Bipyrazone: a new HPPD-inhibiting herbicide in wheat

Bipyrazone, 1,3-dimethyl-4-(2-(methylsulfonyl)-4-(trifluoromethyl) benzoyl)-1H-pyrazol-5-yl 1,3-dimethyl-1H-pyrazole- 4-carboxylate, is a 4-hydroxyphenylpyaunate dioxygenase (HPPD)-inhibiting herbicide. Greenhouse and field experiments were conducted to explore the potential of post-emergence (POST) application of bipyrazone in wheat fields in China. In the greenhouse study, bipyrazone at 10 and 20 g active ingredient (a.i.) ha−1 effectively controlled Descurainia sophia L., Capsella bursa-pastoris (L.) Medic., Lithospermum arvense L. and Myosoton aquaticum L. Whereas, all tested 16 wheat cultivars showed high degree of tolerance to bipyrazone at 375 and 750 g a.i. ha−1. In a dose-response experiment carried on the Shannong 6 wheat cultivar and five weed biotypes, bipyrazone was safe to the wheat cultivar, and C. bursa-pastoris, M. aquaticum and D. sophia were sensitive to this herbicide. The selectivity index (SI) between the Shannong 6 and weeds ranged from 34 to 39. The field experiments confirmed that a mixture of bipyrazone and fluroxypyr-mepthyl is practical for controlling broadleaf weeds, and bipyrazone applied alone at 30 to 40 g a.i. ha−1 can also provide satisfactory control of sensitive broadleaf weeds. These findings suggest that bipyrazone POST application has good potential for broadleaf weed management in wheat fields.

Greenhouse Experiment Design. All the greenhouse experiments were conducted from October 2015 to May 2016 in a controlled greenhouse (natural lighting, daytime temperature 20~25 °C and night temperature 15~20 °C, 75% relative humidity) in the Arboretum of Shandong Agricultural University (36.21°N,117.13°E). Before planting, the wheats and weeds were geminated in a petri dish with two layers of wet filter paper or 1% (m/v) plant agar in an illumination chamber (25/15 °C, 12/12 h day/night) (Model RXZ, Ningbojiangnan Instrument Factory, Ningbo, China). As the radicle became visible, nine wheat seeds or twelve weed seeds were sown below the soil surface in plastic pots (16-cm diameter x 13-cm height). The soil had a pH of 7.67, organic matter of 15.79 g kg −1 and 14.28-2.0-23.15 g kg −1 of N P K, and was passed through a 3-mm sieve. Before herbicide application, the wheat seedlings were thinned to six evenly-sized plants, and weed seedlings were thinned to an appropriate number of plants per pot according to the size of weed. The pots were watered at the bottom, when needed, to maintain the moisture. The seedlings were treated with herbicides at the 3-to 4-leaf stage using a research track sprayer (Model ASS-4, National Agricultural Information Engineering and Technology Center of China) with a Teejet 9503EVS flat-fan nozzle that delivered a 450 L ha −1 spray of solution at a spray pressure of 275 kPa. Twenty-one days after treatment (DAT), the shoots were harvested, oven dried at 80 °C for 72 h and the dry weights recorded. All the experiments in the greenhouse were arranged in a randomized complete block design with four replicates and conducted at least twice.
Weed Control Spectrum. Fourteen species commonly infesting wheat fields in China were selected to conduct the weed control spectrum study. The POST application of bipyrazone at 10 and 20 g a.i. ha −1 was conducted and an untreated control (0.1% mass fraction of Tween 80) was established for each weed species. Other experimental conditions were the same as described in the greenhouse experimental design. Wheat Cultivar Tolerance. Sixteen leading wheat cultivars planted in Northern China were selected to conduct the tolerance experiment. The POST application of bipyrazone at 375 and 750 g a.i. ha −1 was conducted and an untreated control (0.1% mass fraction of Tween 80) was established for each wheat cultivar. Other experimental conditions were the same as described in the greenhouse experiment design.
Selectivity Index (SI). The selectivity index is the ratio between the application rate that caused 10% growth reduction in the crop and the rate that caused 90% growth reduction in the weed 19 . Based on the results of the weed control spectrum and wheat cultivar tolerance experiments, three common and troublesome broadleaf weeds C. bursa-pastoris, M. aquaticum and D. Sophia were selected, along with Shannong 6, one of the most popular wheat cultivars in Northern China, in order to study the SI. To determine the sensitivity of herbicide resistant weeds to bipyrazone, the SI between Shannong 6 and the biotypes of C. bursa-pastoris and D. sophia with TSR to tribenuron-methyl were also determined. Wheat (Shannong 6) was treated with POST application at 0 (0.1% mass fraction of Tween 80), 750, 1500, 3000, 4500, and 6000 g a.i. ha −1 of bipyrazone, and C. bursa-pastoris (R and susceptible (S) biotypes), D. sophia (R and S biotypes) and M. aquaticum were treated with POST application at 0 (0.1% mass fraction of Tween 80), 1.25, 2.5, 5, 10 and 20 g a.i. ha −1 . Other experimental conditions were the same as described in the greenhouse experiment design. Field Experiment. In 2017 and 2018, field experiments were conducted twice at farm fields (36.17°N, 117.16°E, loam with 1.7% organic matter and a pH of 7.1) in Tai'an, Shandong, China to determine the weed control efficacy of bipyrazone, tribenuron-methyl, fluroxypyr-mepthyl and the mixtures of bipyrazone with fluroxypyr-mepthyl against broadleaf weeds in winter wheat fields. The test field was heavily infested with C. bursa-pastoris, D. sophia and G. aparine and the tribenuron-methyl has failed to control C. bursa-pastoris for several years. The wheat (Jimai 20) was sown in a drill by field equipment on 12 October, 2016 and 20 October,   Table 2. Dry weight inhibition of different wheat cultivars treated with bipyrazone POST application relative to the untreated control in the greenhouse study 21 days after treatment. a *significant differences between the two rates at 0.05 level according to Fisher's protected LSD test; NS, not significant; SE, standard error, n = 8.
www.nature.com/scientificreports www.nature.com/scientificreports/ Field experiments were designed as randomized complete blocks with four replicates each of 20 m 2 (5 m × 4 m). The 12 treatments are presented in Table 3 including four rates of bipyrazone, a single rate of tribenuron-methyl, a single rate of fluroxypyr-mepthyl, four rates of a mixture of bipyrazone and fluroxypyr-mepthyl, a hand weeded control and no treatment. The POST application was made on 15 March, 2017 (sunny, −1.7 to 15.5 °C, breeze) and 21 March, 2018 (sunny, 5.8 to 12.9 °C, light breeze) when wheat was at 3-leaf to tillering stages and weeds were at 2-to 5-leaf stages. A total spray volume of 450 L ha −1 was applied with a backpack sprayer (Bellspray Inc., Opelousa, LA) equipped with a single 8002 VS nozzle (Teejet Technologies, Wheaton, IL), and wheat production followed the local common farming practice. The daily air temperature and precipitation at the experimental site after several days of treatment are presented in Fig. 2.
The weed survey was conducted five times in total at 5, 10, 15, 20 and 40 DAT, and a visual estimate was conducted at 5, 10 and 15 DAT. To calculate weed population reduction, all weeds were separated by species, counted and recorded from three 0.33 m 2 quadrats for each plot at 20 DAT. To calculate weed population and biomass reduction, the shoots of all weed plants were cut from three 0.33 m 2 quadrats for each plot at 40 DAT, and all weeds were separated by species, counted, oven dried at 80 °C for 72 h and dry weight recorded. The reduction was calculated according to Eq. 1: where TP is weed number or biomass in the treated plot, and STP is the sum of the number or biomass of the four no treatment plots. The visual wheat damage was evaluated at 5, 10, 20, 40 DAT and harvesting date. Wheat grain yields (adjusted to 13% moisture) were determined by hand harvesting 5 m 2 of each plot and are expressed on a per-hectare basis. Data analysis. The data from repeated greenhouse study were analysed by analysis of variance (ANOVA) using SPSS (Version 17.0; IBM Corporation, Armonk, NY), and the data were pooled because of the non-significant difference between repeated experiments at the 5% significance level. The means were separated using Fisher's protected least-significant difference (LSD) at the 5% significance level.
The dose-response curves were acquired through non-linear regression analysis using the four-parameter log-logistic response Eq. 2 20 : where C is the lower limit of response, D is the upper limit of response, x is the herbicide rate, y is the percentage of dry weight residue, GR 50 is the dose causing 50% growth inhibition and b is the slope of the curve around the GR 50 . Graphs were created by SigmaPlot software (Version 13.0; Systat Software Inc., CA, USA) with the dry weight means (% of residual) and standard errors. According to the regression parameters, the GR 10 and GR 50 values for the wheat and GR 50 and GR 90 for the weeds were calculated based on Eq. 2 21 . The SIs of bipyrazone were calculated as Eq. 3: Where GR 10 equals a 10% growth reduction on Shannong 6 and GR 90 equals a 90% growth reduction on the trial weeds.
The data from the field experiments were tested for homogeneity of variances and normality of distribution, and subjected to arcsine square root transformation as needed before analysis. All data were subjected to ANOVA, and the means were separated using Fisher's protected LSD test at 5% significance level. The data were presented as untransformed means, and analysed separately by year because the weather conditions were different each year.  www.nature.com/scientificreports www.nature.com/scientificreports/
Wheat cultivar tolerance. The POST application of bipyrazone was safe for all 16 tested wheat cultivars under greenhouse conditions and no bleaching symptoms were observed during the whole experiment. The dry weight inhibition of all 16 tested wheat cultivars was below 5% when bipyrazone was applied POST at 375 g a.i. ha −1 , and below 15% when applied POST at 750 g a.i. ha −1 ( Table 2). Selectivity index. The tolerance of Shannong 6 wheat to bipyrazone applied POST was much higher than that of weeds, and D. sophia was more sensitive to bipyrazone applied POST than C. bursa-pastoris and M. aquaticum (Fig. 3). The SI of bipyrazone POST application between Shannong 6 wheat and five biotypes of three weed species were from 34 to 39 (Table 4). Moreover, there was no significant difference in sensitivity (GR 50 value) to bipyrazone between susceptible and resistant biotypes of C. bursa-pastoris and M. aquaticum (Table 4).
Field experiment. Visual estimation found no bleaching on any wheats during all the experiments in both years, and bipyrazone and its mixture with fluroxypyr-mepthyl were safe for wheats when applied POST at all the tested rates under field conditions (data not shown).
As shown in Table 5, although POST application of tribenuron-methy at 22.5 g a. i. ha −1 provided 90.7% population reduction against D. sophia, it showed low weed population reduction against G. aparine (53.1% population reduction) and failed to control C. bursa-pastoris with 8.9% weed population reduction at 20 DAT. Bipyrazone applied alone POST was very effective against C. bursa-pastoris and D. sophia but not G. aparine (weed population reduction <60%) at 20 DAT (Table 5). However, fluroxypyr-mepthyl had greater efficacy against G. aparine (weed population reduction å 97%) than against C. bursa-pastoris and D. sophia at 20 DAT (Table 5). The mixtures of bipyrazone and fluroxypyr-mepthyl were very effective against all tested weeds with a weed population reduction from 84.4% to 100% at 20 DAT (Table 5). Overall, the trend of weed population and biomass reduction at 40 DAT was similar to the weed population reduction at 20 DAT, and the control efficacy in 2018 was a little higher than in 2017 (Tables 5-7). Moreover, all herbicide treatments showed higher weed biomass reduction than weed population reduction at 40 DAT (Table 6 and Table 7).
Wheat grain yields and yield growth rate following different treatments in 2017 and 2018 are presented in Table 8. With increased control of total weeds provided by the herbicide treatments, the grain yield growth rate was 2.2%-22.6% in 2017 and 2018 (Table 7 and Table 8). However, the grain yield was reduced by more than 23.5% by uncontrolled weed growth in the no treatment plots compared with hand weeding control treatment ( Table 8). The wheat grain yields were higher in 2017 than in 2018 (Table 8).

Discussion
Up to now, the most important chemical classes of commercial HPPD-inhibiting herbicides consist of isoxazoles such as isoxaflutole, pyrazoles such as topramezone, and triketones such as mesotrione, tembotrione, bicyclopyrone and sulcotrione 15,22 . Among HPPD-inhibiting herbicides, sulcotrione, a triketone derived from leptospermone from the bottlebrush plant, was the first to have its mode of action identified 23,24 . Meanwhile, triketone derivatives are most widely studied in China because its structure can be further divided into two parts, triketone and aromatic moieties [25][26][27] . Bipyrazone belongs to the same new chemical class of pyrazolones as topramezone 15 , presents the typical bleaching symptoms of an HPPD inhibitor after POST application.
Bipyrazone has the potential for control of broadleaf weeds such as D. sophia, C. bursa-pastoris, L. arvense and M. aquaticum, whereas it has little or no efficacy against grassy weeds like some HPPD-inhibiting herbicides, such as mesotrione 28 . According to the weed control spectrum experiment, the dry weight inhibition was over 85% for bipyrazone applied POST at 20 g a.i. ha −1 against all four broadleaf weed species above. Although the data obtained in the greenhouse may not be the same as that obtained in field conditions due to the complexity and changeability of field environments, it reflects the efficacy of weed control to a certain degree. For example, bipyrazone caused low dry weight inhibition of G. aparine at the tested rate (<20%) under greenhouse conditions, and consistently presented poor control efficacy (<60%) against G. aparine with POST application at all tested rates in field conditions. Study of wheat tolerance showed that bipyrazone POST application was safe for wheat. Also, the SI values for Shannong 6 wheat and five biotypes of three weed species were identified. The selectivity of the herbicide between the crop and the weeds rises with the increase of SI, and a herbicide can be safely used on a crop when the SI value is over 2.0 19,29 . Thus, bipyrazone applied POST was safe for Shannong 6 and effectively controlled C. bursa-pastoris, M. aquaticum and D. sophia with the SI value ranging from 34 to 39.
There was no difference in sensitivity to bipyrazone between susceptible and tribenuron-methyl resistant biotypes of C. bursa-pastoris and M. aquaticum. Nowadays, there are 326 biotypes of 76 wheat field weed species that have evolved resistance to herbicides with different modes of action such as acetolactate synthase (ALS) inhibitors and Acetyl-CoA carboxylase (ACCase) inhibitors, but there is still no report of HPPD-inhibitor resistant weeds in wheat fields 30 . With bipyrazone registration, there is a new mode of action being commercially introduced for the first time in China. Thus, bipyrazone may be an excellent tool to control some broadleaf weeds that have evolved resistance to herbicides with different modes of action. However, careful attention should be paid to preventing or delaying the development of resistance by reducing herbicide selection pressure via alternating between POST   Table 4. Parameters of the four-parameter log-logistic equation used to calculate application rates of bipyrazone causing 10% (GR 10 ) and 50% (GR 50 ) growth reduction of Shannong 6, and 50% and 90% (GR 90 ) growth reduction of five biotypes of weeds, and the selectivity index (SI) between Shannong 6 and five biotypes of weeds in the greenhouse study 21 days after treatment. a R = biotypes of C. bursa-pastoris and M. aquaticum, target-site-resistant to tribenuron-methyl. b y = C + (D-C)/{1+exp[b(log(x)-log (GR 50 ))]}, where C is the lower limit of response, D is the upper limit of response, x is the herbicide rate, y is the percentage of dry weight residue, and b is the slope of the curve around the GR 50 . SE, standard error. c The same letter were not significantly different at 0.05 level according to Fisher's protected LSD test. d SI calculated by SI = GR 10(Shannong 6) / GR 90(weed) . (2020) 10:5521 | https://doi.org/10.1038/s41598-020-62116-6 www.nature.com/scientificreports www.nature.com/scientificreports/ and preemergence (PRE), mixing herbicides with different modes of action, and rotation 31 . Also, increasing crop diversity and using biological control are practices to be encouraged 31 .
In the field experiment, bipyrazone applied POST alone were very effective against C. bursa-pastoris and D. sophia whereas it was not as effective against G. aparine. Although fluroxypyr-mepthyl showed excellent control of G. aparine, its effectiveness against C. bursa-pastoris and D. sophia was not satisfactory. The mixture of bipyrazone and fluroxypyr-mepthyl extended the weed-control spectrum and produced better control of all the weeds mentioned above. Combination of different herbicides not only extends the weed control spectrum but also delays the evolution of resistance better than herbicide rotation 31 . Tribenuron-methy failed to control C. bursa-pastoris in our field experiments, and the infestation of tribenuron-methy resistant C. bursa-pastoris has become a common and serious problem in Shandong and Henan Province, China 4,32 . Luckily, under field conditions, bipyrazone applied alone and its mixture with fluroxypyr-mepthyl also provided high efficacy against tribenuron-methy resistant C. bursa-pastoris as seen in the greenhouse study. The efficacy of all the treatments containing bipyrazone in 2018 was higher than 2017, which may result from the higher temperature 1 to 10 DAT (average temperature of 16.1 °C) in 2018 than 2017 (average temperature of 10.1 °C), which was consistent with the results from the greenhouse study that bipyrazone caused higher dry weight inhibition in 15/20 °C (night/day) than in 10/15 °C (night/day) (data not shown). Several studies have shown that temperature has great influence on herbicide efficacy [33][34][35] . It is reported that mesotrione, a HPPD-inhibiting herbicide widely used in maize fields, POST application showed different control efficacy at different temperature 36 . Temperature in the field may affect the efficiency of bipyrazone applied POST, and the application should be conducted when temperature is ideal for the control of sensitive weeds.
The higher grain yield rate obtained from using a mixture of bipyrazone and fluroxypyr-mepthyl, confirmed the greater total weeds control provided by the mixtures. Bipyrazone, tribenuron-methyl and fluroxypyr-mepthyl, when applied alone, resulted in unsatisfactory grain growth rates since the control of all broadleaf weeds was not   www.nature.com/scientificreports www.nature.com/scientificreports/ warranted due to the limitation of weed control spectrum. In China, the weed flora of wheat fields is very complex; the broadleaf weeds are more common, but, the grassy weeds are spreading quickly 37 . This complexity makes it difficult to manage weeds successfully with a single herbicide. It is essential to expand the weed control spectrum by mixtures of two or three herbicides. Satisfactory yield growth rates were obtained with mixtures of bipyrazone and fluroxypyr-mepthyl at rates of 40 + 120 and 40 + 160 g a.i. ha −1 across years. Therefore, a mixture of bipyrazone and fluroxypyr-mepthyl (mass ratio = 1:3~4) applied POST at 160 to 200 g a.i. ha −1 is recommended for broad weed control in winter wheat fields, based on the results of our study. However, the choice of herbicides and application rates should depend on the actual weed flora in the fields. For example, 30 to 40 g a.i. ha −1 of bipyrazone applied alone can also be recommended when C. bursa-pastoris and D. sophia are the main weeds present.
Although hand weeding produced the highest yield growth rate in both years, it is uneconomical due to the labour and time costs 38,39 . The wheat yield in 2017 was higher than in 2018 because two episodes of heavy rain occurred in the test fields at the flowering stage in 2018 (daily precipitation> 78 mm, data not shown) which greatly reduced the yield.
In conclusion, results collected from both the greenhouse and field experiments suggest that bipyrazone, as a selective POST herbicide, has good potential for the management of broadleaf weeds including D. sophia, C. bursa-pastoris, L. arvense and M. aquaticum in winter wheat production systems in China. Especially with broadleaf weeds in wheat fields being rapidly evolving resistance to herbicides with different modes of action such as ALS inhibitors, protoporphyrinogen oxidase (PPO) inhibitors and synthetic auxins inhibitors 30 , bipyrazone, a new HPPD-inhibiting herbicide, may therefore be a powerful chemical broadleaf-weed-control technology, although it presents a limited weed control spectrum.

Data availability
All data generated or analysed during this study are available from the corresponding author on reasonable request.