LX66, TN18, and 4 common weeds that occur in wheat fields. It is well known that herbicides are more selective between crops and weeds when the SI value is greater than 1.0 33 , and herbicides can be safely used in crops when the SI value increases over 2.0 34 . In this study, we found that QYM201 was safe for JM22, LX66, and TN18 against A. aequalis, A. japonicus, C. bursa-pastoris, and M. aquaticum when POST was applied, with SI values from 5.7 to 16.6. However, the safety of QYM201 for use on other wheat hybrids should be assessed in further experiments in view of the complex distribution of wheat hybrids throughout different areas of China.  Table 2. Dry weight inhibitions (%) and visual injury ratings (%) of trial wheat hybrids treated with QYM201 as a POST relative to the non-treated control in a greenhouse study 28 days after treatment (DAT). a Significant differences between the 2 rates according to Fisher's protected LSD test. *Significant at P < 0.05; **significant at P < 0.01; ***significant at P < 0.001; NS, not significant. b Injury rating scale: 0% = no injury, 0~30% = cotyledon and a few functional leaves showed bleaching in addition to newly-emerged leaves, 30~60% = cotyledon, minority of functional leaves and newly-emerged leaves presented bleaching, 60~100% = most plants showed sever whitening symptoms and some even showed necrosis, 100% = plant death. c ND, not determined.     The 2-year field experiments demonstrated that the herbicide QYM201 had good efficacy against A. aequalis, D. sophia, and M. aquaticum with POST at doses of 90-270 g a.i. ha −1 . Previous field studies further indicate that QYM201 has potential as a POST for weed control. Cheng et al. 35 report that weeds die more slowly than in the glasshouse, which was in accordance with our research. This might be owing to greater weed leaf-age and lower temperatures in the field. Furthermore, no obvious damage to wheat plants was observed during the 2 experimental years in any QYM201 treatments. Moreover, the effect of QYM201 on crop yield was characterized; results showed that wheat yields were higher in 2016 than in 2017 ( Table 5). The differences between the data received might be owing to the lower weed density occurring in the experimental sites in 2016. Other factors such as different environmental conditions could also have caused these differences. Wheat yield was increased for all the QYM201 treatments; furthermore, the wheat yield at 270 g a.i. ha −1 was not much different from that at 180 g a.i. ha −1 . According to our research, all of the facts indicate that the recommended dosage of QYM201 is 90 to 180 g a.i.ha −1 . Field results indicated that the hand-weeding plots had the highest yield among all the treatments; however, the cost of labor make this economically unattractive 36,37 . It is commonly agreed that the combination of chemical measures with other agronomic methods may result in economical and effective control of weeds in wheat fields.
In summary, results from greenhouse and field studies indicated that QYM201 has good potential as an efficient broad-spectrum herbicide for controlling weeds in wheat fields. Under the challenge of controlling multiple herbicide resistance in weeds, the novel structure of this herbicide and its different mode of action could be an ideal option for weed control, especially for resistant weed species in wheat fields.

Greenhouse experiment. Weed seeds of A. aequalis, A. japonicus, Veronica didyma Tenore, D. Sophia, and
Becmannia syzigachne (Steud.) Fern were collected from Jiangsu province and the other 13 weed species were collected from Henan province, China, in 2014 (Table 1). All weed species seed germination rates were >85%. All wheat hybrids used in this study can be found in the agricultural seed market and they are listed in Table 2. Germination rates of all wheat seeds were >80%. All greenhouse conditions involved were similar to those in a previous experiment 38 . Experiments were executed at Shandong Agricultural University, Tai'an, China. Weed and wheat seeds were immersed in a petri dish containing distilled water and placed in a 12 h photoperiod and 20 °C growth chamber (Model RXZ, Ningbojiangnan Instrument Factory, Ningbo, China) to accelerate germination before planting. After visualization of seed radicles, 15-30 seeds were sown below the soil surface per plastic pot (160 mm diameter and 130 mm height). After weed emergence, the seedlings were thinned to 10 plants per plastic pot. At the 3-5 leaf stage the seedlings were treated with QYM201 using an auto spraying tower (Model ASS-4, National Agricultural Information Engineering and Technology Center of China) at a spray pressure of 0.275 MPa with 450 L ha −1 spray volume. All greenhouse experiments had replications and were repeated once.
Effectiveness of weed control. All 18 tested weed species were treated with QYM201 at dosage rates of 90 and 135 g a.i. ha −1 , and an untreated control was designed for each weed species. After 28 days of treatment, the surviving weeds were cut off at the soil surface and placed in a labeled paper bag, put in an oven at 80 °C for 72 h, and finally the dry weights were recorded 39 . Other experimental conditions were consistent with those described above for the greenhouse experiment.
Wheat hybrid tolerance. All wheat hybrids were treated with QYM201 at 360 and 540 g a.i. ha −1 , and a non-treated control was also designed. After 28 days of treatment, wheat plants were cut off and put in an oven at 80 °C for 72 h, and then dry weights were recorded. In addition, the degree of herbicide damage to wheat seedlings was also recorded and expressed as values from 0 to 100%: 0% indicated no damage and 100% indicated total death 31 . Other experimental conditions were consistent with those described above for the greenhouse experiment.

Selectivity index (SI).
The selectivity index refers to the ratio between the concentrations that caused 10% growth inhibition of crops and 90% growth inhibition in weeds 40 Table 6. All treatments were arranged in a randomized complete block design and repeated 4 times. The area of each plot was 20 m 2 (4 m wide and 5 m long). This experiment contained a total of 8 treatments and there were 4 application rates of QYM201 (90, 135, 180, and 270 g a.i. ha −1 ); a single concentration of fenoxapro-P-ethyl (at a rate of 50 g a.i. ha −1 ) and tribenuron-methyl (at a dose of 25 g a.i. ha −1 ), respectively; a hand-weeded control (using hand hoes at 0, 15, 30 and 45 DAT) and an untreated control (Table 4). On March 21, 2016, and March 16, 2017, weeds were sprayed with herbicides at the 7 to 8 leaf stage. The average temperatures on the days of application were 13.2 °C and 9.7 °C, respectively. Herbicides were applied using a backpack sprayer (Bellspray Inc., Opelousa, LA) fitted with a single 8002 VS nozzle (Teejet Technologies, Wheaton, IL) in 450 L ha −1 of water.
Visual estimates for crop injury were performed at 3, 5, 15, and 30 DAT, using a 0% (no crop injury) and 100% (plant death) scale. Visual estimates for weed control were recorded after 45 days of treatment, using a 0% (no weed control) to 100% (complete weed control) scale 30 . In each test plot a random sample area of 0.33 m 2 was surveyed at 3 sample points. The number of healthy plants of 3 weed species at each sample point was investigated at 0, 15, 30, and 45 DAT, and the fresh weight of weeds was recorded while investigating the number of weeds at 45 DAT. At the time of wheat harvesting, 3 samples were taken per plot and weighed to evaluate the grain yield of each plot; the resulting wheat yield was expressed as kg/ha. Statistical analysis. All greenhouse experiment data were subjected to Analysis of Variance (hereafter referred to as ANOVA; Version 22.0; IBM Corporation, Armonk, NY). Data were pooled because there was no significant (P > 0.05) interaction with the 2 replicate treatments, and means were separated using Fisher's protected LSD tests at the 0.05 level. All regression analyses were performed using SigmaPlot software (Version 13.0; Systat Software Inc., CA, USA). To evaluate weed control and assess the dose of QYM201 required for 90% weed control, regression of weed dry matter over herbicide dose was performed using the 4 parameter log-logistic model described by Seefeldt et al. 41 : where b is the slope of the line, c is the lower limit, d is the upper limit. x is the herbicide dose, GR 50 is the dose giving 50% response, and y is the growth response (percentage of the untreated control). GR 10 , GR 50 , and GR 90 values were calculated according to regression parameters 18 . The SI values of QYM201 were calculated by the following equation: (2) crop weed (10,90) 10( ) 90 ( ) where GR 10 is the dose of wheat growth reduction by 10%, and GR 90 is the dose of weeds growth reduction by 90%.
Field experiment data were subjected to ANOVA, and means were separated using Fisher's protected LSD tests at the 0.05 level. Treatment interactions of the 2 years were not significant (P > 0.05), therefore the data were pooled by the year.

Data Availability
All data generated or analyzed in this study are included in the Supplementary Information files.   Table 6. Monthly air temperatures and total precipitation at Tai'an experimental site in Shandong, China, during the period from QYM201 application to wheat harvest in 2016 and 2017.