Comprehensive evaluation of the allelopathic potential of Elymus nutans

Abstract Elymus nutans has been widely planted together with other perennial grasses for rebuilding degraded alpine meadow atop the Qinghai‐Tibetan Plateau. However, the rebuilt sown pastures begin to decline a few years after establishing. One of the possible causes for the degradation of sown grassland may come from allelopathy of planted grasses. The purpose of this study was to examine allelopathic potential of Elymus nutans. Three types of aqueous extract from Elymus nutans and its root zone soil were prepared, and 5 highland crops and 5 perennial grasses were used as recipient plants. Elymus nutans exhibited strong allelopathic potential on germination and seedling growth of 5 crops, but different crops or perennial grasses respond to the extract differently. The pieces aqueous extract have stronger inhibition than whole plant extract and root zone soil extract. Hordeum vulgar var. nudum, Avena sativa, and Festuca sinensis were the most affected, while Chenopodium quinoa and Elymus sibiricus were the least affected. Elymus nutans presented less influence on Poa pratensis and Poa crymophylla than on Festuca sinensis. It is recommended that the species combination of mixture for restoration should be considered for allopathic effects on the coseeding to decrease the seeding rate ratio of Elymus nutans. The annual dicot crop seeds of Chenopodium quinoa and Brassica napus can be used as alternative subsequent crop for the seed field of Elymus nutans monoculture.

that can be used in the alpine meadow region but turned out that only some gramineous perennial grass can adapt to the local natural condition (Zhang et al., 2015(Zhang et al., , 2017. No suitable perennial legumes have been selected to apply to the ecological restoration of degraded meadow (Shang et al., 2017).
Due to the impossibility to restore degraded alpine meadows with native dominant Kobresia plants, mix-seeding(mixture) gramineous perennial grass has been regarded as an ideal option for the revegetation of degraded alpine meadow on the QTP because it increases the diversity and stability of the planted community (Ma et al., 2002;Shi et al., 2009), though the pristine alpine meadow vegetation communities are mainly composed of plant species of Kobresia family, such as Koresia pygmaea and Koresia humilis (Qiao & Duan, 2016). Planting perennial grasslands on degraded alpine meadow can not only increase the land utilization rate and restore the degraded grassland vegetation as soon as possible but also ease the grazing pressure on natural grasslands and prevent grassland degradation and desertification.
The primary grasses being planted for revegetating severely degraded alpine meadow are limited to few graminoid varieties, such as Elymus nutans, Poa crymophila, Poa pratensis, and Festuca sinensis.
Among these germplasms, Elymus nutans is the major seed material for ecological restoration, and the ratio of seeding rate of Elymus nutans in mixture accounts for 50%. This reflects Elymus nutans' relative ease of establishment, high forage production, and sufficient seed supply.
However, the established grassland with perennial grasses begins to decline within 2-4 years, causing economic and ecological loss (Dong et al., 2007). Some sown grasslands are degraded severely (bare land), challenging the sustainable use of revegetated grassland in alpine regions (Shang et al., 2006). It is recorded that most monocropped perennial grasses can be utilized for around ten years in low-altitude areas, but the yield of monoculture of perennial grasses in plateau area begins to decline 3 years after planting which is shorter than expected (Dong et al., 2010).
It is undeniable that redegradation of sown pasture on the plateau may be caused by a combination of biotic and abiotic stresses.
The interspecies (intraspecies) competition may be an important cause (Lin et al., 2018;Uddin et al., 2020). The authors argue that the decline or redegradation of vegetated grassland resulted from interspecies competition between mix-seeding (coseeding) plants and between grasses and unpalatable forbs. The degradation of Elymus nutans monoculture may result from autotoxicity.
Given the fact that much research into plant allelopathy focused on farmland crops and turfgrasses (Yu et al., 2020), and little information about allelopathy of perennial gramineous forage grass is available, we focus our attention on allelopathic effects of Elymus nutans on seed germination and seedling growth of mix-seeding plants and itself. We suppose that the concentration of allelopathicals is below the threshold value of inhibiting in the early stage of the establishment of community or monoculture and is conducive to plants that are invaded or coplanted. With the growth year, the allelopathicals accumulate in the soils, and Elymus nutans not only outcompete coseeding grasses but also release allelopathicals, which enter the soil by leaching and inhibit the germination and the growth of seedlings of itself. The competitiveness of Elymus nutans is weakened, which provides opportunities for invasion of unplatable plants and further accelerates the degradation of seeded pasture and replacement of unwanted vegetation.
The objectives of this study were (a) to evaluate the allelopathical potential of Elymus on highland crops, coseeding grasses, and Elymus itself; and (b) to reveal the potential cause of sown grassland degradation and provide reference for sustainable use and management of revegetated grassland in alpine region. with a small shovel. The plants were cut into aerial parts and belowground parts on site. The root zone soils were collected by shaking the roots parts. All plant and soil materials were put in plastic bags and kept in a refrigerator and taken back to the laboratory for experiment.

| Preparing of extract
Two kinds of plant extract were prepared. The whole plant extract was prepared by soaking whole fresh aboveground parts in distilled water (w/v, 1:4). The pieces aqueous extract were prepared by cutting plants into 1-2 cm and soaking them in distilled water (w/v, 1:4). The containers of soaked plant materials were shaken at 500 rpm for 5 min per 12 hr in 72 hr at room temperature. Soil extract was prepared by soaking fresh root zone soil in distilled water (w/v, 1:2) and shaken at 200 rpm for 24 hr in gyratory shaker at room temperature.
The resulting mixtures were passed through qualitative filter paper and 0.45μm aqueous membrane, respectively. Thus, 0.25 g/ml plant extract and 0.5 g/ml root zone soil extract were obtained. The solutions were stored at 4℃ until use.

| Germination experiment
All seeds were surface-sterilized by soaking in 0.5% sodium hypochlorite solution for 10 min and rinsed several times with distilled water.
Five replicates of 50 seeds were placed in Petri dishes (90 × 15 mm) lined with 2-layer filter paper. The seeds and filter paper were wetted with 3 ml distilled water or extract solution. To maintain moisture, the petri dishes were put in plastic bags and placed in a growth chamber at temperature of 20/16℃ for 12:12 h (light/dark) period. The light intensity was 3,000 lux. A seed was considered germinated when the root protruded ≥2 mm. Germination was counted at 24-hr interval over 15 days, and the first count was carried out on the fourth day.

| Seedling growth experiment
To evaluate the effects of different extract on seedling growth, a completely randomized block design with three replications was applied to conduct seedling growth tests on growth plate (5 × 8 cups; Ø 40 mm; height 80 mm). The media were a mixture of vermiculite/ pearlite (50:50, v/v). Three pregerminated seeds (5 days old) were transplanted into each cup, and ten cups were used as a replicate.
The growth plates were placed in growth chambers with temperature of 20/16℃ for 12:12 h (light/dark) period. The light intensity was 3,000 lux. The moisture content of the media was maintained at a ratio of 1.5:1 solution or water to media on weight basis by spraying different plant extract every three days. After 10 days of growth, the seedling with roots was carefully washed from media. Ten seedlings of a replicate were selected, and the plumule length and radical length of each seedling were measured. The seedlings were ovendried to constant weight at 65℃ and weighed to obtain dry weights.

| Data analysis
Germination force (GF), germination percentage (GP), and germination index (GI) were calculated according to the following equation (Wang et al., 2019): where N 1 , N 2 , N 3 ,…, N n : the number of germinated seeds in the first, second, and final counts, and 1, 2,…, n was the first, second, and final evaluation days.
The allelopathic effect response index (RI) and comprehensive allelopathic effect response index (CE) were calculated using the equation suggested by Williamson and Richardson (1988): where T is the treatment value, C is the corresponding value of control, and RI 1 to RI 6 were allelopathic indexes of GF, GP, GI, root length (RL), shoot length (SL), and dry weight (DW), respectively. Positive values indicate stimulating effects, while negative ones indicate inhibitory activity of the aqueous extract.
Two-way analysis of variance (ANOVA) model was used to test plant species, extract effects, and the species ×extract interaction effects. One-way ANOVA was used to compare treatment effects of different extract on the same recipient plant. Statistically significant difference was assumed at the probability level of 0.05, and means were separated by using LSD.
GF ( % ) = Number of germinated seeds in first one -third of evaluation days∕Total number of Seeds × 100

| Allelopathic potential of Elymus nutans on highland crops
Seed germination (GF, GP, and GI) and seedling growth (SL, DW) of the highland crops were significantly affected by crop species (A), aqueous extract type (B) of Elymus nutans, and their interactions (A × B). RL of the highland crops was significantly influenced by crop species and aqueous extract type but not by their interaction (Table 1).
GF of the tested crop seeds to different extract was dependent on crop varieties and extract types (Figure 1a). Compared with con-

| Autotoxicity of Elymus nutans and its allelopathic potential on perennial grasses
Seed germination and seedling growth of the perennial grasses were significantly affected by species (A), aqueous extract type (B) of Elymus nutans, and their interactions (A × B, Table 2).
The effects of the three types of extract on germination force of perennial grasses are shown in Figure 4a. The control's germination force of Elymus sibiricus was greater than 65% and that of other four grasses was less than 50%, suggesting Elymus sibiricus seed has strong vitality and the emergence is uniform. The pieces aque-

| DISCUSS IONS
Plant allelopathy is a traditional subject, and numerous studies have demonstrated that weeds can exhibit allelopathic effects on economic crops (Nichols et al., 2015). Some perennial grasses, such as Agropyron repens, Lolium perenne, Festuca rubra, and Poa pratensis, have also been proved to be allelopathic to other plants (Fales & Wakefield, 1981;Grummer, 1961). Recently, interest has developed in the allelopathy of agroecosystems, such as cultivated land and commercial forest (Kural & Zkan, 2020;Mushtaq et al., 2020).
However, the role of allelopathic effects of perennial grass on the seeds and seedlings of crops and other grass species remains largely F I G U R E 4 Effects of Elymus nutans extract on germination of highland grasses. Values are mean ± SD (n = 5). Different letters denote significant differences between treatments with a > b > c > d All tested grasses in this study were perennial, different from the aforementioned crops. The highest germination force of control was 67.8%, and the lowest was only 26.2%, reflecting the nonuniformity of germination. The germination rates of control of the five grasses ranged from 63.8% to 81.8%. Except for Elymus sibiricus, which was greater than 80%, the germination rate of other four species was all less than 70%. The control GI of the five grasses was all less than 35%, reflecting the lower vitality of tested perennial grass seeds.
Except that the root zone soil extract did not have a significant effect on the two Poa grasses, other extract treatments caused significant decrease in the germination force, germination rate, and GI of the five grasses. The strongest inhibitory effect was the pieces aqueous extract. Among the five grass species, the least affected was Elymus sibiricus, and the most affected was Festuca sinensis, which was consistent with previous reports (Liang et al., 2020). The effects of the extract on the growth of grass seedlings were mainly manifested in the inhibition of the growth of roots, especially the growth of Elymus nutans and Elymus sibiricus. The three kinds of extract had no significant effect on the seedling growth of Poa pratensis, but other extract had inhibitive effects on the growth of roots and shoots, as well as the DW. Particularly, the effects of the pieces aqueous extract were more apparent. From allelopathic perspective, these may be reasonable explanations for the degradation of mixseeded pasture in alpine area.
Previous studies on autotoxicity of forage mainly focused on alfalfa with less reports of autotoxicity about Gramineae (Ghimire et al., 2019). The present study clearly demonstrated that Elymus nutans had autotoxicity during their germination and seedling growth.
This may help to explain rapid decline of seed yield and aboveground biomass of Elymus nutans monoculture after 3 years.
The differences in the same index for the same crop and forage should be attributed to different extract. The composition of the whole plant extract was analogous to that of rain leaching under natural conditions. The plant pieces aqueous extract contained more components than whole plant extract because sample ground broke plant tissues, and some enzymes, amino acids, inorganic salts, and nitrogen-containing substances entered in the extract. The soil extract from the root zone of Elymus nutans contained root exudates, leachate from aboveground parts, and residues from the decomposition of dead roots in the soil, and soil microorganism-related substances, although the amount may be a little. The composition differences of extract might contribute to the indicator differences of the same crop or forage.

| CON CLUS IONS
Elymus nutans does have allelopathic potential on germination and seedling growth of highland crops or perennial grasses, and the overall effect is inhibited. Different crops or perennial grasses respond differently, and some are sensitive and some are tolerant. The germination force (>80%) and germination rate (>85%) of the control of five crops were relatively higher, reflecting their good germination uniformity. Of the five crops, Hordeum vulgar var. nudum and Avena sativa are susceptible and Chenopodium quinoa is tolerant. Of the five perennial grasses, Elymus sibiricus is the least affected and Festuca sinensis is the most affected. The responses of seed germination and seedling growth of the same crop or grasses vary with the extract from different sources. The pieces aqueous extract has stronger inhibition than others.

Elymus nutans has less allelopathic effects on Poa pratensis and
Poa crymophila than on Festuca sinensis. It is recommended that the co-seeding combination for restoration should consider allopathic

ACK N OWLED G M ENTS
This research was financially supported by the grants from the National Natural Science Foundation of China (31760691) Foundation of Qinghai University (2020-QNY-2). The authors would like thank the anonymous reviewers for their helpful comments.

CO N FLI C T O F I NTE R E S T
None declared.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data and Preregistered, for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at DOI https://doi. org/10.5061/dryad.tht76 hdzv.