The First Case of Short-Spiked Canarygrass ( Phalaris brachystachys ) with Cross-Resistance to ACCase-Inhibiting Herbicides in Iran

: The weed Phalaris brachystachys Link. severely a ﬀ ects winter cereal production. Acetyle-CoA Carboxylase (ACCase)-inhibiting herbicides are commonly used to control this weed in wheat ﬁelds. Thirty-six populations with suspected resistance to ACCase-inhibiting herbicides were collected from wheat ﬁelds in the Golestan Province in Iran. A rapid test performed in Petri dishes and whole-plant dose–response experiments were conducted to conﬁrm and investigate the resistance level of P. brachystachys to ACCase-inhibiting herbicides. The seed bioassay results showed that 0.02 mg ai L − 1 clodinafop-propargyl (CP) and 1.36 mg ai L − 1 of the diclofop-methyl (DM) solution were the optimal amounts for reliably screening resistant and susceptible P. brachystachys populations. In the whole plant bioassay, all populations were found to be resistant to CP, resistance ratios ranging from 2.7 to 11.6, and all of the CP-resistant populations exhibited resistance to DM. Fourteen populations showed low resistance to cycloxydim, and thirteen of these populations were also 2-fold resistant to pinoxaden. The results showed that DM resistance in some P. brachystachys populations is likely due to their enhanced herbicide metabolism, which involves Cytochrome P450 monooxygenases, as demonstrated by the indirect assay. This is the ﬁrst report conﬁrming the cross-resistance of ACCase-inhibiting herbicides in P. brachystachys in Iran.


Introduction
Wheat (Triticum aestivum L.) is one of the most important crops in Iran. Approximately 52% of arable land in Iran is used for wheat cultivation, with a 23% yield reduction caused by weeds [1]. The annual Poaceae species short-spiked Canarygrass (Phalaris brachystachys Link) is a common and troublesome weed in winter cereals in Mediterranean countries [2]. This is a vigorous and prolific weed that can significantly reduce wheat and barley yields and has been shown to decrease wheat yield by 16 to 60% [3,4]. Currently, P. brachystachys is found in the northern part of the Iran, where it infests crops during autumn and winter [5].
The use of herbicides is the most efficient and economical means of controlling grass weeds, and several ACCase-inhibitors have been registered in Iran in the last three decades [6]. The target site of these herbicides is Acetyl-CoA Carboxylase (ACCase; EC 6.4.1.2), which is a key enzyme that catalyzes the primary step in fatty acid biosynthesis [7]. ACCase-inhibiting herbicides are classified into three major families: aryloxyphenoxypropionates (APP), cyclohexanediones (CHD), and phenylpyrazolines The thirty-seven seeds of suspected resistant P. brachystachys populations were collected from winter wheat fields of the Golestan province in Iran during the spring of 2015, 2016 and 2017 (four, twenty-nine and four populations each year, respectively). Additionally, one susceptible population was collected from the same region that had never been treated with herbicides in 2016. Seeds from at least 15 plants that had reached physiological maturity were randomly collected by hand and bulked. The seeds were air dried and stored in paper bags at room temperature until used in the experiment. A global positioning system unit used to take latitude and longitude measurements for each field, and their locations, were mapped (ArcGIS 9.2) ( Figure 1). Information regarding the collection position of each population is shown (Supplementary Materials).

Seed Bioassay
Does-response experiments were conducted using 9-cm diameter plastic Petri dishes. After breaking seed dormancy (seeds were immersed in sulfuric acid (98%) for 3 min then kept in Petri dishes containing moist filter paper with 5 mL distilled water for 72 h in a refrigerator at 4 • C in the dark), five germinated seeds were placed on two sheets of filter paper. Each Petri dish was considered to be one replicate, and the experiment was conducted with three replications for each herbicide and each population. This experiment was repeated three times. A 5-mL aliquot of an aqueous solution of the commercial formulation of DM was applied at 0, 0.1, 0.5, 1, 2, 4, 8, 16 and 32 mg ai L −1 , and CP was applied at concentrations of 0, 0.005, 0.02, 0.04, 0.08, 0.32, 0.64, 1. 28, 5.12, 20.46 and 40.96 mg ai L −1 . For each population, the control treatment (without herbicide solution) with 5 mL distilled water was also considered. Then, the Petri dishes were placed in an incubator at 25 • C. After seven days, the shoot lengths of the coleoptiles in all of the seedlings were measured and expressed as the percentage of the shoot length of coleoptile compared to the control [13].

Whole Plant Dose-Response Assay
The experiment was repeated three times and arranged in a completely randomized design with three replications for P. brachyhstachys populations with different doses of clodinafop-propargyl, diclofop-methyl, cycloxydim and pinoxaden. Seed dormancy was broken as previously described and, then, the germinated seeds were sown in 11-cm plastic pots filled with a mixture (1:2 v/v) of peat and soil and placed in a greenhouse. The pots were irrigated daily as required. Each replicate pot contained five plants. Four weeks after sowing, at the three-to-four-leaf stage (as BBCH scale in [13][14], herbicides were applied at different rates using a calibrated sprayer with a flat-fan nozzle (TeeJet 8001) to deliver 250 L ha −1 of spray solution at 200 kPa. One untreated control (without herbicide application) for each population has been used. The detailed information of the herbicides used for the dose-response assay is presented in Table 1. The plants were harvested and oven dried for 48 h at 70 °C 28 days after herbicide application, and the dry-weight data were recorded. The data were expressed as a percentage of the untreated control. It should be noted that the populations with a high resistance factor based on DM herbicide results were used in the dose-response assay with pinoxaden and cycloxydim herbicides.

Whole Plant Dose-Response Assay
The experiment was repeated three times and arranged in a completely randomized design with three replications for P. brachyhstachys populations with different doses of clodinafop-propargyl, diclofop-methyl, cycloxydim and pinoxaden. Seed dormancy was broken as previously described and, then, the germinated seeds were sown in 11-cm plastic pots filled with a mixture (1:2 v/v) of peat and soil and placed in a greenhouse. The pots were irrigated daily as required. Each replicate pot contained five plants. Four weeks after sowing, at the three-to-four-leaf stage (as BBCH scale in 13-14), herbicides were applied at different rates using a calibrated sprayer with a flat-fan nozzle (TeeJet 8001) to deliver 250 L ha −1 of spray solution at 200 kPa. One untreated control (without herbicide application) for each population has been used. The detailed information of the herbicides used for the dose-response assay is presented in Table 1. The plants were harvested and oven dried for 48 h at 70 • C 28 days after herbicide application, and the dry-weight data were recorded. The data were expressed as a percentage of the untreated control. It should be noted that the populations with a high resistance factor based on DM herbicide results were used in the dose-response assay with pinoxaden and cycloxydim herbicides.

Growth Assay in Combination with CytP450 Inhibitor
Seedlings of different DM-resistant (based on resistance factor) and S populations at the 3-4 leaf stage were treated with DM at the rate of 300 g ai ha −1 with and without amitrole (AM) to study whether metabolism was responsible for resistance in the resistant populations. AM was applied at 13.1 g ha −1 24 h prior to application of DM. Twenty-one days after application, the plants were harvested, and the shoot fresh weight was measured. The experiment was repeated three times and it included ten replicates per repetition.

Statistics Analysis
The data obtained from the Petri dish and pot experiments were fit to a nonlinear log-logistic regression model with four parameters in the "R" statistical software with the "drc" package [14].
In the model, y represents the shoot dry weight or shoot length of coleoptile (percentage of the untreated control) at a herbicide dose of x; c and d denote the lower and upper limits, respectively; b is the slope of the response curve at e; and e denotes GR 50 (or GR 90 ). The effective concentration of herbicide that caused 50% inhibition of the shoot length of coleoptile (EC 50 ) was calculated from the log-logistic regression model, which allowed us to screen resistant and susceptible populations according to the EC 50 Equation (1). From each model, the effective herbicide doses which inhibited plant growth by 50 and 90% (GR 50 and GR 90 ) with respect to the untreated control, were determined for each population. The resistance factor (RF), which is the ratio of the EC 50 , GR 50 or GR 90 of the resistant population to the EC 50 , GR 50 or GR 90 of the susceptible population, was considered as an index in order to compare the resistance levels of tested populations [15].

Seed Bioassay
A difference in the shoot length of the populations was visible after 7 days of incubation. The resistance factors and estimated nonlinear regression parameters for the applied herbicides are shown in Table 2. The four-parameter log-logistic model provided a good fit to the data (p < 0.001; R 2 > 0.96). The results of the Petri dish assays showed that the coleoptile length of the seedlings decreased according to a sigmoidal trend and that the decreasing shoot length of the S population observed at lower concentrations than the other populations. This confirmed that the susceptible population was more sensitive to herbicides than the other populations. Regarding DM, the estimated EC 50 was 1.36 mg ai L −1 for S, while for the other populations it ranged between 1.86 and 6.30 mg ai L −1 ( Table 2). In the Petri dish assays, with the increasing CP concentration, different responses were consistently observed, and all populations had shorter coleoptiles compared to their untreated controls. While 0.02 mg ai L −1 of CP inhibited 50% of the shoot length of the S population, for the other populations, it ranged from 0.07 to 0.29 mg ai L −1 of CP and the resistance factors ranged from 2.77 to 10.27 (Table 2).

Dose-Response Assay
We assessed representatives of all of the different groups of graminicides, such as clodinafop-propargyl, diclofop-methyl, pinoxaden and cycloxydim. The results showed that the susceptible population was considerably controlled by two APP herbicides. The other populations showed resistance to the APP herbicides, but the level of resistance varied substantially. The S population was inhibited by 50% with only 24.22 g ai ha −1 of CP compared with the recommended field amount of 80 g ai ha −1 . The other populations were resistant to the CP field dose, with resistance levels ranging from 2.7 to 11.6-fold based on the GR 50 values (Table 3). Among the 36 populations studied, the Kr15 and Kr16 populations had the largest resistance factor based on their GR 50 values ( Table 3). The estimated GR 50 values indicated different resistance factors (RF = GR 50 R/GR 50 S) to DM for the different populations. The S population was inhibited by 50% with only 279.57 g ai ha −1 of DM, while the amount required to reach GR 50 for the other populations was between 563.14 and 3059.90 g ai ha −1 . The estimated GR 90 for the S population was 866.63 g ai ha −1 , whereas the GR 90 values varied in the other populations from 2934.52 to 22929.67 g ai ha −1 ( Table 4).  For cycloxydim (CHD family herbicides), all populations were found to exhibit low resistance levels ( Table 5). The concentration of cycloxydim that led to a 50% inhibition of shoot growth in the S population was 46.35 g ai ha −1 and the cycloxydim resistance factor for all of the resistant populations was between 2-fold and 3-fold. The lowest GR 90 value for resistant populations was observed in AL21 (295.57 g ai ha −1 ), whereas a higher GR 90 value was recorded in Kr15 (425.97 g ai ha −1 ) (Table 5). Similarly, the pinoxaden herbicide was found to significantly reduce the growth of all the resistant populations, and low resistance levels were recorded for this ACCase-inhibiting herbicide in 13 populations. The pinoxaden GR 50 values of the resistant populations were approximately two times higher than for the S population, and a large reduction of shoot dry weight was found in all the resistant populations (Table 6). b = slope of curve around the dose giving 50% response; 3 SE = standard error; 4 R 2 = 1 − (sum of squares of the regression/corrected total sum of squares); 5 p-value = is the probability level of significance of the resistance factor; 6 GR 50 refers to the herbicides rates required for 50% dry weight reduction compared with the non-treated control; 7 RF 50 = Resistance Factor is calculated as (GR 50 resistant/GR 50 sensitive); 8 GR 90 = refers to the herbicides rates required for 90% dry weight reduction compared with the non-treated control; 9 RF 90 = Resistance Factor is calculated as (GR 90 resistant/GR 90 sensitive). b = slope of curve around the dose giving 50% response; 3 SE = standard error; 4 R 2 = 1 − (sum of squares of the regression/corrected total sum of squares); 5 p-value = is the probability level of significance of the resistance factor; 6 GR 50 refers to the herbicides rates required for 50% dry weight reduction compared with the non-treated control; 7 RF 50 = Resistance Factor is calculated as (GR 50 resistant/GR 50 sensitive); 8 GR 90 = refers to the herbicides rates required for 90% dry weight reduction compared with the non-treated control; 9 RF 90 = Resistance Factor is calculated as (GR 90 resistant/GR 90 sensitive).

Growth Assays in Combination with CytP450 Inhibitor
The responses of P. brachystachys populations to DM, with and without amitrol are shown in Table 7. The present study found that the combination of DM with amitrol was slightly more effective in the AL33, G04 and Kr15 populations than DM alone and pretreatment with amitrole significantly inhibited the growth of these populations compared to populations without amitrole. However, the fresh weight of the S population did not vary and was independent of the amitrole treatment.

Discussion
The seed bioassay method for determining resistant and susceptible populations has been previously utilized [16][17][18]. This method is regarded as the most rapid and simplest way to screen resistant and susceptible populations. In this study, this method was applied for the P. brachystachys populations. In preliminary tests, each APP herbicide was tested. It is necessary to detect resistance as early as possible to avoid the costly consequences of a resistance spread. Seed bioassays have been shown to be a useful and accurate tool for screening a large number of suspected resistant populations. The identification of concentrations that are effective at separating resistant and susceptible populations is important not only for the rapid diagnosis of potential resistance but also for the screening of seeds used for experiments. From this research, it was determined that the seed bioassay could be developed to be a feasible method to identify resistant populations of P. brachystacys. This method has been used to test resistance to ACCase-inhibitors in barnyardgrass (Echinochloa crusgalli) [19] and Johnsongrass (Sorghum halepense (L.) Pers.) [20]. Other researchers described a seed bioassay to detect grass weeds resistant to ACCase-inhibiting herbicides [21].
The dose-response assays confirmed that the P. brachystachys populations were resistant to DM and CP herbicides. The seed assay also confirmed the resistance to APP herbicides. According to the results of both the whole plant and seed bioassay, the resistance factor of most of the populations to CP were considerably higher compared to DM. No precise history of herbicide application in the sampled fields was available. However, Golestan is one of the most important provinces for producing wheat in Iran. The use of these herbicides has been the main approach to control weeds in wheat fields. The high percentage of resistance to CP and DM was expected because these two herbicides have a common basic molecular structure [22], and both have been extensively used to control grassy weeds during wheat cultivation, which is the most frequently grown crop in the area. These results indicate that resistance to these herbicides can be attributed to the use of a wheat monoculture in the sampling areas along with the repeated use of these herbicides for a long period of time [23]. Resistance to APP herbicides has been reported in littleseed canarygrass (Phalaris minor Retz.) [1]. Also, the level of cross-resistance to APP herbicides in Avena spp. has been reported [24]. Notably, most populations were highly resistant to APP herbicides, while their response to PPZ varied. Resistance to APP herbicides is not necessarily associated with resistance to pinoxaden. The AL21 population, which showed high resistance to APP herbicides, was susceptible to pinoxaden. However, the AL04 and Kr15 populations expressed high resistance to CP, with RF values of 9.4 and 11.5, respectively (Table 6). These populations also expressed high resistance to the same chemical class of APP herbicide, DM, with RF values of 8 and 6, respectively, but low cross-resistance was observed to cycloxydim (RF of 2.74 and 3.19, respectively) and pinoxaden (RF of 2.09 and 3.41, respectively) ( Table 5).The reduced control of some P. brachystachys populations by pinoxaden indicates cross-resistance to this herbicide, regardless of the fact that this herbicide has been used in Iran for the last few years. The whole-plant dose-response assays showed that the cross-resistance levels of ACCase inhibitors varied among P. brachystachys populations. APP presented the highest RF values, while the cross-resistance corresponding to CHD and PPZ herbicides was low.
The differences in the cross-resistance patterns in these resistant populations indicate that resistance evolved independently and that each resistant population has likely been exposed to a different selection pressure. Additionally, the differences indicate that more than one resistance mechanism is likely involved in these P. brachystachys resistant populations.
To test the hypothesis that enhanced DM metabolism is conferred by CytP450, a known CytP450-inhibitor, amitrole, was tested. Amitrol has long been used as an indicator of the involvement of P450 in metabolic resistance to ACCase herbicides [25][26][27]. These results of this experiment suggest that CytP450 monooxygenase-mediated metabolism could be present in these populations and contribute to the resistance phenotype. These results indicate that CytP450 is involved in DM-resistance in G04, Kr15 and AL33 populations of P. brachystachys and metabolic resistance could be the mechanism responsible for resistance in these populations ( Table 7).
The consecutive use of different herbicides with the same mode of action in wheat fields in the Golestan province led to the selection of resistant P. brachystachys individuals, and their numbers have increased within the populations. Today, resistant populations have been established in several parts of the province, and if the current weed/crop management method does not change, increasing selection pressure will result in further infestation of resistant populations. However, crop rotation and, consequently, different weed management methods would be the best way to control resistant P. brachystachys populations in this region. The results of this study clearly indicate that pinoxaden and cycloxydim have become ineffective at controlling some of the APP resistant P. brachystachys populations and these herbicides should not be considered as alternative herbicides for the effective control of resistant populations. Our results regarding this species are in agreement with the results reported by others regarding the different levels of cross-resistance patterns of different weeds resistant to the three groups of ACCase-inhibiting herbicides [18,24]. The Italian ryegrass (Lolium multiflorum) with DM resistance was also cross-resistant to pinoxaden [28]. Additionally, there was a level of cross-resistance to APP, CHD, and PPZ in bristly dogstail grass (Cynosurus echinatus) populations [27]. Hood canarygrass (Phalaris paradoxa) populations have also been reported to have cross-resistance to the APP, CHD and PPZ herbicides [7]. The insensitivity of the ACCase target site is the most common mechanism of resistance to ACCase-inhibiting herbicides [28]. However, resistance likely did not develop via a single mechanism; rather, multiple mechanisms, including enhanced metabolism, an altered target site, and other uncharacterized mechanisms, may be involved [29].

Conclusions
This is the first study confirming the cross-resistance of the aryloxyphenoxypropionates, cyclohexanediones and phenylpyrazolines herbicides in P. brachystachys in Iran. The CytP450 monooxygenase data in the present study indicate that a metabolic mechanism is probably involved in conferring cross-resistance among ACCase-inhibiting herbicides in some P. brachystachys populations. However, the resistance level cannot only be explained by herbicide metabolism to non-toxic forms, and other additional mechanisms should be studied. ACCase enzyme activity and gene analysis are needed to identify the resistance mechanisms in these populations. A goal for further research is the identification of the resistance mechanisms that are involved in ACCase inhibitor herbicides. We plan to study these mechanisms in the future; meanwhile, due to the results of the present study, resistance to ACCase inhibitors in P. brachystachys from Iran has been identified. In further studies, we will elucidate the resistance mechanisms of resistance by biochemical and molecular methods.