Introduction
The continued reliance on herbicides for weed control has resulted in an increased number of weed species evolving resistance to herbicides. Of great concern are non–target site resistance mechanisms, which can confer resistance to a range of different site-of-action (SOA) herbicides (Dücker et al. Reference Dücker, Lorentz, Hull, Anderson, Moss and Beffa2016; Keshtkar et al. Reference Keshtkar, Mathiassen, Moss and Kudsk2015; Yu and Powles Reference Yu and Powles2014; Yuan et al. Reference Yuan, Tranel and Stewart2007). Rigid ryegrass (Lolium rigidum Gaudin), a genetically diverse, obligate-outcrossing species has been extensively studied and reported as having the capacity to evolve resistance to different SOA herbicides, including populations with cross-resistance to the thiocarbamate and sulfonylisoxazoline herbicides prosulfocarb and pyroxasulfone (15(K3): inhibitors of very-long-chain fatty-acid synthesis) (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018; Busi and Powles Reference Busi and Powles2016). Research into the possibility of L. rigidum evolving resistance to more diverse SOA herbicides has provided some understanding of the likelihood of resistance evolution to herbicides not yet discovered (Busi et al. Reference Busi, Gaines, Walsh and Powles2012). The current practice of managing L. rigidum in Australia in wheat (Triticum aestivum L.) relies heavily on the use of PRE herbicides, including trifluralin (3(K1): inhibitors of microtubule assembly), prosulfocarb, and pyroxasulfone (Boutsalis et al. Reference Boutsalis, Gill and Preston2014; Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2020a). A greater reliance has been placed on PRE herbicides due to significant and widespread resistance to the herbicides diclofop-methyl (1(A): inhibitors of acetyl CoA carboxylase) and chlorsulfuron (2(B): inhibitors of acetolactate synthase) (Broster and Pratley Reference Broster and Pratley2006).
Clomazone (13(F4): inhibitors of deoxy-d-xylulose phosphate [DXP] synthase) is registered in Australia for the control of broadleaf weeds in opium poppy (Papaver somniferum L.), cotton (Gossypium hirsutum L.), and vegetable crops. Clomazone has most commonly been used for the control of broadleaf and grass weeds in rice (Oryza sativa L.) (Ferhatoglu and Barrett Reference Ferhatoglu and Barrett2006). Clomazone is not registered for L. rigidum control in Australia, and as a result, resistance to clomazone remains rare. Resistance to clomazone was reported by Tardif and Powles (Reference Tardif and Powles1999) in the population SLR31, despite the fact that the population had never before been treated with this herbicide. In contrast to clomazone, the isoxazolidinone herbicide bixlozone (13(F4)) will provide an additional SOA for the control of L. rigidum populations in wheat in Australia.
Several herbicides require in vitro biochemical conversion to exert herbicidal effects in plants. This process involves the metabolic activation of the herbicide into an active metabolite (Kern et al. Reference Kern, Peterson, Miller, Colliver and Dyer1996) and the oxidation of the parent compound through specific cytochrome P450s (CYP450s) (Casida et al. Reference Casida, Gray and Tilles1974; Fuerst Reference Fuerst1987). Herbicides that require activation through this process include the thiocarbamates and isoxazolidinones (Casida et al. Reference Casida, Gray and Tilles1974; Ferhatoglu and Barrett Reference Ferhatoglu and Barrett2006; Weimer et al. Reference Weimer, Buhler, Balke, Caseley, Cussans and Atkin1991). The competition in P450-mediated reactions between herbicides and insecticides as well as oxidative desulfuration can confer protection against herbicides such as clomazone (Durst et al. Reference Durst, Salaun, WerckReichhart, Zimmerlin, DePrado, Jorrin and GarciaTorres1997; Ferhatoglu et al. Reference Ferhatoglu, Avdiushko and Barrett2005; Fuerst Reference Fuerst1987). Organophosphate insecticides are highly diverse and reactive molecules that possess a phosphorous atom with a covalent bond to either sulfur or oxygen. To be active, both clomazone and bixlozone require metabolic conversion to the active keto-variant (5-ketoclomazone), which inhibits DXP synthase, leading to the biosynthesis of isopentenyl pyrophosphate in plastids (Ferhatoglu and Barrett Reference Ferhatoglu and Barrett2006). In cotton, phorate and disulfoton enhanced protection through inhibiting P450-mediated bioactivation and preventing the production of 5-ketoclomazone (Ferhatoglu et al. Reference Ferhatoglu, Avdiushko and Barrett2005).
Metabolic resistance to herbicides is frequently associated with increased levels of CYP450 activity leading to enhanced detoxification of these primary metabolites (Siminszky Reference Siminszky2006). Both clomazone and bixlozone are at risk of P450-mediated metabolism of the primary keto variant 5-OH clomazone (Ferhatoglu et al. Reference Ferhatoglu, Avdiushko and Barrett2005), a precursor of the active metabolite 5-ketoclomazone (Ferhatoglu and Barrett Reference Ferhatoglu and Barrett2006). Studies in corn (Zea mays L.) and soybean [Glycine max (L.) Merr.] have linked the degradation of major clomazone metabolites and hydroxylated derivatives through specific P450s (ElNaggar et al. Reference ElNaggar, Creekmore, Schocken, Rosen and Robinson1992; Ferhatoglu et al. Reference Ferhatoglu, Avdiushko and Barrett2005). Resistance to clomazone has been reported in late watergrass [Echinochloa phyllopogon (Stapf) Koso-Pol.] and has been linked to cytochrome P450-mediated metabolism that confers resistance to other SOA herbicides (Yasuor et al. Reference Yasuor, TenBrook, Tjeerdema and Fischer2008, Reference Yasuor, Zou, Tolstikov, Tjeerdema and Fischer2010). In contrast, no cases of resistance to clomazone have been reported in blackgrass (Alopecurus myosuroides Huds.), which has evolved diverse metabolic resistance mechanisms (Keshtkar et al. Reference Keshtkar, Mathiassen, Moss and Kudsk2015). The evolution of metabolic resistance is of significant concern and has been reported to endow cross-resistance to a diverse range of herbicides (Beckie and Tardif Reference Beckie and Tardif2012; Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2018). Metabolic resistance has also been shown to evolve resistance to chemically unrelated herbicides (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2019; Busi and Powles Reference Busi and Powles2016).
To date, there are no reports of resistance to bixlozone, but there is a risk of cross-resistance evolution from prosulfocarb for other herbicides that require activation through P450s. A previous study by Busi et al. (Reference Busi, Gaines, Walsh and Powles2012) highlight the importance of research investigating the likelihood and potential of resistance evolution to diverse SOA herbicides in L. rigidum. The current research investigates possible resistance mechanisms to bixlozone in three recurrently selected L. rigidum populations from southern Australia with known resistance to thiocarbamate herbicides and multiple other SOA herbicides.
Materials and Methods
Plant Material
Lolium rigidum populations 375-14 (R), 198-15 (R), and EP162 (R) used in this study were collected from cropping fields across southern Australia as described by Brunton et al. (Reference Brunton, Boutsalis, Gill and Preston2018). A well-characterized L. rigidum population, SLR4, was used as the susceptible (S) control (Boutsalis et al. Reference Boutsalis, Gill and Preston2012). Seeds of each population were weighed (0.2 g = 50 to 60 seeds) and spread onto the surface of 9.5 cm by 8.5 cm by 9.5 cm punnet pots (Masrac Plastics, Dry Creek, SA, Australia) containing cocoa peat potting mix, as described by Boutsalis et al. (Reference Boutsalis, Gill and Preston2012).
Inhibitor and Herbicide Application
Before herbicide application (3 h), the insecticide phorate (Thimet, Barmac Industries, Stapylton, QLD, Australia) was applied to the soil surface as described by Busi et al. (Reference Busi, Gaines and Powles2017) at a dose of 0.076 g pot−1, corresponding to 10 kg ha−1 phorate (Table 1). Pots treated with the inhibitor only were also included. Herbicides were applied directly on seed and soil using a laboratory spray cabinet equipped with flat-fan nozzles (Hardi ISO F-110-01, Hardi, Adelaide, SA, Australia) delivering 118 L ha−1 water at a pressure of 2.54 kPa. Control pots were not treated with any herbicide or inhibitor. The experiment was conducted outdoors under natural growing conditions in winter during the normal growing season (May to July). Pots were watered as needed to maintain potting mix near field capacity. There were three replicates for each herbicide dose, and pots were arranged in a randomized complete block design, and the experiment was repeated.
Active ingredients, formulations, and manufacturers of herbicides used in dose–response experiments.
Generation of RS1 and RS2 Families
Seed from the resistant populations (375-14, 198-15, and EP162) were treated with the recommended field rate of bixlozone (500 g ha−1) in 2018, and survivors were collected. Four surviving plants of each R population were grown in a single 30-cm-diameter pot containing standard potting mix. Plants were grown outdoors under natural growing conditions during the months May to November 2018 and watered as required. Before plants flowered, pots were encased in a 1.2-m-long polypropylene sleeve supported by a wire mesh cage that was open at the top. This pollen sleeve was used to minimize the risk of cross-pollination from other plants present in the area. Seeds were harvested separately from each population and labeled RS1 for each population. To generate the RS2 population, the seeds of progeny of RS1 were further treated with the recommended field rate of bixlozone in 2019, and survivors were grown in a single 30-cm pot as described earlier during the months May to November 2019. At maturity, seeds from all plants within each pot were pooled to create the RS2 families. RS2 populations were then selected for inclusion in dose–response experiments in 2020.
Dose–Response Experiment and Seedling Growth
Dose–response experiments were conducted on S, R, RS1, and RS2 populations and repeated. Two PRE herbicides were applied (Table 1) directly onto the seed following the method described by Boutsalis et al. (Reference Boutsalis, Gill and Preston2012). The herbicide bixlozone was applied to the S biotype at 15.62, 31.25, 62.5, 125, 250 and 500 g ha−1 and to the R biotypes at 125, 250, 500, 1,000, 2,000, 4,000, and 8,000 g ha−1, with the recommended label rate for L. rigidum control in Australia at 500 g ha −1. Clomazone was applied to the S biotype at 15, 30, 60, 120, 240, and 480 g ha−1 and to the R biotypes at 120, 240, 480, 960, 1,920, 3,840, and 7,680 g ha−1, with the recommended label rate being 240 to 480 g ha−1. The resistant parent and recurrently selected RS1 and RS2 populations were tested during the normal growing season (May to July) 2020. Pots were assessed at 28 d after herbicide treatment, and plants that had emerged and grown to the 2-leaf stage were counted. Percentage survival was assessed as the number of plants growing in the treated pots compared with the average number present in the control pots.
Statistical Analysis
Following ANOVA, the data for both experimental runs were pooled, as there was no statistical difference between the runs. The data were analyzed using a log-logistic equation (GraphPad Prism v. 8.0, GraphPad Software, San Diego, CA, USA) fit to the percentage emergence data (Seefeldt et al. Reference Seefeldt, Jensen and Fuerst1995). The normalized three-parameter logistic regression model was fit, where y represents plant survival (%), x is the log-dose of the herbicide used, LD50 is the herbicide dose required to cause 50% reduction in plant emergence, and b denotes the slope of the curve. LD50 parameter estimates from the log-logistic analysis were used to calculate the resistance index (RI), which is the resistant:susceptible ratio of the LD50.
$${\rm{y}}\;{\rm{ = }}\;{{{\rm{100}}} \over {{\rm{1}}\;{\rm{ + }}\;{\rm{10}}{\;^{({\rm{log}}\;{\rm{L}}{{\rm{D}}_{{\rm{50}}}}\;{\rm{ - }}\;{\rm{x}})\; \times \;{\rm{b}}}}}}$$
To compare the LD50 values for L. rigidum populations in the presence of the insecticide phorate, a t-test was conducted with the null hypothesis of no difference between resistant versus susceptible populations, or their ratio was equal to 1 (Ritz et al. Reference Ritz, Kniss and Streibig2015).
Results and Discussion
Herbicide Response of Recurrent Selection in Lolium rigidum Populations
The susceptible L. rigidum population SLR4 was completely controlled by bixlozone at the recommended field rate of 500 g ha−1 with an LD50 of 43 g ha−1 (Table 2; Figure 1). Furthermore, SLR4 was completely controlled by clomazone at the recommended field rate of 480 g ha−1 with an LD50 of 56 g ha−1 (Table 2). The bixlozone rates required for 50% mortality (LD50) for the resistant populations EP162, 375-14, and 198-15 were 108, 253, and 433 g ha−1, respectively (Table 2). Lolium rigidum population EP162 had a lower LD50 for bixlozone compared with the two other resistant populations. The RI for bixlozone was 2.5- to 10.1-fold greater than that of the S population SLR4 (Table 2). The LD50 values for populations EP162, 375-14, and 198-15 treated with clomazone were higher than that of the S population at 214, 319, and 551 g ha−1, respectively giving an RI of 3.8 to 9.8 compared with the S population.
Pooled dose–response data of bixlozone and clomazone dose required for 50% mortality (LD50) of the resistant parent, RS1, RS2 crosses, and susceptible Lolium rigidum populations (95% confidence intervals in parentheses) and resistance index (RI).a
a RS1, first-generation recurrent selection; RS2, second-generation recurrent selection.
b RI values calculated as the ratio between the LD50 of the resistant populations compared with the mean LD50 of the susceptible population (SLR4).
c P-value indicates significant difference in LD50 values between L. rigidum populations treated with a particular herbicide compared with SLR4.
Response of SLR4 (solid line, ●), resistant parent (dotted and dashed line, ■), recurrent progeny RS1 (dotted line, ▲), and RS2 (dashed line, ▼) of Lolium rigidum populations EP162 (A), 375-14 (B), and 198-15 (C) treated with varying rates of bixlozone. Each data point is the mean of six replicates, and bars represent the standard error of the mean.
The response of the RS1 survivors showed a significant (P < 0.01) increase in LD50 as compared with the parent populations. The RS1 populations EP162 RS1 and 375-14 RS1 showed 29% and 37% survival to bixlozone at 500 g ha−1, respectively, while 198-15 RS1 showed 75% survival at this rate (Figure 1). The populations EP162 RS1, 375-15 RS1, and 198-15 RS1 recorded RIs for bixlozone that were 7.8-, 8.8-, and 17.5-fold greater than that of the S population, respectively (Table 2). The RS1 populations also showed a significant (P < 0.01) increase in LD50 for clomazone (Table 2), with RIs to clomazone for populations EP162 RS1, 375-14 RS1, and 198-15 RS1 being 6.3-, 7.9-, and 11.3-fold compared with SLR4, respectively.
Further selection with bixlozone in the second generation (RS2) resulted in a significant increase (P < 0.01) in LD50 for bixlozone in all populations. All RS2 L. rigidum populations had LD50 values greater than the recommended field rate of 500 g ha−1 and were 579, 793, and 1,157 g ha−1 for EP162 RS2, 375-14 RS2, and 198-15 RS2, respectively. This increase in LD50 in the RS2 populations was reflected in 54%, 84%, and 98% survival at the recommended rate of bixlozone (Figure 1). The RIs for the RS2 populations were 13.5-, 18.4-, and 26.9-fold greater than that of the S population for EP162 RS2, 375-14 RS2, and 198-15 RS2, respectively (Table 2). Furthermore, all RS2 populations treated with clomazone had LD50 values greater than the recommended field rate 480g ha−1 and were 544, 690, and 879 g ha−1 for EP162 RS2, 375-14 RS2, and 198-15 RS2, respectively, giving RIs of 9.7-, 12.3-, and 15.7-fold compared with the S population.
The parental populations EP162, 375-14, and 198-15 with known cross-resistance to thiocarbamate, chloroacetamide, and sulfonylisoxazoline herbicides (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2019) displayed reduced sensitivity to the isoxazolidinone herbicides bixlozone and clomazone. Cross-resistance to the isoxazolidinone herbicide clomazone was previously reported by Tardif and Powles (Reference Tardif and Powles1999) in L. rigidum population SLR31, which had a long and complex history of exposure to herbicides. In this study, we show a similar level of resistance to bixlozone and clomazone in parent populations EP162, 375-14, and 198-15, as previously reported in E. phyllopogon by Guo et al. (Reference Guo, Iwakami, Yamaguchi, Uchino, Sunohara and Matsumoto2019) (Table 2). Furthermore, the response of the RS1 and RS2 populations demonstrated that recurrent selection of multiple-resistant L. rigidum populations with a full herbicide dose significantly increased the level of resistance to bixlozone and clomazone (Table 2).
The L. rigidum parental populations in this study had not been exposed to bixlozone in the field; their only exposure to bixlozone was in pots during generations RS1 and RS2. These findings highlight the capacity for L. rigidum to evolve resistance to herbicides in the absence of direct selection (Busi and Powles Reference Busi and Powles2013; Keshtkar et al. Reference Keshtkar, Mathiassen, Moss and Kudsk2015). Furthermore, observed resistance to clomazone suggests the possibility of a similar P450 mechanism conferring resistance to both herbicides.
Effect of Phorate on Isoxazolidinone Herbicides
The susceptible population SLR4 was completely controlled by both bixlozone and clomazone applied at the recommended field rate. In the absence of herbicide, phorate at 10 kg ha−1 had no effect on the germination percentage of all L. rigidum populations when compared with nontreated control (data not shown). The LD50 for L. rigidum population SLR4 treated with bixlozone was 47 g ha−1 and ranged from 122 to 455 g ha−1 for the resistant parent populations (2.6- to 9.5-fold resistance compared with SLR4) (Table 3). The addition of phorate significantly (P < 0.01) increased the LD50 for bixlozone in all populations compared with the herbicide-only treatment. In the presence of phorate, the LD50 for SLR4 was 1,627 g ha−1 (Table 3) and ranged from 2,188 to 4,061 g ha−1 for the resistant populations, giving RIs of 46.6- to 86.4-fold compared with SLR4 in the absence of phorate (Table 3). The L. rigidum populations SLR4, EP162, 375-14, and 198-15 treated with phorate and clomazone displayed a significant (P < 0.01) antagonistic response similar to the response to bixlozone. In the presence of phorate, the LD50 value for SLR4 was 1,575 g ha−1 (28.1-fold increase) and ranged from 2,044 to 5,127 g ha−1 with RIs of 36.5- to 91.5-fold compared with SLR4 in the absence of phorate (Table 3).
Pooled dose–response data of isoxazolidinone herbicides bixlozone and clomazone with or without phorate required for 50% mortality (LD50) of resistant and susceptible Lolium rigidum populations (95% confidence intervals in parentheses) and resistance index (RI).
a RI values calculated as the ratio between the LD50 of the resistant populations compared with the mean LD50 value of the susceptible population (SLR4) without phorate.
b P-value indicates the significance of differences in LD50 values between L. rigidum populations treated or not treated with phorate.
In this study, we report major difference between L. rigidum populations in response to bixlozone and clomazone in the presence of the P450 inhibitor phorate. As reported previously, P450 enzymes are involved in the activation (thiocarbamates) or hydroxylation (isoxazolidinones) of the parent compound into the bioactive form of clomazone, 5-ketoclomazone (Ferhatoglu and Barrett Reference Ferhatoglu and Barrett2006; Fuerst Reference Fuerst1987; Norman et al. Reference Norman, Liebl and Widholm1990), and inhibition of P450s by phorate would therefore reduce the activity of these herbicides. Reduced activation has been reported as a possible mechanism conferring resistance to the thiocarbamate herbicide triallate (Kern et al. Reference Kern, Peterson, Miller, Colliver and Dyer1996). The antagonism between phorate and bixlozone or clomazone observed in the susceptible population SLR4 and the three resistant populations (Table 3) indicates involvement of P450s in herbicide activation (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2020b; Busi et al. Reference Busi, Gaines and Powles2017), but lack of herbicide activation is not the mechanism of resistance to these herbicides. Whether resistance to bixlozone in L. rigidum is based on a similar mechanism involved in resistance to thiocarbamates remains to be determined.
In a previous study, this resistant population (EP162) displayed no antagonism between phorate and the thiocarbamate herbicide prosulfocarb (Brunton et al. Reference Brunton, Boutsalis, Gill and Preston2020b), which suggested loss of activation as the mechanism of resistance in this population. The response of these populations is consistent with recent studies in which clomazone injury in cotton was reduced with phorate (Culpepper et al. Reference Culpepper, York, Marth and Corbin2001). Research with clomazone-resistant arabidopsis (Arabidopsis thaliana L.) revealed that upregulation of two P450 genes CYP81A15 and CYP81A21 was correlated with (>10-fold) resistance to clomazone (Guo et al. Reference Guo, Iwakami, Yamaguchi, Uchino, Sunohara and Matsumoto2019), suggesting P450-mediated metabolism of clomazone as a possible mechanism. It is possible that a similar mechanism could be selected in L. rigidum.
Understanding the risk of resistance evolution to new herbicide discoveries requires early examination of the activity of herbicides and identifying the likelihood of metabolic resistance in weed populations so that resistance can be managed proactively (Busi et al. Reference Busi, Gaines, Walsh and Powles2012; Yu and Powles Reference Yu and Powles2014). The evolution of metabolic resistance and unexpected failures of key herbicides has been well reported in weed species globally (Hall et al. Reference Hall, Tardif and Powles1994; Mangin et al. Reference Mangin, Hall and Beckie2016; Preston et al. Reference Preston, Tardif, Christopher and Powles1996; Tardif and Powles Reference Tardif and Powles1999). Resistance evolution to bixlozone and clomazone in L. rigidum highlights the significant challenges in managing this species effectively while maintaining the sustainability of diverse SOA herbicides into the future. Furthermore, the resistance shift observed in both herbicides following two generations of recurrent selection at full field rates demonstrates the capacity of L. rigidum to rapidly evolve resistance without any prior exposure. The mechanism of resistance to clomazone and bixlozone in L. rigidum remains unknown; however, it is clear there are existing resistance mechanisms present in multiple-resistant populations. In a field context, use of this herbicide could rapidly select for resistant individuals, unless a diverse array of herbicide, non-herbicide, and cultural weed control tactics are being implemented (Beckie Reference Beckie2006).
Acknowledgments
The authors acknowledge the Grains Research and Development Corporation for funding this research and FMC and Barmac Industries for the supply of herbicide and inhibitor for experiments. No conflicts of interest have been declared.
