Plant Material and Plants Preparation

Seed samples of Italian ryegrass suspected of being resistant to quizalofop were collected in 2010 from alfalfa fields after farmers' complaints to local extension services about poor herbicide efficacy. Seeds were collected from at least 10 to 15 plants (Panozzo et al. 2015) that had survived a quizalofop treatment at seven sites located in Ravenna province (Emilia-Romagna region, Italy) at least 2 km apart from each other. All alfalfa fields were cultivated for dual forage and seed production and treated with quizalofop every year after the first forage cut. Two susceptible populations were included in the experiments: the standard commonly used in our laboratory (Rigid ryegrass [Lolium rigidum Gaudin] S-204L) and another collected in the same area as the putative resistant ones (Lolium multiflorum S-389). After collection, seeds were cleaned, stored in double paper envelopes, and exposed to the natural indoor fluctuations of temperature and humidity. Experiments were conducted on the experimental farm of Padova University at Legnaro, Italy (45.35°N, 11.95557°E).

Plants used in all experiments were grown from seeds vernalized for 7 d at 4 C to break dormancy and to obtain simultaneous germination. Vernalized seeds were put in petri dishes containing 0.6% (wt/vol) agar and placed in a germination cabinet at 25/15 C (day/night temperature) and 12-h photoperiod with neon tubes providing a photosynthetic photon flux density (PPFD) of 15 to 30 μmol m⁻² s⁻¹. Germinated seedlings at a similar growth stage were transplanted into plastic trays (32.5 by 26.5 by 9.5 cm) for the greenhouse experiments or into pots for the outdoor dose–response experiment (15 by 15 by 20 cm). Plastic trays and pots were filled with a standard potting mix (60% silty loam soil, 15% sand, 15% perlite, 10% peat). Plants were watered as needed to keep the substrate at or near field capacity.

Whole-Plant Resistance Testing

Preliminary Greenhouse Screenings. Thirty seedlings at a similar growth stage were transplanted into plastic trays and placed in a heated greenhouse in which the average daily minimum and maximum temperatures were 16.5 and 27.7 C, respectively. The experimental layout was a completely randomized design with two replicates of 30 plants each. Herbicides were applied at the three- to four-leaf stage (BBCH 13 to 14; Hess et al. 1997) as commercial formulations plus adjuvants (when required) using a precision bench sprayer with a boom equipped with three flat-fan hydraulic nozzles (TeeJet XR11002-VK; Teejet Technologies, Springfield, IL), 0.5 m apart, delivering a spray volume of 300 L ha⁻¹ at a pressure of 215 kPa and speed of 0.75 m s⁻¹. A spray volume of 200 L ha⁻¹ and TeeJet TP11001-VH hydraulic nozzles were used for glyphosate treatments. An untreated control for each population was included in all experiments.

Italian ryegrass populations were screened twice, once during winter 2011 and once during spring 2012. Three ACCase inhibitors were tested at the recommended field dose (1×) and at three times that rate (3×). However, for glyphosate, the recommended field dose (1×) and double that rate (2×) had previously been identified as suitable for glyphosate-resistance screenings (Collavo and Sattin 2012). The other four herbicides with different SoAs were tested at the recommended field dose only (Table 1).

Table 1.

Herbicide common and trade names, herbicide rate in active ingredients (ai) or acid equivalents (ae), name of respective herbicide manufacturer, and manufacturer's city, state, Web site, and recommended field dose (1×).

a 

*, recommended field dose in Italy (1×); **, minimum label dose recommended at the time the populations were sampled for annual weed species.

In the greenhouse, light was supplemented during a 14-h photoperiod using 400 W metal-halide lamps, which provided a PPFD of about 150 μmol m⁻² s⁻¹. At 4 wk after treatment (WAT), plant survival was recorded. Plants were assessed as being dead if they showed no active growth, regardless of color or other appearance (Panozzo et al. 2015). Populations were classified as resistant when more than 20% of plants survived the herbicide dose at 1× and highly resistant when survivors were more than 20% at a herbicide dose of 1× and more than 10% at herbicide dose of 3× (Panozzo et al. 2015).

Outdoor Dose–Response Bioassay

Quizalofop efficacy on the seven populations of Italian ryegrass plus a susceptible population (S-204L) was also tested in an outdoor dose–response experiment during April to May 2012. Plants were treated at BBCH stages 13 to 14, and the experimental layout was a completely randomized design with three replicates of nine plants each. Herbicide doses ranged from 2.3 to 2,400 g ai ha⁻¹. Each population was sprayed with eight doses plus an untreated check, and the dose range varied based on the results obtained from the greenhouse experiments. Spraying equipment was as described in the previous section. Average daily minimum and maximum temperatures during the experiment were 8.7 and 18.4 C, respectively.

Plant survival and fresh weight per pot were recorded at 4 WAT. Plants were assessed as being dead if they showed no active growth, regardless of color or other appearance (Panozzo et al. 2015). Survival and fresh weight were expressed as a percentage of the untreated control. The 50% lethal dose (LD₅₀) and LD₈₀ (based on plant survival), 50% growth reduction (GR₅₀), and GR₈₀ (based on fresh weight), and their relative standard errors, were calculated using a nonlinear regression analysis performed with the macro BIOASSAY97 (version 2.651, Onofri A, Borgo XX Giugno 74, University of Perugia, Italy) running in Windows (Microsoft, Redmond, WA) Excel. The macro is based on a log–logistic equation to fit the data:

where Y is the fresh weight or survival; C and D are the lower and upper asymptotes at higher and zero doses, respectively; I₅₀ is the herbicide dose resulting in a 50% inhibition in plant survival (LD₅₀) or biomass (GR₅₀); and b is the slope. To calculate the 80% inhibition in survival or growth, I₈₀ was been used in the log–logistic equation.

The procedure estimates the standard error of the parameters and performs the Box–Cox power transformation family. Data were analyzed as described in Seefeldt et al. (1995) by regressing all the considered curves together using independent parameters. The complex model with independent parameters for each population was then compared with relatively simplified models having common parameters among curves. The lack-of-fit F test was performed at each step, and the simplification stopped when a significant lack of fit occurred. For biological reasons, and to improve the estimates of other parameters, the upper asymptote was forced to 100, whereas the lower asymptote was not constrained to allow an appropriate adjustment of the regression curves with the observed data of resistant or susceptible populations. The resistance indexes—RI = LD₅₀ (or LD₈₀, GR₅₀, GR₈₀) of the resistant population (R)/LD₅₀ (or LD₈₀, GR₅₀, GR₈₀) of the susceptible population (S)—for each putative resistant population were calculated.

ACCase Gene Sequencing

Plant material for molecular analyses was collected from the second greenhouse screening. Fresh leaf tissue from 10 plants per population that survived a quizalofop treatment at the dose 1× was analyzed. Amino acid substitutions in the carboxyl transferase (CT) domain of the ACCase gene responsible for target-site resistance were investigated. A bulk of leaves of the untreated susceptible S-204L and S-389 were used as wild-type controls.

The leaf samples were ground to powder using an electric drill (Skil, Breda, The Netherlands) equipped with a plastic pestle (Sigma-Aldrich, Saint Louis, MO). Genomic DNA was extracted following the CTAB method (Doyle and Doyle 1987) and then quantified through spectrophotometer analysis using a NanoDrop 2000C (Thermo Scientific, Wilmington, DE). The polymerase chain reaction (PCR) analysis was performed using a T1 Thermocycler (Biometra, Göttingen, Germany). An 1,100-base pair region of the ACCase CT domain was amplified using the primers LOL_FOR (5′-CTGTCTGAAGAAGACTATGGCCG-3′) and LOL_REV_CT (5′-ATGCATGGGTAGGCTTGATCCAG-3′). The reaction volume was 50 μL including 200 ng of genomic DNA, 0.6 μM of each ACCase primer, 0.2 mM of each deoxynucleotide triphosphate, 2.5 mM MgCl₂, 10 μm of 5× Colorless GoTaq Flexi buffer (Promega Corporation, Fitchburg, WI), and 1.25 μm of GoTaq G2 Hot Start polymerase (Promega). Amplification was performed using the following program: denaturation step for 2 min at 95 C, 35 cycles of 30 s at 95 C, 30 s at 58 C, and 75 s at 72 C, followed by a final extension step of 5 min at 72 C. The PCR product was directly purified with the NucleoSpin Gel and PCR Clean UP kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions and sequenced using the primers LOL_FOR and LOL_FOR_SEQ (5′-GAGGTGGCTCAGCTATGTTCCTG-3′) with an ABI 3730 XL sequencer (Applied Biosystem, Foster City, CA) by BMR Genomics (Padova, Italy). Sequences were edited with FinchTV 1.4.0 software (Geospiza Inc., Seattle, WA) and nucleotide sequences were manipulated using MEGA 5.05 software (Tamura et al. 2007). The amino acid positions in the CT domain of the ACCase gene, known to endow herbicide resistance, were investigated (Kaundun 2014).

Effect of Growth Stage on Quizalofop Efficacy

To quantify the effect of weed age/phenological stage on quizalofop efficacy, a dose–response experiment was performed in the greenhouse during February to April 2012 on two quizalofop susceptible Lolium populations (S-204L and S-389) treated at three different phenological stages: three to four leaves (BBCH scale 13 to 14), hereafter, called A; tillering (BBCH scale 14 to 21), hereafter, called B; and regrowth (shoots were cut about 3 cm above the soil surface, and the regrowth was treated 12 d later), hereafter, called C. The experimental layout was a completely randomized design with three replicates of 15 plants (plants/tray/treatment). The herbicide was applied at eight doses calculated using a geometric progression ranging from 4.7 to 150 g ai ha⁻¹. An untreated check was also included. Spraying equipment was as described above. The average daily minimum and maximum temperatures during the experimental period were 16.5 and 27.7 C, respectively. Plant survival and fresh weight were recorded at 4 WAT. Plants were assessed as being dead if they showed no active growth, regardless of color or other appearance (Panozzo et al. 2015).

Data were recorded and analyzed as reported above and LD₅₀, LD₉₀, GR₅₀, GR₉₀, and their relative standard errors were calculated. For biological reasons, and to improve the estimates of other parameters, the upper and lower asymptotes were forced to 100 and 0, respectively. A tolerance index (TI; de Mol et al. 2015) was calculated to express variable susceptibility at different phenological stages. It was defined as the ratio between the LD₅₀ (or LD₉₀, GR₅₀, GR₉₀) of a specific phenological stage (i.e., tillering or regrowth) and the LD₅₀ (or LD₉₀, GR₅₀, GR₉₀) of the “optimum” phenological stage treatment (i.e., three to four leaves).

Field Experiments

The field experiments were repeated twice, once in 2011 and once in 2012, to evaluate the efficacy of quizalofop on a resistant population of Italian ryegrass. The two alternative herbicides authorized in Italy for alfalfa crops (propyzamide and imazamox) were also tested, as well as herbicides reported to be used in the field history. The experiment was set up at a commercial farm near Ravenna, Italy (44.516667°N, 12.85°E; 8 m above sea level) where population 375 was originally sampled. The alfalfa-based cropping system was the same as described above. The experiment was repeated in two adjacent fields for 2 consecutive yr (the fourth and fifth years of continuous alfalfa cultivation). Field records showed that the use of ACCase inhibitors had been continuous for at least 5 yr, and glyphosate was occasionally sprayed on not actively growing alfalfa, that is, at the end of winter. The experimental layout was a randomized block design with four replicates; the size of each plot was 20 m² (2.5 by 8 m).

Quizalofop, propyzamide, and imazamox (Table 1) were sprayed at the recommended field doses. Untreated plots were also included in the experiments. Herbicides were applied as commercial formulations (plus adjuvants when required) using a backpack sprayer with a 2.5 m boom equipped with five flat-fan hydraulic nozzles (Teejet 110-03 VK-XR), spaced 0.5 m apart, delivering a spray volume of 300 L ha⁻¹ at a pressure of 250 kPa, and sprayed at a speed of 1.1 m s⁻¹. Herbicides were applied in early February when alfalfa was not actively growing or in May, 10 d after the first forage cut.

Herbicide efficacy, in relation to the untreated control, was calculated in terms of both number of ears per square meter and the biomass of the Italian ryegrass. The number of ears per square meter was determined by counting the number of ears in four randomly placed quadrates of 0.5 m by 0.5 m, for a total sampled area of 1 m² plot⁻¹. Biomass was estimated using the visual estimated biomass (VEB) scale in the central portion of each plot. A visual comparison of plant biomass between treated and untreated plots was done giving a score from 10 (plants not affected by the herbicide) to 0 (plants clearly dead) to each treated plot (Panozzo et al. 2015). Weed assessments were done in April and June for treatments applied in February, whereas only in June for treatments applied in May.

The effects of control strategies on the infestation level were calculated using Abbott's formula:

Statistica 7 (version 7.0, StatSoft, Tulsa, OK) software was used to analyze the data. Preliminary analysis was done to normalize the data using Bartlett's and Levene's tests, but because of their variability, normalization was not possible, even by applying the most common data transformations {e.g., arcsen(P), arcsen[],, etc.}.

Characterization of Resistant Populations

Preliminary Greenhouse Screenings. The effect of screening replication was tested using ANOVA, which indicated that data from the two experiments were not significantly different (P < 0.05); therefore, data for each treatment were pooled across experiments. Five of seven populations were resistant to quizalofop (Table 2), whereas the other two (376 and 380) had less than 20% of survivors at the recommended field dose (Table 1). Resistant populations were selected on different farms through several quizalofop treatments to the regrowth of Italian ryegrass using a dose recommended for three- to four-leaf stage. It should be stressed that all populations came from alfalfa crops grown for dual production, and several thousand hectares were affected.

Table 2.

Plant survival (percentage ± SE) of Lolium spp. populations calculated based on two greenhouse screenings for each herbicide and dose tested.

The pattern of cross-resistance to other ACCase inhibitors differed among populations (Table 2). The results indicated that three of five populations resistant to the FOP herbicide were controlled by pinoxaden, a herbicide belonging to a different chemical family of ACCase inhibitors, the phenylpyrazolins (DEN). Populations 375 and 377 were cross-resistant to quizalofop and pinoxaden, whereas a few plants of population 388 survived the treatment with pinoxaden. Populations 378 and 379 were highly resistant only to quizalofop (Table 2). This is valuable information because pinoxaden is one of the most used graminicides in wheat, which is the crop usually rotated with alfalfa. A few individuals of population 375 and population 388 survived cycloxydim treatments (Table 2), suggesting that a resistance mechanism related to an altered ACCase enzyme is involved because cycloxydim is not metabolizable (Keshtkar et al. 2015). Chlortoluron controlled Italian ryegrass more than 90% and equal to susceptible checks (Table 2). The mix of two ALS inhibitors tested (mesosulfuron + iodosulfuron) adequately controlled all populations, and the few survivors were heavily injured and most likely unable to produce seeds. At dose 720 g ae ha⁻¹ (2×), glyphosate controlled Lolium species 100% (Table 2). This dose proved to be suitable for controlling Lolium spp. populations in Italy (Collavo and Sattin 2012). Summarizing, all quizalofop-resistant populations were controlled by herbicides having SoA different from ACCase inhibitors. However, the few survivors detected after the treatment with 360 g ae ha⁻¹ of glyphosate, representing the low dose commonly used to control Lolium spp., confirm that the efficacy of this important herbicide needs careful monitoring to maintain its control even in arable crops that are not genetically modified (Collavo and Sattin 2014). In this situation, it is fundamental to reduce Italian ryegrass seed set and to keep efficacy close to 100% to limit the selection of resistant alleles (Manalil et al. 2011; Neve and Powles 2005a).

Dose–Response Bioassays

The response of Lolium spp. to increasing quizalofop doses was adequately fitted by the log–logistic equation without any data transformation, with most standard errors of parameters being one order of magnitude lower than the parameters values. Plant survival and fresh weight gave similar results; therefore, only plant survival data are reported. The lack-of-fit F test on plant survival indicated that it was not possible to simplify the regressions to a model with a common lower asymptote and slope for all populations. The same results were obtained when grouping susceptible and resistant populations, so a single-curve approach was used.

Overall, data were consistent with the results previously obtained from the preliminary screening. According to the RIs (Table 3), populations 376 and 380 were considered susceptible because the RI based on the LD₅₀ was between 1 and 1.5, whereas LD₈₀ was between 1.9 and 2.5 and less than 20% of the plants survived the recommended field dose. However, a few plants (16.7 ± 5.56% and 11.1 ± 5.56% for populations 376 and 380, respectively; data not shown) survived the dose 4× (300 g ai ha⁻¹), indicating that the selection process was ongoing in these populations. Populations 375, 377, 378, and 388 were highly resistant to quizalofop, whereas population 379 showed a lower level of resistance. For population 375, it was not possible to calculate either the LD₅₀ or the LD₈₀ values because of the high plant survival rate (> 50%) at the highest dose sprayed (2,400 g ai ha⁻¹); therefore, the LD values are given as > 2,400 g ai ha⁻¹ (Table 3). No relation was detected between the slope and the resistance status.

Table 3.

Plant survival: lethal dose, the herbicide dose that reduce 50% of plant survival (LD₅₀ and LD₈₀) ± SE and slope (b) calculated through nonlinear regression analysis of quizalofop dose–response experiment. Resistant indexes (RIs) calculated based on susceptible check S-204L LD₅₀ or LD₈₀. Upper asymptote was fixed at 100.

Effect of Growth Stage on Quizalofop Efficacy

Plant survival and fresh weight gave similar results in this study, therefore only data on plant survival are presented and discussed. The susceptible rigid ryegrass S-204L, treated at stage A responded differently in the dose–response tests held in the greenhouse at different growth stages and the outdoor experiment. The LD₅₀ was 2.5 and 9.6 g ha⁻¹ in the indoor and outdoor experiments, respectively. This difference is likely caused by the diverse climatic conditions in the two environments, the herbicides usually being more active in a greenhouse.

As expected, in general, the older the plants, the less susceptible they were to the herbicide (Figure 1). In fact, large differences in quizalofop efficacy were recorded between phenological stages A and B for both populations, whereas the differences in efficacy between plants treated at stages B or C were smaller for population S-204L and negligible for population S-389 (Figure 1). The TI based on the LD₉₀ showed that to control the regrowth, it was necessary to use three to four times the herbicide dose suitable for younger plants (Figure 1). A few studies (Christoffoleti et al. 2005), albeit indirectly, have previously stressed the effect of weed age on herbicide efficacy. Kudsk (2014) also recently highlighted that herbicide rate and plant age were important, but often underappreciated, factors.

Figure 1.

Lethal dose, the herbicide dose that reduce 50% of plant survival (LD₅₀ [▪] and LD₉₀ [♦]) of Lolium spp. susceptible checks (S-204L and S-389) treated with quizalofop ethyl ester at different phenological development stages (A, three to four leaves, BBCH scale 13 to 14; B, tillering, BBCH scale 23 to 26; C, regrowth, BBCH scale 25 to 27). Vertical bars represent standard errors, values refer to tolerance indices calculated using the LD₅₀ or LD₉₀ of the correct phenological development stage (A).

The results indicate that the more advanced the phenological stage of application, the more difficult it was to control susceptible biotypes of Italian ryegrass with quizalofop. In field conditions, it is unlikely that weeds are found at a uniform phenological stage. This variability leads to a difference in the amount of active ingredient that reaches the target (per unit weight or leaf area). Unfortunately, in field conditions, the importance of the phenological stage of weeds is frequently overlooked, which often results in sublethal application rates. This may accelerate the selection of nontarget-site (polygenic) herbicide-resistant mechanisms, such as enzyme overexpression and detoxification, which might initially be hidden by the presence of target-site mutations that usually induce highly resistant biotypes. Several studies have demonstrated that recurrent selection with reduced rates of the P450-metabolisable ACCase herbicide diclofop-methyl results in rapid herbicide resistance evolution in rigid ryegrass (Busi et al. 2012; Manalil et al. 2011; Neve and Powles 2005a,b). If a herbicide is used at a sublethal dose, the more tolerant individuals will survive and possibly reproduce. In this way, resistance alleles will become progressively enriched in successive generations, resulting in a polygenic-based shift toward more resistant individuals. Evidence of sublethal drug dose selection for polygenic resistance is also known for bacteria (Olofsson and Cars 2007), fungi (Shaw 2006), and insects (Roush and McKenzie 1987). In rigid ryegrass, this type of selection was also described using glyphosate (Busi and Powles 2009). It can, therefore, be inferred that, despite the LD₉₀ always being below the recommended field rate for quizalofop (75 g ha⁻¹; Figure 1), repeated treatments in fields heavily infested with the regrowth of Italian ryegrass (stage C and beyond) have progressively selected quizalofop-resistant populations. This was confirmed by empirical observations of the alfalfa fields in which the number of quizalofop-treatment survivors progressively increased over the years.

Field Experiments

Bartlett's and Levene's tests revealed that the data were not normally distributed. Despite various types of data transformation, it was not possible to normalize the data; therefore, ANOVA could not be used and only standard errors (which do not require normally distributed data to be applied) are reported on the graphs. The data collected in the 2 yr were very similar and were, therefore, averaged.

Quizalofop efficacy was always very low. Only propyzamide gave satisfactory control of quizalofop-resistant Lolium, if the treatment was applied early in the season (i.e., late winter). The same herbicide did not provide a good control when applied in May because it is known to be poorly active on adult plants (Clay et al. 2006) and the environmental conditions in late spring are less favorable for exerting its herbicidal activity (Figure 2).

Figure 2.

Herbicide efficacy of the field treatments calculated using Abbott's formula, number of ears (A) and visual estimation of biomass (VEB) (B) of Italian ryegrass. All data are expressed as percentages of the untreated control. Treatments were applied in February and in May, 10 d after the first cut. Number of ears was recorded only in June for both treatments (applied in February and May), whereas VEB was recorded in April (dark bars) and June (light bars) for treatments applied in February but only in June (light bars) for treatments applied in May. Vertical bars represent standard errors calculated on the mean value of two experiments.

Weed phenological stage/age influenced the efficacy of all herbicides applied. The VEB recorded in June showed no differences between the plots of the untreated control and quizalofop or imazamox applied in February (i.e., VEB = 100%) because ryegrass regrew from survivors after the forage cut (Figure 2B). A slight decrease in the number of ears was observed in imazamox plots (−23%) sprayed in February (Figure 2A). Fewer ears were counted in all plots treated in May (Figure 2A). The efficacy of the treatments on the regrowth (i.e., May treatment) generally increased compared with the untreated control but remained unsatisfactory.

This study documents that even cropping systems, such as alfalfa, which are considered at low-risk of resistance evolution, if “stressed” by an intensive production system (i.e., dual seed–forage production and intense herbicide use) can be affected by herbicide resistance, particularly when no rotation of SoA is adopted in the overall cropping system. In Europe, no herbicide-resistant weeds have so far been reported in alfalfa crops for forage production only (about 1.5 M ha). It is also clear that herbicide resistance risk increases when dense infestations of species highly prone to evolve herbicide-resistant populations, such as Lolium spp., are not properly managed.

Despite alfalfa–wheat rotation being proposed in the literature for managing herbicide-resistant Lolium spp. (Doole and Pannell, 2008), the results from this study highlight associated issues. This is likely due to the dual forage and seed alfalfa production system studied. Alfalfa–wheat rotation can be adopted, if herbicides other than FOP herbicides are used in wheat and propyzamide is used in alfalfa. Other rotations between alfalfa and winter crops, such as barley (Hordeum vulgare L.) or some vegetables are precluded because only ACCase inhibitors are allowed to control Lolium spp. in these crops.

Herbicide resistance management is important to prevent weed seed production (Lutman et al. 2013) and to keep the infestation at a manageable level. Depletion of the soil weed seed bank is an excellent strategy to combat weeds, especially herbicide-resistant ones (Goggin et al. 2012). Different chemistries (i.e., ALS- and PSII-inhibiting herbicides are still effective), as well as nonchemical agronomic practices, should be used to increase diversity in the system. Lolium spp. can be managed effectively by avoiding continuous direct seeding or reduced tillage. Potential Lolium infestation can also be reduced through stale seedbed preparation when alfalfa is rotated with cereals, and the latter are sown late in the season. Curative chemical measures for FOP-resistant Lolium spp. in alfalfa include a timely and careful use of propyzamide or imazamox when alfalfa is not actively growing.