Introduction
The ryegrass or Lolium genus, native to the Mediterranean region of southern Europe, northwest Africa, and southwest Asia (Beddows Reference Beddows1973; Hubbard Reference Hubbard1968), comprises eight species (Bararpour et al. Reference Bararpour, Norsworthy, Burgos, Korres and Gbur2017). Rigid ryegrass (Lolium rigidum Gaudin), perennial ryegrass (Lolium perenne L.), and Italian ryegrass [Lolium perenne L. ssp. multiflorum (Lam.) Husnot] are considered highly valuable as forage, turf, and cover crops (Matzrafi et al. Reference Matzrafi, Preston and Brunharo2021). Factors contributing to the spread of weedy Lolium spp. across six continents (Bararpour et al. Reference Bararpour, Norsworthy, Burgos, Korres and Gbur2017) include assisted breeding programs targeting warmer regions (Matzrafi et al. Reference Matzrafi, Preston and Brunharo2021) and climate change. Climate change is likely to create suitable potential areas for Lolium species (Castellanos-Frias et al. Reference Castellanos-Frías, De Leon, Bastida and González-Andújar2016). Additionally, the presence of genetically diverse variants has accelerated acclimation (Matzrafi et al. Reference Matzrafi, Preston and Brunharo2021). Due to self-incompatibility, these three species naturally hybridize, leading to high genetic diversity and overlapping phenotypic traits (Pasquali et al. Reference Pasquali, Palumbo and Barcaccia2022). Extensive genetic diversity facilitates rapid evolution of adaptive traits that help Lolium spp. succeed in diverse agroclimatic and geographic regions, escape cultivation, and develop into feral and/or weedy biotypes (Jhala et al. Reference Jhala, Beckie, Mallory-Smith, Jasieniuk, Busi, Norsworthy and Geddes2021). The introduction of L. perenne ssp. multiflorum in the United States dates back to the early colonial days (Holt Reference Holt, Holt and Lewis1976), but its expansion as a pasture crop across the country likely occurred in the early 1930s (Evers Reference Evers1995). Today, it is widely grown as a winter pasture crop due to its high palatability and nutritional value (Undersander and Casler Reference Undersander and Casler2014). However, feral biotypes of L. perenne ssp. multiflorum have become a problematic weed in many parts of the world including the United States.
In a survey conducted in 2023 by the Weed Science Society of America across the United States and Canada, L. perenne ssp. multiflorum has been reported as the 2nd most troublesome weed in winter cereal grains, 5th in spring cereal grains, and 10th among all grass crops, pasture, and turf weeds (Van Wychen Reference Van Wychen2023). Moreover, L. perenne ssp. multiflorum is one of the most competitive weed species in winter wheat (Triticum aestivum L.), corn (Zea mays L.), and vegetable crops (Appleby et al. Reference Appleby, Olson and Colbert1976; Bell Reference Bell1995; Nandula Reference Nandula2014). In a study conducted in California, L. perenne ssp. multiflorum at a density of 600 to 1,000 plants m−1 of crop row caused a 100% yield loss in broccoli (Brassica oleracea L. var. botrytis L.) (Bell Reference Bell1995). Another study conducted in Oregon on wheat found that a density of 400 plants m−2 resulted in up to a 92% yield loss (Appleby et al. Reference Appleby, Olson and Colbert1976). In Mississippi, L. perenne ssp. multiflorum has been reported to cause a 49% reduction in corn yield at a density of just 4 plants m−1 of crop row (Nandula Reference Nandula2014). However, managing herbicide-resistant L. perenne ssp. multiflorum significantly increases production costs for affected crops. For instance, successfully managing glyphosate-resistant (GR) L. perenne ssp. multiflorum in soybean [Glycine max (L.) Merr.] can cost more than US$100 ha−1 (Mississippi Soybean Promotion Board 2021).
Under southeast U.S. climatic conditions, L. perenne ssp. multiflorum behaves as a winter annual, typically germinating in the fall (October to November) when soil temperatures average between 10 and 18.3 C (Undersander and Casler Reference Undersander and Casler2014; Russell Reference Russell2022). Additionally, a second flush of L. perenne ssp. multiflorum is often observed in early spring (January) in parts of the southern United States (Bagavathiannan et al. Reference Bagavathiannan, Maity, Ackroyd, Flessner, Rubione and VanGessel2021), which underscores the importance of season-long weed management. Lolium multiflorum can also be considered a satellite weed for winter wheat, as it emerges around planting time, typically in the second week of October, and flowers from May to July, leaving behind a substantial soil seedbank (Hancock Reference Hancock2011). Cechin et al. (Reference Cechin, Schmitz, Hencks, Vargas, Agostinetto and Vargas2021) reported that more than 95% of L. perenne ssp. multiflorum seeds, regardless of soil depth, become unavailable after approximately 1.5 yr, indicating its short seedbank persistence in soil. However, L. perenne ssp. multiflorum is a prolific seed producer, meaning that 5% of seeds are adequate for its persistent perpetuation. The standard management practice in the southeastern United States for controlling L. perenne ssp. multiflorum in winter wheat typically begins with a preplant burndown using thifensulfuron and tribenuron (WSSA Group 2) (FirstShot® SG), combined with nonselective herbicides such as glyphosate (WSSA Group 9) or paraquat (WSSA Group 22). For delayed preemergence to provide overlapping residual activity, a mix of flumioxazin (Group 14) and pyroxasulfone (Group 15) is recommended. To manage escaped L. perenne ssp. multiflorum, late-season postemergence (rescue) treatments using WSSA Group 1 herbicides, such as a premix of pinoxaden + fenoxaprop (Axial® Bold) combined with the Group 2 herbicide pyroxsulam, are advised (Russell Reference Russell2022).
Given their effectiveness, economic benefits, rapid action, and flexibility, herbicides are the most common choice for farmers to manage weeds, including L. perenne ssp. multiflorum in wheat and other crops (Duke Reference Duke2015; Russell Reference Russell2022; Singh et al. Reference Singh, Maity, Abugho, Swart, Drake and Bagavathiannan2020). However, the growing herbicide resistance issue in L. perenne ssp. multiflorum has complicated management by increasing complexity and expense. Worldwide, 75 cases of HR have been reported for L. perenne ssp. multiflorum across eight modes of action (MOAs) seven cropping systems (Heap Reference Heap2024). In the United States, L. perenne ssp. multiflorum has been reported to be herbicide resistant (HR) to six MOAs, including acetyl-coenzyme A carboxylase (ACCase) inhibitors (WSSA Group 1), acetolactate synthase (ALS) (WSSA Group 2) inhibitors, enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (WSSA Group 9), glutamine synthetase inhibitors (WSSA Group 10), very-long-chain fatty-acid (VLCFA) inhibitors (WSSA Group 15), and photosystem I (PSI) inhibitors (WSSA Group 22) (Heap Reference Heap2024). Glyphosate had been an effective preplant treatment for growers for control of L. perenne ssp. multiflorum in the southern United States. However, a GR biotype with 3-fold resistance was reported in Mississippi in 2005, while another resistant biotype was reported in Tennessee in 2012 (Heap Reference Heap2024). Resistance to fluazifop-butyl, a member of the aryloxyphenoxypropionate (FOP) family, in L. perenne ssp. multiflorum was reported in California in 2019 (Tehranchian et al. Reference Tehranchian, Nandula, Matzrafi and Jasieniuk2019) and resistance to clethodim, a member of the cyclohexanedione (DIM) family, has been confirmed in southeastern states, including Mississippi and North Carolina (Nandula et al. Reference Nandula, Giacomini, Lawrence, Molin and Bond2020).
After the development of GR and ACCase-resistant biotypes of L. perenne ssp. multiflorum, growers increasingly relied on ALS-inhibiting herbicides, particularly in wheat. However, resistance to chlorsulfuron was reported in Arkansas in 1995 (Heap Reference Heap2024) and resistance to pyroxsulam was confirmed in North Carolina in 2007 (Chandi et al. Reference Chandi, York, Jordan and Beam2011). ALS resistance is challenging due to the stronger selection pressure exerted by ALS herbicides than other modes, which facilitates the rapid selection and spread of resistance in weeds, including L. perenne ssp. multiflorum populations (Jones et al. Reference Jones, Taylor and Everman2021; Tranel and Wright Reference Tranel and Wright2002). Despite the continued reliance on pyroxsulam, glyphosate, clethodim, and fluazifop-butyl in wheat, soybean, corn, and preplant herbicide programs across Alabama, recent reports from neighboring states have documented cases of multiple and cross-resistance in L. perenne ssp. multiflorum. These findings signal an urgent need for proactive resistance management strategies in Alabama (Figure 1).
Geographic distribution of herbicide-resistant Lolium perenne ssp. multiflorum in the southern United States for acetolactate synthase (ALS), acetyl-CoA carboxylase (ACCase), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and photosystem I electron divertor (PSI) inhibitors. Colored shapes indicate resistance to a given mode of action reported within a specific state, but they do not conform to exact geographic coordinates. Information from Chandi et al. (Reference Chandi, York, Jordan and Beam2011), Heap (Reference Heap2024), Kuk and Burgos (Reference Kuk and Burgos2007), Nandula et al. (Reference Nandula, Poston, Eubank, Koger and Reddy2007), Salas et al. (Reference Salas, Burgos, Mauromoustakos, Lassiter, Scott and Alcober2013), and Taylor (Reference Taylor2015).
Although there are several reports of herbicide failures from growers in Alabama, no herbicide resistance survey has been conducted in Alabama to date. The wide range of genetic and phenological diversity exhibited by L. perenne ssp. multiflorum not only enables L. perenne ssp. multiflorum to adapt to any harsh field conditions but also helps select HR biotypes when exposed to similar MOAs repeatedly (Mortimer Reference Mortimer1997). Therefore, the objectives of our study were to survey, screen, and confirm the distribution of HR L. perenne ssp. multiflorum in Alabama.
Materials and Methods
Collection and Storage of Plant Materials
Lolium multiflorum populations were collected from late spring to early summer of 2023 through a semi-stratified survey (Garetson Reference Garetson2017) designed to target wheat, soybean, peanut (Arachis hypogaea L.), cotton (Gossypium hirsutum L.), and cornfields, as well as field borders, across Alabama (Figure 2). Some of the plants were survivors of in-season herbicide applications, identified based on the herbicide failure reports from growers. Others were assumed to be either potential survivors of season-long herbicide applications or late-emerged plants. A total of 20 to 25 seed heads from plants growing within an area of 10 to 15 m2 were collected and combined to form each population. A distance of 3.2 to 4.8 km was maintained based on the previous weed survey between collection sites to ensure diversity among the populations (Bagavathiannan and Norsworthy Reference Bagavathiannan and Norsworthy2016).
Geocoordinates of 65 Lolium perenne ssp. multiflorum populations collected from crop and non-cropped areas in Alabama in 2023.
A total of 65 L. perenne ssp. multiflorum populations were collected late in the summer, by which point most populations had already completed their life cycle. As L. perenne is also common in the southeastern United States, all the Lolium populations collected were examined and confirmed to be L. perenne ssp. multiflorum following the methods of Maity et al. (Reference Bagavathiannan, Maity, Ackroyd, Flessner, Rubione and VanGessel2021) and Bararpour et al. (Reference Bararpour, Norsworthy, Burgos, Korres and Gbur2017), largely based on the plant height, length of awn, leaf blade width, and plant growth habit. The seed heads were hand threshed immediately after collection, and the seeds were stored at room temperature (20 to 22 C) until they were needed for further experiments. Due to limited seed quantity and high seed dormancy, only 44 populations from Escambia, Baldwin, Lee, Henry, Dallas, Walker, Franklin, Cullman, Limestone Bibb, and Jackson counties were included in the herbicide screening study.
Herbicide Efficacy
Herbicide efficacy trials were conducted in the spring of 2024 at the Plant Science Research Center Greenhouse Complex (32.58°N, 85.48°W) located at Auburn University, Auburn, AL. Out of 44 populations, one population (AL-64) from northern Alabama without any known history of herbicide exposure was tested at field rate for all herbicides used in the study and, after results were analyzed, it was declared to be the susceptible standard. Each efficacy trial was conducted with two replications per population, each consisting of 24 plants. Populations were grown in 48-cell inserts placed inside trays (53.34 cm by 27.94 cm). Each tray was divided lengthwise into four sections, with each section containing 12 cells, for a total of 48 cells per tray. One section of 12 cells was allotted to each population, with two plants maintained per cell, allowing four populations to be grown adjacent to each other in a tray. Potting mix (Miracle-Gro® Potting Mix, Scotts Miracle-Gro, Marysville, OH) was used as growing medium, which was watered regularly as needed and fertilized with a soluble fertilizer (Osmocote® Pro 19-5-8, ICL Specialty Fertilizers, Charleston, SC) at the recommended rate at 2 wk after planting (WAP). Plants were maintained at a temperature range of 22 to 28 C. Photoperiod of 12 h was maintained, to compensate on cloudy days, supplemental light was provided by 1,000-W metal-halide bulbs delivering approximately 400 µmol m−² s−1.
Herbicide treatments included (1) ACCase inhibitors: fluazifop-butyl (Fusilade® DX, Syngenta, Greensboro, NC) and clethodim (Section® Three, Winfield Solutions, St Paul, MN); (2) the ALS inhibitor pyroxsulam (PowerFlex® HL Herbicide, Corteva Agriscience, Indianapolis, IN); and (3) the EPSPS inhibitor glyphosate (Roundup PowerMAX® 3 Herbicide, Bayer CropScience, St Louis, MO). All herbicides were applied at their recommended label rates: clethodim (283 g ai ha−1), fluazifop-butyl (213 g ai ha−1), pyroxsulam (233 g ai ha−1), and glyphosate (1,133 g ae ha−1). Herbicides were applied when the plants were at the 3- to 4-leaf seedling stage (∼3- to 4-wk old), using a two-nozzle sprayer powered by a CO2 cylinder and equipped with flat-fan nozzles (TeeJet® XR110015, TeeJet Technologies, Wheaton, IL) calibrated to deliver a spray volume of 140 L ha−1 at 289 kPa at a speed of 4.8 km h−1. Injury ratings (based on a scale of 0% to 100%, where 0% means no injury, and 100% means completely dead) and plant mortality were recorded at 3 wk after treatment (WAT) to estimate the level of resistance, as described in Singh et al. (Reference Singh, Maity, Abugho, Swart, Drake and Bagavathiannan2020). Non-treated controls were included for initial screening. Survival rates of L. perenne ssp. multiflorum populations to respective herbicides were visualized using a modified box plot (violin plot) with the ggstatsplot package in R v. 4.3.0 (Patil Reference Patil2021; R Core Team 2023).
Dose–Response Assay
Following the initial assessment, populations with the highest survival rate and lowest injury from all four herbicides were selected for further dose–response assays to confirm the resistance in comparison to a susceptible biotype. The selected populations were AL-61 for glyphosate, AL-62 for pyroxsulam, and AL-65 for both clethodim and fluazifop-butyl-P-butyl. The susceptible population (AL-64) selected in the initial screening was used as a common standard for all herbicides. Plants for the dose–response studies were grown in 10.16-cm round pots using Miracle-Gro® Potting Mix (Scotts Miracle-Gro). Emergence was excellent, and thinning was carried out 1 wk after emergence to maintain a population of five plants per pot. The dose–response screening was then conducted using a spray chamber (Generation 4 Research Track Sprayer, DeVries Manufacturing, Hollandale, MN) equipped with a flat-fan nozzle (TeeJet® XR110015, TeeJet Technologies) calibrated to deliver a spray volume of 140 L ha−1 at 276 kPa at a speed of 4.8 km h−1. The experiment was a completely randomized design with four replications, conducted across two independent trials. Eight rates were selected for resistant populations (0.5×, 1×, 2×, 4×, 8×, 16×, 32×, and 64×) and six (0.0625×, 0.125×, 0.25×, 0.5×, 1×, and 2×) for susceptible populations. At 4 WAT, plants were evaluated for injury percentages, followed by the shoot biomass collection. Shoot biomass was dried at 65 C for 3 d to record dry biomass.
Data for dose–response trials were analyzed in RStudio. Levene’s test was used to check for the homogeneity of two independent trials. No significant difference was found, so data were pooled across the two runs for further analysis. The visual injury was regressed against herbicide dose using a three-parameter logistic regression equation (Equation 1) with the drc package (Knezevic et al. Reference Knezevic, Streibig and Ritz2007; Ritz et al. Reference Ritz, Baty, Streibig and Gerhard2015) described later:
$$Y = d + \exp \left\{ {b\left[ {\log \left( x \right) - e} \right]} \right\}$$
In the equation, Y is the response variable (i.e., relative dry weight); x is the applied dose of respective herbicide; d is the upper limit; b is the relative slope around e; and e is GR50, which is the dose of herbicide required for 50% growth reduction. The herbicide dose required to reduce growth by 50% (GR50) was estimated, and the resistance index (R/S) was calculated as the ratio of GR50 values of resistant populations to that of the susceptible standard.
Target Site–Based Resistance Detection Studies
To investigate potential target-site resistance mechanisms associated with ACCase-, ALS-, and EPSPS-inhibiting herbicides, RNA was isolated from young leaf samples (approximately 0.1 g) of four L. perenne ssp. multiflorum populations: AL-61, AL-62, AL-65, and AL-64 (S). For resistant populations, plants surviving from the highest dose in the dose–response assay were used. The RNA extraction was performed using the Zymo-Spin™ II RNA Kit (Zymo Research, Irvine, CA), following the manufacturer’s guidelines. The RNA quality and quantity were evaluated through gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). High-quality RNA was converted into complementary DNA (cDNA) using the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA) via reverse transcriptase-polymerase chain reaction (RT-PCR). Sections covering known resistance-conferring mutations of the ACCase, EPSPS, and ALS genes were amplified using specific forward and reverse primers (Brunharo and Hanson Reference Brunharo and Hanson2018; Nandula et al. Reference Nandula, Giacomini, Lawrence, Molin and Bond2020; Tehranchian et al. Reference Tehranchian, Nandula, Matzrafi and Jasieniuk2019; Table 1).
The six primer pairs used to detect single-nucleotide polymorphisms in Lolium perenne ssp. multiflorum populations.
PCR amplification was conducted in a 25-μl reaction volume using plant cDNA. The PCR reaction mixture consisted of 1× standard reaction buffer, 200 µM dNTPs, 0.5 µM of both forward and reverse primers, 250 ng of cDNA, and 0.125 U Taq DNA polymerase (New England Biolabs). The thermal-cycling program included an initial activation step at 95 C for 30 s, followed by 35 cycles of 20 s at 95 C, 1 min of annealing at 58 to 62 C (depending on the primer), and 1 min at 68 C, with a final extension step of 5 min at 68 C. The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide and run in Tris-acetate-EDTA buffer. The PCR product was sent to Eurofins Genomics (eurofinsgenomics.com) for PCR cleanup and Sanger sequencing. BioEdit (Hall Reference Hall1999) software was used to align the sequences and identify mutations.
Results and Discussion
Herbicide Resistance
Glyphosate
Out of the 44 populations subjected to the field rate of glyphosate, 21% survived with varying levels of injury (Table 2), among which three populations were from southern Alabama (Baldwin County) and six populations were from northern Alabama (Cullman and Limestone counties). Among the populations, 34% were classified as putatively resistant, and 66% as potentially resistant. Injury percentages among survivors in putative resistant populations ranged from 15% to 75% with a mean of 46%, while survival rates ranged from 45% to 83% with an average of 67%, which is higher compared with survival rates across all populations (17%) (Figure 3). All the populations from southern Alabama were classified as potentially resistant and were from peanut- and corn-cropping situations. Populations from northern Alabama soybean and cotton rotations varied from potentially to putatively resistant. Glyphosate has been extensively used in Alabama as a burndown herbicide before wheat, soybean, corn, and cotton planting, as well as for GR crops, potentially applying significant selection pressure for glyphosate resistance in L. perenne ssp. multiflorum populations. To our knowledge, this is the first report of glyphosate-resistant L. perenne ssp. multiflorum from Alabama.
Resistance levels of Lolium perenne ssp. multiflorum populations collected from Alabamaa.
a A total of 44 populations of L. perenne ssp. multiflorum were subjected to 1× screening.
b Putatively resistant defined as populations with <20% injury.
c Potentially resistant defined as populations with 21–79% injury.
d Susceptible defined as populations with 80–100% injury.
Percent survival of Lolium perenne ssp. multiflorum populations in response to different herbicides. The y axis represents the number of survivors in 44 different populations. This violin plot is a modified box plot in which the box represents the upper and lower quartiles, and the central line represents the median. The violin area shows the proportion of populations with survivors for each herbicide.
The AL-61 population from Cullman County (GR soybean crop) was selected for dose–response study based on its high survival rate (76%) and low injury (18%) when treated with glyphosate at 1,133 g ae ha−1. Dose–response assays revealed that the GR50 (herbicide dose causing 50% growth reduction) for the susceptible population was 16 g ae ha−1, while it was 3,075 g ae ha−1 for the resistant population, indicating a 192-fold increase in resistance compared with the susceptible population (Table 3; Figures 4 and 5).
GR50 values and herbicide resistance levels in Lolium perenne ssp. multiflorum populations collected from Alabamaa.
a GR50, the dose of herbicide required for 50% growth reduction; R, resistant population for respective herbicide; RMSE, root square mean error; RI, resistance index, which is ratio of GR50 for the resistant and the susceptible populations; S, susceptible population for respective herbicide; SEM, standard error of the mean.
b Means compared within herbicide. Letters represent significant differences identified by separation of means using Tukey’s honest significant difference (HSD) test (α = 0.05).
Dose–response of the resistant and susceptible Lolium perenne ssp. multiflorum populations to (A) glyphosate (population AL-61), (B) fluazifop-butyl (population AL-65), (C) clethodim (population AL-65), and (D) pyroxsulam (population AL-62). Injury (%) was based on visual assessment of respective treated populations compared with non-treated L. perenne ssp. multiflorum. Population AL-64 that was confirmed susceptible in the initial screening was used as the standard susceptible population for all herbicides.
Dose–response of resistant Lolium perenne ssp. multiflorum populations, encompassing both resistant (R) and susceptible (S) populations, to varying rates of four herbicides: glyphosate, fluazifop-butyl, clethodim, and pyroxsulam. Herbicide application rates range from 1× to 8× the recommended label rate for resistant populations, and from 0.065× to 2× the recommended rate for the susceptible population. The populations tested include AL-61, AL-62, and AL-65, which are resistant (R), and AL-64, which is known susceptible (S). NT, non-treated control plants.
Globally, there have been 29 reports of GR L. perenne ssp. multiflorum (Heap Reference Heap2024). In the United States, the first report of resistance came from Oregon in 2004, with a 5-fold resistance observed in orchard settings (Perez-Jones et al. Reference Perez-Jones, Park, Colquhoun, Mallory-Smith and Shaner2005). In the southeastern United States, the first case was reported in Mississippi (2005), followed by Arkansas (2008), North Carolina (2009), Tennessee (2012), and Louisiana (2014) (Heap Reference Heap2024). In California, resistance in L. perenne ssp. multiflorum has been documented ranging from 2- to 15-fold compared with susceptible populations (Jasieniuk et al. Reference Jasieniuk, Ahmad, Sherwood, Firestone, Perez-Jones, Lanini and Stednick2008). Similarly, Salas et al. (Reference Salas, Dayan, Pan, Watson, Dickson, Scott and Burgos2012) reported 7- to 13-fold glyphosate resistance in Arkansas based on visual injury comparisons with susceptible populations. The extreme sensitivity of susceptible populations to glyphosate (GR50 = 16 g ae ha−1) is an obvious factor contributing to the high level of resistance. The GR50 for susceptible L. perenne ssp. multiflorum populations was as high as 171 g ae ha−1 in Arkansas (Dickson et al. Reference Dickson, Scott, Burgos, Salas and Smith2011) and 230 g ae ha−1 in California (Karn et al. Reference Karn, Beffa and Jasieniuk2018). In this study, 11× of 1,133 g ae ha−1 would be required to reduce the biomass of the resistant population 90%. Additionally, the significantly shorter awn length in putative GR populations may indicate a potential fitness cost (Yanniccari et al. Reference Yanniccari, Vila-Aiub, Istilart, Acciaresi and Castro2016).
Pyroxsulam
Out of the 44 total populations, 11 survived the field rate of pyroxsulam (233 g ai ha−1; Table 2). Five of the 11 populations were from southern Alabama (Escambia and Baldwin counties), 2 from central Alabama (Bibb and Dallas counties), and 4 from northern Alabama (Walker, Limestone, and Cullman counties). Populations from southern and central Alabama were from field borders as well as in fields of peanut, corn, and wheat; however, all putatively resistant populations from northern Alabama were from fields of cotton and soybean. Among these surviving populations, 45% were classified as putatively resistant and 65% as potentially resistant. The average survival rate across potentially and putatively resistant populations was 56% and across all populations it was 28% (Figure 3). Injury observed in potentially and putatively resistant populations ranged from 10% to 73%. The putatively resistant population (AL-62) for dose–response was selected based on a high survival (90%) and low injury (10%) rate from Bibb County collected from a field border. The resistance index, calculated from GR50 values, indicated a 90-fold resistance in the resistant population compared with the susceptible (Table 3; Figure 4).
Globally, 31 cases of resistance to ALS-inhibiting herbicides have been reported in L. perenne ssp. multiflorum, with 22% of these specifically resistant to pyroxsulam (Heap Reference Heap2024). In the Texas Blacklands region, Singh et al. (Reference Singh, Maity, Abugho, Swart, Drake and Bagavathiannan2020) reported that 93% of the populations (n = 64) was resistant to the ALS herbicide mesosulfuron-methyl, with resistance levels as high as 37-fold. The 90-fold resistance observed in this study could be attributed to the frequent use of ALS-inhibiting herbicides in Alabama wheat production and the high sensitivity of the susceptible standard used. However, with 14% of the populations classified as potentially resistant and 75% still susceptible, resistance evolution appears to be slow in progress or the current L. perenne ssp. multiflorum management strategies in the region are still effective. This presents an opportunity for timely intervention and precautionary measures to prevent further resistance development.
Clethodim
Only 2% (n = 1 out of 44) of the populations collected from northern Alabama (Cullman County) were found to be putatively resistant to clethodim (Table 2). The mean survival rate across all populations was 4.48% (Figure 3). Dose–response studies revealed GR50 values of 705.24 g ai ha−1 for the resistant population and 0.95 g ai ha−1 for the susceptible population, indicating a 738-fold resistance in the resistant population (Table 3), which requires 12× the field rate to reduce 90% biomass of the resistant population.
Clethodim is a postemergence herbicide frequently used to manage GR L. perenne ssp. multiflorum. The soybean field in which the resistant population was collected had a history of repetitive use of clethodim to control annual grass weeds (personal communication with the wheat grower). Previously, resistance to clethodim was documented in the U.S. Pacific Northwest, where 5% of populations were resistant, with another 8% developing resistance (Rauch et al. Reference Rauch, Thill, Gersdorf and Price2010). In the southeastern United States, Nandula et al. (Reference Nandula, Giacomini, Lawrence, Molin and Bond2020) observed up to 10-fold resistance in populations from Mississippi and up to 40-fold resistance in populations from North Carolina. In two additional populations, resistance levels could not be calculated because biomass reduction was only 17% to 30% at the highest tested dose (2,170 g ha−1).
The extremely high resistance level (738-fold) observed in this study could be attributed to the high sensitivity of the susceptible standard, which showed a 93% biomass reduction at the lowest dose tested (17 g ha−1). Furthermore, as glyphosate has become less effective in controlling L. perenne ssp. multiflorum, the use of clethodim has increased in Alabama as a burndown in soybean and cotton. Despite this, clethodim resistance was detected in only one population from northern Alabama, and the mean survival rate across all populations was low (4.48%). This suggests that while resistance is present, it remains rare, indicating that the systems may be integrated sufficiently to select against rapid resistance evolution to clethodim. Strong preventive measures are essential to prevent the spread of clethodim resistance, and the integration of other MOAs will be necessary to reduce the selection pressure exerted by clethodim.
Fluazifop-Butyl
Eleven percent of the total population survived fluazifop-butyl field rate, among which one population collected from a corn field in Baldwin County in southern Alabama was potentially resistant; the remaining four populations were from northern Alabama (Cullman County) from soybean fields (Table 2). Of the surviving populations, 45% were classified as putatively resistant and 55% as potentially resistant. Survival rates in putatively and potentially resistant populations ranged from 46% to 100% with a mean of 79%. A mean survival rate of 24% was observed across the populations (Figure 3). Population (AL-65) was selected for dose–response studies based on its high survival rate (98%) and low injury (13%); the same population was also used for dose–response studies for clethodim. The dose–response study indicated a 14-fold resistance in AL-65 compared with the susceptible (Table 3). No significant differences in seed traits were observed between fluazifop-butyl-resistant and fluazifop-butyl-susceptible populations.
Fluazifop-butyl is recommended in Alabama for controlling annual and perennial grasses in cotton, soybean, and peanut (Alabama Cooperative Extension System, 2023a, 2023b, 2023c). Resistance to diclofop-methyl, a herbicide from the same aryloxyphenoxypropionate family as fluazifop-butyl, has become widespread (Heap Reference Heap2024; Singh et al. Reference Singh, Maity, Abugho, Swart, Drake and Bagavathiannan2020), largely due to its frequent use in wheat for grass weed control. This overreliance on diclofop has exerted extreme selection pressure on weeds, including L. perenne ssp. multiflorum. Previous studies have reported that both target-site and non–target site resistance mechanisms confer resistance to diclofop-methyl, which also extends resistance to fluazifop-butyl and other ACCase-inhibiting herbicides in L. perenne ssp. multiflorum (Kaundun et al., Reference Kaundun, Hutchings, Dale and McIndoe2012, Reference Kaundun, Bailly, Dale, Hutchings and McIndoe2013).
Fluazifop-butyl resistance has been reported in California, where the labeled field rate reduced L. perenne ssp. multiflorum biomass by only 14% (Tehranchian et al. Reference Tehranchian, Nandula, Matzrafi and Jasieniuk2019). In the current study, the population (AL-65) that has the highest level of resistance to fluazifop-butyl also exhibited cross-resistance to clethodim, a common phenomenon among ACCase-inhibiting herbicides (Takano et al. Reference Takano, Ovejero, Belchior, Maymone and Dayan2020). Although resistance to fluazifop-butyl in Alabama is not as widespread as the resistance to glyphosate or pyroxsulam, it remains a viable option for controlling L. perenne ssp. multiflorum. However, proactive measures are essential to avoid overreliance on fluazifop-butyl and to ensure its continued efficacy through judicious use.
Cross-Resistance and Multiple Resistance
Fourteen percent of the populations (n = 6) exhibited putative multiple resistance to either the EPSPS inhibitor (glyphosate) and ALS inhibitor (pyroxsulam) or EPSPS inhibitor (glyphosate) and ACCase inhibitor (fluazifop-butyl) (Table 4). Two populations (AL-21 and AL-58) exhibited putative multiple resistance to ALS and EPSPS inhibitors from the Baldwin–peanut fields and Limestone–cotton fields, respectively. AL-21 was resistant to (injury = 13%) against pyroxsulam; however, it exhibited potential resistance (injury = 45%) against glyphosate, whereas AL-58 was putatively resistant (injury = 18%) to glyphosate and showed a moderate level of resistance (injury = 75%) to pyroxsulam. Four populations (AL-20, AL-59, AL-61, and AL-66) exhibited putative multiple resistance to ACCase and EPSPS inhibitors. Out of the four populations, one was from southern Alabama (Baldwin County), and three from Northern Alabama (Cullman County). The population from southern Alabama was from a cornfield, while all populations from northern Alabama were from soybean fields. All the populations were potentially resistant to both MOAs, except AL-61, which was putatively resistant to both MOAs.
Cross- and multiple resistance in 44 Lolium perenne ssp. multiflorum populations collected from Alabama.
a ACCase, acetyl-coenzyme A carboxylase; ALS, acetolactate synthase; dim, cyclohexanedione family; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase; fop, aryloxyphenoxypropionate family.
b Cross-resistance refers to resistance to different herbicide families with the same mode of action (MOA); multiple resistance refers to resistance to two or more unique herbicide MOAs. Resistance was determined based on plant survival (1–79% injury) at the recommended label rate for the given herbicide.
One population (AL-65) expressed cross-resistance to both aryloxyphenoxypropionate (fluazifop-butyl) and cyclohexanedione (clethodim) families of ACCase inhibitors, as well as multiple resistance to ACCase (clethodim and fluazifop-butyl), ALS (pyroxsulam), and EPSPS inhibitors (glyphosate). The resistant population was collected from a soybean field in Cullman County. It exhibited the highest survival rate in response to clethodim and fluazifop-butyl and had a target-site mutation (Asp-2078-Gly) in the ACCase gene. However, non–target site resistance may also be involved in conferring herbicide resistance in L. perenne ssp. multiflorum, as reported by previous literature (Kaundun et al., Reference Kaundun, Hutchings, Dale and McIndoe2012, Reference Kaundun, Bailly, Dale, Hutchings and McIndoe2013). According to the Heap (Reference Heap2024) database, 28% of total HR cases (n = 36) reported against L. perenne ssp. multiflorum in the United States involve multiple resistance. In North Carolina, L. perenne ssp. multiflorum has been reported resistant to ALS, ACCase, EPSPS, and PSI inhibitors (Heap Reference Heap2024). Similarly, in California, resistance to EPSPS, ACCase, and PSI inhibitors has been documented (Tehranchian et al. Reference Tehranchian, Nandula, Matzrafi and Jasieniuk2019). Lolium perenne ssp. multiflorum populations in California have also shown cross-resistance to ACCase-inhibiting herbicides, including fluazifop-butyl and clethodim (Tehranchian et al. Reference Tehranchian, Nandula, Matzrafi and Jasieniuk2019), which is consistent with the findings of this study.
Target-Site Mutation Detection
Sequencing of the ACCase region in the AL-65 population revealed a Asp-2078-Gly mutation (Figure 6B), which has previously been documented in L. perenne ssp. multiflorum populations from Leicestershire, UK (Kaundun Reference Kaundun2010) and is associated with resistance to ACCase-inhibiting herbicides. In the ALS region of the AL-62 population, a novel Arg-421-Thr mutation was identified (Figure 6A), which has not been reported in previous studies. It was a transversion, which refers to a point mutation in DNA in which a single (two-ring) purine (A or G) is changed for a (one-ring) pyrimidine (T or C), or vice versa. Additionally, the EPSPS gene region of the AL-61 population exhibited a Pro-106-Ser mutation (Figure 6C), a mutation also reported by Brunharo and Hanson (Reference Brunharo and Hanson2018) in GR L. perenne ssp. multiflorum populations.
Multiple sequence alignments in the Lolium perenne ssp. multiflorum populations from BioEdit: (A) ALS gene of AL-62, (B) ACCase gene of AL-65, and (C) EPSPS gene of AL-61. Sequences from resistant populations are highlighted with yellow rectangles, while susceptible population (AL-64) respective gene sequences are highlighted with green rectangles. Other sequences from NCBI are included for cross-validation.
The herbicide resistance in L. perenne ssp. multiflorum populations from Alabama was confirmed across all four herbicides evaluated, with molecular data corroborating these findings. However, resistance levels and distribution varied considerably among the tested herbicides. Fluazifop-butyl and pyroxsulam resistance was more widespread than glyphosate and clethodim resistance. Despite these differences, significant resistance was evident for all herbicides, likely influenced by the high sensitivity of the susceptible control population. To mitigate further spread of herbicide resistance in Alabama, it is critical to implement an integrated weed management strategy. Proactive measures such as using overlapping residual herbicides, establishing cover crops, timely postemergence herbicide applications, and integrating emerging technologies like harvest weed seed control are urgently required in the region.
Acknowledgements
We are grateful to Forrest Davis for his assistance with initial screening and to Cade Grace for helping in the collection of L. perenne ssp. multiflorum populations from northern Alabama.
Funding
This work is partially supported by the Foundational Knowledge of Agricultural Production Systems, project award no. 2023-09643, from the U.S. Department of Agriculture’s National Institute of Food and Agriculture; the Alabama Agricultural Experiment Station and the Hatch program of the National Institute of Food and Agriculture, U.S. Department of Agriculture; and the startup fund provided to the corresponding author by Auburn University.
Competing interests
The authors declare no conflicts of interest.
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