A method for mixed sowing of orchardgrass and companion species for weed control and application thereof

Abstract

The application provides a method for mixed sowing of dactylis glomerata and companion species for weed control and application thereof, and belongs to the technical field of pasture ecological planting and green weed control, wherein dactylis glomerata is used as a dominant species, and companion pastures such as bromus inermis, trifolium repens and medicago sativa are used; through optimization of a mixed sowing ratio, a seeding amount, a fertilization and mowing system, and by using interspecific competition advantages and root system allelopathy of dactylis glomerata, the germination and growth of weeds are inhibited; under the condition of less or no application of chemical herbicides, the method can make dactylis glomerata maintain a community advantage for a long time, continuously reduce weed biomass and population density, realize ecological safety, long-acting and stable weed control, and is suitable for artificial grassland planting, degraded grassland improvement and green feed production, and has ecological and production benefits.

Description

Technical Field

[0001] This invention relates to the field of forage cultivation and grassland weed control technology, specifically to a method for mixed sowing of orchardgrass and associated species for weed control and its application. Background Technology

[0002] In existing technologies, the mixing ratio of orchardgrass and associated species is mostly determined based on experience, without establishing a quantitative adaptation scheme that takes into account the competitive characteristics of orchardgrass. In practical applications, two extreme problems often occur: first, orchardgrass is too competitive, inhibiting the growth of associated species, resulting in a homogenized mixed community and decreased stability; second, the proportion of associated species is too high, failing to leverage the weed-suppressing advantage of orchardgrass, reducing the total yield and stress resistance of the grassland, and lacking differentiated mixing parameters for different soil and climate conditions, which limits the productivity and adaptability of the mixed grassland.

[0003] Although existing research has confirmed that orchardgrass can inhibit the germination and growth of weeds and associated species, and it is speculated that allelopathic effects exist, the specific components, secretion pathways, and action thresholds of the allelopathic substances remain unclear. Due to the lack of understanding of the allelopathic mechanism, it is impossible to actively enhance the weed control ability of orchardgrass through technical means. It still relies on natural competition to achieve the inhibition effect, which is inefficient and unstable, and difficult to replace chemical herbicides.

[0004] Weed control in existing orchardgrass pastures mainly relies on two methods: first, chemical herbicides, which easily cause soil pollution, pesticide residues in pastures, and herbicide resistance in weeds, contradicting the requirements of green agriculture; second, relying on the natural competitive advantages of orchardgrass, but this effect is affected by factors such as planting years and mixed planting ratios, resulting in poor stability and an inability to specifically control early-stage weeds. The lack of green control technologies that incorporate the characteristics of orchardgrass leads to high weed control costs and significant environmental risks.

[0005] Therefore, developing a mixed-sowing method for orchardgrass that is based on a scientifically calculated mixing ratio and combines high yield with ecological control functions has become a key need to address current technical challenges. Summary of the Invention

[0006] The purpose of this invention is to provide a method for mixed sowing of orchardgrass and associated species for weed control and its application. By optimizing the mixed sowing ratio, seeding rate, fertilization management and mowing system of orchardgrass and associated forage grasses, orchardgrass can maintain its dominance in the mixed sowing community for a long time without the application or with a significant reduction in the use of chemical herbicides, thereby achieving continuous suppression of weed populations.

[0007] A method for intercropping orchardgrass with associated species for weed control utilizes the competitive effect of orchardgrass on associated species and the allelopathic effect of its root exudates to inhibit the germination and growth of weed seeds, comprising the following steps: (1) Select plots with pH 5.5 to 7.5, good drainage, medium or higher soil organic matter, and no severe salinization or compaction. The previous crop should be leguminous forage grass or root crops. (2) The dominant species is orchardgrass, and the companion species are one or more of the following: awnless brome, white clover, alfalfa, etc. (3) Based on the number of effective seeds, orchardgrass accounts for 30% to 90%, and associated species account for 10% to 70% in total; the seed usage of each species is calculated based on 3 to 6 kg of effective seeds / ha of orchardgrass sown alone. (4) Sow in spring or during the rainy season by broadcasting, at a depth of 1-2 cm, and compact after sowing; (5) Apply 20-60 kg / ha of nitrogen fertilizer and 30-60 kg / ha of phosphorus fertilizer as base fertilizer, and remove noxious weeds within 2-3 weeks after emergence; (6) When the average height of the orchardgrass plant reaches 15cm, it should be cut for the first time, leaving a stubble height of 2cm. After that, it should be cut 2 to 3 times a year. (7) After using the above mixed sowing method and management measures for 2 to 3 consecutive years, the dry matter ratio of orchardgrass in the community was consistently higher than the designed ratio, and the cumulative amount of dry matter and number of wild weeds were significantly reduced, thus achieving long-term ecological suppression of weed populations.

[0008] Furthermore, between steps (3) and (4) are: before sowing, soaking the seeds of *Pleurotus ostreatus* in a 50%-75% concentration of rhizosphere sensitization extract for 6-12 hours to promote germination; soaking the seeds of associated species in a 10%-20% low concentration of *Pleurotus ostreatus* rhizosphere sensitization extract for 2-4 hours; and synergistically coating the pretreated seeds. The coating agent is in the following proportions by weight: 10-15 parts of concentrated *Pleurotus ostreatus* rhizosphere sensitization extract powder, 5-8 parts of gelatinous *Bacillus* inoculant, 2-4 parts of phosphate-solubilizing *Bacillus megaterium* inoculant, 1-2 parts of rhizobium inoculant, 3-5 parts of humic acid, 2-3 parts of controlled-release nitrogen fertilizer, 1-3 parts of nano-chitosan, and 1-2 parts of nano-silica modified film-forming agent. The seed-coating ratio is 1:40-1:50.

[0009] Furthermore, before step (5), the following are also included: additional application of 15-30 t / ha of bio-organic fertilizer and 750-1500 kg / ha of silicon-calcium-magnesium soil conditioner as base fertilizer; after the first mowing, topdressing with 30-45 kg / ha of nitrogen fertilizer and 15-30 kg / ha of potassium fertilizer. After each subsequent mowing, a machine vision imaging device combined with soil nitrogen, phosphorus and potassium sensors is used to monitor the dry matter ratio of orchardgrass and the content of available nutrients in the soil in real time, and the amount and method of topdressing are dynamically adjusted based on the monitoring data. Topdressing is carried out by variable fertilization using drones. When the orchardgrass ratio is ≥80%, topdressing with 15-20 kg / ha of nitrogen fertilizer is applied; when the ratio is 50%-80%, topdressing with 20-30 kg / ha of nitrogen fertilizer and 10-15 kg / ha of phosphorus fertilizer is applied; when the ratio is <50%, drones are used to directionally reseed 10%~20% of the base amount of the co-coated orchardgrass seeds in the corresponding area, and topdressing with 30-40 kg / ha of nitrogen fertilizer is applied.

[0010] Furthermore, the associated forage grass is a leguminous forage grass or a grass forage grass; the leguminous forage grass is selected from alfalfa or white clover, and the grass forage grass is selected from awnless brome.

[0011] Furthermore, the first mowing is carried out 2 months after sowing, and the second mowing is carried out 1 month after the first mowing. After mowing, water and fertilizer management of the grassland should be maintained.

[0012] Furthermore, at the time of the first mowing, when the designed proportion of orchardgrass was 40%, 50%, and 70%, the average observed tiller density proportions were 72%, 81%, and 82%, respectively.

[0013] Furthermore, in the aforementioned mixed-sowing method, the cumulative total dry matter yield of orchardgrass and alfalfa mixed-sowing reaches 76.3 g / m². 2 .

[0014] Furthermore, the preparation method of the allelopathic extract of the orchardgrass rhizosphere is as follows: select healthy orchardgrass plants that have grown for 60-90 days, collect roots and 0-20cm of rhizosphere soil, add sterile water at a material-to-liquid ratio of 1:5-1:10, first extract by ultrasonication at 200W for 30 minutes, then extract by shaking at 4℃ for 24-48 hours, filter with double-layer filter paper to obtain the stock solution, and dilute with sterile water to the target concentration; the concentrated powder is prepared by vacuum freeze-drying of the stock solution, and then loaded onto straw biochar with a particle size of 50-100 mesh at a mass ratio of 1:5-1:10 to prepare an allelopathic substance-biochar composite carrier. The total allelopathic substance content in the stock solution is ≥10mg / g, and the total allelopathic substance content in the allelopathic substance-biochar composite carrier is ≥8mg / g.

[0015] Furthermore, the orchardgrass variety is a highly allelopathic orchardgrass variety screened through transcriptome and metabolome sequencing. The screening indicators are: rhizosphere phenolic acid allelopathic substance content ≥1.2mg / g, terpene substance content ≥0.8mg / g, and no significant inhibitory effect on awnless brome, white clover, and alfalfa.

[0016] This invention also proposes the application of the above-mentioned method for mixed planting of orchardgrass and associated species for weed control in the establishment of temperate perennial grasslands, weed control, and high-yield cultivation of forage grasses.

[0017] The present invention has the following technical effects: Achieving green and ecological control of weeds eliminates or significantly reduces the use of chemical herbicides, avoids soil pollution and pesticide residues in pasture caused by chemical agents, and prevents weeds from developing resistance, thus meeting the development needs of green agriculture.

[0018] By improving the stability and forage production efficiency of mixed-sowing communities and maximizing the resource utilization efficiency of the community through scientific mixing ratios and the selection of companion species, orchardgrass can maintain its community dominance in the long term, significantly improving the total yield and stress resistance of the pasture. This solves the problems of monoculture or poor weed suppression in traditional mixed-sowing communities.

[0019] To achieve the artificial and targeted enhancement of the allelopathic effect of oriental fescue, the rhizosphere allelopathic extract of oriental fescue was applied to seed pretreatment before sowing for the first time. The optimal concentration of the extract was determined, which solved the technical pain points of the difficulty in artificially controlling allelopathic effects and the low efficiency and poor stability of weed suppression.

[0020] It combines good ecological and production benefits. Mixed-sowing communities can effectively reduce soil erosion, increase soil organic matter content, and improve the soil environment. At the same time, it can stably produce high-quality forage grass, which is suitable for artificial grassland establishment, degraded grassland improvement and green fodder production, realizing the coordinated development of ecological protection and forage grass production.

[0021] A standardized management system for mixed orchardgrass planting has been established. By optimizing the entire process of sowing, fertilization, and mowing, and combining dynamic topdressing and reseeding strategies, the weed control effect of mixed orchardgrass planting is made long-term and stable, which is convenient for promotion and application in actual production.

[0022] By combining IoT, machine vision, and drone technologies, weed control and grassland management can be intelligently monitored and simplified throughout the entire process, significantly improving the efficiency of large-scale planting and reducing labor costs. The compound microbial agents and allelochemicals form a synergistic effect, which not only enhances the weed suppression effect but also improves the soil micro-ecology. The controlled release of nanomaterials and biochar improves the utilization rate of allelochemicals: nanomaterial coating and biochar loading technologies greatly improve the stability and slow release of allelochemicals, increasing the utilization rate of allelochemicals from about 50% to over 80%, and reducing the cost of using allelochemical extracts. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0024] Figure 1 This is a schematic diagram of the process for the mixed sowing of oriental fescue and associated species for weed control according to the present invention. Figure 2 Images showing experimental sowing in pots; Figure 3 This is a comparative diagram showing the effects of the extract on seed germination. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0026] Example 1 like Figure 1 The diagram shown illustrates the overall process of the mixed sowing method of orchardgrass and associated species for weed control according to the present invention. The following example uses a farm in Sichuan Province. The soil pH is 6.8, drainage is good, organic matter content is 2.5%, and there are no salinization or compaction problems. The previous crop was alfalfa, a legume forage crop.

[0027] The dominant species was orchardgrass, with awnless bromegrass and white clover as companion species. The sowing ratio was calculated based on the effective seed count, with orchardgrass accounting for 70%, awnless bromegrass for 20%, and white clover for 10%. Seed usage was calculated based on an effective seed quantity of 5 kg / ha for orchardgrass sown alone. The orchardgrass seed usage was calculated as 5 kg / ha multiplied by 70%, which is 3.5 kg / ha. The awnless bromegrass seed usage was calculated by converting the baseline orchardgrass seed quantity into the total companion species usage, and then allocating it according to the proportions. The result was 5 kg / ha multiplied by 20% and then divided by 70%, approximately 1.43 kg / ha. The white clover seed usage was calculated similarly, 5 kg / ha multiplied by 10% and then divided by 70%, approximately 0.71 kg / ha.

[0028] Seed pretreatment consists of three steps. The first step is extract preparation. Roots and 0-20cm rhizosphere soil from healthy orchards grown for 75 days are collected and added to sterile water at a material-to-liquid ratio of 1:8. The extract is first ultrasonically extracted at 200W for 30 minutes, then constantly shaken at 4℃ for 36 hours. After filtration through double-layer filter paper, the extract is centrifuged at 8000r / min for 15 minutes. The supernatant is then filtered through a 0.22μm organic filter membrane to obtain the stock solution. The stock solution is then freeze-dried under vacuum to prepare a concentrated powder, which is then loaded onto 80-mesh straw biochar at a mass ratio of 1:8 to prepare an allelochemical-biochar composite carrier. The total allelochemical content in this carrier is ≥8mg / g. The second step is seed soaking. Orchard seeds are soaked in a 60% concentration extract for 10 hours, while awnless bromegrass and white clover seeds are soaked in a 15% concentration extract for 3 hours. The third step is synergistic coating, in which the pretreated seeds are mixed and coated. The coating agent formula is as follows by weight: 12 parts of concentrated powder of rhizosphere chemotherapeutic extract of duckgrass, 6 parts of gelatinous Bacillus agent, 3 parts of phosphate-solubilizing Bacillus megaterium agent, 1.5 parts of rhizobium agent, 4 parts of humic acid, 2.5 parts of controlled-release nitrogen fertilizer, 2 parts of nano-chitosan, and 1.5 parts of nano-silica modified film-forming agent. The seed-coating ratio is controlled at 1:45 during the coating process.

[0029] Sowing should be carried out in late March when the soil moisture is good. Drones are used for large-scale planting areas, while mechanical sowing is used for small plots. The sowing depth is uniformly controlled at about 1.5 cm. After sowing, the soil is immediately compacted with a roller to ensure close contact between the seeds and the soil.

[0030] Base fertilizer is applied once during land preparation before sowing. The types and amounts of fertilizer applied include 40 kg / ha of nitrogen fertilizer (calculated as pure nitrogen), 45 kg / ha of phosphate fertilizer (calculated as phosphorus pentoxide), 20 tons / ha of bio-organic fertilizer, and 1000 kg / ha of silicon-calcium-magnesium soil conditioner. During the seedling management stage, within two weeks after emergence, a small number of germinating noxious weeds, including barnyard grass and Polygonum tinctoria, are manually removed.

[0031] Topdressing management is divided into two stages. After the first mowing, 35 kg / ha of nitrogen fertilizer (calculated as pure nitrogen) and 20 kg / ha of potassium fertilizer (calculated as potassium oxide) are applied using a drone-based variable-rate fertilization method. Subsequent topdressing uses a machine vision imaging system combined with soil nitrogen, phosphorus, and potassium sensors to monitor the dry matter percentage of orchardgrass and the available nitrogen, phosphorus, and potassium content in the soil every 15 days. The monitoring data is transmitted to the terminal system and a topdressing plan is automatically generated. The entire process is implemented through drone-based variable-rate fertilization. For example, if the orchardgrass percentage is 75% after the first mowing in the second year and the available phosphorus content in the soil is low, then 25 kg / ha of nitrogen fertilizer and 12 kg / ha of phosphorus fertilizer (calculated as phosphorus pentoxide) are applied. If the orchardgrass percentage drops to 45% in a certain monitoring, then drones are immediately used to precisely and directionally reseed 15% of the baseline amount of orchardgrass seeds coated as described above at a rate of 5 kg / ha in that area, and then 35 kg / ha of nitrogen fertilizer is applied.

[0032] Harvesting Management The first mowing is carried out when the average height of the orchardgrass reaches 15 cm, leaving a stubble height of 2 cm. The annual mowing plan is formulated according to the climate and pasture growth, and mowing is carried out three times a year, once in spring, once in summer, and once in autumn.

[0033] Implementation effect After three years of continuous use of the above-mentioned mixed sowing method and supporting management measures, four core implementation effects were observed. First, regarding community stability, the proportion of orchardgrass in the total dry matter of the community remained stable at 70% to 75%, consistent with and slightly exceeding the designed proportion of 70%, fully demonstrating that the dominant position of orchardgrass in the community was stably maintained. Second, regarding weed suppression, compared with the plot before implementation or adjacent monoculture plots, the dry matter accumulation of free-growing weeds, including crabgrass and foxtail, in this mixed-sowed grassland decreased by more than 70%, and the number of weeds decreased by more than 80%. The synergistic effect of allelochemical-biochar composite carrier and compound microbial agent significantly improved the weed suppression effect compared to traditional methods. Third, regarding soil and seed growth effects, the compound microbial agent increased the available phosphorus content in the soil by 35% and the available nitrogen content by 20%. Nanomaterial coating increased the germination rate of orchardgrass seeds by 18% and the germination rate of associated seeds by 12%, significantly improving the soil microecological environment. Fourth, in terms of operational efficiency, machine vision-based intelligent monitoring replaces manual inspection, increasing the detection efficiency of orchardgrass by 90%. Drone seeding, topdressing, and precision reseeding improve overall field operation efficiency by 85%, significantly reducing labor costs for large-scale planting. This model has successfully established a community structure with orchardgrass as the dominant species, and awnless bromeliad and white clover coexisting stably, achieving long-term ecological suppression of weed populations and essentially eliminating dependence on chemical herbicides during the planting process.

[0034] The allelopathic extract of *Festuca macrantha* in this invention has a significant weed-suppressing effect. Its core function comes from the phenolic acids, terpenes and alkaloids contained in the extract. These substances work together through different mechanisms to interfere with the germination of weed seeds and the growth of seedlings, thereby achieving targeted inhibition of weeds.

[0035] Preferably, the present invention uses high performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS) to qualitatively and quantitatively identify the effective components of the allelopathic extract of *Festuca macrantha* rhizosphere, clarifying the core active substances and minimum effective concentrations. This provides a repeatable and detectable technical basis for the standardized production, quality testing, and efficacy verification of the extract, and solves the technical problems of unclear effective components and non-standardized production processes in allelopathic extracts.

[0036] The main weed-suppressing substances in the rhizosphere allelopathic extract of *Orchardgrass sinensis* are phenolic acids, monoterpenes / sesquiterpenes, and quinoline alkaloids. Phenolic acids are the main active ingredients, accounting for more than 65% of the total allelopathic substances, terpenes account for 20%–25%, and alkaloids account for 8%–12%. The qualitative identification results and content ranges of each core substance are shown in Table 1. Based on the original extract prepared from the 0-20cm rhizosphere soil of healthy *Orchardgrass sinensis* plants grown for 75 days, the total allelopathic substance content was ≥10 mg / g.

[0037] Table 1 Various allelochemicals interfere with key physiological processes in weed seed germination and seedling growth through synergistic effects. The main mechanisms of action are as follows, and different substances have complementary inhibitory targets on weeds, achieving a dual blockade of weed germination and growth: Phenolic acids: These substances mainly exert their effects by inhibiting the activity of key enzymes in seed germination, interfering with cell membrane permeability, and hindering the establishment of the photosynthetic system. Ferulic acid and coumaric acid can significantly inhibit the activity of α-amylase and lipase in weed seeds, reducing the hydrolysis efficiency of starch and fat in the seeds, causing a lack of energy and nutrient supply for seed germination, and delaying or even blocking the germination process. Gallic acid and p-hydroxybenzoic acid can damage the cell membrane structure of root cells in weed seedlings, increase cell membrane permeability, leading to the leakage of intracellular electrolytes, loss of the root cells' ability to absorb water and fertilizer, and inhibiting the formation of chloroplast grana in seedlings, reducing chlorophyll synthesis, preventing normal photosynthesis in seedlings, and ultimately leading to yellowing and wilting.

[0038] Terpenes: Their core function is to interfere with plant hormone metabolism and inhibit cell division and elongation. Linalool and eucalyptol can inhibit the synthesis of gibberellin (GA) in weed seedlings while promoting the accumulation of abscisic acid (ABA), disrupting the GA / ABA balance, causing the stems of weed seedlings to elongate and the root growth to be inhibited; caryophyllene can act on the microtubule system of weed cells, inhibit the formation of the spindle, and hinder the process of cell mitosis, causing seedling cell proliferation to stop and plant growth to slow down or even stop.

[0039] Quinoline alkaloids: These primarily exert their inhibitory effects by suppressing key enzymes in respiration and interfering with nitrogen metabolism. Cinchona alkaloids and quinoline acid can inhibit the activity of succinate dehydrogenase and cytochrome oxidase in the mitochondria of weeds, reducing the efficiency of mitochondrial aerobic respiration, decreasing ATP synthesis, and causing energy deficiency in various physiological activities of weed seedlings. Simultaneously, they can inhibit the activity of glutamine synthetase, hindering the absorption and assimilation of nitrogen by weeds, leading to insufficient amino acid synthesis and impaired protein synthesis.

[0040] Synergistic effect: When phenolic acids, terpenes, and alkaloids work together, they can block energy metabolism, substance synthesis, cell proliferation, and hormone balance in weeds at multiple targets. Their inhibitory effect is much higher than that of a single substance, and the minimum effective concentration of each substance decreases with each other, achieving high efficiency in weed suppression at low concentrations. At the same time, this synergistic effect has no significant inhibitory effect on orchardgrass and the companion species (awnless brome, white clover, and alfalfa) selected in this invention, but only produces targeted inhibition of weeds. This is also the core basis for the use of a 50%-75% high concentration extract of orchardgrass seeds and a 10%-20% low concentration extract of companion species in this invention.

[0041] The weed-suppressing effect of allelochemicals in this invention is not a single direct allelochemical inhibition, but a triple inhibition mechanism consisting of direct allelochemical inhibition, indirect microecological inhibition, and soil environment synergy. Through multi-dimensional synergy, long-term targeted inhibition of weeds is achieved, while strengthening the ecological advantages of orchardgrass mixed-sowing communities.

[0042] Allelochemicals in the rhizosphere of *Orchardgrass simonii* can selectively enrich beneficial microorganisms such as phosphate-solubilizing bacteria, nitrogen-fixing bacteria, and gelatinous Bacillus in the soil, promoting the colonization of these beneficial microorganisms in the rhizosphere and forming a dominant microbial community. Simultaneously, they inhibit the reproduction of harmful microbial communities and pathogenic microorganisms in the rhizosphere of weeds, indirectly suppressing weeds by regulating the soil microecological structure through allelochemicals. Furthermore, allelochemicals loaded with biochar can form allelochemical microdomains in the soil, which can continuously and slowly release allelochemicals, prolonging the duration of allelochemical inhibition. At the same time, biochar itself can improve soil porosity, water and fertilizer retention capacity, and enhance the interspecific competitive advantage of *Orchardgrass simonii*, achieving a dual effect of allelochemical inhibition and soil improvement.

[0043] The nano-chitosan added to the seed coating agent of this invention can form a breathable and water-permeable nanoscale protective film on the seed surface, reducing the damage from soil pathogens and adverse environments during seed germination, and significantly improving the stress resistance and germination rate of orchardgrass and its associated seeds. At the same time, nano-chitosan, nano-silica and other nanomaterials have strong adsorption properties, which can effectively adsorb heavy metal ions in the soil, reduce the degree of heavy metal pollution in the soil, improve the soil physicochemical environment, and synergize with the ecological benefits of soil and water conservation and soil organic matter improvement of orchardgrass mixed-sowing communities, further enhancing the ecological value of mixed-sowing grasslands.

[0044] This invention determined the minimum effective concentration (based on the concentration of substances in the extract) of each core allelochemical on the germination and growth of common weeds (barnyard grass, foxtail grass, barnyard grass, and Polygonaceae weeds) through pot experiments. When the concentration of each substance in the extract reaches the following range, significant inhibition of weed germination rate (inhibition rate ≥30%) can be achieved, providing quantitative indicators for the concentration dilution and quality testing of the extract: Gallic acid: ≥0.3 mg / g, ferulic acid: ≥0.25 mg / g, coumaric acid: ≥0.2 mg / g; Linalool: ≥0.1 mg / g; Eucalyptol: ≥0.08 mg / g; Cinchonasaline: ≥0.05mg / g.

[0045] When the original extract of orchardgrass rhizosphere allelopathic solution is diluted with sterile water to 50%-75%, the concentration of each core allelopathic substance is higher than the minimum effective concentration mentioned above, which can achieve an inhibitory effect on weed germination rate of 30.5%-42.7% and root length of 62%-82%. When diluted to 10%-20%, the concentration of each substance is in the range that it does not inhibit the germination of associated species, but can only slightly activate the germination of associated species seeds. This is also the scientific basis for the use of different concentrations of extract for orchardgrass and associated species in this invention.

[0046] Example 2 Under potted conditions, mixed sowing of orchardgrass with associated species suppresses free-growing grasses. 1. Experimental conditions and materials The experiment was conducted in a greenhouse using plastic flowerpots with a diameter of 18 cm and a depth of 12 cm. The potting soil consisted of a mixture of 60% field soil, 20% humus, and 20% river sand, and was sterilized at high temperature. The orchardgrass variety "Chuandong" was selected, and the accompanying species were awnless bromegrass, white clover, and alfalfa.

[0047] 2. Processing Settings Treatments were set up including monoculture of orchardgrass, monoculture of associated species, and mixed sowing with orchardgrass comprising 40%, 50%, and 70% respectively, using orchardgrass at a concentration of 0.4 g / m². 2 The sowing quantity for each species is calculated based on the available seeds.

[0048] 3. Sowing and Management Sow the mixed seeds evenly on the surface of the potting soil, cover with 1-2 cm of soil, and press lightly. Water twice a week to keep the soil moist. Do not use chemical herbicides; only remove individual large, free-growing weeds when they are severely affecting the growth of the pasture.

[0049] 4. Harvesting and Measurement The orchardgrass was first mowed when its average height reached about 15cm, leaving a stubble height of 2cm. The first and second mowings were spaced about 4 to 5 weeks apart. At each mowing, the dry matter accumulation of orchardgrass, associated species, and native grass species was measured, and the number of tillers or growing points was counted.

[0050] 5. Results During the second mowing, in the mixed sowing treatments with designed proportions of 40%, 50%, and 70%, the actual dry matter content of orchardgrass reached approximately 72%, 85%, and 90%, respectively, which was significantly higher than the designed proportions. The number of free-growing grass seeds and the dry matter content in the pots were significantly lower than those in the control pots without orchardgrass sowing, indicating that the method of the present invention has a significant effect on inhibiting free-growing grass seeds under potted conditions.

[0051] Example 3 Weed control effect of mixed orchardgrass grassland under field conditions 1. Test site and natural conditions The experimental site is located in a temperate semi-arid region with an average annual precipitation of about 330 mm, mainly concentrated in June to August. The soil is sandy loam with a pH of about 6.6.

[0052] 2. Experimental Design Treatments were set up with monocultures of orchardgrass, awnless bromegrass, white clover, and alfalfa, and mixed treatments with orchardgrass comprising 10%, 30%, 50%, 70%, and 90% respectively. A blank control without any forage grasses was also included. The plot area was 0.4m × 0.5m, and a randomized block design was used with 4 replicates per treatment.

[0053] 3. Sowing and fertilization The sowing rate for each treatment was calculated based on a single sowing rate of 4 kg / ha for orchardgrass. Urea was applied at a rate of 10 kg / ha before sowing, and nitrogen fertilizer application was controlled in conjunction with the nitrogen-fixing effect of the previous alfalfa crop.

[0054] 4. Field Management Within two weeks of emergence, manually remove any noxious weeds that are significantly taller than the pasture grass. After that, no further weeding or herbicides should be applied. Harvest twice in the first year and three times in the second year, each time when the orchardgrass is about 15cm tall on average, leaving a stubble height of 2cm.

[0055] 5. Measurement and Results The dry matter accumulation of orchardgrass, associated forage grasses, and free-growing grasses was measured at each harvest, and the tillering or growing point density at the end of the growing season was investigated. The results showed that the dry matter accumulation of orchardgrass in the second year was significantly higher than that in the first year in both monoculture and mixed-sowing treatments. When the designed proportion of orchardgrass in the mixed sowing was ≥50%, the dry matter accumulation and density of free-growing grasses were significantly lower than those in treatments with a lower proportion of orchardgrass or no orchardgrass mixed sowing. The control area had the highest biomass of free-growing grasses, indicating that the method of this invention can effectively suppress weed communities under field conditions.

[0056] Example 4 The experimental site is located at Gansu Agricultural University (36°05´N, 103°41´E, 1604m). The average annual temperature is 9.1℃, and the average annual precipitation is 250-350mm, with most rainfall concentrated in June to August, accounting for 50%-70% of the annual precipitation. The average annual sunshine duration is 2446 hours, the frost-free period is 180 days, and the soil organic matter content is 9.58g.kg. -1 The pH value is 6.63, and the total nitrogen content is 0.99 g / kg. -1 The effective phosphorus content is 1.6 mg / kg. -1 .

[0057] The seeding rate for mixed-sown grasslands was based on the single-sowing rate of orchardgrass, and the actual seeding rate of each grass species in the mixed-sowing was calculated according to different mixing ratios. The sowing depth was 1 cm, with 4 replicates. The experiment adopted a randomized block design. In the early stage of pasture growth, weeds were removed by hoeing or manual weeding. No measures were taken to control pests and diseases during the experiment.

[0058] Before sowing, the germination rate and thousand-grain weight of different grass species were measured. Sowing was carried out in early July 2014, with orchardgrass seeding rate of 4 kg / ha. Orchardgrass was mixed with other grass species at germination ratios of 9:1, 7:3, 5:5, 3:7, and 1:9. Monoculture of different grass species and sowing without forage served as controls. The experiment included 4 monocultures, 15 mixed sowings, and 1 sowing without forage, for a total of 20 treatments.

[0059] The grassland was mowed twice in 2014, on August 13th and September 16th, 2014, during the establishment phase. Three mowings were conducted the following year, on July 5th, August 17th, and September 25th, 2015. In 2015, when the orchardgrass reached a height of 10-15cm, the first mowing was carried out, leaving a 2-3cm stubble for nutritional value analysis. The number of tillers, growing points, and branches per plant were measured during the mowing.

[0060] 1) Dry matter (DM): The sample was weighed fresh, dried at 65℃ to constant weight, pulverized and then tested.

[0061] 2) Tiller number / growing point / branch number: Count the number of tillers, growing points and branch density of the forage grass before cutting.

[0062] Data statistical analysis was performed using GENSTAT 12.1 statistical analysis software, and the GAOVA method was used for comparison. Office 2010 application software was used to process the measurement data and create graphs. P<0.05 or P<0.01 was marked as significant.

[0063] Dry matter (kg / hm) under different treatments in different years 2The comparison revealed significant differences (P<0.01 or 0.05) in dry matter accumulation among different treatments in the same year. In 2014, the dry matter accumulation of orchardgrass was lower in mixed-sowing combinations compared to monoculture. In 2015, except for the treatment '50%CF+50%L', the dry matter accumulation of orchardgrass in other mixed-sowing combinations was lower than that in monoculture. In both 2014 and 2015, the dry matter accumulation of other grass types in different mixed-sowing combinations was lower than that in monoculture. In 2015, monoculture alfalfa had the highest accumulation, followed by monoculture orchardgrass and white clover, with awnless bromegrass having the lowest. The differences in dry matter accumulation among different treatments in different years were highly significant (P<0.01).

[0064] In terms of the dry matter accumulation of mixed forages, orchardgrass mixed with alfalfa has the highest dry matter accumulation; orchardgrass mixed with white clover has the second highest dry matter accumulation; and orchardgrass mixed with awnless bromegrass has the lowest dry matter accumulation.

[0065] With increasing planting years, the cumulative weed amount gradually decreased. The amount of weeds in the second year was less than that in the first year across different treatments. Meanwhile, the treatment without pasture sowing had the highest cumulative weed amount. In 2015, there was a highly significant difference (P<0.01) in the cumulative weed amount among different treatments. The cumulative weed amount of each mixed sowing combination was lower than that of their respective monocultures. Among the different monocultures, monoculture of orchardgrass had the lowest cumulative weed amount, and the number of weeds gradually decreased with increasing planting time.

[0066] Significant differences were observed in the number of tillers of orchardgrass among different treatments (P<0.05 or P<0.01). The number of tillers gradually increased with the extension of planting time; after overwintering, the number of tillers at the final harvest of monoculture orchardgrass in 2014 was the same as the initial number of tillers in 2015. With the increase of the mixed planting ratio, the number of tillers of orchardgrass gradually decreased.

[0067] The density of tillers, growing points, or branches per unit area differed significantly among different treatments (P<0.01). At the end of 2015, the density of other grasses was highest when the proportion of orchardgrass in the mixed sowing combination was 10%; and as the proportion of orchardgrass decreased, the number of tillers / growing points / branches of awnless brome, white clover, and alfalfa gradually increased.

[0068] Regarding free-growing grasses, at the initial count in 2014, there was no significant difference in the number of weed plants per unit area among the different treatments (P>0.05), with the fewest weeds in the monoculture treatment of orchardgrass. At the end of mowing in 2015, there was a significant difference in the number of weed plants among the different treatments (P<0.01); in the mixed planting combination, the number of weed plants was the highest when the proportion of orchardgrass was 10%; in the monoculture treatment of orchardgrass, the number of weed plants was 0.

[0069] The coefficient of variation of orchardgrass tiller density varied significantly (P<0.01) under different mowing times and mixed sowing conditions. In the first mowing of 2014, although the designed percentages ranged from 10% to 90%, the actual percentage of orchardgrass tillers in the total density (tillers, growing points, and branches) ranged from 36.5.9% to 88.2%, and in the second mowing, it ranged from 36.5% to 94.4%. In the first mowing of 2015, the mixed sowing percentages of '90%CF+10%L' and '50%CF+50%L' were both below 90% and 50%, respectively. However, at the final harvest in 2015, the tillering ratio of orchardgrass was higher than the experimental orchardgrass mix ratio in all mixed planting combinations. At the same mix ratio, orchardgrass mixed with awnless bromegrass produced the highest tillering, followed by orchardgrass mixed with leguminous forage grasses. The results indicate that with increasing planting time, the tillering density of orchardgrass is significantly higher than the tillering capacity of other grasses.

[0070] The tillering variation rate of orchardgrass differed significantly at different harvesting times (P<0.01). The tillering variation coefficient gradually decreased with increasing orchardgrass mix ratio. The variation rate gradually increased with prolonged planting time; at the end of 2015, the variation rate was highest when orchardgrass was mixed with awnless bromegrass, followed by when mixed with white clover, and lowest when mixed with alfalfa.

[0071] Example 5 Qualitative and quantitative identification of allelochemicals in rhizosphere allelochemical extract of Imperata cylindrica 1. Experimental Objective Qualitative and quantitative analyses of the rhizosphere allelopathic extract of *Festuca macrantha* were conducted using analytical techniques such as HPLC-MS and GC-MS to identify the types and contents of the core allelopathic substances, providing a basis for the standardized preparation and quality testing of the extract.

[0072] 2. Test materials Tested duckgrass: such as Figure 2 As shown, the experiment was conducted by sowing seeds in pots. The roots and 0-20cm soil around the rhizosphere of healthy orchardgrass plants (of the Chuandong variety) that had grown for 75 days were collected. Reagents: sterile water, chromatographic grade methanol, acetonitrile, formic acid, n-hexane, and standards (gallic acid, ferulic acid, coumaric acid, p-hydroxybenzoic acid, linalool, eucalyptol, caryophyllene, cinchona alkaloid, quinoline acid, purity ≥98%). Instruments: High performance liquid chromatography-mass spectrometry (HPLC-MS, Agilent 1290-6460), gas chromatography-mass spectrometry (GC-MS, Agilent 7890B-5977B), high-speed refrigerated centrifuge (12000 r / min), vacuum freeze dryer, ultrasonic cleaner, 0.22 μm organic filter membrane.

[0073] 3. Test methods 3.1 Preparation of the stock solution of the chemiluminescent extract of Imperata cylindrica root The extract stock solution was prepared according to the method of the present invention: healthy orchardgrass plants that had grown for 75 days were selected, and roots and 0-20cm soil from the rhizosphere were collected. Sterile water was added at a material-to-liquid ratio of 1:8, and the extract was extracted at a constant temperature of 4℃ with shaking for 36 hours. After filtration through double-layer filter paper, the extract was centrifuged at 8000r / min for 15 minutes. The supernatant was then filtered through a 0.22μm organic filter membrane to obtain the extract stock solution. A portion of the stock solution was freeze-dried under vacuum to prepare a concentrated powder for the detection of total allelochemical content.

[0074] 3.2 Qualitative Analysis of Allelochemicals Phenolic acids and alkaloids: HPLC-MS was used with a C18 column (250 mm × 4.6 mm, 5 μm), a mobile phase of methanol-0.1% formic acid aqueous solution (gradient elution), a flow rate of 1.0 mL / min, a column temperature of 30 ℃, and an injection volume of 20 μL. Mass spectrometry was performed using an electrospray ionization (ESI) source in positive / negative ion mode. The phenolic acids and alkaloids were identified by comparing their retention time and mass-to-charge ratio (m / z) with those of the standards.

[0075] Terpenes: GC-MS was used with an HP-5MS capillary column (30m × 0.25mm, 0.25μm), high-purity helium as the carrier gas, a flow rate of 1.0mL / min, and a programmed temperature ramp (initial temperature 60℃, hold for 2min, ramp to 250℃ at 5℃ / min, hold for 10min); the injection port temperature was 250℃, the ion source temperature was 230℃, and electron impact ionization (EI, 70eV) was used. Terpenes were identified by searching the NIST mass spectrometry library and comparing with standards.

[0076] The transcriptome sequencing results are as follows: Three key genes involved in the synthesis of allelochemicals in orchardgrass were identified through transcriptome sequencing: phenylalanine ammonia-lyase gene, terpene synthase gene, and alkaloid synthase gene. The expression levels of these genes in the orchardgrass roots were more than 5 times higher than in the aboveground parts, providing target genes for marker-assisted breeding of highly allelochemical orchardgrass varieties. Metabolome-transcriptome association analysis showed a significant positive correlation between the expression level of the phenylalanine ammonia-lyase gene and the content of phenolic acid allelochemicals. 2 =0.89, which can be used as a screening marker for high phenolic acid duckgrass varieties.

[0077] 3.3 Quantitative Analysis of Allelochemicals Standard curves for each core allelochemical standard were plotted using the external standard method. The content of each substance in the original extract was calculated based on the standard curves. The total allelochemical content was the sum of the contents of phenolic acids, terpenes, and alkaloids. The total allelochemical content in the concentrated powder was tested to ensure that it met the requirement of ≥10 mg / g.

[0078] 3.4 Repeatability Validation Three parallel experiments were conducted to prepare three original extract solutions. Qualitative and quantitative analyses were performed on each solution, and the relative standard deviation (RSD) of the content of each substance was calculated to verify the repeatability of the detection method and the extract preparation process.

[0079] 4. Test Results 4.1 Qualitative Results Four major phenolic acids (gallic acid, ferulic acid, coumaric acid, and p-hydroxybenzoic acid), three major terpenoids (linalool, eucalyptol, and caryophyllene), and two major quinoline alkaloids (cinchona alkaloid and quinoline acid) were detected in the rhizosphere allelopathic extract of *Festuca macrantha*. No allelopathic substances with significant inhibitory effects on *Festuca macrantha* and its associated species were detected. Figure 3 This is a comparative diagram showing the effects of the extract on seed germination.

[0080] 4.2 Quantitative Results Based on the original extract, the contents of each core allelochemical and the total contents of allelochemicals are shown in Table 2 (average, n=3): Table 2 The total content of allelochemicals in the concentrated powder is 27.58 mg / g, which is far higher than the ≥10 mg / g requirement of this invention, thus meeting the requirements for production and application.

[0081] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the embodiments of the present invention. The spirit and scope of the technical solution.

Claims

1. A method for mixed planting of orchardgrass and associated species for weed control, characterized in that, The germination and growth of weed seeds can be inhibited by utilizing the competitive effect of orchardgrass on associated species and the allelopathic effect of its root exudates, including the following steps: (1) Select plots with pH 5.5 to 7.5, good drainage, medium or higher soil organic matter, and no severe salinization or compaction. The previous crop should be leguminous forage or root crops. (2) The dominant species is orchardgrass, and the companion species are one or more of awnless brome, white clover, and alfalfa; (3) Based on the number of effective seeds, orchardgrass accounts for 30% to 90%, and associated species account for 10% to 70% in total; the seed usage of each species is calculated based on 3 to 6 kg of effective seeds / ha of orchardgrass sown alone. (4) Sow in spring or during the rainy season by broadcasting, at a depth of 1-2 cm, and compact after sowing; (5) Apply 20-60 kg / ha of nitrogen fertilizer and 30-60 kg / ha of phosphorus fertilizer as base fertilizer, and remove noxious weeds within 2-3 weeks after emergence; (6) When the average height of the orchardgrass plant reaches 15cm, it should be cut for the first time, leaving a stubble height of 2cm. After that, it should be cut 2 to 3 times a year. (7) After using the above mixed sowing method and management measures for 2 to 3 consecutive years, the dry matter ratio of orchardgrass in the community was consistently higher than the designed ratio, and the cumulative amount of dry matter and number of wild weeds were significantly reduced, thus achieving long-term ecological suppression of weed populations. Between steps (3) and (4) is the following: before sowing, soak the seeds of *Hemiberlesia argyi* in a 50%-75% concentration of rhizosphere chemosympathetic extract for 6-12 hours to promote germination; soak the seeds of associated species in a 10%-20% low concentration of *Hemiberlesia argyi* rhizosphere chemosympathetic extract for 2-4 hours; and perform synergistic coating on the pretreated seeds. The coating agent is in the following proportions by weight: 10-15 parts of concentrated *Hemiberlesia argyi* rhizosphere chemosympathetic extract powder, 5-8 parts of gelatinous *Bacillus* inoculant, 2-4 parts of *Bacillus megaterium* inoculant, 1-2 parts of rhizobium inoculant, and 3-5 parts of humic acid. The mixture contains 2-3 parts controlled-release nitrogen fertilizer, 1-3 parts nano-chitosan, and 1-2 parts nano-silica modified film-forming agent, with a seed coating ratio of 1:40-1:

50. The concentrated powder of the allelopathic extract from the rhizosphere of *Festuca macrantha* is prepared by vacuum freeze-drying the original solution and then loading it onto straw biochar with a particle size of 50-100 mesh at a mass ratio of 1:5 to 1:10 to prepare an allelopathic substance-biochar composite carrier. The total allelopathic substance content in the original solution is ≥10 mg / g, and the total allelopathic substance content in the allelopathic substance-biochar composite carrier is ≥8 mg / g. Before step (5), the following additional steps are taken: applying 15-30 t / ha of bio-organic fertilizer and 750-1500 kg / ha of silicon-calcium-magnesium soil conditioner as base fertilizer; applying 30-45 kg / ha of nitrogen fertilizer and 15-30 kg / ha of potassium fertilizer after the first mowing; and after each subsequent mowing, using a machine vision imaging device combined with soil nitrogen, phosphorus and potassium sensors to monitor the dry matter ratio of orchardgrass and the content of available nutrients in the soil in real time, and dynamically adjusting the amount and method of topdressing based on the monitoring data. Topdressing is carried out by variable fertilization using drones; when the orchardgrass ratio is ≥80%, apply 15-20 kg / ha of nitrogen fertilizer; when the ratio is 50%-80%, apply 20-30 kg / ha of nitrogen fertilizer and 10-15 kg / ha of phosphorus fertilizer; when the ratio is <50%, use drones to sow 10%~20% of the base amount of the co-coated orchardgrass seeds in the corresponding area, and apply 30-40 kg / ha of nitrogen fertilizer.

2. The method for mixed planting of orchardgrass and associated species for weed control according to claim 1, characterized in that, The associated species are leguminous forage grasses or grasses; the leguminous forage grasses are selected from alfalfa or white clover, and the grasses are selected from awnless brome.

3. The method for mixed planting of orchardgrass and associated species for weed control according to claim 1, characterized in that, The first mowing is carried out 2 months after sowing, and the second mowing is carried out 1 month after the first mowing. After mowing, maintain water and fertilizer management for the grassland.

4. The method for mixed planting of orchardgrass and associated species for weed control according to claim 3, characterized in that, At the time of the first cut, when the design proportion of orchardgrass was 40%, 50% and 70%, the average observed tiller density proportions were 72%, 81% and 82%, respectively.

5. The method for mixed planting of orchardgrass and associated species for weed control according to claim 1, characterized in that, In the aforementioned mixed-sowing method, the cumulative total dry matter yield of orchardgrass and alfalfa mixed-sowing reached 76.3 g / m². 2 .

6. The method for mixed planting of orchardgrass and associated species for weed control according to claim 1, characterized in that, The preparation method of the rhizosphere chemosensitive extract of *Orchardgrass sinensis* is as follows: Select healthy *Orchardgrass sinensis* plants that have grown for 60-90 days, collect roots and soil from the rhizosphere 0-20cm, add sterile water at a material-to-liquid ratio of 1:5-1:10, first extract by ultrasonication at 200W for 30 minutes, then extract by shaking at 4℃ for 24-48 hours, filter with double-layer filter paper to obtain the stock solution, and dilute with sterile water to the target concentration.

7. The method for mixed planting of orchardgrass and associated species for weed control according to claim 1, characterized in that, The orchardgrass variety was selected through transcriptome and metabolome sequencing for high allelopathicity. The screening criteria were: rhizosphere phenolic acid allelochemical content ≥1.2 mg / g, terpene content ≥0.8 mg / g, and no significant inhibitory effect on awnless brome, white clover, and alfalfa.

8. The application of the mixed planting method of orchardgrass and associated species for weed control as described in any one of claims 1-7 in the establishment of temperate perennial grasslands, weed control and high-yield cultivation of forage grasses.

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