Weed Control and Physiological Responses in Poplar Plantations: Assessing Glyphosate’s Impact

Abstract

The presence of weeds and changes in temperature and precipitation due to global climate change can negatively affect the growth, development, and adaptation of poplars to new places. Experiments were conducted at the Experimental Estate of the Institute of Lowland Forestry and Environment to test glyphosate’s efficacy and phytotoxicity and to assess the impact of glyphosate on physiological parameters in different stages of poplar plantations. A test with glyphosate was set up by a random block system with four replications at three localities, each characterized by different physical soil properties. Glyphosate efficacy was evaluated after 15 and 30 days, while phytotoxicity was evaluated according to the EWRC scale. Net photosynthesis (A), stomatal conductance (gs), transpiration rate (E), and intercellular CO₂ concentration (Ci) were measured, and water use efficiency (WUE) was computed. Annual and perennial broadleaf weeds, grasses, and woody shrubs and bushes were identified. The dominant weed species across all poplar plantations were Solidago gigantea L., Solidago speciosa L., and Poa pratensis L. Glyphosate was highly effective against the major weed species, with the total efficacy ranging from 94.29 to 97.67%. The results showed significant differences in all observed gas exchange parameters, except for transpiration rate (E), between the different-aged poplar plantations. The younger poplars showed lower gas exchange rates in the treatment under the environmental conditions of the studied sites. Weed suppression resulted in altered microhabitats for poplar development at various ages, causing variation in the physiological parameters compared to the control.

1. Introduction

Due to climate change, global temperatures will increase by at least 2 °C in the near future [1]. Global warming and shifts in rainfall patterns drive current climate change, increasing the frequency of heavy rain episodes separated by long dry spells [2]. Under these circumstances, abiotic stresses are likely to become more significant, posing new challenges to ecosystems and agricultural practices. Higher temperatures, drought, and cold stress can negatively affect tree physiology, development, and growth [3,4], which is related to the genetic variation of plants. Finding sustainable methods to maintain and enhance the productivity, quality, resistance, and resilience of forest plantation ecosystems in response to climate change is crucial. This approach will contribute to achieving the United Nations Sustainable Development Goals, reduce the strain on forests, and promote global forest economy [5,6].

Leaf gas exchange is a fundamental aspect of plant physiology that directly affects plant growth. Gases exchange between the whole leaf, a part of the leaf, or the entire plant, with the atmosphere supporting photosynthetic CO₂ uptake and transpiration [7]. As species have different abilities to respond to environmental fluctuations, all responses are linked to the species’ genetic structure. Leaf morphology and anatomical structure, such as thickness, specific leaf mass and stomatal density, can change significantly as plants grow, impacting photosynthesis [8]. Some studies found that rates of photosynthesis and stomatal conductance decrease with the heights or ages of many species [9], but certain research results contradict these fundamental patterns, showing no changes in these rates. In summary, there exists a link between plant physiological processes and plant ages or sizes, although the correlations vary widely depending on the species and site conditions [8].

Poplars are among the fastest-growing trees in temperate latitudes and have been widely used in short-rotation forestry as a source of biomass for biofuels and biomaterials [10,11,12]. The global area of planted poplars has increased to 31.4 million hectares, with the largest plantations occurring in Canada and China, while in Europe, the main plantation areas are in France and Italy [13]. Beyond the economic interest of bioenergy from biomass, the ability to reduce greenhouse gas concentrations and prevent climate change provides an additional motivation for cultivating bioenergy crops like poplar [14]. Based on their economic and ecological benefits, they are commonly planted for afforestation, aesthetic purposes, and the protection of farmsteads and agricultural fields (riparian regions and shelterbelts). Also, they are used for environmental benefits, including phytoremediation, soil carbon sequestration, reduction in sediment run-off, and offering wildlife habitat [15]. Populus spp. offer fast growth rates and high productivity, so proper site preparation is essential to ensure their survival and promote early growth, as the species are particularly sensitive to competition and resource availability [16]. The investment process in poplar wood production involves present financial investments for future economic benefits, with seasonal characteristics. Plantations gradually transfer their value to the products during harvesting, and challenges may arise in a framework of forecasts, investment calculation, and managing human labour and mechanical work [17]. They show high tolerance to various environments and are important subjects in global climate change research [18]. Water availability has a significant impact on poplar biomass productivity, and the degree of drought tolerance plays an important role in determining productivity in selected clones. Although many poplar species are native to areas where there is high soil moisture [19], hybrid clones are being used in many areas where soil moisture may be limiting. Monclus et al. [20] studied 33 poplar genotypes under water deficit conditions and found significant reductions in leaf biomass, as well as a decrease in the maximal sizes of individual leaves due to drought. Several physiological and morphological traits of poplar clones have been observed and are important in drought tolerance, including stomatal sensitivity to water stress, sensitivity of leaf expansion, leaf abscission, and root/shoot ratio changes [21]. However, the growth and productivity of poplars can be significantly impaired by the presence of weeds, which can outcompete them for primary resources such as water, nutrients, light, and space [22,23]. They may also interfere with the trees by releasing toxins, modifying soil and air temperatures, and harboring pests. Climate change, growing populations, and environmental degradation have intensified the demand for limited water resources. Consequently, the increased pressure from water competition with weeds in a field can worsen conditions such as turgor loss, decrease in photosynthesis, stomatal closure and transpiration, halting cell enlargement and metabolic activities and resulting in suppressed plant growth and development and, eventually, reduced production performance [22]. During dry periods, weeds and other unwanted vegetation often compete with young poplar plants. It is a prudent measure to continue controlling the weeds until canopy closure starts to occur and the trees start to dominate or suppress weed growth and establish a woodland environment [24]. Weeds dry the soil by extracting the moisture through their roots and transpiring it from their leaves into the atmosphere [25]. They also have aggressive roots that can outcompete the shallow root systems of poplars for soil water access. As a result, the poplar trees may experience water stress, particularly during drought or limited water availability in the soil. This can lead to the reduced growth, decreased photosynthetic activity, and overall diminished health and vigor of the poplar trees. The sensitivity of poplar trees to the presence of weeds is a critical issue that warrants attention, as it can have significant implications for forest management and ecosystem health. The critical period during weed competition that affects tree growth and survival is thought to be the first year after planting, typically extending for an additional 2 to 4 years. Best practices in silviculture for tree establishment recommend maintaining a weed-free area with a minimum diameter of 1.2 m around each tree for at least the first 3–5 years after planting [26]. Therefore, effective weed control is critical for the successful establishment and growth of poplars. There are several methods for weed control in poplar plantations, such as mechanical, manual, mulching, biological, and chemical control [27]. Nowadays, herbicide application is a popular option and is more cost-effective, due to high manpower costs, labour shortages, and large areas [28,29]. In the forestry sector of the Republic of Serbia, three active substances with herbicidal effects are registered for use: glyphosate, triclopyr, and quizalofop-P-tefuryl [30]. Among these, only glyphosate has been registered for weed control in forest plantations [31]. Glyphosate is the most heavily used and successful herbicide globally [32], used to control weeds in both agricultural and non-agricultural areas [33]. It has been described as a “once in a century herbicide” due to its high efficacy, environmental safety and low cost [34]. Glyphosate inhibits the enzyme 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS), which is crucial for the shikimic acid pathway that produces amino acids such as tyrosine, tryptophan, and phenylalanine [35]. This enzyme is present in plants and microorganisms, but not in animal cells, which is why glyphosate is believed to be harmless to humans and animals. However, over the past decade, concerns have grown about the potentially wide-ranging side effects of glyphosate use on human health and the environment. In 2015, the International Agency for Research on Cancer (IARC) classified glyphosate as “probably carcinogenetic to humans (Group 2A)” [33,36]. Nevertheless, in 2016, the U.S. Environmental Protection Agency (EPA) and the European Food Safety Authority (EFSA) concluded in 2017 that “glyphosate is not likely to be carcinogenetic to human”. These differing opinions arise from the varying methodologies used in the studies [37]. Despite the evidence regarding its carcinogenicity and toxicity, the European Union extended the use of glyphosate in Europe until 2034 [38]. However, glyphosate is a valuable herbicide in sustainable forest management practices in terms of weed control, tree growth promotion, economic efficacy, and low toxicity to non-target organisms. For these reasons, this study aimed to investigate the efficacy of the herbicide glyphosate on weed competition and the physiological responses of poplar plantations of different ages in terms of photosynthetic activity.

2. Materials and Methods

2.1. Study Area and Herbicide Application

The study was conducted in 2021, at three locations (one-, two-, and three-year-old poplar plantations) at the Experimental Estate of the Institute of Lowland Forestry and Environment (45°17′48.2″ N 19°53′30.8″ E) at Kać, Serbia. The experimental plots differed in the physical characteristics of the soil. The soil type at the first location was fluvisol with loam sandy form, containing 84.96% sand and clay fractions of 15.04%. The second location also had soil-type fluvisol but with a loam form, a sand content of 34.04% and clay fractions of 65.96%, while the third location had the highest sand content (99.68%) and the lowest clay fractions (0.32%), with soil-type fluvisol with sandy form [39,40]. The experimental trail was set up by a completely random block system with four replications, with elementary plots of 40 m² (2 m × 20 m), including untreated (control) plots. Chemical weed control was performed with the application of herbicide glyphosate at a rate of 2880 g ha⁻¹ over the whole experimental area, to suppress weed competition and to test its efficacy. Glyphosate doses can vary based on the weed species, application time, environmental conditions, and soil type. The chosen (2880 g ha⁻¹) and higher doses of glyphosate in our country are used for weed control on non-agricultural areas and forest plantations [31]. The glyphosate was applied after weed emergence, using a back sprayer “Solo” with a consumption of 350 L of water per hectare. Efficacy and phytotoxicity assessments were performed after 15 (first assessment) and 30 (second assessment) days of application (Figure 1).

Weeds were identified by Flora of SR Serbia I-IX [41], Flora of Serbia I [42], Flora Europeae I-V [43], and Ikonographie der flora des Sudostlichen Mitteleuropa [44], and then counted using a 1 m² frame randomly placed in the treated and untreated plots. The coefficient of efficacy, Ce (%), of glyphosate was calculated by using the following equation (Equation (1)) [45]:

$$\mathrm{C}\mathrm{e}={\displaystyle \frac{\mathrm{A}-\mathrm{B}}{\mathrm{A}}}\times 100$$

(1)

where A is the average number of weeds in the control, and B is the average number of weeds in the treated plot.

In this way, the efficacy of the herbicide is shown as a relative ratio between the number of destroyed weeds and the weed number in the control, where 0% means no efficacy and 100% means total weed control. The phytotoxicity of glyphosate on the poplar plants was visually assessed using the European Weed Research Council (EWRC) scale from 1 to 9 (

Table 1. Scale of phytotoxicity according European Weed Research Council (EWRC).

EWRC Score	Crop Tolerance	Damaged Plants (%)
1	No effect	0
2	Very slight effects; some stunting and yellowing; just visible	1
3	Slight effects; stunting and yellowing; effects reversible	2
4	Substantial chlorosis and/or stunting; most effects probably reversible	5
5	Strong chlorosis/stunting; thinning of stand	10
6	Increasing severity of damage	25
7	Increasing severity of damage	50
8	Increasing severity of damage	75
9	Total loss of plants and yield	100

).

2.2. Leaf Gas Exchange Assessment

The leaf gas exchange parameters were measured in June 2021 (30 days after the treatment), under field conditions on one-, two- and three-year-old poplar seedlings (Populus × euramericana clone I-214), to evaluate the variability in physiological characteristics. For these measurements, fully developed leaves from the same orientation were chosen. Net photosynthesis (A [µmol m⁻² s⁻¹]), stomatal conductance (gs [mmol m⁻² s⁻¹]), transpiration rate (E [mmol m⁻² s⁻¹]), and intercellular CO₂ concentration (Ci [µmol mol⁻¹]) were measured with an LCPro + portable photosynthesis system (ADC Bioscientific, Ltd., Hoddesdon, UK). These parameters are powerful indicators of the physiological status of plants under experimental or natural growth conditions [46]. Therefore, the leaf gas exchange measurements were used to calculate the water use efficiency (WUE, in μmol CO₂ mol⁻¹ H₂O) [47] as the ratio between A and E [48,49]. The measurements were taken under photosynthetic active radiation (PAR) of 1000 µmol m⁻² s⁻¹ [48,50], while the air humidity, temperature, and CO₂ concentration were assessed during measurement. All measurements were made on three plants per treatment in the period between 09:00 and 11:00 in the morning, in sunny and clear weather. All poplar plants had fully developed leaves without disease, and were exposed to full sunlight in the upper one-third of the canopy.

2.3. Meteorological Data (Temperature and Precipitation)

In 2021, the global average temperature was 1.11 ± 0.13 °C higher than the 1850–1900 average. According to six different data sets, 2021 ranks between the fifth and seventh warmest years ever recorded worldwide. The regions with above-normal precipitation totals in 2021 were Eastern Europe, Southeast Asia, northern South America, and parts of southeastern North America [51].

Monthly meteorological data for temperature and precipitation in the study area for 2021 were obtained from the Republic Hydrometeorological Service of Serbia www.hidmet.gov.rs (acessed on 9 August 2024). The average monthly temperature and precipitation during the experiment are displayed in Figure 2. The meteorological data showed that the year was characterized by a significant range in temperatures, and notable variability in precipitation. The winter months were relatively cold, with January (3.3 °C) and December (3 °C) being the coldest, while February (5.3 °C) showed a slight warming trend. Precipitation during this period was moderate to high, especially in December (88.9 mm). A steady warming trend occurred in spring, when May had significantly warmer temperatures compared to March (6.2 °C) and April (9.6 °C), marking the transition to summer. During May, when the herbicide application was performed, the average air temperature was 16 °C, with an average precipitation of 62.90 mm/m². June, July, and August were characterized by high temperatures. In June, when physiological measurements were made on the poplar seedlings, an extremely warm and dry month was recorded; the average air temperature was 23.30 °C, with a precipitation amount of 23.90 mm/m². Based on the data extracted from the Republic Hydrometeorological Service of Serbia, July was the hottest month (25.50 °C), with the maximum amount of precipitation (114.4 mm/m²). A decline in temperature was observed in autumn, with September still being a relatively warm month (17.9 °C), while November approached winter conditions.

2.4. Statistical Analysis

The significance of the differences between the controls and the treatments was assessed using Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons, based on the one-way analysis of variance (ANOVA). The analyses aimed to detect significant differences between the controls and the treatments at the level p < 0.05. The post hoc test was performed separately for each group. All statistical analyses were conducted with the STATISTICA 13 software package (TIBCO Software Inc., 2020, Palo Alto, CA, USA).

3. Results and Discussion

Weeds are often the primary cause of slow development in forest plantations. Typically, a reduction in tree growth is strongly tied to weed biomass, the dry weight of their crowns and roots. Forest plantations are great habitats for floristically rich and diversified weed flora due to their large inter-row spaces and open canopies during the early stages of development [52]. In forest environments, weeds grow relatively faster, taking over the space of young plants and overshadowing and stifling them. Furthermore, they deplete water and nutrients, harming the cultivated plants.

At all three locations (the one-, two-, and three-year-old poplar plantations), the weed vegetation was determined to establish the glyphosate efficacy. The experimental field was characterized by the abundant presence of diverse weed species, encompassing annual and perennial broadleaf weeds, grasses, and some woody shrubs. Also, some invasive species were recorded, and they can rapidly dominate an area, making it difficult for native tree species to establish themselves. These species often have aggressive growth habits and extensive root systems that compete directly with poplar seedlings for resources. The number of weeds, efficacy and phytotoxicity of the herbicide glyphosate during the all-efficacy assessment in the one-, two-, and three-year old poplar plantations are shown in

Table 2. Efficacy and phytotoxicity of glyphosate in one-year-old poplar plantation.

		First Assessment			Second Assessment
	Weed Species	Control	Glyphosate		Control	Glyphosate
		No/m2	No/m2	Ce (%)	No/m2	No/m2	Ce (%)
Monocotyledons	Avena fatua L.	5.50 ± 0.58	0	100	2.00 ± 0	0	100
	Dactylis glomerata L.	2.75 ± 0.96	0	100	2.50 ± 0.58	0	100
	Lolium spp. L.	3.75 ± 1.71	0	100	2.75 ± 1.71	0	100
	Poa pratensis L.	4.00 ± 2.16	0	100	2.25 ± 1.26	0	100
Dicotyledons	Amorpha fruticosa L.	2.00 ± 0.82	0.25 ± 0.5	87.5	1.50 ± 0.58	0.25 ± 0.5	83.33
	Aristolochia clematitis L.	4.00 ± 2.45	0.25 ± 0.5	93.75	3.00 ± 1.41	0	100
	Asclepias syriaca L.	5.50 ± 1	0.50 ± 0.58	90.9	3.25 ± 0.96	0.50 ± 0.58	84.61
	Clematis vitalba L.	0.25 ± 0.5	0	100	0.25 ± 0.5	0	100
	Erigeron canadensis L.	3.50 ± 1.91	0	100	2.00 ± 1.15	0	100
	Euphorbia cyparissias L.	4.50 ± 1.91	0.75 ± 0.96	83.33	1.75 ± 1.71	0	100
	Euphorbia helioscopia L.	2.00 ± 0	0	100	1.25 ± 0.5	0	100
	Galium aparine L.	1.25 ± 0.96	0	100	2.00 ± 1.41	0	100
	Rubus caesius L.	0.25 ± 0.5	0	100	0.50 ± 0.58	0	100
	Solidago gigantea L.	10.00 ± 2.45	0	100	8.00 ± 2	0	100
	Solidago speciosa L.	4.75 ± 3.4	0	100	2.75 ± 1.89	0	100
	Symphytum officinale L.	0.50 ± 0.58	0	100	0.25 ± 0.5	0	100
	Vicia villosa Roth.	0.75 ± 0.96	0	100	0.75 ± 0.96	0	100
	Viola arvensis Murr.	5.75 ± 1.5	0	100	5.25 ± 1.89	0	100
Horsetail	Equisetum arvense L.	1.50 ± 1.29	0.50 ± 0.58	66.66	1.00 ± 0.82	0.25 ± 0.5	75.00
	Total number of weeds	62.50	2.25		43.00	1.00
	Total efficacy	-	96.40%		-	97.67%
	Phytotoxicity	-	1		-	1

No/m²—number of weeds per square meter; Ce (%)—efficacy coefficient; mean ± standard deviations sd. First and second assessments were conducted 15 and 30 days after glyphosate treatment.

,

Table 3. Efficacy and phytotoxicity of glyphosate in two-year-old poplar plantation.

		First Assessment			Second Assessment
	Weed Species	Control	Glyphosate		Control	Glyphosate
		No/m2	No/m2	Ce (%)	No/m2	No/m2	Ce (%)
Monocotyledons	Apera spica-venti L. P. Beauv.	2.50 ± 1.73	0	100	1.50 ± 1.29	0	100
	Avena fatua L.	2.75 ± 0.96	0	100	2.00 ± 0.82	0	100
	Lolium perenne L.	3.25 ± 1.71	0	100	2.25 ± 0.96	0	100
	Poa pratensis L.	4.25 ± 1.82	0	100	2.00 ± 0	0	100
Dicotyledons	Amorpha fruticosa L.	2.00 ± 0.82	0.50 ± 0.58	75.00	1.00 ± 0.82	0.25 ± 0.58	75.00
	Asclepias syriaca L.	1.75 ± 0.5	0	100	1.00 ± 0.82	0.25 ± 0.5	75.00
	Cirsium arvense L.	0.75 ± 0.5	0	100	0.50 ± 0.58	0	100
	Clematis vitalba L.	0.50 ± 0.58	0	100	1.00 ± 1.15	0	100
	Cornus sanguinea L.	1.25 ± 0.5	0.50 ± 1	60.00	0.75 ± 0.5	0.25 ± 0.5	66.66
	Erigeron annus L.	3.50 ± 1.29	0	100	3.50 ± 1.29	0	100
	Erigeron canadensis L.	2.00 ± 0.82	0	100	1.50 ± 0.58	0	100
	Galium aparine L.	2.25 ± 0.5	0	100	2.50 ± 0.57	0	100
	Robinia pseudoacacia L.	2.25 ± 1.29	0.25 ± 0.5	88.88	1.00 ± 0.82	0.25 ± 0.5	75.00
	Rubus caesius L.	0.75 ± 0.96	0	100	0.25 ± 0.5	0	100
	Solidago gigantea L.	6.50 ± 1.73	0.75	88.5 ± 0.96	7.50 ± 1.91	0	100
	Solidago speciosa L.	6.25 ± 0.5	0	100	3.25 ± 1.26	0	100
	Symphytum officinale L.	0.50 ± 0.58	0	100	0.25 ± 0.5	0	100
	Vitis vinifera L.	0.75 ± 0.5	0.50	33.33 ± 0.58	0.50 ± 0.58	0	100
	Total number of weeds	43.75	2.50		32.25	1.00
	Total efficacy	-	94.29%		-	96.90%
	Phytotoxicity	-	1		-	1

No/m²—number of weeds per square meter; Ce (%)—efficacy coefficient; mean ± standard deviation sd. First and second assessments were conducted 15 and 30 days after glyphosate treatment.

and

Table 4. Efficacy and phytotoxicity of glyphosate in three-year-old poplar plantation.

		First Assessment			Second Assessment
	Weed Species	Control	Glyphosate		Control	Glyphosate
		No/m2	No/m2	Ce (%)	No/m2	No/m2	Ce (%)
Monocotyledons	Apera spica-venti L. P. Beauv	3.00 ± 1.41	0	100	1.75 ± 0.96	0	100
	Dactylis glomerata L.	7.00 ± 1.41	0	100	3.75 ± 0.95	0	100
	Poa pratensis L.	4.50 ± 1	0	100	2.75 ± 0.96	0	100
Dicotyledons	Achillea millefolium L.	1.50 ± 1.29	0	100	1.25 ± 1.50	0	100
	Amorpha fruticosa L.	1.75 ± 0.95	0	100	1.25 ± 0.5	0	100
	Asclepias syriaca L.	1.75 ± 0.5	0.25 ± 0.5	85.71	1.25 ± 0.5	0.25 ± 0.5	80.00
	Clematis vitalba L.	0.25 ± 0.5	0	100	0.25 ± 0.5	0	100
	Cornus sanguinea L.	0.50 ± 0.58	0.25 ± 0.5	50.00	0.50 ± 0.5	0.25 ± 0.5	50.00
	Daucus carota L.	1.00 ± 0.82	0.25 ± 0.5	75.00	0.50 ± 0.57	0	100
	Erigeron annus L.	2.75 ± 1.26	0.75 ± 0.5	72.72	2.50 ± 1.73	0	100
	Erigeron canadensis L.	2.50 ± 0.58	0	100	1.50 ± 0.58	0	100
	Galium aparine L.	2.75 ± 0.5	0	100	2.00 ± 0.82	0	100
	Geranium disectum L.	2.50 ± 0.57	0	100	1.75 ± 0.96	0	100
	Myosotis arvensis L.	0.75 ± 0.5	0	100	1.00 ± 0.96	0	100
	Solidago gigantea L.	9.25 ± 1.89	0.50 ± 0.58	94.59	8.25 ± 1.71	0	100
	Solidago speciosa L.	6.50 ± 1	0	100	4.25 ± 0.96	0	100
	Trifolium campestre Shreb.	2.00 ± 0	0	100	1.25 ± 0.5	0	100
	Vicia villosa Roth.	0.50 ± 0.58	0	100	0.50 ± 0.58	0	100
	Vitis vinifera L.	0.50 ± 1	0.25 ± 0.5	50.00	0.50 ± 1	0.25 ± 0.5	50.00
Horsetail	Equisetum arvense L.	1.25 ± 0.5	0.50 ± 0.58	60.00	1.25 ± 0.5	0.50 ± 0.58	60.00
	Total number of weeds	52.50	2.75		38.00	1.25
	Total efficacy	-	94.76%		-	96.71%
	Phytotoxicity	-	1		-	1

No/m²—number of weeds per square meter; Ce (%)—efficacy coefficient; mean ± standard deviation sd. First and second assessments were conducted 15 and 30 days after glyphosate treatment.

. The values presented in all Tables as ± indicate standard deviations (sd).

The differences in the composition of the weeds were identified through research carried out at the three different locations, differing by soil characteristics. One soil type, fluvisol, was identified in all three locations, and based on the total sand content, three forms were distinguished: sandy loam, loam, and sandy forms. Fluvisols as soil types are characterized by high texture variability, pronounced stratification, and poor humus content [53]. The difference in the physical properties of the soil influenced the varying composition of the weed vegetation across the examined areas, and also the action and behavior of the herbicide glyphosate. In all poplar plantations, broadleaf weeds were more dominant, while grasses were present to a lesser extent.

In the one-year-old poplar plantation, a total of 19 weed species were identified and determined. The specific weeds were present due to the open canopy and relatively unoccupied ground space, and varied depending on the location, soil type, and local climate. The most abundant weed species at the first poplar plantation were Solidago gigantea L. (10.00 weeds m⁻²), Viola arvensis Murr. (5.75 weeds m⁻²), Asclepias syriaca L., and Avena fatua L. (5.50 weeds m⁻²). The total weediness at the control plot was 62.50 weeds m⁻². At the second assessment, the most abundant species were S. gigantea (8.00 weeds m⁻²) and V. arvensis (5.25 weeds m⁻²), with a total weediness at the control of 43.00 weeds m⁻². The number of weeds in the control was higher after the first assessment, but as time progressed, the competition for resources (water, light, nutrients) led to a decrease in the number of individual weeds. This decrease can occur due to several natural processes and ecological factors. These factors act over time, even in the absence of human intervention, causing fluctuations in weed density.

In addition to ferns, herbaceous annual and perennial weeds andwoody weeds like shrubs and bushes were also recorded.

Compared to the control, the weed density per unit area was significantly reduced after glyphosate treatment. Glyphosate was applied for total weed control during the period of intensive growth. Due to its good translocation into rhizomes and roots, its efficacy on the weeds was high [52]. After the first assessment, the herbicide glyphosate showed good efficacy (>90%) on most present weeds. Satisfactory efficacy (75%–90%) was expressed on Amorpha fruticosa L. and Euphorbia cyparissias L., while low efficacy was expressed on the field horsetail, Equisetum arvense L. (66.66%). The total efficacy of the glyphosate after the first assessment was a good 96.40%. The highest efficacy during the second assessment of the glyphosate was shown on 16 weed species (100%), while satisfactory efficacy was expressed on the shrub A. fruticosa and the broadleaf species A. syriaca. As well as during the first assessment, low efficacy was expressed on E. arvense (Table 2). Glyphosate as a total herbicide did not show a phytotoxic effect on the one-year-old poplar seedlings.

At the second location, the most abundant weeds were Solidago gigantea L., Solidago speciosa L., and Poa pratensis L. The species with low abundance were Cirsium arvense L., Clematis vitalba L., Rubus caesius L., Symphytum officinale L., Vitis Vinifera L., and Cornus sanguinea L. (at the second assessment). The total weediness at the control plot after the first assessment was 43.75 weeds m⁻², and after the second assessment it was 32.25 weeds m^-2. During 2021, at the first assessment, glyphosate showed very high efficacy on the majority of the weed species, while satisfactory efficacy was shown on A. fruticosa (Ce 75%), Robinia pseudoacacia L. (Ce 88.88%), and S. gigantea (Ce 88.50%). The glyphosate efficacy was somewhat lower on the shrub C. sanguinea (Ce 60%) and the vigorous climber V. vinifera (33.33%). After the second assessment, satisfactory efficacy was expressed on the shrubs A. fruticosa and R. pseudoacacia, and on the perennial invasive weed A. syriaca. Low efficacy was expressed only on C. sanguinea (Ce 66.66%) (Table 3). Woody weeds are extremely resilient and can regenerate rapidly, making mechanical destruction difficult, which is why herbicide is used. The total efficacy of glyphosate at the two-year-old poplar plantation ranged from 94.29% to 96.90%, which represents good efficacy. According to Clay et al. [54], glyphosate application in the spring and autumn provided excellent or very good control of all species except for young plants of Elytrigia repens (L.) Nevski, Festuca longifolia Thuill., and Molinia caerulea (L.) Moench and older plants of Festuca rubra L.

In the three-year-old poplar plantation, 20 weed species were recorded. The most abundant species were S. gigantea (9.25 weeds m⁻²), D. glomerata (7.00 weeds m⁻²), S. speciosa (6.25 weeds m⁻²), and P. pratensis (4.50 weeds m⁻²). As in the first and second locations, shrubs (A. fruticosa and C. sanguinea), a vigorous climber (V. vinifera), and a flowerless perennial plant (E. arvense) were also determined. The total weediness after the first and second assessments at the control was 52.50 and 38.00 weeds m⁻². High efficacy (>90%) of the systemic herbicide glyphosate was shown against the most annual, perennial broadleaf and grass species (Table 4). Satisfactory efficacy was shown on A. syriaca (80%–85.71%), Daucus carota L., (75%) and Erigeron annus L. (72.72%). After the assessments, low efficacy was expressed on E. arvense (60%), C. sanguinea (50%), and V. vinifera (50%). Glyphosate showed a good total efficacy in the range of 94.76%–96.71% (Table 4). Most woody species tend to regrow later in the first year of plantation, creating a significant challenge due to the limited availability of selective herbicides. This limitation becomes more problematic when regulations, herbicide resistance, or ecosystem sensitivities limit the number of available herbicides. As the only registered herbicide in forest plantations in Serbia, the recorded low glyphosate efficacy against woody species underscores the necessity of integrating comprehensive weed control measures. This holistic approach not only minimizes weed resistance, but also preserves biodiversity and maintains the ecological health of forest ecosystems. Kogan and Alister [24] stated that the recommended glyphosate rate for brush and tree control varies depending on the target species and stage of growth. The rate varies significantly depending on the species’ susceptibility to glyphosate, with ranges of 1.8–2.1 kg a.i. ha⁻¹ (more susceptible species), 2.5–2.88 kg a.i. ha⁻¹ (moderately susceptible), and 3.3–4.0 a.i. ha⁻¹ (more tolerant species). If the weed density is really high or the plants are larger than what is necessary for the best outcomes, the highest rate should be preferred.

The natural variability in photosynthetic gas exchange is crucial for understanding the growth potential of poplar plants and their responses to environmental stresses, including competition from weeds. Net photosynthesis and related gas exchange parameters have been recognized as determinants of plant productivity. Various studies by different authors show that examining these parameters can provide useful information about growth potential and plant productivity [55,56]. Plant photosynthesis is affected not only by environmental factors but also by plant age, a key factor affecting the physiological processes of photosynthesis. However, the implications of stand age in plant photosynthesis remain inadequately understood [57]. The impact of age on photosynthesis can vary between species and even between different genotypes within a species.

With regard to the leaf gas exchange parameters, significant differences between the control and the treatment were detected for all observed parameters—A (net photosynthesis), gs (stomatal conductance), Ci (intercellular concentration of CO₂), and WUE (water use efficiency)—except for parameter E (transpiration).

In the two- and one-year-old poplar plantations, the obtained data of parameter A showed significant differences between the control and the treatment. The highest average values of A were detected in the two-year-old poplar plantation, where the A value at the control was 19.74 µmol m⁻² s⁻¹, and at the treatment was 18 µmol m⁻² s⁻¹. Conversely, the one-year-old poplar leaves exhibited the lowest net photosynthesis (A) values, measured at 16.04 µmol m⁻² s⁻¹ in the control and 10.73 µmol m⁻² s⁻¹ in the treatment (Figure 3). Significant differences were observed between groups in these seedlings, particularly when comparing the two- and three-year-old poplar plantations. The impact of the treatment (glyphosate) on the net photosynthetic rates (A) was most pronounced in the youngest plantation, whereas its effect was least noticeable in the oldest one. Some research has stated that glyphosate indirectly impacts photosynthesis by inhibiting the biosynthesis of chlorophylls, carotenoids, and amino or fatty acids. As a competitive inhibitor of the enzyme EPSPS, glyphosate blocks the shikimate pathway, inhibiting the biosynthesis of the secondary metabolites critical for photosynthesis, including quinones [58,59]. It has been reported that this compound reduces the levels of chlorophyll a and b in plants, impedes electron transfer in PS II photosynthesis, and reduces the photosynthesis rate in Imperata cylindrical L., Arundo donax L. [60], or glyphosate-resistant soya (Glycine Willd.) [61]. Research on similar species like willow (Salix miyabeana) has shown that glyphosate exposure results in decreased chlorophyll content, changes in the photosynthetic apparatus, and an accumulation of hydrogen peroxide, which further impairs photosynthesis by inducting oxidative stress [62]. Among woody plants, poplar trees are known to show a high photosynthetic capacity, although they also exhibit high genotypic variation within the genus [63]. Some types of weeds could potentially increase the photosynthesis of trees indirectly. For example, certain nitrogen-fixing weeds can improve soil fertility by adding nitrogen, which could benefit nearby trees. Leguminous weed plants have long been used in many countries as cover crops for soil and water conservation, especially on steep land, and to improve soil structure [64]. Overall, while weeds around young poplar trees may have both positive and negative effects on their photosynthesis, excessive weed growth is more likely to have a negative impact, particularly if it shades the tree or competes intensely for resources, impacting growth and productivity.

Environmental factors like temperature, light intensity, and CO₂ are often regarded as the primary influencers of photosynthetic rates, and have both direct and indirect effects on stomatal behavior. The stomatal conductance (gs) for water and CO₂ depends on stomatal characteristics such as density (number of stomata per unit leaf area) and aperture (width of pore). The number, size, and distribution of stomata can vary both between and within species, and are typically influenced by the plant’s growth conditions [65].

A significant difference in stomatal conductance (gs) was observed between the control and the treatment in the three-year-old and two-year-old poplar plantations (Figure 4). The highest gs value was recorded in the two-year-old poplar plantation, measuring 0.40 mmol m⁻² s⁻¹ in the control, while in the treatment, the gs value was lower (0.24 mmol m⁻² s⁻¹). The stomatal conductance exhibited a slightly lower trend the in three-year-old poplars, with a gs value of 0.29 in the control and 0.24 mmol m⁻² s⁻¹ in the treatment. However, there was no discernible difference in the gs parameter between the control and the treatment in the one-year-old poplar plantation. The results also show that treatment significantly decreased the gs parameters in all poplar plantations, with the highest reduction recorded in the youngest poplars. Decreased stomatal conductance (gs) in the presence of herbicides can negatively impact carbon assimilation, and therefore photosynthesis [66]. Studies have shown that reduced gs in Hordeum vulgare L., and Lolium perenne L. exposed to glyphosate can constrain photochemistry, leading to a decreased electron transport rate [62]. Ferrell et al. [67] reported that the application of glyphosate, imazapic, and halosulfuron reduced both leaf net carbon assimilation and stomatal conductance (gs) in Cyperus esculentus L. The lack of a discernible difference in gs between the control and the treatment in the one-year-old poplars can likely be attributed to their underdeveloped stomatal regulatory mechanisms. The regulation of stomatal conductance (gs) is an effective mechanism for optimizing the balance between water loss and carbon uptake in plants [68]. A precise representation of the stomatal regulation of transpiration and CO₂ assimilation is essential for predicting how terrestrial ecosystems will respond to global change.

The photosynthesis and transpiration processes of terrestrial ecosystems, including poplar biomass production systems, greatly influence global carbon and hydrologic cycles by facilitating the large-scale exchange of water and carbon between the land and the atmosphere [69]. Transpiration intensity is controlled not only by water deficit, but also by the influence of plant genetics. Also, the process of transpiration can be affected by light, wind, temperature, soil water, and relative humidity [70].

Comparing the intensity of transpiration in all poplar plantations, no statistically significant differences were recorded between the control and the treatment (Figure 5). The intensity of transpiration showed the same trend of changes as the A, where the highest value of E was recorded in the two-year-old poplar plantation (the E value in the control was 3.55 mmol m⁻² s⁻¹, and in the treatment was 3.43 mmol m⁻² s⁻¹). According to Van Oorschot [71], specific inhibitors of the photosynthetic process had a more pronounced effect on the photosynthetic activity than on the transpiration rate. The impact of herbicidal treatments on transpiration is important for reducing water competition from weeds [72]. Increased water competition from weeds leads to stomatal closure, reducing photosynthesis and transpiration, disrupting metabolic processes, and suppressing plant growth and development, eventually reducing production performance [22]. Additionally, managing weed density can indirectly mitigate water stress by enhancing soil moisture retention and modifying the microclimate, thereby contributing to reduced transpiration. Understanding these dynamics is crucial for effective weed management strategies in tree ecosystems.

The obtained results indicate that different conditions and factors can affect the intercellular concentrations of CO₂ between the examined localities. Notably, statistically significant differences in the intercellular concentration of CO₂ (Ci) between the control and the plants in the treatment were observed in the two- and one-year-old poplar plantations (Figure 6). However, these changes compared to the control are significant solely within the youngest poplar plants, specifically in the control, where Ci was 199.22 µmol mol⁻¹, and in the treatment, where Ci was 125.97 µmol mol⁻¹. Despite recording the highest average values of intercellular CO₂ concentration, no statistically significant differences were discerned between the investigated groups in the three-year-old poplar plantation. Understanding how stomata react to both internal and external CO₂ concentrations is crucial for understanding gas exchange between plants and the atmosphere. The presence of weed vegetation can create complex ecological interactions that ultimately influence stomatal responses to CO₂ levels in plants. Despite numerous studies investigating stomatal responses to CO₂ levels, challenges persist in fully grasping this phenomenon due to its susceptibility to various influencing factors, as well as questions whether the stomata on diverse leaf surfaces exhibit uniform responses to gas exchange and how factors such as stomatal density and genotype influence this mechanism [73].

Different water use strategies affect the characteristics of Populus genotypes, such as total water consumption, water use efficiency, and response to drought, and in turn, their impacts on the water availability in the ecosystem [74]. In terms of plant growth, the water use efficiency (WUE) is defined as the aboveground biomass production per unit area per unit water loss (evapotranspired), while from the leaf physiology, the WUE is the ratio of the rate of CO₂ assimilation during photosynthesis to the rate of water transpired [69]. This parameter serves as an important tool for evaluating ecosystem response to climate change.

The highest average values of water use efficiency (WUE) were recorded in the poplar plants in the control of the one-year-old plantation (6.12 µmol·CO₂·mmol⁻¹·H₂O), whereas the lowest WUE value was noted in the treatment group consisting of seedlings of the same age (3.94 µmol·CO₂·mmol⁻¹·H₂O). Statistically significant differences between the control and the treatment were recorded only in the two- and one-year-old plantations (Figure 7). Regardless of weed density, the rates of WUE in all poplars were high when compared to the treatment. Generally, water use efficiency is often considered an optimal strategy in environments where water is limited, considering that the presence and competition of weeds and crops for resources (including water) can affect crop production [75]. Research on the water use efficiency (WUE) of poplar plantations provides valuable insights into how much water the plants require to assimilate a certain amount of carbon. It also aids in assessing how climate change-driven hydrological shifts will affect the carbon budgets of poplar plantation ecosystems in the future [76].

4. Conclusions

The sensitivity of poplar plantations of different ages to weed vegetation is a critical issue that warrants attention, as it can have significant implications for forest management and ecosystem health. Based on the results, this study identified a diverse range of weed species, including annual and perennial broadleaf weeds, grasses, and some woody shrubs and bushes. Specifically, 19 weed species were identified in the one-year-old plantation, 18 in the two-year-old plantation, and 20 in the three-year-old poplar plantation. Among these, Solidago gigantea L., Solidago speciosa L., and Poa pratensis L. had a higher density at the poplar plantations. The tested herbicide, glyphosate, significantly reduced the weed abundance compared to the control, demonstrating high efficacy against weed species. Glyphosate did not show phytotoxic effects on the poplar seedlings (Table 2, Table 3 and Table 4). The results of the leaf gas exchange parameters noted significant differences between the control and the treatment in the rate of net photosynthesis (A), the stomatal conductance (gs), the intercellular concentration of CO₂ (Ci), and the water use efficiency (WUE) in all plantations. The impact of treatment on A, gs, Ci, and WUE was most pronounced in the youngest plantation (one-year-old), and least noticeable in the oldest one, suggesting potential age-related resilience or adaptation over time. By suppressing the weeds, changes in the microhabitats for the development of the poplars at various ages occurred, leading to differences in most important physiological parameters compared to the control.