Glyphosate-Resistant Italian Ryegrass ( Lolium perenne L. spp. Multiﬂorum ) Control and Seed Suppression in Mississippi

: Italian ryegrass is a major weed problem in wheat ( Triticum aestivum L.) production worldwide. Two separate studies were conducted in Stoneville, Mississippi to evaluate: (1) the e ﬃ cacy of herbicides available to Mississippi producers for controlling glyphosate-resistant (GR) Italian ryegrass (control study), and (2) fall burndown herbicide seed suppression study. Results of the control study showed that ﬂufenacet / metribuzin EPOST followed by (fb) pinoxaden LPOST (standard treatment) provided 93% control of GR Italian ryegrass. Some other treatments provided comparable Italian ryegrass control (92% to 97%) as the standard treatment in 2017. Italian ryegrass control in the seed suppression study was 100%, 100%, 67.5%, 97%, and 99.5% from the application of the following treatments: (1) S -metolachlor + ﬂumioxazin + paraquat in October–November fb glyphosate + clethodim in January–February fb gramoxone as needed (weed-free check); (2) S -metolachlor + ﬂumioxazin + paraquat in October–November; (3) ﬁeld cultivator (disk) in October–November; (4) glyphosate + clethodim in January–February; and (5) ﬁeld cultivator in October–November fb glyphosate + clethodim in January–February, respectively. The remaining Italian ryegrass from the application of treatments 3, 4, and 5 produced 65,700; 1008; and 9 seeds m − 2 , respectively. Seed suppression study highlights the importance of 100% control that is required to manage GR Italian grass. management


Introduction
Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot) is among the top ten most abundant and problematic weeds for wheat production in the mid-southern United States [1,2]. Ryegrass weed spp., has a high capacity for evolving complex herbicide resistance patterns, making them one of the most troublesome weeds to control [3]. Hybrids of cultivated Italian ryegrass species and Italian ryegrass have been reported to escape along roadsides which have become problematic to control. Carey [4] and Van Wychen [5] reported that Italian ryegrass is the most troublesome and second most common weed for wheat producers in Mississippi. Italian ryegrass competitive indices including leaf development rate and plant height were reported to be greater than wheat [6]. Stone, et al. [7] reported that failure to control Italian ryegrass in wheat resulted in greater ryegrass root density, hence, creating an environment for an increased competition of ryegrass for moisture and nutrients [8].
Historically, Italian ryegrass has been controlled in crop production systems and along roadsides with herbicides [9]. Since 1998, populations of Italian ryegrass have been documented to be resistant to glyphosate, one of the most commonly used herbicides for burndown applications. Powles, et al. [10] reported the first evidence of glyphosate-resistance (GR) in rigid ryegrass. In an orchard in Australia (Orange, New South Wales, Australia), a rigid ryegrass (Lolium rigidum Gaud.) population was exposed to two to three applications of glyphosate year −1 for 15 yr and exhibited a 7 to 11-fold greater resistance than susceptible populations [11]. In a fruit orchard in Chile, rigid ryegrass populations exposed to three glyphosate applications every year for 8 to 10 years exhibited 2 to 4-fold greater resistance than susceptible populations [12]. In 2003, a 5-fold level of resistance to glyphosate was documented in Oregon [13]. The first GR Italian ryegrass in row crop production in the United States was documented by Nandula, et al. [14] and found two separate populations to be resistant to glyphosate rates up to 0.84 and 1.68 kg ae ha −1 [14]. In the mid-southern United States populations of GR Italian ryegrass have been documented in Arkansas, Louisiana, North Carolina, and Tennessee [15].
In wheat production, Italian ryegrass control has heavily relied on diclofop, an acetyl-CoA-carboxylase (ACCase-) inhibiting herbicide that only controls grass weed species [9]. Control options have become limited due to diclofop-resistant Italian ryegrass [16]. Aside from glyphosate, populations of Italian ryegrass in Mississippi have been found resistant or multiple-resistant to glyphosate, acetolactate synthase-inhibitors (ALS), and most recently, clethodim, an ACCase-inhibiting herbicide [15,17]. With populations evolving resistance to one or multiple herbicide chemistries, the ability for Italian ryegrass to mature and return seed back to the soil seedbank is becoming more prominent. In Arkansas, Bararpour, Norsworthy, Burgos, Korres, and Gbur [3] reported populations of Italian ryegrass plants producing 20,000 to 45,000 seed plant −1 . Natural infestations of Italian ryegrass of Arkansas were ±323 plants m −2 ; furthermore, with interference due to densities of this magnitude wheat yield reductions were reported to be as high as 72% over 6 years [18].
Herbicide control options continue to be the most effective option for producers because of time and money. Bararpour, et al. [19] reported 93% control of GR Italian ryegrass with flufenacet + metribuzin at early POST (EPOST) fb pinoxaden late POST (LPOST) on one-to two-leaf Italian ryegrass. In a seed suppression study, Bararpour,et al. [20] reported 100% control and seed suppression with treatments containing S-metolachlor + flumioxazin + paraquat in October-November fb glyphosate + clethodim in January-February fb paraquat as needed; and S-metolachlor + flumioxazin + paraquat in October-November. These studies emphasize the importance of achieving 100% control of Italian ryegrass and ways to prevent seed returns to the soil seedbank. The high capacity of seed production will lead to increased seed deposition into the soil seedbank and may increase the evolution of herbicide-resistant populations of Italian ryegrass [3]. Combining these factors will ultimately lead to herbicide failure and cause technologies that are available to producers to collapse.
Heavy reliance on weed control programs by the use of herbicides may cause more herbicide chemistries to fail in providing acceptable control. Italian ryegrass continues to cause problems for producers in fall and spring burndown applications. The use of tillage, delayed planting, increased seeding rates, narrow rows, and crop rotation are some of the cultural practices for controlling herbicide-resistant Italian ryegrass [21]. Therefore, research should be conducted by incorporating cultural practices with herbicide programs to help mitigate Italian ryegrass seed production, thereby, decreasing the return of seed to the soil seedbank to delay the evolution of herbicide resistance, and to preserve herbicide technologies that are available. The objective of this study was to evaluate the efficacy of herbicides available to Mississippi producers for controlling GR Italian ryegrass. Additionally, we investigate the weed management strategy targeting GR Italian ryegrass seed production or seed deposition to the soil seedbank in Mississippi in order to facilitate the solution of current weed problems (preventing/reducing the occurrence of herbicide resistance, reducing weed fecundity and soil seedbank).

Glyphosate-Resistant Italian Ryegrass Control Study
A two-year field study was conducted (2016-2017 and 2017-2018) at the Mississippi State University Delta Research and Extension Center in Stoneville, MS on Sharkey clay (very-fine, smectitic, thermic Chromic Epiaquerts) with 1.7% organic matter and pH 7.1. The experimental design was a randomized complete block design with 12 treatments and four replications (Table 1). Research plots contained a uniform, natural infestation (±39 plants m −2 ) of GR Italian ryegrass. The plot size was 3 × 10 m. The herbicide treatments, application rates, and application timings, trade names, and manufacturer information are listed in Table 1. A CO 2 -pressurized backpack sprayer with four 110,015 TTI nozzles (TeeJet Technologies, Springfield, IL, USA) mounted on a handheld boom calibrated to deliver 140 L ha −1 at 276 kPa was used for applying the treatments. Italian ryegrass control at five different timings was recorded 3, 5, 7, 11, and 15 weeks after treatment application (WAT). Weed control was scored on a scale of 0 to 100 where 0 is no weed control and 100 being complete weed control or plant death.

Glyphosate-Resistant Italian Ryegrass Seed Suppression Study
Seed suppression study was conducted based on the reasoning that the evolution of herbicide-resistant weeds from seed deposition (input) of the un-controlled weeds into soil seedbank is greater than the output (seed loss from soil seedbank through germination and weed control programs, predation, decay, etc.). Italian ryegrass seed suppression study was conducted in 2016-2017 at the Delta Research and Extension Center in Stoneville, MS on Sharkey clay (very-fine, smectitic, thermic Chromic Epiaquerts) with 1.7% organic matter and pH 7.1. The experimental design was a randomized complete block design with six treatments and four replications ( Table 2). The plot size was 3 × 10 m. The herbicide treatments, application rates, application timings, trade names, and manufacturer information are listed in Table 2. A CO 2 -pressurized backpack sprayer with four 110,015 TTI nozzles (TeeJet Technologies, Springfield, IL, USA) mounted on a handheld boom calibrated to deliver 140 L ha −1 at 276 kPa was used for applying the treatments. Weed control was scored on a Agronomy 2020, 10, 162 4 of 10 scale of 0 to 100 where 0 is no weed control and 100 being complete weed control or plant death. Additionally, the number of plant m −2 , number of spikes m −2 , and the total number of seeds m −2 were also recorded from each replication. The total number of seeds m −2 were calculated by [(number of spikes m −2 ) × (number of spikelets per spike) × (number of seeds per spikelet)].

Statistical Analysis
Statistical analysis was performed in SAS (SAS Institute Inc., Cary, NC, USA) using GLM procedure. In the Italian ryegrass control study, weed control data collected at five different timing were treated as dependent variables and herbicide treatments were treated as independent variables. Additionally, weed control data from 2016-2017 and 2017-2018 for Italian ryegrass control study were analyzed separately, because of significant differences in the year. For Italian ryegrass seed suppression study percent weed control, plants m −2 , number of spikes m −2 , and the total number of seeds m −2 were dependent variables and number of herbicide treatments were independent variables. For the data that met the assumptions of ANOVA, means were separated using Fisher's protected Least Significant Difference (LSD) (α = 0.05).
Single herbicide application program included mesosulfuron EPOST (T1), T2 (flufenacet/metribuzin + pinoxaden EPOST), T7 (pyroxasulfone EPOST), and T8 (mesosulfuron LPOST). Only T2 provided >90% GR Italian ryegrass control during two years of study. The application timing of T2 was EPOST targeted Agronomy 2020, 10, 162 7 of 10 at one-to two-leaf stage of Italian ryegrass. Flufenacet herbicide in flufenacet/metribuzin + pinoxaden is reported to have herbicide residual activity and inhibit the synthesis of fatty acids for wax and phospholipid formation in plant leaves [22]. Metribuzin herbicide in T2 is a photosynthesis inhibitor; whereas, pinoxaden inhibits the enzyme Acetyl-CoA-Carboxylase (ACCase), resulting in interruption of synthesis of fatty acids and impacting the formation of biomembranes [23,24]. The multi-mode of action and herbicide residual activity provided by T2 resulted in the highest GR Italian ryegrass control. Several studies have reported 84 to 96% Italian ryegrass control with flufenacet/metribuzin herbicides [16,19,20,[25][26][27][28]. Glyphosate-resistant Italian ryegrass control in 2017-2018 using mesosulfuron and pyroxasulfone ranged between 45% to 85% (Figure 2A-E). Mesosulfuron is a member of sulfonylurea group of herbicides and its mode of action is inhibition of acetolactate synthase (ALS); whereas, pyroxasulfone herbicide blocks lipid biosynthesis through inhibition of several very-long-chain fatty acids [29,30]. Kuk and Bugos [29] reported that the resistance mechanism of Italian ryegrass to mesosulfuron is partly due to an alteration in the target enzyme, ALS. Mesosulfuron LPOST (T8) was applied to GR Italian ryegrass when it had three-to four-tillers and it is possible that ALS inhibition was not 100%, therefore control ranged from 66% to 70%. Hulting, Dauer, Hinds-Cook, Curtis, Koepke-Hill and Mallory-Smith [28] studied Italian ryegrass control with pyroxasulfone, flufenacet, and flufenacet + metribuzin and reported that pyroxasulfone applications controlled Italian ryegrass between 65% to 100% and flufenacet and flufenacet + metribuzin treatments provided similar control as that of pyroxasulfone.
Multiple herbicide application programs included T3, T4, T5, T6, T9, T10, and T11. Flufenacet/metribuzin EPOST fb pinoxaden LPOST (T11) was the only herbicide application program that provided 93% and 87% control of GR Italian ryegrass in 2016-2017 and 2017-2018, respectively. Flufenacet + metribuzin was reported to control the Italian ryegrass population ranging from 66% to 97% [8,16]. Pinoxaden is an ACCase-inhibiting herbicides and has been reported previously to control diclofop-resistant Italian ryegrass [19]. A single application of pinoxaden resulted in 58% Italian ryegrass control; whereas, metribuzin fb pinoxaden showed Italian ryegrass control ranging between 84% to 89% [19]. Mesosulfuron EPOST fb mesosulfuron LPOST (T4) and metribuzin EPOST fb mesosulfuron LPOST (T5) showed GR Italian ryegrass control ranging between 65% to 97% which is similar to that reported by Bararpour, Korres, Burgos, Hale, and Tseng [19]. Ryegrass control by application of metribuzin EPOST fb metribuzin LPOST ranged between 79% to 94% [25]. Among all single and dual herbicide application programs T2 and T11 showed >87% control at 15WAT over two years of the study period. It is important to note that resistance to ACCase and ALS inhibitors in Italian ryegrass present a serious threat to wheat growers. Herbicide treatment program including flufenacet/metribuzin and pinoxaden either applied EPOST or in a combination of EPOST and LPOST provided GR Italian ryegrass control; however, this control was highly variable and inconsistent. Limited herbicide options for Italian ryegrass control in wheat, resistance to most currently available herbicides, and diminishing prospects for novel modes of action being commercialized emphasize the need for 100% seed suppression of GR Italian ryegrass [9].

Glyphosate-Resistant Italian Ryegrass Seed Suppression Study
In the seed suppression study, T1 had S-metolachlor + flumioxazin + paraquat applied in October-November fb an application of glyphosate and clethodim in Jan-Feb and paraquat applied as need showed 100% GR Italian ryegrass control ( Figure 3A). Similarly, T2 had S-metolachlor + flumioxazin + paraquat applied in October-November also provided 100% GR Italian ryegrass control. Treatment 3 was a field cultivator treatment and GR Italian ryegrass control was lowest (68%) ( Figure 3A). Treatment 4 and T5, glyphosate + clethodim without and with field cultivator showed 97% and 99.5% control, respectively. Plots that received T3 (field cultivation) had 18 GR Italian ryegrass plants m −2 , which produced 292 spikes m −2 (each spike had 25 spikelets and each spikelet had 9 seeds) further producing 65,700 seeds m −2 ( Figure 3B-D). Similarly, T4 with 97% control had 1 GR Italian ryegrass plant m −2 which accounted for 18 spikes m −2 (each spike had 14 spikelets and each spikelet had four seeds) resulting in producing 1008 seeds m −2 . Plots that received T5 (field cultivator in October-November fb glyphosate + clethodim in January-February with 99.5% GR Italian ryegrass control) average 0.009 plants m −2 . The remaining GR Italian ryegrass plants were able to produce 0.75 spike m −2 . Each spike had six spikelet's and each spikelet had two seeds. Therefore, the remaining GR Italian ryegrass plants produced 9 seeds m −2 . The untreated control had 39 GR Italian ryegrass plants m −2 which produced 454 spike m −2 (each spike had 27 spikelets and each spikelet had 8 seeds); therefore, GR Italian ryegrass plants in the untreated check plot produced 98,064 seeds m −2 ( Figure 3B-D).
Agronomy 2020, 10, x FOR PEER REVIEW 8 of 10 had 9 seeds) further producing 65,700 seeds m −2 ( Figure 3B-D). Similarly, T4 with 97% control had 1 GR Italian ryegrass plant m −2 which accounted for 18 spikes m −2 (each spike had 14 spikelets and each spikelet had four seeds) resulting in producing 1008 seeds m −2 . Plots that received T5 (field cultivator in October-November fb glyphosate + clethodim in January-February with 99.5% GR Italian ryegrass control) average 0.009 plants m −2 . The remaining GR Italian ryegrass plants were able to produce 0.75 spike m −2 . Each spike had six spikelet's and each spikelet had two seeds. Therefore, the remaining GR Italian ryegrass plants produced 9 seeds m −2 . The untreated control had 39 GR Italian ryegrass plants m −2 which produced 454 spike m −2 (each spike had 27 spikelets and each spikelet had 8 seeds); therefore, GR Italian ryegrass plants in the untreated check plot produced 98,064 seeds m −2 ( Figure  3B-D).  Table 2. Letters show the least squared difference between the treatments at α = 0.05.
One of the most critical issues for weed scientists today is the management of herbicide-resistant weeds in a long-term spatiotemporal scale. An efficient weed management program should focus on eliminating crop-weed interference but also maintain this result for as long as possible by preventing/delaying the occurrence of herbicide-resistant weeds. One of the ways of doing this is to stop weed seed deposition. We believe that if the input (seed deposition to the soil seedbank from the remaining or un-control weeds) is greater (>) than the output (seed loss from soil seedbank through germination and weed control programs, predation, decay, etc.), the resistant-weeds will be evolved and the technology or herbicide control program will be lost.
Therefore, one of the best ways to preserve the technology or to maintain the technology for a longer period is to stop weed seed deposition to the soil seedbank (no seeds means no weeds or no herbicide resistance weeds). In this study, it seems that weed control programs that provided around 90% control were a good program. However, the seed suppression study showed that the plots which received a treatment that controlled 97% of GR Italian ryegrass, deposited >1008 seed to the soil seedbank. This means that resistance will be developed in a matter of time since the input (seed deposition to the soil seedbank) is greater than the output (seed withdrawal from soil seedbank).  Table 2. Letters show the least squared difference between the treatments at α = 0.05.
One of the most critical issues for weed scientists today is the management of herbicide-resistant weeds in a long-term spatiotemporal scale. An efficient weed management program should focus on eliminating crop-weed interference but also maintain this result for as long as possible by preventing/delaying the occurrence of herbicide-resistant weeds. One of the ways of doing this is to stop weed seed deposition. We believe that if the input (seed deposition to the soil seedbank from the remaining or un-control weeds) is greater (>) than the output (seed loss from soil seedbank through germination and weed control programs, predation, decay, etc.), the resistant-weeds will be evolved and the technology or herbicide control program will be lost.
Therefore, one of the best ways to preserve the technology or to maintain the technology for a longer period is to stop weed seed deposition to the soil seedbank (no seeds means no weeds or no herbicide resistance weeds). In this study, it seems that weed control programs that provided around 90% control were a good program. However, the seed suppression study showed that the plots which received a treatment that controlled 97% of GR Italian ryegrass, deposited >1008 seed to the soil seedbank. This means that resistance will be developed in a matter of time since the input (seed deposition to the soil seedbank) is greater than the output (seed withdrawal from soil seedbank). Weed