Allelopathic Potential of Nicotiana glauca Aqueous Extract on Seed Germination and Seedlings of Acacia gerrardii

Abstract

Nicotian glauca is a noxious invasive shrub in Saudi Arabia, and it is well known for its harmful impact on biodiversity, competing with native plants using various strategies. Among their strategies is their allelochemical activity, i.e., the use of chemicals to dominate and compete. The present study aims to evaluate the allelopathic potential of N. glauca aqueous extracts from leaves, flowers, and twigs on Acacia gerrardi seed germination and seedling growth. Petri dishes containing extracts from N. glauca parts with different concentrations were incubated in a growth chamber. The results indicated that extracts and concentrations negatively impact seed germination and hypocotyl. Relative to the control, the leaf extract with concentrations of 60 and 100 g/L reduced the seed germination rate by 81.11% and 93.33%, respectively. Flower extracts of similar concentrations decreased germination by 81.91 and 92.55%, while the twig extracts declined germination by 79.56 and 95.69%, respectively. The leaf, flower, and twig extracts (100 g/L) decreased hypocotyl radicle by 76.54, 78.05, and 65.75%, respectively. Overall, the concentrations of 20, 60, and 100 g/L showed the lowest growth indices. Generally, aqueous extracts, particularly 100 g/L, impacted the germination and protrusion of A. gerrardii hypocotyl. In conclusion, our study draws attention to the harmful impact of N. glauca on A. gerrardii regeneration.

1. Introduction

Invasive plants play a primary role in decreasing the abundance of native plant species due to their allelopathic effects, therefore contributing to biodiversity loss and damage to ecosystem services at different environmental levels [1]. Also, invasive plants have a great ability to change plant communities because of their rapid growth and high reproduction ability. Furthermore, they impact the environment by affecting soil seed banks and nutrient cycles [2,3].

In Saudi Arabia, Acacia gerrardii Benth. (Talh tree) is a wild native tree that tolerates drought and salinity stresses, and it enhances the fertility of deteriorated soils in arid and semi-arid lands. A. gerradii is considered the most important and major source of nectar and pollen grain for bees in the Arabian Peninsula [4]. N. glauca is an invasive shrub that is 5 m tall [5]. The species is native to South America, particularly Bolivia and Argentina [6]. N. glauca is considered one of the allelopathic and toxic invasive shrub species that recently invaded Saudi Arabia, especially mountainous areas in the south-western parts of the country, causing degradation in soils and vegetation composition in those areas. This shrub can grow in a broad range of disturbed habitats, including roadsides, rocky areas, and coastal beaches [7,8]. In natural habitats, the shrub increased in terms of both population and area by dominating and competing with the native plants [2] and having determinant impacts on native plant germination and seedling growth [9].

Invasive plants suppress neighboring plants by releasing poisonous compounds, a mechanism called allelopathy [10]. Allelopathy is an interference mechanism through which plants release different molecules that may affect seed germination, plant physiology, growth, and the survival of other plants [11]. It is well known that invasive plants, such as N. glauca, have a wide range of trait strategies that help them establish new habitats. Among these characteristics is the ability to produce allelopathic substances that can directly inhibit the coexistence of other plant species [12,13]. The allelochemicals produced by plants can be spread by rainfall water, which flows through the soil surface and then impacts plants’ growth and development [14]. Allelopathic substances were reported to be responsible for suppressing the successful germination and establishment of many other invasive plant species. Allelopathic compounds can have direct effects on plant germination and growth, or they can have indirect effects on plant root–soil interactions, including mycorrhizal associations [15]. Allelochemical substances comprise several different types of chemical compounds; however, most are phenolic compounds that have been found to influence the seed germination of many plants, reducing or even completely inhibiting their germination capacity [16]. Moreover, phenolic allelochemical compounds impact different biochemical and physiological processes in plants like photosynthesis, cell division, enzyme activities, plant water relations, and nutrient movement [17,18]. The actions of allelopathic substances on seed germination can be illustrated through the interruption of plant cellular processes and the modification of membrane permeability [19]. Also, allelopathic substances repress plant phytohormones such as indole acetic acid and gibberellins, which are important for plant cell enlargement [20]. Ref. the study conducted by [21] stated that reserve mobilization mechanisms usually occur during the early stages of seed germination when plants are exposed to allelopathic stress.

It was found that aqueous extracts obtained from plant species (N. glauca, Artemisia princeps var. orientalis Pilocarpus goudotianus), which have great phenol substances, prevent the germination of seeds and the cotyledons of different plants [22,23]. The allelochemical compounds found to have the greatest germination-inhibiting capacity include flavonols, catechols, tannins, and cinnamic acids [24]. These substances are released into the soil through different mechanisms, such as root system exudation, the leaching of shoot parts, and the decomposition of plant litter [25]. The N. glauca shrub produces allelochemical substances, such as alkaloids, steroids, tannins, flavonoids, and saponins, in the leaves, twigs, and flowers [26]. The aqueous extracts of N. glauca were reported to affect the germination and growth of many plants, such as Medicago sativa L., Triticum aestivum L., and Juniperus procera [9,27].

In Saudi Arabia, N. glauca is considered one of the main threats to the native tree cover in general and to A. gerrardii in particular due to their competitiveness and allelopathic activity. To our knowledge, the allelopathic impacts of N. glauca on A. gerrardii are completely unreported, even though allelopathy has been thoroughly researched. Therefore, this study aims to evaluate the allelopathic impact of N. glauca aqueous extracts obtained from leaves, flowers, and twigs on the seed germination of the wild A. gerrardii tree and its early post-emergence. This is the first study to evaluate the impact of N. glauca on A. gerrardii.

2. Materials and Methods

2.1. Plant Materials Collection

Fresh leaves, flowers, and twigs were collected in October 2022 from healthy trees of the invasive N. glauca growing in the Al-Baha region (19°39′48′′ N, 41°21′45′′ E), south-western part of Saudi Arabia. Each plant part (leaves, flowers, and twigs) was kept separately in small paper bags. The plant materials were brought directly by car to the forestry lab at the Faculty of Food and Agriculture Sciences, King Saud University, Saudi Arabia. The leaves, flowers, and twig samples were oven-dried at 50 °C for 72 h, and then grinded using a blinder device and kept in paper bags under lab conditions until use. The ripened pods of A. gerrardii were also collected manually at the same time from healthy A. gerrardii trees growing in the same habitat. The collected pods were kept in small bags for further processing.

2.2. Purification, Treatment, and Viability Test of A. gerrardii Seeds

A. gerrardii seeds were separated from the seed pods physically and sieved to obtain a homogeneous seed sample size. Because A. gerrardii seeds have a hard coating, and due to the likelihood of seed dormancy occurrence, mechanical scarification was applied using sandpaper sheets (9 × 11 inch, silicon with 150 grits). Seed scarification was performed (for 10 min) manually by placing seeds between two sandpaper sheets and rubbing them to create seed abrasion that can facilitate seed water imbibition. Furthermore, the viability of the seeds was tested via the floating method by placing them in a deep dish containing distilled water for 20 min to distinguish the viable and non-viable seeds, where the non-viable seeds floated. Thus, floating seeds were counted as non-viable and then discarded [28]. In addition, t seed germination test (viability test) was conducted, where 200 seeds of A. gerrardii were pretreated by soaking in hot (boiled) water for six minutes to break the seed coat, and immediately, 20 seeds were distributed over double Whatman filter paper No.1 in Petri dishes (90 mm) according to [29]. The filter papers were wetted with 10 mL of distilled water. The germinated seeds were monitored daily, and after 2 weeks of the experiment, the total number of germinated seeds was counted [28]. Germination test on A. gerrardii reached 96% seed viability.

2.3. Preparation of Aqueous Extracts and Germination Bioassay

Various concentrations of the aqueous extract from leaves, twigs, and flowers of N. glauca were prepared separately by soaking the plant powder of each plant part in distilled water at concentrations of 20, 60, and 100 g/L. The suspension was shaken for 24 h at 20 °C. Then, it was centrifuged (3000 rotations) for 30 min; thereafter, extracts were filtered using 2 layers of Whatman filter paper No. 1. The stock of extracts for each plant part (leaves, flowers, and twigs) was kept separately in a refrigerator at 4 ºC till further analyses.

Twenty seeds of A. gerrardii were placed and distributed in Petri dishes (90 mm) lined with 2 layers of Whatman filter paper No. 1, and then 10 mL of each concentration of N. glauca parts (leaves, twigs, and flowers) or water (control) was put on the filter paper. For each treatment, five replications were prepared, with a total of 60 Petri dishes (3 plant parts × 4 treatments × 5 replications). The Petri dishes were then incubated in a growth chamber adjusted at 20 °C and 12 h light/dark cycle, with 75 ± 5 µmol m^–2 s ^–1 light intensity. Seed germination was counted daily for 1 month, and seeds were considered when they developed radicals of >1 mm in length [30]. In addition, other parameters were calculated and measured as follows:

• Germination percentage (GP): GP was measured using the formula below:

  $$\mathrm{GP} =\frac{\mathrm{Number} \mathrm{of} \mathrm{germinated} \mathrm{seeds}}{\mathrm{Total} \mathrm{number} \mathrm{of} \mathrm{seeds}}\times 100$$

  (1)
• Coefficient of velocity of germination (CVG) or germination speed (GS): CVG was computed according to the formula below [31].

  $$\mathrm{Germination} \mathrm{speed}={ \mathrm{N}}_1/{\mathrm{T}}_1+{ \mathrm{N}}_2/{\mathrm{T}}_2+{ \mathrm{N}}_3/{\mathrm{T}}_3+\dots +{ \mathrm{N}}_k/{\mathrm{T}}_k$$

  (2)

  where N₁, N₂, N₃, …, N_k represent the number of germinated seeds perceived at time (days) T₁, T₂, T₃, ⋯⋯, T_k after sowing, while k represents the total number of time intervals.
• Mean germination time (MGT): MGT was measured according to the formula described in [32].

  $$\mathrm{MGT} \left(\mathrm{days}\right)=\sum { \mathrm{T}}_{\mathrm{i} }{\mathrm{N}}_{\mathrm{i} }/\mathrm{S}$$

  (3)

  where T_i is the number of days from the beginning of the test, N_i is the number of germinated seeds per day, and S is the total number of germinated seeds.
• Germination rate index [33]: GRI was measured using the following formula:

  $$\mathrm{GRI}= \mathrm{G}1/1+\mathrm{G}2/2+\dots +\mathrm{Gx}/\mathrm{x}$$

  (4)

  where G1 represents the percentage of germination in the first day after seed sowing, and G2 represents the percentage of germination in the second day after seed sowing.
• First day of germination (FDG) and last day of germination (LDG) were recorded.
• Time spread of germination (TSG): TSG was calculated by different of time intervals between the first and last day of germination [34].
• Radicle length (RL), shoot length (SL), and total length (TL) of hypocotyl were measured using a ruler.
• Vigor Index (VI): = VI was measured using the formula described by [35].

  $$\mathrm{VI}=\left[\mathrm{average} \mathrm{of} \mathrm{shoot} \mathrm{length} \left(\mathrm{mm}\right)+\mathrm{average} \mathrm{of} \mathrm{root} \mathrm{length} \left(\mathrm{mm}\right)\right] \ast \mathrm{GP}$$

  (5)
• Fresh weights of young hypocotyls were determined using electrical balance.

2.4. Estimation of Total Phenols and Tannins of N. glauca Aqueous Extract

Total phenolic content was determined using the Folin–Ciocalteu reagent [36]. In brief, the extract of each plant part sample (0.5 mL of 100 g/L) was mixed with 2 mL Folin–Ciocalteu reagent (diluted 1:10 with de-ionized water) and 4 mL liquid Na₂CO₃ (7.5% w/v). The resulting mixture was kept in the incubator for 30 min at room temperature. The absorbance was read at 765 nm using a spectrophotometer device (SHIMADZU, Kyoto, Japan, UV1800). The equation of a standard curve of gallic acid (as a standard phenolic compound) was used to determine the total phenolic content [36].

On the other side, tannin contents were determined using the method described in [37]. According to this method, 50 µL of the aqueous extract from each plant part (leaves, flowers, and twigs) was placed in a tube containing 1.6 mL of disallowed water and 50 µL of Folin–Ciocalteu reagent. The mixture was kept in the dark for 30 min at room temperature. After that, 300 µL of 35% sodium carbonate solution was added to the mixture. Sample absorbance was measured at 700 nm using a spectrophotometer (SHIMADZU, Kyoto, Japan, UV1800). The standard was prepared using different concentrations (250–750 µg/mL) of tannic acid. The determination of the total tannin content was performed in triplicate.

2.5. Statistical Analysis

The experiments of bioassay were factorial (plant parts × concentration) in Completely Randomized Design (CRD). The collected data were subjected to analysis of variance [38], and means were separated from each other at p < 0.05 by applying the Least Significant Difference (LSD) test. Statistical analysis was applied using the Statistix 10.0 software program. Moreover, Excel software program was used to generate graphs. For the correlation between plant aqueous extracts, concentration treatments, and germination parameters (GP%, MGT, GRI, CVG, FDG, and LDG), analysis of simple regression was applied. Moreover, correlation among all studied parameters was also performed to identify parameters’ relationship.

3. Results

3.1. Effects of Aqueous Extracts on Germination Indices of A. gerrardii

All extracts from different parts of invasive N. glauca showed significant negative impacts on A. gerrardii seed germination in a concentration-dependent manner (Figure 1A–C). For leaves’ extract, after 4 weeks, the percentage of germinated seeds was 90% in the control group, but for 20 g/L, 60 g/L, and 100 g/L, seed germination percentages were 40%, 17%, and 6%, respectively (Figure 1A). Relative to control, 60 and 100 g/L leaf extracts reduced germination by 81.1 and 93%, respectively. The aqueous extracts of the flowers negatively reduced the GP by 48.93% 81.91, and 92.55 for 20 g/L, 60 g/L, and 100 g/L, respectively, while for the control group, the germination percentage was 94% (Figure 1B). For aqueous twig extract, the germination percentages for 20 g/L, 60 g/L, and 100 g/L were 50%, 19%, and 4%, respectively; however, GP was 93% in the control group (Figure 1C).

The results indicate significant differences when using the aqueous extracts of invasive N. glauca on the A. gerrardii seeds’ germination indices. The application of these extracts also slowed down the germination speed, particularly for the seeds that were treated with high concentrations compared to the untreated seeds. The germination speed index was also high in the control germinated seeds, as the germination index decreased with the increase in the extract concentration.

Table 1. Effects of different concentrations of aqueous leaf, flower, and twig extracts of invasive N. glauca on MGT, CVG, and GRI of A. gerrardi.

Plant Part	Concentration (g/L)	Germination Parameters
		MGT (Day)	CVG	GRI (Day)
Leaves	0	8.06 ± 0.25 d	12.58 ± 0.25 a	13.92 ± 0.09 ab
	20	10.13 ± 1.06 cd	10.27 ± 1.02 bc	4.69 ± 0.60 c
	60	19.30 ± 1.43 ab	5.31 ± 0.38 ef	1.03 ± 0.23 d
	100	24.90 ± 0.50 a	4.14 ± 0.05 fg	0.23 ± 0.03 d
Flowers	0	8.04 ± 0.19 d	12.39 ± 0.32 a	14.62 ± 0.48 a
	20	12.22 ± 1.13 cd	8.43 ± 0.75 cd	4.74 ± 0.45 c
	60	14.43 ± 1.10 bc	7.08 ± 0.55 de	1.37 ± 0.31 d
	100	21.80 ± 1.07 a	4.65 ± 0.18 fg	0.31 ± 0.05 d
Twigs	0	8.82 ± 0.17 cd	11.29 ± 0.22 ab	13.21 ± 0.47 b
	20	11.33 ± 1.04 cd	8.96 ± 0.92 cd	5.19 ± 0.61 c
	60	14.18 ± 3.60 bcd	4.53 ± 1.15 fg	1.10 ± 0.28 d
	100	14.20 ± 5.45 bcd	2.81 ± 1.16 g	0.18 ± 0.08 d

Data are displayed as means and ± standard errors (SE) of mean at significant level of p < 0.05. The different superscript letters within each parameter indicated significant difference (p < 0.05).

shows the results of the impact of the leaf, flower, and twig aqueous extracts (at different concentrations) on the mean germination time (MGT), coefficient of velocity of germination (CVG), and germination rate index [39] of A. gerrardii seeds. At 60 and 100 g/L, the leaf and flower extracts significantly increased the MGT of A. gerrardii seeds (p = 0.0001), while in the case of using the twig extracts, it slightly increased. Therefore, when the concentration of the extract increases, the seed germination rate decreases. The leaf and flower extracts at 60 and 100 g/L decreased the rapidity of seed germination compared to the control. At 20 g/L, the values of CVG were the highest for all parts compared to 60 and 100 g/L. The GRI was lower for all concentrations for all parts compared to the control. However, all aqueous extracts of the different plant parts at all concentrations reduced the germination rate of the seeds per day. Generally, the GP%, GRI, and CVG were negatively correlated with the increase in the extract concentration, but the MGT and FDG were positively correlated (Figure 2). The GP% was correlated positively with the CVG and GRI, but was negatively correlated with the MGT, FDG, and LDG (Figure 2).

In

Table 2. Effects of different concentrations of aqueous leaf, flower, and twig extracts of invasive N. glauca on FDG, LDG, and TSG of A. gerrardi.

Plant Part	Concentration (g/L)	Germination Parameters
		FDG	LDG	TSG
Leaves	0	2.60 ± 0.24 d	14.00 ± 0.94 c	11.40 ± 0.97 abc
	20	6.00 ± 1.14 cd	15.20 ± 0.86 c	9.20 ± 1.49 bcd
	60	14.40 ± 2.82 b	23.40 ± 0.67 ab	9.00 ± 2.46 bcd
	100	24.80 ± 0.48 a	25.00 ± 0.54 a	0.20 ± 0.20 e
Flowers	0	3.00 ± 0.00 d	15.00 ± 0.44 c	12.00 ± 0.44 ab
	20	6.00 ± 0.00 cd	18.20 ± 1.11 bc	12.20 ± 1.11 ab
	60	10.40 ± 1.74 bc	18.40 ± 0.92 bc	8.00 ± 2.25 cd
	100	21.60 ± 1.02 a	22.00 ± 1.14 ab	0.40 ± 0.24 e
Twigs	0	3.00 ± 0.00 d	15.00 ± 0.44 c	12.00 ± 0.44 ab
	20	5.40 ± 0.81 cd	19.20 ± 1.39 abc	13.80 ± 1.01 a
	60	10.80 ± 2.80 bc	17.60 ± 4.45 bc	6.80 ± 1.93 d
	100	12.60 ± 5.21 b	13.20 ± 5.45 c	0.60 ± 0.60 e

Data are displayed as means and ± standard errors (SE) of mean at significant level of p < 0.05. The different superscript letters within each parameter indicated significant difference (p < 0.05).

, the aqueous extracts of N. glauca leaves, flowers, and twigs showed significant differences on the first day of germination (FDG) (p = 0.0001), last day of germination (LDG) (p = 0.0140), and time spread of germination (TSG) (p = 0.00001) on the A. gerrardi seeds. However, 60 g/L and 100 g/L of leaf extracts delayed the first germination day until the second and third weeks, respectively. However, the first day of germination for the control and 20 g/L of extracts occurred in the first week of the experiment. Moreover, 90% of seed germination occurred in the second week for the control and 20 g/L of extracts. However, 60 and 100 g/L of extracts significantly delayed germination until the beginning of the fourth week. Similarly, 60 and 100 g/L of flower and twig extracts delayed the beginning of germination to the second and third week, respectively. Regarding the TSG, 100 g/L of leaf, flower, and twig extracts significantly decreased the seed germination speed compared to 20 and 60 g/L. Moreover, the intervals between the first and last germination days increased when the extract concentrations decreased. However, the control and 20 g/L of extract showed the highest values of TSG.

3.2. Effects of Aqueous Extracts on A. gerrardii Hypocotyl Indices

The A. gerrardii hypocotyl treated with aqueous leaf, flower, and twig extracts (at concentrations of 20, 60, and 100 g/L) led to significant reductions in the radicle length (RL), shoot length (SL), and total length (TL) (p = 0.02, 0.0001, and 0.01, respectively) compared to the counterparts’ control extracts (Figure 3). Leaf extracts at 60 and 100 g/L significantly inhibited the protrusion of radicles (0.94 and 0.76 cm, respectively), shoots (1.90 and 1.53 cm, respectively), and total length (2.84 and 2.30 cm, respectively) of the hypocotyl compared with the counterparts’ control extracts. Flower and twig aqueous extracts at 60 and 100 g/L showed significant inhibition in all hypocotyl parameters compared to the control (Figure 3). However, the leaf and flower aqueous extracts showed a greater impact on the RL in comparison to the twig aqueous extracts. Moreover, the hypocotyl TL showed a positive correlation with all other hypocotyl parameters (RL, SL, HW, and VI) and GRI (Figure 4).

Overall, 20, 60, and 100 g/L of leaf, flower, and twig aqueous extracts showed the lowest radicle, shoot, and total length compared to the control. It is clear that plant extracts for all parts had a negative impact on the hypocotyl parameters, and the impact increased with the increasing concentration. The parts’ extracts at 100 g/L showed severe inhibition on the hypocotyl growth parameters (Figure 3 and Figure 5).

The results indicated that the leaves’, flowers’, and twigs’ aqueous extracts had significant negative impacts on the hypocotyl weight [12] (p = 0.001) and vigor index (VI) (p = 0.02). However, reductions in the HW and VI was observed in concentrations of 60 and 100 g/L in comparison to the control (Figure 3).

In comparison to the control, the leaf extract induced a reduction in the HW by 51.5% for 100 g/L and 48.8% for 60 g/L. The flower extract decreased the weight by 52.96 and 54% for 100 and 60 g/L, respectively. For the twig aqueous extracts, the HW was, respectively, reduced by 25.45% and 17.85% at 100 g/L and 60 g/L. The hypocotyls in the control had a higher VI compared to the hypocotyls in all extract concentrations (Figure 3). The reduction in the VI was highly correlated with the aqueous concentrations in all plant part extracts.

3.3. Total Phenols and Tannins in Aqueous Extract Sources of Invasive N. glauca

The results of our study stated that the accumulation of the total phenol and tannin contents significantly differs in the aqueous sources of plant parts. The phenolic compounds showed significant variation among the plant parts (p = 0.0001), with the greatest value recorded in the leaves, followed by flowers and twigs, respectively. However, the tannins showed the highest accumulation in flowers, followed by the leaves and twigs (p = 0.0001) (

Table 3. Total phenolic and tannin contents (mg/g dry weight) of aqueous extracts of different parts of N. glauca plant.

Plant Organ	Compounds
	Total Phenols	Tannins
Leaves	4.30 ± 0.394 a	2.61 ± 0.021 b
Flowers	3.63 ± 0.037 a	4.17 ± 0.019 a
Twigs	0.61 ± 0.027 b	0.75 ± 0.013 c

Data are displayed as means ± standard errors (SE). The different superscript letters within each parameter indicated significant difference (p < 0.05).

).

4. Discussion

The results clearly showed that all plant parts used in this study had allelopathic effects. Many studies have reported that invasive N. glacua contains allelochemical compounds in all of its parts [40]. Plant species in the family Solanaceae have the potential to produce and release allelochemical compounds from their vegetative parts (leaves, twigs, roots, flowers, and bark) [41]. However, chemical compounds released by invasive plant species, such as N. glauca, affect co-existing native plants either during germination or growth, resulting in the alteration of local plant community structures [13].

The percentage of final seed germination of A. gerradii differed according to the extract concentrations, where 60 and 100 g/L of aqueous leaf, flower, and twig extracts showed the lowest germination rate compared to the seeds treated with 20 g/L and the control treatments. This decrease in germination rate is ascribed to the high concentration of inhibiting allelochemical substances contained in the aqueous extracts. The allelochemical compounds weaken or inhibit seed endosperm, decreasing or stopping plant germination entirely [38,42]. Our results are in accordance with those of [43], which reported that Nicotiana tabacum L. extracts negatively affected corn seeds by decreasing the germination rate. The tobacco plant contains secondary metabolites, such as alkaloids and phenols, and all have different effects on the seed germination percentage. The authors of [44] stated that dry leaf and twig extracts of N. glauca had adverse effects on L. sativa germination. Similarly, in laboratory conditions, aqueous and powder extracts obtained from N. tabacum had an inhibitory impact on the germination of Amaranthus retroflexus L. (redroot plants), where the high extract concentration declined and delayed the germination process in this plant [45].

The high accumulation of allelopathic substances is considered one of the factors that had obvious negative impacts on plant germination indices, such as FDG and CVG [46]. This effect is seen in high-concentration treatments.

The results of this study were in line with the findings reported by many researchers. For example, the authors of [47] stated that the effects of invasive plant allelopathic substances on seed germination depend on extract concentrations. Allelochemical compounds typically impair the regeneration of many plant species, according to research workers [46]. They discovered that dried leaf extracts of Eucalyptus camaldulensis inhibited and delayed seed germination and seedlings in A. gerrardii, with concentration-dependent inhibitory effects. Many studies have indicated that invasive weeds possess an inhibitory effect. For example, Asphodelus tenuifolius and Fumaria indica, two invasive weeds, have leaf powder extracts that negatively impact maize germination, resulting in lower germination percentages and germination indices [47]. Further, leaches of leaves and stem extracts of invasive Acasia saligna caused considerable declines in the germination seedling growth of wheat (Triticum aestivum L.), radish (Raphanus sativus L.), barley (Hordeum vulgare L.), and arugula (Eruca sativa L.) [48].

As can be seen, the phenolic allelochemical compounds found in the aqueous extracts of N. glauca delay the germination process of A. gerrardii and destroy the seed embryo. This can be noticed in the decrease in the germination indices (Table 2). It has been stated that the increased content of phenolic allelochemical compounds highly impacts germination indices in several plant species around the globe [49,50].

Accordingly, the leaves, flowers, and twigs of invasive N. glauca contain allelopathic compounds that negatively affected the A. gerrardii hypocotyl weight, vigor index, radicle, shoot, and total length. This phytotoxicity impact may be due to the occurrence and production of reactive oxygen species, which affect plant cell degradation, leading to oxidative stress in the plant cell. Allelochemical compounds interfere with plant cells, resulting in a higher production of reactive oxygen species (ROS) [38], which eventually leads to oxidative stress in plants. ROS may also oxidize the macromolecules in the plant cell, such as proteins and lipids and thus weaken and suppresses plant cell function [51]. Several different compounds enriched with allelopathic effects are produced in different invasive plant parts and tissues, including leaves, stems, and roots; however, their amount and accessibility vary from one plant part to another [52]. Leaves are main sources of influential allelopathic chemicals, while roots release less effective chemicals or phytotoxins [53]. The effects are exacerbated and become more pronounced with increasing concentrations (Table 1 and Table 2, and Figure 1, Figure 2 and Figure 3). Many researchers have reported that plant parts vary in the content of allelochemical compounds [54]. Thus, it can be stated that leaf extracts may have greater concentrations of allelochemical compounds. According to the results stated by [24] the allelochemical compounds exist more in the leaves than in other parts of the plant. This indicates the presence and interference of allelochemical compounds with the tested plants (Table 3). The rate of reduction correlated positively with the increase in the extract concentration [9].

In our study, we observed that aqueous extracts (concentrations of 60 and 100 g/L) obtained from invasive N. glauca leaves, flowers, and twigs resulted in a reduction in the hypocotyl indices, reaching more than 90%. The degree of inhibition was severe on the hypocotyl radicle because the radicle is considered the most sensitive plant part to toxic substances (Figure 3) [55]. According to a report, several allelopathic substances induce reactive oxygen species production in lettuce seedlings, which, in turn, causes oxidative stress in plant cells [37]. This leads to lipid peroxidation, which damages and even kills plant cells. Furthermore, such allelochemicals impact the mitosis of the recipient plant, resulting in a reduced number of cells during the division stage [56]. Additionally, exposure to high concentrations of N. glauca aqueous extracts caused the radicles to weaken and darken. Similar allelopathic impacts of invasive N. glauca on the growth parameters of many plant species have previously been reported. The roots of plants are more susceptible to allelopathic substances than the plants’ vegetative parts [41,57]. Allelochemical substances can impact the apical tissues of plant roots through decreased mitosis, which can result in the destruction of root development and decreased root length [58]. According to an article [9], 60 g/L of aqueous leaf extract of invasive N. glauca severely inhibited the growth of Juniperus procera’s radicle, shoot, and roots. The allopathic extracts obtained from the Artistolochia esperanzae plant also suppressed the growth of sesame roots [59]. Furthermore, leaf, flower, and twig extracts of N. glauca at 60 and 100 g/L reduced the hypocotyl weight by around 50% compared to the control. Also, the hypocotyl vigor index was decreased by the concentration increase. According to [43], the root aqueous extracts of invasive N. glauca inhibited corn (Zea mays L.) growth. However, their leaches from the vegetative part inhibited the development of different plants such as L. sativa [44]. The results of this study are in line with the findings of other studies, which proposed that the aqueous extracts obtained from N. glauca’s leaf, flower, and twig extracts contain water-soluble compounds [26,60] and that they can cause inhibition effects on the target plant [27]. Growth inhibitors extracted from invasive N. glauca parts induce physiological problems during one or more stages of plant development life cycles [22]. Based on reference [61], allelochemicals can inhibit cell division by directly disrupting the process of cell elongation, which alters the balance of cell enzymes.

Phenols and tannins are known to act as allelochemical compounds in N. glauca [62]. Our result clearly stated the presence of phenolic and tannin compounds in the aqueous extracts of invasive N. glauca with the highest concentrations in leaves and flowers. These results agreed with the findings reported in [63], where it was stated that the phenol content in invasive N. glauca varies between plant parts, with high concentrations in the leaves and flowers. In addition, tannin compounds are considered one of the most available secondary metabolites in invasive N. glauca and mostly occur in the roots, leaves, and flowers. The study conducted in [63] reported that the accumulation of tannins in invasive N. glauca occurred in its roots and vegetative parts and was mainly concentrated in the flowers.

5. Conclusions

This study indicates that aqueous extracts (from leaves, flowers, and twigs) of invasive N. glauca negatively impacted A. gerrardii germination. These effects increased with the increasing concentration, particularly in the leaf and flower extracts, because these parts have higher contents of phenolic compounds. These allelopathic substances of N. glauca may inhibit A. gerrardii’s ability to naturally regenerate in their natural habitat, resulting in a monoculture stand where invasive N. glauca predominate over native plants. Our study highlights the allelopathic problems of invasive plants, with an example of the allelopathic impact of N. glauca on A. gerrardii germination and growth. Our findings urge further investigation to identify the allelochemicals and their mode(s) of action on A. gerrardii as well as an intensive field investigation to monitor plant growth behavior.