Morphological and physiological adaptations underpin drought resilience in contrasting populations of annual ryegrass (Lolium rigidum)

Annual ryegrass (Lolium rigidum Gaud.), originally introduced as a pasture species, has evolved into the most problematic and economically damaging weed in Australian grain cropping systems¹. It is now widely distributed across the western, southern, and northern grain production regions of Australia², where it competes aggressively with crops for light, nutrients, and water^3,4. Its prolific growth and adaptability have been estimated to cause annual yield losses of 352,258 tons exceeding $114 million across 119 million hectares of all grain growing regions⁵.

The invasiveness and persistence of annual ryegrass can be attributed to several biological traits, including high genetic diversity, phenotypic plasticity, rapid growth, and high seed production. These traits, coupled with its capacity for efficient seed dispersal and local environmental adaptation, allow annual ryegrass to thrive in a wide range of agroecological conditions^4,1. Recent surveys confirm that Australian grain growers consider annual ryegrass as the most adaptable and difficult to control weed in their systems⁶. Adding to its threat is the rapid and widespread evolution of herbicide resistance¹. Over 60% of Australian annual ryegrass populations are now resistant to one or more herbicide modes of action⁷, including glyphosate, acetyl coenzyme a carboxylase inhibitor, and acetolactate synthase inhibitors⁸. Emerging evidence suggests that some herbicide resistance mechanisms may also influence plant responses to abiotic stresses. Enhanced metabolic activity, altered antioxidant systems, and modified physiological regulation associated with certain resistance traits may contribute to improved tolerance to environmental stresses including drought⁹. However, the extent to which herbicide resistance mechanisms directly confer cross-tolerance to drought stress remains poorly understood and requires further investigation. This resistance, combined with widespread distribution and biological adaptations, makes annual ryegrass an ever-increasing challenge to grain production systems.

In addition to herbicide resistance, adaptability of annual ryegrass to abiotic stress, particularly drought, may further contribute to its persistence under climate change. Drought is one of the most limiting environmental factors for plant growth, negatively affecting germination, photosynthesis, nutrient uptake, and biomass accumulation^10,11. In response, plants adopt various physiological strategies, including osmotic adjustment, enhanced stress tolerance mechanisms and structural modifications^11,12,13. While drought responses in crop species have been widely studied, there is limited knowledge about how major weeds, like annual ryegrass, respond to moisture stress. This gap is particularly concerning given that drought-prone environments are becoming more common, and weeds that can withstand these conditions may pose an even greater threat to cropping systems. Climate projections for Australian grain production regions indicate increasing temperatures, greater rainfall variability, higher evaporative demand, and more frequent or intense drought conditions under future climate scenarios¹⁴, potentially intensifying weed–crop competition and influencing weed adaptation patterns.

Annual ryegrass poses a significant challenge in agricultural systems due to its resilience and adaptability, particularly in drought-prone environments¹⁵. Hence, understanding how annual ryegrass survives in water limited environments is crucial for developing targeted and effective weed control strategies in cropping systems. Drought stress influences plant survival, growth, and competition by limiting water availability, disrupting metabolic processes, and reducing overall biomass production¹⁰.

Although annual ryegrass has been extensively studied in terms of its growth patterns and herbicide responses, significant gaps remain in understanding its adaptations to drought stress. Identifying the mechanisms that regulate its water use and inhibit its growth can improve agricultural productivity and economic outcomes. This can be achieved by studying its growth and morphological responses to drought stress. Furthermore, limited information is available on whether different physiological traits, such as plant water status and photosynthetic efficiency, play a critical role in mitigating drought stress in annual ryegrass. Addressing these gaps is crucial for developing targeted drought management strategies and understanding the resilience of annual ryegrass in water-limited environments. This study was conducted to compare the morpho-physiological responses of herbicide-resistant and susceptible annual ryegrass populations under varying soil moisture levels to better understand their drought resilience mechanisms. We hypothesized that annual ryegrass would exhibit morphological, physiological and phenological adjustments in response to declining soil moisture availability, and that these responses may vary between herbicide-resistant and susceptible populations.

Experimental design and setup

A pot experiment was conducted, twice between July 2024 and February 2025, to evaluate the drought response of two contrasting annual ryegrass populations. The experiment followed a completely randomized factorial design consisting of 2 annual ryegrass populations × 5 soil moisture levels × 5 biological replicates, repeated over 2 independent experimental runs under controlled glasshouse conditions at Centre for AgriBioscience (AgriBio), La Trobe University, Melbourne, Australia. Environmental parameters were maintained at 22/16℃ (day/night) temperatures, 40% relative humidity, and a 16/8 h light/dark photoperiod. Although photosynthetically active radiation was not continuously monitored, plants were maintained under uniform glasshouse conditions with consistent natural and supplemental lighting throughout the study.

Two populations, herbicide resistant and herbicide susceptible (details in Table 1), were selected for this study. The seeds were collected from naturally occurring populations of annual ryegrass in Australian grain production systems. The species was identified by the research team, including experienced weed scientists, based on established morphological characteristics described in the literature¹. Annual ryegrass is a widespread and non-protected weed species in Australia, and no specific permits were required for seed collection. All procedures complied with relevant institutional, state and national guidelines. No endangered or protected species were involved in this study. Seed collection was conducted in accordance with farm biosecurity and hygiene protocols. Voucher specimens were not deposited, as this study involved a well-characterized agricultural weed species. Herbicide resistance status was previously confirmed by Plant Science Consulting (Adelaide) following protocols by Broster et al.¹⁶. Prior to sowing, germination percentage rates were evaluated in a growth chamber (25/15°C, 14/10 h light/dark) using Petri dishes.

The soil used was sourced from the Agriculture Reserve at La Trobe University, Bundoora (37.72°S, 145.05°E). After air-drying for 3 days, the soil was sieved (5 mm sieve) and characterized as heavy clay loam with pH 6.0, electrical conductivity 0.10 dS m^− 1, organic matter 3.04%, nitrogen 62 kg ha^− 1, phosphorus 87 kg ha^− 1 and potassium 412 kg ha^− 1 (tested by Nutrient Advantage Laboratory Services, Incitec Pivot Limited, Werribee 3030, Victoria). Before placing the seeds, 5.4 L pots were saturated with tap water to establish maximum soil water holding capacity (WHC), calculated following Bajwa et al.¹²; see details in supplementary material S1). Pots were left to equilibrate for 5 days before sowing. Five seeds of each population were planted per pot; seedlings were thinned to one uniform seedling per pot at the 1–2 leaf stage post-emergence.

Drought treatments were initiated two weeks after emergence and maintained until harvest. Five soil moisture levels were imposed: 100%, 75%, 50%, 25%, and 12.5% WHC, by weighing pots every 2–3 days (later extended to 7-day intervals) and adding precise amounts of water maintained throughout the experimental period. The adjustment to 7-day interval was made because evapotranspiration rates stabilized after canopy establishment and soil moisture loss became slower during later growth stages. Regular monitoring ensured that soil moisture remained close to the target WHC throughout the experiment. Plant biomass was accounted for in weighing adjustments based on destructive sampling of additional plants. Each treatment had five biological replicates, and the experiment was repeated once over time.

Observations

Morphological and growth parameters

Growth related parameters such as the number of leaves, tillers, and plant height were recorded at 3 and 7 weeks after drought treatment. These intervals were chosen for tillering and peak vegetative growth stages. Observations were also made to note the date of first flower for each replicate, and readings were conducted from week 7 to week 12 after drought onset. These data were later used to determine the number of days to flower. Moreover, plant height data were also recorded at harvest in addition to periodic data similar to other growth-related parameters. At harvest (14 weeks after imposing drought), shoots were cut at the soil surface and weighed for fresh weight. Roots were carefully removed from the soil, gently washed, and root length was measured using a ruler. Samples (shoot and root) were oven-dried at 76 °C for 72 h for dry biomass determination.

Physiological parameters

Specific leaf area was recorded after 7 weeks post drought initiation. One representative fully expanded leaf was collected from each biological replicate within each treatment combination and scanned using a Li-Cor LI-3100 C area meter to record leaf area. After drying the leaf at 60 °C for 72 h, dry weight was recorded. The specific leaf area was calculated as a ratio of leaf area to leaf weight¹⁷; see details in supplementary material S1). Relative leaf water content was also recorded after 7 weeks post drought initiation. A fully expanded leaf was excised and weighed immediately (fresh weight), then hydrated in sealed bags for 24 h to obtain turgid weight, followed by drying for 72 h at 60 °C to obtain dry weight. Relative leaf water content was calculated using the formula by Fariñas et al.¹⁸; see details in supplementary material S1). SPAD readings were taken at 7 weeks after drought treatment using a handheld device (SPAD 502 Plus chlorophyll meter – Konica Minolta, Tokyo, Japan). Three readings were taken from the uppermost fully expanded leaf between 10:00–12:00 under full light conditions.

Reproductive parameters

At harvest (14 weeks after imposing drought), reproductive traits were recorded, including number of spikelets per plant, spike length and number of seeds per plant. For each plant, five spikes were randomly selected to measure spike length and spikelets per spike. Total seed production per plant was estimated by collecting all mature spikes and threshing them after drying.

Statistical analysis

All data were analyzed using the analysis of variance (ANOVA) under a factorial design to assess the effects of drought level, population, and their interaction using Statistix 8.1. Prior to ANOVA, assumptions of normality and homogeneity of variance were formally assessed using the Shapiro–Wilk test and Levene’s test, respectively. Residual diagnostics were also visually inspected to confirm compliance with ANOVA assumptions. While data of most parameters followed a normal distribution, the data for shoot and root fresh and dry weights and root length were subjected to log transformation [log (x + 1)] to satisfy assumptions of normality and homogeneity of variance. The ANOVA and subsequent mean comparisons were performed on transformed data; however, mean values presented in tables are based on original (back-transformed) data to maintain biological relevance. Where the interaction between factors was significant (p < 0.05), treatment means were compared using the least significant difference (LSD) test and interpreted based on interaction means only, with no separate comparison of main effects. In cases where interactions were non-significant, main effects were interpreted using mean separation with uppercase lettering. The interaction between experimental runs and treatments was tested, and where non-significant (p > 0.05), data were pooled across runs to improve statistical power. All statistical inferences were made at the 5% significance level.

This study highlights the adaptability of annual ryegrass to a wide range of soil moisture conditions, including both severe drought and waterlogging conditions. While germination stage was negatively affected under drought (see supplementary material S1), established plants demonstrated a strong capacity to survive and reproduce across extreme moisture regimes. The ability of this weed to sustain growth and complete its life cycle, even under such stress, highlights its ecological plasticity, a key factor contributing to its invasiveness and persistence in diverse agricultural environments. The observed drought tolerance is supported by a range of morphological, phenological and physiological adaptive adjustments, some of which varied between the two populations tested.

Morphological, growth and phenology parameters

Morphological responses to drought varied markedly between the herbicide resistant and susceptible populations, particularly at early growth stages (Tables 2 and 3). At 3 weeks, the resistant population exhibited strong growth stimulation under moderate moisture limitation, with leaf number increasing by ~ 132% at 50% WHC (31.1 leaves) compared with well-watered conditions (13.4 leaves), whereas the susceptible population remained relatively stable (13–19 leaves) (Table 2). A similar trend was observed for tillering, where the herbicide resistant population produced ~ 153% more tillers at 50% WHC (8.6 tillers) than at 100% WHC (3.4 tillers), while the susceptible population showed minimal responsiveness (Table 2). By 7 weeks, drought effects plateaued and inherent population differences became dominant, with the resistant population producing ~ 89% more leaves and ~ 62% more tillers than its susceptible counterpart across drought treatments (Table 2). Plant height responses were stage-dependent: early growth showed slight elongation under severe stress (~ 17% increase at 12.5% WHC), while at harvest, moderate moisture (50–75% WHC) maximized plant height (up to 58 cm), representing ~ 35–38% greater height than under severe drought (~ 42 cm) (Table 3). Overall, the resistant population demonstrated greater morphological plasticity, particularly under moderate moisture limitation (50–75% WHC). The enhanced vegetative growth observed under moderate moisture limitation may partly reflect compensatory growth or hormetic-type responses, where mild stress stimulates physiological and developmental processes before inhibitory effects dominate under more severe drought conditions.

Flowering initiation time showed a strong population × drought interaction (p < 0.05), reflecting contrasting adaptive strategies (Table 3). The resistant population flowered significantly later under 100% WHC (85.7 days) but accelerated flowering under drought, with up to 35% reduction at 50% WHC (55–59 days), indicating a drought escape strategy (Table 3). Under field conditions, such accelerated flowering may facilitate earlier seedbank replenishment before terminal drought intensifies, potentially contributing to persistence of annual ryegrass populations in drought-prone agroecosystems. In contrast, the susceptible population flowered earlier overall (51–62 days) and exhibited less variation across drought levels. This pattern may partly reflect inherent life-history differences and a comparatively shorter developmental cycle in the susceptible population rather than solely a drought-induced response. These results also suggest that the resistant population has greater phenological flexibility, enabling adjustment of reproductive timing under stress. Populations of annual ryegrass in Western Australia are known to have adapted to flower early in short growing season¹⁹. This response, reported across multiple weed species, reflects an adaptive survival strategy in which water limitation triggers reduced vegetative growth and an accelerated shift to flowering and seed production, prioritizing reproductive investment under uncertain future resource availability and enabling life-cycle completion before stress intensifies and senescence occurs^20,21. While this may ensure seed production under adverse conditions, it often comes at the cost of reduced biomass and seed yield, as observed in the results of this study.

These results on plant morphology and phenology indicate that Lolium rigidum displayed vigorous vegetative growth during the early to mid-phases of imposed drought stress, as indicated by increased leaf production up to 7 weeks after stress initiation. The herbicide resistant population exhibited more robust growth and delayed senescence, traits often associated with improved photosynthetic efficiency and drought tolerance¹¹. In this study, the herbicide resistant population consistently produced more tillers than the herbicide susceptible population, suggesting a potential advantage in the resource allocation and root function²². Interestingly, plants grown at moderate moisture limitation (50% WHC) showed a spike in tillering during the early stages of drought (3 weeks), indicative of compensatory growth (see photos in supplementary material S2S4). This initial growth surge, however, was unsustainable as stress intensified, tiller production declined sharply by week 7 under severe drought (12.5% WHC), reflecting a shift from adaptive response to physiological inhibition²³.

The results of this study indicate that annual ryegrass achieved optimal growth under moderate soil moisture conditions (50–75% WHC), rather than under saturated or severely water-limited conditions. This has also been observed across several other weed species, for example, in prickly lettuce (Lactuca serriola L.), plants maintained at 75% WHC exhibited the highest plant height, shoot diameter, leaf area, fresh weight and biomass compared with 50%, 25% and fully saturated (100% WHC) soils, indicating that intermediate moisture supports better shoot development than either extreme of dryness or saturation²⁴. The decline in plant height likely reflects limited cell enlargement resulting from reduced turgor pressure during drought stress¹⁰. However, such growth suppression should be viewed not only as a stress symptom but also as an adaptive strategy that enables plants to conserve resources under adverse conditions²⁵.

In studies of other weeds, such as parthenium weed (Parthenium hysterophorus L.) and giant ragweed (Ambrosia trifida L.), similar patterns emerge where moderate soil moisture supports higher growth traits than either drought or excessive wetness^12,26. These patterns align with fundamental soil physics that when soil is excessively wet (near 100% WHC), large pores fill with water and oxygen diffusion into the root zone becomes severely limited, leading to root hypoxia, reduced nutrient uptake and impaired growth^27,28. Conversely, at intermediate soil moisture (50–75% WHC), there is enough water for turgor-driven expansion and metabolic activity while also leaving sufficient air-filled pore space for oxygen diffusion to roots. This balance of water supply and root-zone aeration explains why many weeds show peak morphological response under intermediate moisture rather than at the wettest or driest soil conditions.

Biomass accumulation and root length

Drought stress progressively reduced biomass in both populations (no significant effect of population: p > 0.05), but with notable differences in magnitude and response patterns (Table 4). While statistically non-significant, shoot fresh weight declined by 44% from well-watered (19.2 g) to moderate moisture limitation (10.7 g), and by up to 44% under severe stress averaged across two populations (Table 4). Root fresh and dry weights showed more pronounced reductions (up to 90%) under severe drought averaged across two populations. However, moderate moisture limitation enhanced root elongation, with root length increasing by 20% at 75% WHC (22.5 cm) compared with well-watered conditions (18.8 cm) (Table 4), suggesting an adaptive response to improve water foraging. Severe drought (12.5% WHC), however, reduced root length by 37%. Despite substantial percentage differences, several biomass parameters remained statistically non-significant, likely reflecting the inherently high biological variability characteristic of annual ryegrass populations and the sensitivity of biomass traits to environmental variation. The relatively large standard error observed for root fresh weight under well-watered conditions (100% WHC) reflects the inherently high biological variability of annual ryegrass, particularly for root growth under non-limiting moisture conditions, rather than measurement error. As these data did not satisfy the assumptions of ANOVA, log transformation was applied prior to statistical analysis, and all statistical inferences were based on the transformed data, while original means are presented to facilitate biological interpretation.

Drought-induced reductions in shoot and root biomass observed in this study are consistent with the well-documented inhibitory effects of water deficit on plant growth, primarily through reduced cell expansion, photosynthesis, and assimilate production¹⁰. Although population differences were not statistically significant, the substantial declines in fresh and dry weights (up to 64% for shoots and ~ 90% for roots) indicate that severe drought strongly constrained carbon allocation to both above- and below-ground tissues. The root system, pivotal for water and nutrient uptake, showed altered development under drought. Contrary to typical trends where drought encourages deeper rooting^12,29, this study observed reduced root length under severe drought, likely indicating a shallow rooting strategy in annual ryegrass, similar to perennial ryegrass (Lolium perenne L.)^30,31. This may reflect a carbon saving adaptation, producing smaller roots in drier soil patches to reduce maintenance costs and root respiration^32,33. Reduction in root length under water limiting conditions was also reported in common waterhemp (Amaranthus rudis Sauer)³⁴. Further investigation into root-shoot dynamics and water-use efficiency could clarify the physiological basis for this deviation.

The adaptation of plants to certain environments occurs through the selection of a population that grows well in a certain prevailing condition³⁵. Herbicide resistance in weeds is often associated with a fitness cost where resistant plants may have a disadvantage in terms of growth than susceptible ones³⁶. These costs arise because of the genetic changes that confer resistance with normal physiological processes. However, the extent and nature of fitness costs may vary depending on the resistance mechanism, genetic background, and environmental conditions of the populations^36,37.

Interestingly, in the current study, instead of showing a fitness cost, the resistant population showed vigorous growth by producing more leaves and tillers than the susceptible one. These results are consistent with findings of Wang et al.³⁸ and Beres et al.³⁹ who examined and developed the transgenic rice lines (Oryza sativa) and Arabidopsis thaliana to overexpress 5-enolpyruvyl shikimate phosphate synthase (EPSPS), respectively. The EPSPS gene is responsible for glyphosate resistance in transgenic crops and some weeds³⁸. The overexpression of EPSPS gene showed fitness advantages with higher germination rates, photosynthetic efficiency, enhanced tryptophan and EPSPS protein concentration as well as 48–125% more seeds produced per plant as compared to the control plant without the transgene³⁸. Although, increased fitness associated with overexpression of EPSPS was only identified in 2 out of 7 transgenic lines³⁹. In the current study, the resistant population tended to allocate more resources towards vegetative growth, such as more leaves, tillers and early flowering. In contrast, the susceptible population showed relatively consistent growth and produced more flowers. This shows the comparative strategy of herbicide resistant and susceptible populations of annual ryegrass when grown in similar conditions. These observations suggest that the resistant population possessed traits associated with improved performance under moisture stress conditions. However, direct mechanistic links between herbicide resistance and drought tolerance was beyond the scope of this study and requires further molecular and physiological validation.

Physiological traits

Specific leaf area did not differ between populations and was unaffected by soil moisture levels as well as their interaction with populations (P > 0.05; see supplementary material S1). This suggests that specific leaf area was less sensitive to water availability compared with morphological and biomass traits, which showed clear responses to drought. The lack of significant effects may be due to the conservative nature of this trait, which is often governed by inherent structural and genetic constraints rather than short-term environmental variation. In addition, relatively high within-treatment variability may have masked subtle treatment differences, while compensatory adjustments such as reduced leaf expansion and increased leaf thickness under drought could have resulted in minimal net change. Similar stability in specific leaf area under varying environmental conditions has been reported in previous studies, highlighting its limited plasticity relative to other functional traits^40,41.

Relative water content declined by 14% from well-watered to severe drought conditions, but the resistant population maintained 6% higher relative water content than the susceptible population (Table 5), indicating superior water retention capacity. SPAD readings showed a significant interaction (p < 0.05), with drought generally increasing greenness index which represents chlorophyll concentration (Table 5). SPAD values increased by 18% under 25% WHC compared with well-watered conditions (100% WHC). Notably, the resistant population exhibited a stronger incremental increase under stress, suggesting a compensatory physiological response, whereas the susceptible population showed more moderate but stable changes (Table 5). These results indicate that while the susceptible population relied more on physiological adjustment, the resistant population maintained better water status under stress.

Relative water content declined under drought, but the herbicide-resistant population maintained higher tissue hydration, indicating improved water retention and drought tolerance, a trait linked with persistence in annual ryegrass under stress⁴. Overall, increasing drought led to reduced leaf relative water content, with similar findings observed in transgenic drought tolerant perennial ryegrass⁴². The increase in SPAD values under drought likely reflects a concentration effect associated with reduced leaf expansion and smaller leaf area rather than increased chlorophyll biosynthesis per se, as widely reported in water-stressed grasses¹¹. Together, these responses suggest that the resistant population combines superior water conservation with adaptive physiological adjustment, enhancing its performance under water-limited conditions. These responses likely involve a combination of drought tolerance mechanisms, such as improved tissue water retention, and drought avoidance strategies including accelerated flowering and altered resource allocation patterns^10,11.

Reproductive traits

Reproductive responses were highly sensitive to drought. The number of spikelets per plant and spike length were not affected by population (P > 0.05). Averaged across both populations, spikelets per plant increased by 44% under moderate moisture limitation (75% WHC) compared with well-watered conditions (336.1) but declined under severe stress (278.5) (Table 5). Spike length also peaked under moderate moisture limitation (~ 14% increase at 75% WHC) and declined by ~ 23% under severe drought (Table 5). Seed production exhibited strong interaction effects and high variability between runs, highlighting environmental sensitivity. In the first run, the susceptible population produced up to ~ 280% more seeds than the resistant population under optimal conditions (Table 6). In the second run, maximum seed production occurred under moderate moisture limitation (75% WHC), particularly for the resistant population. Across both runs, severe drought reduced seed production by 30–50% across both populations. These findings suggest that while the susceptible population may have higher reproductive potential under favorable conditions, the resistant population showed more stable performance under stress. Reproductive traits, particularly seed production, are known to be highly sensitive to subtle environmental variation between experimental runs despite controlled glasshouse conditions¹⁹. Minor differences in seasonal radiation, vapor pressure deficit, or plant-to-plant variability may have contributed to the observed inter-run differences.

The highest seed production in annual ryegrass was at 75% WHC, which has been observed in other weeds such as thorn apple (Datura stramonium L.)⁴³, itchgrass (Rottboellia cochinchinensis (Lour.) Clayton)⁴⁴ and parthenium weed¹². Both populations managed to complete their life cycle under high-stress conditions, but with significantly reduced reproductive output. Another Lolium species, L. perenne has been reported to tolerate very high moisture stress (75% lower than soil field capacity), while still maintaining its growth and producing seeds⁴⁵. The persistence of even low seed production under drought poses a significant challenge for weed management, as it can contribute to long-term seedbank replenishment. The ability to survive and reproduce under such conditions highlights the ecological threat posed by annual ryegrass, particularly in drought-prone regions.

Limitations of the current study and considerations for future research

Lolium rigidum is a highly genetically diverse species which is reflected in significant variation in growth patterns of individual plants within different treatment groups. While this variability increased experimental variance, the large number of replicates and repeated trials provided a robust and reliable dataset. Despite these efforts, the study relied on manual watering and monitoring soil moisture levels. Although pots were weighed regularly and adjusted carefully to maintain target moisture levels, minor fluctuations around the intended WHC thresholds may have occurred due to manual watering procedures. This introduces potential for human error and labor-intensive processes, particularly in maintaining precise drought and waterlogging conditions. Future research should consider using automated irrigation systems and soil moisture sensors to improve accuracy and reduce labor input. Moreover, this study focused only on the plant morphological, growth and physiological responses, further studies into biochemical regulatory mechanisms would be needed to better understand the adaptive strategies that enable annual ryegrass to tolerate such drought and other environmental conditions⁴⁶. Future studies should also consider quantifying formal phenotypic plasticity indices and biomass allocation ratios to better characterize adaptive responses under varying environmental conditions.

In addition, this study was conducted under controlled conditions and does not fully capture the complexity of field environments. The findings, while informative, are limited in their extrapolation to broadacre agricultural systems where multiple biotic and abiotic interactions influence plant behavior. Moreover, the current focus on annual ryegrass in isolation does not reflect its interactions with crop species in real-world agroecosystems. Future studies should focus on evaluating the competitive dynamics between annual ryegrass and crop plants under varying moisture regimes to better understand weed–crop competition interactions. Similarly, further research is needed to evaluate the response of annual ryegrass populations to other abiotic stresses, such as heat, salinity, or nutrient imbalances, particularly in the context of climate variability. Furthermore, understanding how environmental stress conditions affect the efficacy of weed control strategies, especially herbicide performance, will be critical for optimizing integrated weed management approaches under changing climatic conditions.

In conclusion, this study demonstrates that annual ryegrass exhibits considerable resilience and adaptability across a wide range of soil moisture conditions. Moderate moisture limitation (50–75% WHC) frequently enhanced vegetative growth, root development, and reproductive traits, whereas severe drought (12.5–25% WHC) consistently reduced plant performance. The herbicide-resistant population exhibited greater phenological plasticity and better water retention, indicating enhanced stress tolerance. In contrast, the susceptible population showed stronger physiological responses and higher reproductive output under favorable conditions but greater sensitivity to environmental variability. These contrasting strategies suggest that herbicide-resistant populations may possess adaptive traits associated with improved performance under moisture-limited conditions, although the underlying mechanisms require further investigation.