Allelopathic Effects of Aqueous Leaf Extracts from Four Shrub Species on Seed Germination and Initial Growth of Amygdalus pedunculata Pall

This study aimed to screen out the shrub species which can promote the seed germination and seedling growth of Amygdalus pedunculata Pall. and offer insight for ecological environment governance of the coal mines subsidence area in Mu Us Sandy Land, Yulin City of Shaanxi Province. The indoor bioassay method was used to study the aqueous leaf extracts from Amorpha fruticosa Linn., Hedysarum mongolicum Turez., Sabina vulgaris Ant., and Hippophae rhamnoides Linn. under different concentration gradients to examine seed germination, initial growth, and physiological and biochemical of two Amygdalus pedunculata varieties (YY-1 from Yuyang County (YY) and SM-6 from Shenmu County (SM), Shaanxi Province, China). The results showed that with aqueous leaf extracts concentrations at lower concentrations of 0.025 (T1) and 0.05 g·mL−1 (T2) from A. fruticosa, H. mongolicum, and S. vulgaris significantly promoted seed germination and seedling growth of two A. pedunculata varieties. Moreover, H. rhamnoides aqueous leaf extracts had the strongest inhibitory effect on seed germination and seedling growth of A. pedunculata, and death occurred at concentrations of 0.15 (T4) and 0.20 g·mL−1 (T5). The enzyme activity and chlorophyll content of the A. pedunculata leaves decreased with an increase in the aqueous leaf extracts concentration of the four shrubs; the change trend of malondialdehyde content was the opposite. Root activity of the A. pedunculata increased and then decreased. The H. mongolicum and S. vulgaris are the most suitable mixed tree species for YY-1, while H. mongolicum and A. fruticosa are the most suitable mixed tree species for SM-6 at a relatively low density with more security. The results provide a theoretical basis and technical support for the establishment of an artificial mixed forest of A. pedunculata in the coal mine subsidence area of Mu Us Sandy Land.


Introduction
Amygdalus pedunculata Pall. is a perennial deciduous shrub and one of the most important ecologically economical tree species.It is widely cultivated in the coal mining subsidence area of Mu Us Sandy Land.For pure forests of A. pedunculata, influenced by local conditions, seedlings are susceptible to sand burial, and sandy soil itself lacks moisture and nutrients [1].Forest biodiversity is low, causing slow growth of A. pedunculata [2][3][4]; thus, it is difficult to realize the shrub's ecological and economic benefits in the short term.At the same time, residents have neglected the management of A. pedunculata, so its resource advantages have not been fully realized.Therefore, farmers and local governments are extremely concerned about improving the stand quality of A. pedunculata, improving its ecological and economic benefits, and having better governance over the ecological environment in the coal mining subsidence area of Mu Us Sandy Land.These issues warrant urgent investigation.
Put simply, a mixed-mode forestry ecological system offers multiple benefits.Many studies have shown that reasonable collocation of mixed tree species increases the diversity of forest communities, effectively improves woodland soil fertility, promotes root growth, and improves nutrient absorption and utilization [5][6][7].Collocation can also effectively inhibit weeds and insect pests, enhance trees' photosynthetic capacity, increase soil enzyme activity and microbial diversity, promote tree growth, and enhance forest stand stability [8][9][10][11].These improvements enhance trees' adaptability via mixed transformation, ecosystem restoration, and potentially improved forest productivity [12].Therefore, in the planting area of A. pedunculata, it will be beneficial to introduce suitable native shrub tree species with reciprocal effects to transform pure forests into mixed forests.
The coal mines subsidence area in Yulin of Shaanxi province, China, is located in the southern margin of Mu Us Sandy Land.The main suitable shrub tree species for planting include Amorpha fruticose Linn., Hedysarum mongolicum Turez., Sabina vulgaris Ant., and Hippophae rhamnoides Linn.Among them, A. fruticosa, H. mongolicum, and H. rhamnoides have an outstanding rhizobium symbiotic nitrogen fixation system to effectively improve soil nitrogen and fertility while increasing soil organic matter, thereby improving sandy land [13][14][15], excluding nutrient competition for A. pedunculata.S. vulgaris is one of the only species of evergreen shrub in the region and plays an irreplaceable role in enhancing the ecological winter landscape [16].More importantly, four species of shrub have extensive root systems, leading to stronger tolerance to cold, drought, barrenness, salt alkali, and sand burial [17,18]; hence, these species can adapt to the environment of sandy areas.The selection leaves of these four species of shrub as associated donor species therefore exhibit a strong typicality and representativeness.
Allelopathy is defined as the beneficial or unfavorable effects of metabolic secretions of plants or microorganisms on other plants or microorganisms in the environment [19].Moreover, allelopathy more commonly refers to the inhibitory effects of one species on another.However, Ninkovic [20] and Glinwood et al. [21] proposed the term 'allelobiosis' to indicated that the exchange of phytochemicals was informative for receiving plants, and the response of receiving plants affects their growth strategy, and their relationship to herbivores and their natural enemies.They argued that allelopathy and allelobiosis were any chemical-mediated interactions between plants, whether positive or negative.Therefore, allelopathy is widely distributed in ecosystems and plays an important role in species' rational allocation, forest tending, weed control, pest control, and disease prevention and control [22][23][24].
Seed germination is the primary and inevitable stage of plant survival and reproduction, and the seedling stage is also a relatively fragile stage during plant growth [25].In these stages, the inhibition or promotion of other plants will affect the seed germination or seedling growth.Many studies have shown that allelochemicals affect the activities of enzymes, malondialdehyde, chlorophyll and root activity in plants, and these physiological and biochemical indicators were important indicators of plant growth.
In nature, the mutual competition of tree species will inevitably lead to competition for sunlight, water, and nutrients.At the same time, compound allelochemicals can inhibit and promote cell membrane permeability, photosynthesis, nutrients and enzyme activities through different release pathways, such as leaching, volatilization, plant residue or litter decomposition, and so on.[19].
However, there is a dearth of research on the allelopathic effects between the endemic economic trees species of A. pedunculata and other shrub species in Yulin region.At present, there was no relevant literature which reported on the growth pattern of the chosen companion species for mixing with A. pedunculata, and also no reference to its competitiveness.
If the A. pedunculata seeds are able to germinate normally and grow healthily in the sand area under the allelopathy and allelobiosis, the survival and natural reproduction of tree species can be guaranteed, and the diversity and stability of community structure can be restored.Therefore, in order to screen out the shrub species which can promote the growth of the A. pedunculata, it is necessary to study the effects of these shrub aqueous leaf extracts on the seed germination and seedling growth of the A. pedunculata.
This study may provide a theoretical basis and technological support for the cultivation of an A. pedunculata mixed forest in the coal mine subsidence area of Mu Us Sandy Land in Yulin City, Shaanxi Province, playing a reciprocal symbiotic role for economic tree species A. pedunculata and associated shrub species to promote ecological environment restoration and enhanced economic benefits.

Seed and Plant Material
Receptors were seeds and seedlings of A. pedunculata (YY-1 from Yuyang County (YY) and SM-6 from Shenmu County (SM), Shaanxi Province, China).The basic layout of seed sampling points is shown in Table 1; the seeds' morphological characteristics appeared in Table 2.The methods were according to Yan et al. [26] and Wang et al. [27]: the thousand weight of seeds and seed kernels were to randomly select 1000 seeds and seed kernels of two A. pedunculata varieties with disease-free pests and full-grain, and weighed them with an electronic balance (FA3104N, Shanghai Jinghai, Shanghai, China), this was repeated 3 times, and then we calculated the average value.The single volume of seed and seed kernel were determined by the volumetric method.Kernel rate was the percentage of seed kernel weight and seed weight.Means in the same column followed by different letters denote significant differences between YY-1 and SM-6 of Amygdalus pedunculata (p < 0.05).
Donors were the leaves of A. fruticosa, H. mongolicum, S. vulgaris, and H. rhamnoides, which were collected from the natural forest of Xiaotuhao township, Yulin City, Shaanxi Province, China in October 2016.Fresh and healthy plants were selected and leaf samples were taken.The leaf samples were separated, air-dried, milled, and spared.

The Aqueous Leaf Extracts Preparation
The experiment was conducted on aqueous extracts from leaves of A. fruticosa, H. mongolicum, S. vulgaris, and H. rhamnoides were prepared by the methods of Le Rouzic et al. [28]: soaking 200 g of plant powder in 1000 mL of distilled water and then keeping the mixtures at room temperature for 20-25 • C for 48 h.The mixtures were then filtered through 300 muslin cloth, and the samples were centrifuged using a micro centrifuge (TG18G-II, Hunan Kaida, Hunan, China) at 3500 rpm for 20 min.The supernatants are hereafter referred to as the undiluted extracts.The extracts obtained were diluted with distilled water to final concentrations of 0 (CK), 0.025 (T1), 0.05 (T2), 0.1 (T3), 0.15 (T4) and 0.2 g•mL −1 (T5), respectively.The aqueous leaf extracts were stored in a refrigerator (HX-30-620E, Dongguan Haoxin, Dongguan, China) at 4 • C until use.

Seed Germination Experiment
The method according to Zhang et al. is as follows [25].Plump and uniform A. pedunculata seeds (kernels) without any pest infestation were carefully selected and soaked in distilled water for 1 day before being disinfected with a 1% KMnO 4 (potassium permanganate) solution for 15 min.The seeds were then rinsed with sterile distilled water and air-dried in a clean bench.Two layers of filter paper were placed into autoclaved petri dishes containing 5 mL of distilled water or different concentrations of the prepared aqueous leaf extracts.Then, 25 seeds of each cultivar of A. pedunculata were laid uniformly in each petri dish (12 cm in diameter).All treatments had four replications.The prepared petri dishes were incubated in an illuminated incubator at 18/23 • C (night/day) at a light intensity of 3000 Lx with a 12 h light/dark photoperiod and humidity of 45%-60%.In addition, the number of germinated seeds was noted every 24 h for 10 days, according to the method of Wang et al. [29].The germination potential, germination rate, and germination index were calculated using the Equations ( 1)-( 3) as follows: Germination potential(%) = the numbers o f seed germination in the third day the total numbers o f seeds × 100 Germination rate(%) = the numbers o f seed germination in the tenth day the total numbers o f seeds × 100 (2) where Gt is the number of seeds emerging on a given day, and Dt is the time after setting the seeds for germination.

Seedling Growth Experiment
After the germination test, 60 strains of consistently growing seedlings were randomly selected from each treatment.Plants were cultivated in 32 plastic plug holes, each of which contained 10 g (1:1) of a sterilized vermiculite and perlite mixture.The plugs were placed randomly in a cultivate frame-type artificial climate chamber for one month under the same culture conditions as for seedling growth.During the seedling growth experiments, 10 mL of different concentrations of shrub aqueous leaf extracts were poured into different treatments every 5 days, and the same volume of distilled water was poured into the control.The length of each seedling and root was measured.The activity of catalase (CAT) was determined by ultraviolet spectrophotometry (UV-2450, Shimadzu Corporation, Kyoto, Japan) [30], peroxidase (POD) was determined by the methyl catechol method [31], and superoxide dismutase (SOD) was determined by the nitro blue tetrazolium photoreduction method [32].The malondialdehyde (MDA) content was determined by the 2-thiobarbituric acid reaction method [33].Fresh leaf tissue (0.5 g) was homogenized in an ice bath in 10 mL of chilled 0.05 mol•L −1 phosphate buffer (pH 7.8) containing 1% (w/v) soluble polyvinyl pyrrolidine (PVP).The homogenates were centrifuged at 4 • C for 20 min at 15,000 rpm, and the resulting supernatants were used for the determination of MDA.The malondialdehyde content reaction mixture consisted of 0.2 mL enzyme extract and 5 mL 0.5% glucosinolates barbituric acid (TBA), the final solution was analyzed with the spectrophotometer at 3 different wavelengths (532 nm, 600 nm, and 450 nm).Total chlorophyll contents of fresh brassica leaves were determined via the method detailed in Arnon [34].Fresh leaf tissue (0.5 g) was placed in 10 mL 80% acetone, until the leaf completely became white, then we added 80% acetone to 25 mL, the final solution was analyzed with the spectrophotometer at 3 different wavelengths (663 nm, 646 nm, and 470 nm).Root activity was assessed using the triphenyl tetrazolium chloride reduction method [31].Fresh root tissue (0.5 g), 5 mL 0.4% triphenyl tetrazolium chloride (TTC), 5 mL 0.067 mol•L −1 phosphate buffer put in 37 • C incubator in the dark for 2 h, and then added to 2 mL 1 mol•L −1 H 2 SO 4 .Finally we put out of the roots into another 10 mL test tube, added to 10 mL methanol, put in 30-40 • C incubator in the dark, until the roots completely became white.The final solution was analyzed with the spectrophotometer at a wavelength of 485 nm.
The allelopathy index (RI), obtained from Equations ( 4) and ( 5) [35], was used to indicate the allelopathic effects of aqueous leaf extracts on seedlings' seed germination, seedling growth, and physiological characteristics: where T is the treatment value and C is the control value.A positive RI value indicated a promotional effect of allelochemicals on A. pedunculata, whereas a negative value indicated allelopathic inhibition.
The synthetic inhibiting effect (SE) value was used as an indicator to evaluate allelopathic effects, calculated as the average RI value of each treatment index [25].

Statistical Analysis
All analyses were conducted using a completely randomized design.Data were statistically processed in SPSS 22.0 (SPSS Inc., Chicago, IL, USA), using one-way ANOVA with the lowest standard deviations test for analysis.Comparisons with p < 0.05 were considered significantly different.Figures were created in OriginPro 9.0 (Origin Lab Corporation, Northampton, MA, USA).

Enzyme Activity
YY-1 and SM-6 showed an increasing trend of CAT activities at concentrations of T1 to T3 for aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris (Figure 3a,b), but POD and SOD activity showed a decreasing trend with increasing aqueous leaf extracts concentrations (Figure 3cf).The results indicated that the stress level of A. pedunculata was unable to regulate the enzyme itself at low concentrations for these shrub aqueous leaf extracts; thus, the seedling of A. pedunculata grew better.In addition, at the same concentration, H. rhamnoides aqueous leaf extracts on CAT, POD, and SOD activity of YY-1 and SM-6 were significantly lower than that of A. fruticosa, H. mongolicum, and S. vulgaris (Figure 3a-f), implying that the YY-1 and SM-6 seedlings suffered greater stress from H. rhamnoides aqueous leaf extracts and died at high concentrations (T4 and T5).

Enzyme Activity
YY-1 and SM-6 showed an increasing trend of CAT activities at concentrations of T1 to T3 for aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris (Figure 3a,b), but POD and SOD activity showed a decreasing trend with increasing aqueous leaf extracts concentrations (Figure 3c-f).The results indicated that the stress level of A. pedunculata was unable to regulate the enzyme itself at low concentrations for these shrub aqueous leaf extracts; thus, the seedling of A. pedunculata grew better.In addition, at the same concentration, H. rhamnoides aqueous leaf extracts on CAT, POD, and SOD activity of YY-1 and SM-6 were significantly lower than that of A. fruticosa, H. mongolicum, and S. vulgaris (Figure 3a-f), implying that the YY-1 and SM-6 seedlings suffered greater stress from H. rhamnoides aqueous leaf extracts and died at high concentrations (T4 and T5).c,d), and superoxide dismutase (SOD) (e,f) of the target A. pedunculata species of YY-1 and SM-6 seedlings exposed to six concentrations 0 (CK), 0.025 (T1), 0.05 (T2), 0.1 (T3), 0.15 (T4), and 0.2 g•mL −1 (T5) of aqueous leaf extracts from the donor species-Amorpha fruticosa (A.f.), Hedysarum mongolicum (H.m.), Sabina vulgaris (S.v.), and Hippophae rhamnoides (H.r.).N means no value (all the plants death and no relevant indicators have been detected).Different little letters indicate significant difference (p < 0.05).Vertical bars represent standard errors (n = 4).

Malondialdehyde Content
The treatment with A. fruticosa, H. mongolicum, S. vulgaris, and H. rhamnoides increased MDA contents in seedlings of YY-1and SM-6 at all concentration (Figure 4a,b).Among the four-sampled species, H. rhamnoides aqueous leaf extracts showed the strongest allelopathy promotional effects on MDA contents in seedlings of YY-1 (T3, 177.73% increase, p < 0.05) and SM-6 (T3, 143.26% increase, p < 0.05), indicating that H. rhamnoides aqueous leaf extracts had stronger effects on the lipid membrane peroxidation of YY-1 and SM-6.Cell membrane permeability was also greater and cell damage was higher, leading to poorer growth of the A. pedunculata seedling.The aqueous leaf extracts of A. fruticosa, H. mongolicum, and A. fruticosa did not exceeded their own range for the degree of membrane damage of YY-1 and SM-6, so the seedlings grew better.

Chlorophyll Content
The chlorophyll contents of YY-1 and SM-6 showed similar trends to POD and SOD activity.The chlorophyll content in seedling of YY-1 and SM-6 decreased with an increase in the concentration of four species of shrub aqueous leaf extracts and showed an inhibitory effect (Figure 4c,d).The variance analysis showed that aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris had no significant difference (p > 0.05) on the chlorophyll content in the SM-6 seedling.In addition, H. rhamnoides aqueous leaf extracts was significantly lower on the chlorophyll content of YY-1 (T3, 69.51% decrease, p < 0.05) and SM-6 (T3, 18.56% decrease, p < 0.05) than that of the control, illustrating a stronger inhibitory effect on YY-1 and SM-6 seedlings in terms of light absorption and utilization efficiency; hence, seeding growth and death at high concentrations were restrained.

Root Activity
The effects of A. fruticosa, H. mongolicum, and S. vulgaris aqueous leaf extracts on the root activity of YY-1 and SM-6 under five concentration gradients showed an initial increase followed by a decrease with increasing extracts concentration (Figure 4e,f).These results suggest that the stress level gradually increased on the root system of YY-1 and SM-6, which may inhibit root growth.As dehydrogenase activity gradually increased, root activity decreased.The treatments with A. fruticosa

Chlorophyll Content
The chlorophyll contents of YY-1 and SM-6 showed similar trends to POD and SOD activity.The chlorophyll content in seedling of YY-1 and SM-6 decreased with an increase in the concentration of four species of shrub aqueous leaf extracts and showed an inhibitory effect (Figure 4c,d).The variance analysis showed that aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris had no significant difference (p > 0.05) on the chlorophyll content in the SM-6 seedling.In addition, H. rhamnoides aqueous leaf extracts was significantly lower on the chlorophyll content of YY-1 (T3, 69.51% decrease, p < 0.05) and SM-6 (T3, 18.56% decrease, p < 0.05) than that of the control, illustrating a stronger inhibitory effect on YY-1 and SM-6 seedlings in terms of light absorption and utilization efficiency; hence, seeding growth and death at high concentrations were restrained.

Root Activity
The effects of A. fruticosa, H. mongolicum, and S. vulgaris aqueous leaf extracts on the root activity of YY-1 and SM-6 under five concentration gradients showed an initial increase followed by a decrease with increasing extracts concentration (Figure 4e,f).These results suggest that the stress level gradually increased on the root system of YY-1 and SM-6, which may inhibit root growth.

Synthetic Inhibiting Effect
Eleven indicators, including germination potential, germination rate, germination index, length of seedling and root, enzyme activity and so on, were used to analyze the allelopathy SE value to reflect the total allelopathic intensity of four species of shrub aqueous leaf extracts on YY-1 and SM-6.Aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris at concentrations of T1 to T3 were positive and had promotional effects on YY-1 and SM-6 (Figure 5a,b).At the concentration of T2, the SE values of YY-1 peaked at 0.06, 0.12, and 0.09, respectively.The SE values of the four species of shrub aqueous leaf extracts on YY-1 were as follows: H. mongolicum > S. vulgaris > A. fruticosa > H. rhamnoides (Figure 5a).The SE values of four species of shrub aqueous leaf extracts at the concentration of T1 to T3 on SM-6 were as follows: H. mongolicum > A. fruticosa > S. vulgaris > H. rhamnoides (Figure 5b).In addition, SE values of H. rhamnoides aqueous leaf extracts at all tested concentrations on YY-1 and SM-6 were negative, indicating an inhibitory effect.and H. mongolicum produced significant promotional effects on the root activity of YY-1 (265.67% and 241.79% increases, respectively, p < 0.05) and SM-6 (288.66% and 236.08% increases, respectively, p < 0.05) at the concentrations of T3.However, H. rhamnoides aqueous leaf extracts had the strongest inhibitory effect on root activity of YY-1 (32.34% decrease, p < 0.05) and SM-6 (28.87% decrease, p < 0.05) at the concentrations of T3.

Synthetic Inhibiting Effect
Eleven indicators, including germination potential, germination rate, germination index, length of seedling and root, enzyme activity and so on, were used to analyze the allelopathy SE value to reflect the total allelopathic intensity of four species of shrub aqueous leaf extracts on YY-1 and SM-6.Aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris at concentrations of T1 to T3 were positive and had promotional effects on YY-1 and SM-6 (Figure 5a,b).At the concentration of T2, the SE values of YY-1 peaked at 0.06, 0.12, and 0.09, respectively.The SE values of the four species of shrub aqueous leaf extracts on YY-1 were as follows: H. mongolicum > S. vulgaris > A. fruticosa > H. rhamnoides (Figure 5a).The SE values of four species of shrub aqueous leaf extracts at the concentration of T1 to T3 on SM-6 were as follows: H. mongolicum > A. fruticosa > S. vulgaris > H. rhamnoides (Figure 5b).In addition, SE values of H. rhamnoides aqueous leaf extracts at all tested concentrations on YY-1 and SM-6 were negative, indicating an inhibitory effect.

Discussion
The seed germination potential could reflect germination speed and uniformity of the seed germination of the receptor plant.The germination rate determines individual survival and population quantity, while the germination index generally reflects the receptor plant seeds' germination process [24].In this study, four species of shrub leaf extracts at a concentration of T1 showed significant promotional effects on the germination potential, germination rate, and germination index of YY-1 and SM-6 (excluding the germination rate of SM-6).Inhibitory effects were enhanced at high concentrations (T4 and T5).The germination index of SM-6 showed a stronger inhibitory effect than that of YY-1.In addition, H. rhamnoides leaf extract had the strongest inhibitory effect on seed germination indicators of A. pedunculate, and death occurred at concentrations of T4 and T5. A. fruticose, H. mongolicum, and S. vulgaris leaf extracts showed significant promotional effects

Discussion
The seed germination potential could reflect germination speed and uniformity of the seed germination of the receptor plant.The germination rate determines individual survival and population quantity, while the germination index generally reflects the receptor plant seeds' germination process [24].In this study, four species of shrub leaf extracts at a concentration of T1 showed significant promotional effects on the germination potential, germination rate, and germination index of YY-1 and SM-6 (excluding the germination rate of SM-6).Inhibitory effects were enhanced at high concentrations (T4 and T5).The germination index of SM-6 showed a stronger inhibitory effect than that of YY-1.In addition, H. rhamnoides leaf extract had the strongest inhibitory effect on seed germination indicators of A. pedunculate, and death occurred at concentrations of T4 and T5. A. fruticose, H. mongolicum, and S. vulgaris leaf extracts showed significant promotional effects on seedling and root length at concentrations of T1 and T2 for YY-1 and SM-6, but the inhibitory effect was enhanced as leaf extract concentrations increased.H. rhamnoides leaf extract had the strongest inhibitory effect on the length and weight of YY-1 and SM-6 seedlings and roots.
Four species of shrub leaf extracts tested for seed germination and seedling growth indicators on YY-1 and SM-6 showed significant promotional effects at low concentrations and significant inhibitory effects at high concentrations.These findings concurred with those of Arowosegbe and Afolayan [36], who reported that stem and root extracts at low concentrations had stimulatory effects on the seedling growth of lettuce and tomatoes, but leaf extracts more potent than stem and root extracts completely inhibited seed germination and tomato seedling growth at a high concentration (10 mg•mL −1 ).However, the effects of H. rhamnoides leaf extracts on seed germination and seedling growth of YY-1 and SM-6 aligned with the results of Mahmoud et al. [37], who stated that jatropha extracts suppressed seed germination and seedling growth in wheat as found in ex-situ bioassay, but seed germination and initial growth at low concentrations (0.5% and 5%) had higher tolerance than at high concentrations (10% and 20%).
Aliskan and Terzi [38] identified seed size as an important factor affecting allelopathic sensitivity.Smaller seeds have fewer mineral nutrient reserves and rely on the absorption of material components from external leachate.In this study, the allelopathic effects and concentration response of the four species of shrub leaf extracts on seed germination and seedling growth of YY-1 and SM-6 may be related to differences between YY-1 and SM-6 seeds: YY-1 seeds (single-seed volume ≈ 1532.09mm 3 ; single-kernel volume ≈ 371.34 mm 3 ; thousand-seed weight ≈ 746.27 g; thousand-kernel weight ≈ 183.76 g) are larger and heavier than those of SM-6 (single-seed volume ≈ 984.99 mm 3 ; single-kernel volume ≈ 250.01 mm 3 ; thousand-seed weight ≈ 448.46 g; thousand-kernel weight ≈ 121.08 g) (Table 2).Therefore, SM-6 appears to be more dependent on the absorption of four species of shrub leaf extracts than YY-1 during seed germination and seedling growth.Thomaszewishi and Thimann [39] found that most allelochemicals inhibit growth by inhibiting gibberellin and auxin receptor levels.Yu and Matsui [40] discovered that allelochemicals resist the absorption of nutrients such as K + , Ca 2+ , Mg 2+ , and PO4 3− by changing the plant's absorptive capacity.Therefore, the differences in seed germination and seedling growth indicators in SM-6 and YY-1 for four species of shrub leaf extract may be related to the allelochemicals in different leaf extracts.
Enzyme activity (CAT, POD, and SOD) and chlorophyll content decreased with an increase in concentrations of four species of shrub leaf extracts.The trend for MDA content was opposite, root activity tended to increase first and then decrease.These results are consistent with research findings of Zeng et al. [41], who used the allelochemical secalonic acid F (SAF) treated with Sorghum Vulgare Pers., Bidens Pilosa L., and Echinochloa crus-galli (L.) Beauv.The receptor plant roots' biological membrane permeability increased significantly; SOD and POD activity decreased significantly.MDA content increased substantially, chlorophyll content decreased, and SAF demonstrated membrane peroxidation that damaged the structure of the entire lipid membrane and weakened plant tissue protection.Meanwhile, Roshchina and Roshchina [42] reported that some polyphenols can inhibit SOD and POD activity in plants, leading to an increase of active oxygen and activation of lipid peroxidation.Chlorophyll can absorb and transfer light energy.The content and composition of chlorophyll determines plants' absorption and utilization efficiency of different light; therefore, it is often considered an important indicator of photosynthetic physiology [25].Many studies have indicated that allelochemicals can affect every aspect of plant growth and plants' physiological and biochemical processes [43][44][45][46][47].An increase in inhibitory allelochemicals such as terpenes, furanones, phenol abscisic acid, coumarin, and so on, can hinder seed germination and seedling growth through physiological and biochemical processes [48].

Conclusions
The aqueous leaf extracts of A. fruticosa, H. mongolicum, and S. vulgaris at low concentrations (T1 and T2) showed significant promotional effects on seed germination and seedling growth indicators for Amygdalus pedunculata Pall.At high concentrations (T4 and T5), the inhibitory effects were enhanced as aqueous leaf extracts concentrations increased.In addition, H. rhamnoides aqueous leaf extracts had the strongest inhibitory effect on seed germination and seedling growth of YY-1 and SM-6.Enzyme activity (CAT, POD, and SOD) and chlorophyll content decreased with an increase in the concentrations of four species of shrub aqueous leaf extracts; however, the trend of MDA content was opposite (root activity tended to increase first and then decrease).The SE values of aqueous leaf extracts from four species of shrub on YY-1 were as follows: H. mongolicum > S. vulgaris > A. fruticosa > H. rhamnoides (T1 to T3).For SM-6, SE values were as follows: H. mongolicum > A. fruticosa > S. vulgaris > H. rhamnoides (T1 to T3).Therefore, H. mongolicum and S. vulgaris appear to be the most suitable mixed tree species for YY-1, while H. mongolicum and A. fruticosa are most suitable for SM-6, and at a relatively low density mixed more securely.H. rhamnoides should not be mixed with YY-1 and SM-6.

Figure 2 .
Figure 2. Effect of shrub aqueous leaf extracts on length of seedling and root of Amygdalus pedunculata.(a-d).Data (Mean ± Standard Error, n = 42) of the seedling length (a,b) and root length (c,d) of the target A. pedunculata species of YY-1 and SM-6 exposed to six concentrations 0 (CK), 0.025 (T1), 0.05 (T2), 0.1 (T3), 0.15 (T4), and 0.2 g•mL −1 (T5) of aqueous leaf extracts from the donor species-Amorpha fruticosa (A.f.), Hedysarum mongolicum (H.m.), Sabina vulgaris (S.v.), and Hippophae rhamnoides (H.r.)-Plants were cultivated in 32 plastic plug holes, each of which contained 10 g (1:1) of a sterilized vermiculite and perlite mixture.The plugs were placed randomly in a cultivate frame-type artificial climate chamber for one month.During the seedling growth experiments, 10 mL of different concentrations of shrub aqueous leaf extracts were poured into different treatments every 5 days, and the same volume of distilled water was poured into the control.N means no value (all the plants death and no relevant indicators have been detected).Different little letters indicate significant difference (p < 0.05).Vertical bars represent standard errors (n = 4).

Figure 2 .
Figure 2. Effect of shrub aqueous leaf extracts on length of seedling and root of Amygdalus pedunculata.(a-d).Data (Mean ± Standard Error, n = 42) of the seedling length (a,b) and root length (c,d) of the target A. pedunculata species of YY-1 and SM-6 exposed to six concentrations 0 (CK), 0.025 (T1), 0.05 (T2), 0.1 (T3), 0.15 (T4), and 0.2 g•mL −1 (T5) of aqueous leaf extracts from the donor species-Amorpha fruticosa (A.f.), Hedysarum mongolicum (H.m.), Sabina vulgaris (S.v.), and Hippophae rhamnoides (H.r.)-Plants were cultivated in 32 plastic plug holes, each of which contained 10 g (1:1) of a sterilized vermiculite and perlite mixture.The plugs were placed randomly in a cultivate frame-type artificial climate chamber for one month.During the seedling growth experiments, 10 mL of different concentrations of shrub aqueous leaf extracts were poured into different treatments every 5 days, and the same volume of distilled water was poured into the control.N means no value (all the plants death and no relevant indicators have been detected).Different little letters indicate significant difference (p < 0.05).Vertical bars represent standard errors (n = 4).

Table 1 .
The basic layout of seed sampling points for experimental materials.