CN112189525B - Method for promoting leguminous plant growth by using arbuscular mycorrhizal fungi and rhizobia - Google Patents
Method for promoting leguminous plant growth by using arbuscular mycorrhizal fungi and rhizobia Download PDFInfo
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Abstract
The invention discloses a method for promoting leguminous plant growth by using arbuscular mycorrhizal fungi and rhizobia, which can improve the yield and quality of leguminous plant grassland and cannot damage grassland ecosystem. According to the method, by inoculating the arbuscular mycorrhizal fungi microbial inoculum and rhizobia, nutrient absorption of leguminous plants can be promoted, diversity and stability of communities are improved, the utilization rate of space soil fertilizers can be improved, nitrogen fixation of leguminous plants is promoted, the absorption of soil nutrients by host plants is assisted, soil can be loosened, soil hardening is eliminated, the soil structure is improved, the using amount of chemical fertilizers and pesticides can be reduced, the loss of artificially applied chemical nitrogen fertilizers is prevented, environmental pollution is reduced, ecological safety is protected, the utilization rate is higher compared with that of the chemical fertilizers, the action time is long, the organic matter and nutrient content of the soil can be increased, the soil fertility is improved, and the emission of greenhouse gases is reduced, so that the virtuous cycle of an agricultural grassland ecosystem is promoted. Is suitable for popularization and application in the technical field of pasture planting.
Description
Technical Field
The invention relates to the technical field of pasture planting, in particular to a method for promoting leguminous plant growth by utilizing arbuscular mycorrhizal fungi and rhizobia.
Background
In recent years, as the problems of various natural disasters, serious overloading of grassland livestock carrying capacity, excessive reclamation, excessive digging, traveling and other human interference are increased, the grassland ecosystem is damaged, the development of grassland animal husbandry is seriously influenced, and along with the rapid development of current world economy, the requirements of people on animal foods such as meat, dairy products and the like are continuously expanded, greater requirements are provided for the grassland productivity, the bottleneck problem of serious shortage of high-quality forage materials in meat and milk production in China is fundamentally solved for adapting to the development of times and considering ecology and production, and meanwhile, the ecological safety problems of serious decline of ecological functions and the like are restrained, the planting industry structure is required to be adjusted, and the grassland animal husbandry is developed.
The existing method for promoting the growth of the leguminous plants is mainly realized by fertilizing, but the method for promoting the growth of the grasslands of the leguminous plants by fertilizing has high production cost, much manpower and material resources are required to be input, the yield increasing effect of the grasslands is not obvious due to too little fertilizing amount, soil acidification and soil hardening can be caused due to too much fertilizing, a large amount of enriched fertilizers flow into nearby water along with rainfall to cause environmental damage, and the ecological system is disturbed.
Disclosure of Invention
The invention aims to provide a method for promoting leguminous plants to grow by using arbuscular mycorrhizal fungi and rhizobia, which can improve the yield and the quality of the grassland of the leguminous plants and cannot damage the grassland ecosystem.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for promoting the growth of leguminous plants by utilizing arbuscular mycorrhizal fungi and rhizobia comprises the following steps of:
A. selecting seeds; selecting seeds of the leguminous forage with full seeds;
B. and (3) sterilization treatment: sterilizing the selected herbage seeds of the bean family;
C. germination and culture: placing the sterilized leguminous forage seeds on sterilized vermiculite for germination culture until the forage seeds grow seedlings, continuing to culture for 25 days to obtain leguminous forage seedlings, and watering once every 1-3 days in the germination culture process;
D. transplanting: transplanting leguminous forage seedlings into a plurality of flowerpots, wherein the specific method comprises the following steps: firstly, selecting soil 15cm below the ground, filtering the soil by using a screen, and then mixing the filtered soil and river sand according to the weight ratio of 1: (2-4) mixing the raw materials according to the proportion, sterilizing at high temperature, cooling to serve as a substrate, filling the substrate with the volume of 2/3 flowerpots into each flowerpot, spreading a layer of arbuscular mycorrhizal fungi compound microbial inoculum on the surface of the substrate, covering the substrate with the thickness of 2cm, transplanting leguminous grass seedlings into each flowerpot, enabling the leguminous grass seedlings to be identical in number and growth vigor, absorbing the rhizobia microbial inoculum by using an injector, inoculating rhizobia according to the amount of 1.5-3ml of each grass seedling plant, placing the flowerpot transplanted with the leguminous grass seedlings in a greenhouse for culturing, adopting a random arrangement method, changing the positions once every two weeks, adopting natural illumination, the temperature in the daytime being 20-30, the temperature at night being 15-25 ℃, and pouring water once every 2-3 days in the process of culturing in the greenhouse; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week.
Further, in the step A, the selected leguminous forage grass seeds are white clover forage grass seeds or alfalfa seeds.
Further, in the step B, the procedure of sterilizing the selected leguminous grass seeds and gramineous grass seeds respectively is as follows: soaking the seeds of the leguminous forage in 75% ethanol for 1min, washing with distilled water, soaking in 5% sodium hypochlorite for 2min, and washing with distilled water.
Further, in step C, water was poured every 2 days during the germination cultivation.
Further, in the step D, the aperture of the screen is 2mm, and the weight ratio of the filtered soil to the sand is 1: 3, performing high-temperature sterilization treatment at 121 ℃ for 2 hours, and cooling to obtain the matrix.
Further, in the step D, the number of seedlings of the leguminous forage grass transplanted in each flowerpot is 6.
Further, in step D, the injector sucks the rhizobium inoculant to inoculate rhizobium in an amount of 2ml per pasture grass seedling plant.
Further, in the step D, the arbuscular mycorrhizal fungi agent is obtained by the following method: selecting high-quality sorghum seeds, carrying out aseptic seedling culture in a growth chamber for 15 days, preparing a sterile plastic pot with the diameter of 10cm, filling the sterile plastic pot into 2/3 matrix, adding a layer of composite microbial inoculum with the thickness of 1-2cm, wherein the composite microbial inoculum is a culture matrix with spores and hyphae, the composite microbial inoculum comprises Glomus intraradiculis (Glomus intraradices), Glomus mosseae and Glomus younghusbandii (Glomus etuneticam), then adding a layer of matrix with the thickness of 2cm, transplanting sorghum seedlings into the sterile plastic pot to serve as host plants, culturing 4-6 strains per pot, and culturing 4 months of potting soil to obtain the arbuscular mycorrhizal fungal inoculum.
Further, the rhizobium inoculant is obtained by adopting the following method: selecting rhizobium meliloti ACCC18007 and rhizobium meliloti ACCC17676, inoculating to YMA inclined plane for activation, culturing at 25 deg.C for 3 days, transferring to a triangular flask containing YMA culture solution, shaking at 28 deg.C for 48h with a shaking table 150, and culturing to logarithmic growth phase to obtain liquid rhizobium inoculant with bacterial count of 1 × 109One per ml.
Further, in the step D, the Hoagland culture solution is composed of calcium nitrate tetrahydrate, potassium nitrate, ammonium nitrate, anhydrous potassium dihydrogen phosphate, magnesium sulfate heptahydrate, an iron salt solution and a trace element solution, and the contents of the components are as follows: 1180 mg.L-1 of calcium nitrate tetrahydrate, 505 mg.L-1 of potassium nitrate, 80 mg.L-1 of ammonium nitrate, 68 mg.L-1 of anhydrous potassium dihydrogen phosphate (pH is 6.0), 493 mg.L-1 of magnesium sulfate heptahydrate, 22.5 mg.L-1 of iron salt solution and 5.151 mg.L-1 of trace element solution.
The invention has the beneficial effects that: the method for promoting leguminous plant growth by utilizing arbuscular mycorrhizal fungi and rhizobia, disclosed by the invention, has the advantages that the arbuscular mycorrhizal fungi microbial inoculum and the rhizobia are inoculated, the arbuscular mycorrhizal fungi microbial inoculum and the rhizobia can generate symbiotic relation with host plants to form arbuscular mycorrhizal and rhizobia, a symbiotic system formed by the arbuscular mycorrhizal fungi, the rhizobia and the leguminous plants degrades and absorbs nutrients through root exudates and litter of the leguminous plants, available nitrogen and phosphorus nutrition in soil is increased, nutrient absorption of the leguminous plants can be promoted, intra-species and inter-species compatibility of pasture is improved, competition is minimized, diversity and stability of a community are improved, space soil fertilizer utilization rate can be improved, nitrogen fixation of the leguminous community is promoted, a large part of nitrogen is transmitted to adjacent non-nitrogen-fixing plants through a hypha network, nitrogen is contributed to the whole plant community, the host plants are assisted in absorbing soil nutrients, the arbuscular mycorrhizal fungi can loosen soil, and soil, The organic fertilizer has the advantages of eliminating soil hardening, improving soil structure, reducing the using amount of chemical fertilizers and pesticides, preventing the loss of artificially applied chemical nitrogen fertilizers, reducing environmental pollution, protecting ecological safety, being higher in utilization rate compared with chemical fertilizers, long in action time, simple and feasible, increasing the content of organic matters and nutrients in soil, improving fertility betterment soil and reducing the emission of greenhouse gases, thereby promoting the virtuous cycle of a grassland agricultural ecosystem, further promoting the improvement of the production and quality of the whole grassland and providing excellent pasture for livestock.
Drawings
FIG. 1 is a graph of the effect of four examples on aboveground biomass of alfalfa;
FIG. 2 is a graph of the effect of four examples on the phosphorous content of alfalfa overground parts;
figure 3 is a graph showing the effect of four examples on the aerial nitrogen content of alfalfa,
FIG. 4 is a graph of the effect of four examples on alfalfa nodule count;
FIG. 5 is a graph of the effect of four examples on aboveground biomass of Trifolium repens;
FIG. 6 is a graph of the effect of four examples on the number of white clover nodules.
Detailed Description
The existing method for promoting the growth of the leguminous grassland is mainly realized by fertilizing, but the method for promoting the growth of the leguminous grassland by fertilizing has high production cost, a lot of manpower and material resources are needed to be invested, the yield increasing effect of the grassland is not obvious due to too little fertilizing amount, soil acidification and soil hardening are caused due to too much fertilizing, a lot of enrichment of the fertilizer flows into nearby water along with rainfall to cause environmental damage, and the ecological system is disturbed. In order to solve the above problems, the present invention provides a method for promoting the growth of leguminous plants using arbuscular mycorrhizal fungi and rhizobia. Specifically, the method for promoting the growth of leguminous plants by utilizing arbuscular mycorrhizal fungi and rhizobia comprises the following steps:
A. selecting seeds; selecting leguminous forage seeds with full seeds;
B. and (3) sterilization treatment: sterilizing the selected leguminous forage seeds;
C. germination and culture: placing the sterilized leguminous forage seeds on sterilized vermiculite for germination culture until the forage seeds grow seedlings, continuing to culture for 25 days to obtain leguminous forage seedlings, and watering once every 1-3 days in the germination culture process;
D. transplanting: transplanting leguminous forage seedlings into a plurality of flowerpots, wherein the specific method comprises the following steps: firstly, selecting soil 15cm below the ground, filtering the soil by using a screen, and then mixing the filtered soil and river sand according to the weight ratio of 1: (2-4) mixing the raw materials according to the proportion, sterilizing at high temperature, cooling to serve as a substrate, filling the substrate with the volume of 2/3 flowerpots into each flowerpot, spreading a layer of arbuscular mycorrhizal fungi compound microbial inoculum on the surface of the substrate, covering the substrate with the thickness of 2cm, transplanting leguminous grass seedlings into each flowerpot, enabling the leguminous grass seedlings to be identical in number and growth vigor, absorbing the rhizobia microbial inoculum by using an injector, inoculating rhizobia according to the amount of 1.5-3ml of each grass seedling plant, placing the flowerpot transplanted with the leguminous grass seedlings in a greenhouse for culturing, adopting a random arrangement method, changing the positions once every two weeks, adopting natural illumination, the temperature in the daytime being 20-30, the temperature at night being 15-25 ℃, and pouring water once every 2-3 days in the process of culturing in the greenhouse; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week.
Since soil microbial communities are one of the important drivers affecting plant community productivity and community architecture. Arbuscular Mycorrhizal Fungi (AMF) and rhizobiam (Rhizobium) are two important symbiotic microorganisms, and can have symbiotic relationship with host plants to form Arbuscular mycorrhiza and Rhizobium, assist the host plants to absorb soil nutrients, increase the effectiveness of nitrogen and phosphorus in soil and jointly promote the growth of the host plants. AMF and rhizobia can simultaneously provide nutrients necessary for growth to host plants, the host plants can provide photosynthetic products to the AMF and rhizobia, and the AMF and rhizobia can generate forward or reverse interaction on plant communities by regulating the distribution of carbohydrates of the plants and assisting the complementary effect of nutrient absorption of the plants, and the interaction is very important in maintaining high yield stability of the grassland. Research shows that the symbiont formed by rhizobia and leguminous plants absorbs nitrogen through root systems and transmits a large part of nitrogen to adjacent non-nitrogen-fixing plants through hypha networks, so that the nitrogen is contributed to the whole plant community. A large amount of arbuscular mycorrhizal fungi and rhizobia grow around the root system of the leguminous plants, and the arbuscular mycorrhizal fungi and rhizobia form a symbiotic system of the leguminous plants and the arbuscular mycorrhizal fungi and rhizobia in a long-term evolution process, so that rich nutrition is provided for the growth of the leguminous plants. Based on the method, the arbuscular mycorrhizal fungi and the rhizobia are inoculated, the arbuscular mycorrhizal fungi and the rhizobia can have a symbiotic relationship with host plants to form arbuscular mycorrhizal roots and rhizobia, available nitrogen and phosphorus nutrition in soil is increased, nutrient absorption of leguminous plants can be promoted, intra-species and inter-species compatibility of pasture is improved, competition is reduced to the minimum, diversity and stability of communities are improved, space soil fertilizer utilization rate can be improved, leguminous nitrogen fixation is promoted, a large part of nitrogen is transmitted to adjacent non-nitrogen fixation plants through a hypha network, nitrogen is contributed to the whole plant community, the host plants are assisted to absorb the soil nutrients, the arbuscular mycorrhizal roots and the rhizobia can loosen soil, soil hardening is eliminated, the soil structure is improved, the using amount of chemical fertilizers and pesticides can be reduced, loss of artificially applied chemical nitrogen fertilizers is prevented, environmental pollution is reduced, ecological safety is protected, and the fertilizer utilization rate is higher than that of chemical fertilizers, The method has long action time, is simple and easy to implement, can increase the content of organic matters and nutrients in soil, improve the fertility of the soil and reduce the emission of greenhouse gases, thereby promoting the virtuous cycle of a grassland agricultural ecosystem, further promoting the improvement of the production and the quality of the whole grassland and providing excellent pasture for livestock.
Further, in the step D, the arbuscular mycorrhizal fungi agent is obtained by the following method: selecting high-quality sorghum seeds, carrying out aseptic seedling culture in a growth chamber for 15 days, preparing a sterile plastic basin with the diameter of 10cm, filling 2/3 substrate, adding a layer of composite microbial inoculum with the thickness of 1-2cm, wherein the composite microbial inoculum is a culture substrate with spores and hyphae, the composite microbial inoculum comprises Glomus intraradicans (Glomus intraradices), Glomus mosseae (Glomus mosseae) and Glomus younghusbandii (Glomus etuneticam), the composite microbial inoculum can be purchased in a arbuscular mycorrhizal fungi germplasm resource library (BGC), then adding a layer of substrate with the thickness of 2cm, transplanting sorghum seedlings into the sterile plastic basin as host plants, and culturing 4-6 strains per basin for 4 months to obtain the arbuscular mycorrhizal fungal inoculum. The rhizobium inoculant is obtained by adopting the following method: selecting rhizobium meliloti ACCC18007 and rhizobium meliloti ACCC17676, inoculating to YMA inclined plane for activation, culturing at 25 deg.C for 3 days, transferring to a triangular flask containing YMA culture solution, shaking at 28 deg.C for 48h with a shaking table 150, and culturing to logarithmic growth phase to obtain liquid rhizobium inoculant with bacterial count of 1 × 109One per ml.
In addition, in the step D, the Hoagland culture solution is composed of calcium nitrate tetrahydrate, potassium nitrate, ammonium nitrate, anhydrous potassium dihydrogen phosphate, magnesium sulfate heptahydrate, an iron salt solution and a trace element solution, and the content of the components is as follows: 1180 mg.L of calcium nitrate tetrahydrate-1505 mg/L potassium nitrate-1Ammonium nitrate 80 mg.L-168 mg. L of anhydrous potassium dihydrogen phosphate (pH 6.0)-1493 mg. L magnesium sulfate heptahydrate-122.5 mg. L of iron salt solution-15.151 mg.L of trace element liquid-1。
Comparative example 1
Firstly, selecting full-seed white clover (sea method) seeds and alfalfa (WL168) seeds; then, soaking the selected white clover (sea method) seeds and alfalfa (WL168) seeds in 75% ethanol for 1min, washing the seeds with distilled water, soaking the seeds in 5% sodium hypochlorite for 2min, washing the seeds with distilled water, placing the seeds on sterilized vermiculite for germination culture, watering the seeds once every 2 days in the culture process to ensure sufficient water, continuously culturing the seeds for 25 days after the seedlings of the pasture grass emerge to obtain white clover and alfalfa seedlings, then transplanting the white clover and alfalfa seedlings into a plurality of flowerpots, firstly, selecting soil 15cm below the ground, filtering the soil through a screen, and then filtering the filtered soil and river sand according to the weight ratio of 1: 3, mixing the components in proportion, performing high-temperature sterilization treatment and cooling the mixture to be used as a substrate, then filling the substrate with the volume of 2/3 in each flowerpot, respectively transplanting the Trifolium repens and alfalfa seedlings into each flowerpot, wherein the number of the Trifolium repens or alfalfa seedlings transplanted in each flowerpot is the same and the growth of the Trifolium repens or alfalfa seedlings is the same, finally placing the flowerpot transplanted with the grass seedlings in a greenhouse for culturing, adopting a random arrangement method, changing the positions once every two weeks, adopting natural illumination, the temperature in the daytime is 20-30, the temperature at night is 15-25 ℃, in the process of culturing in the greenhouse, watering is performed once in the early stage for two days, and watering is performed once in the later stage for 3 days to ensure sufficient water; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week. Harvesting plants at the end of growing season, separating from the base diameter, deactivating enzymes of the two parts at 105 deg.C for 30min, oven drying at 70 deg.C to constant weight, and measuring aboveground biomass. Carefully washing the plant root system with clear water, and taking care to preserve the integrity of the root system and the root nodule. Carefully picking off root nodules by using tweezers, and recording the number of effective nodules of each plant and the total number of nodules. The contents of total phosphorus and total nitrogen were measured by molybdenum-antimony anti-absorptiometry and kjeldahl method, respectively.
Comparative example 2
Firstly, selecting full-seed white clover (sea method) seeds and alfalfa (WL168) seeds; then, soaking the selected white clover (sea method) seeds and alfalfa (WL168) seeds in 75% ethanol for 1min, washing the seeds with distilled water, soaking the seeds in 5% sodium hypochlorite for 2min, washing the seeds with distilled water, placing the seeds on sterilized vermiculite for germination culture, watering the seeds once every 2 days in the culture process to ensure sufficient water, continuously culturing the seeds for 25 days after the seedlings of the pasture grass emerge to obtain white clover and alfalfa seedlings, then respectively transplanting the white clover and alfalfa seedlings into a plurality of flowerpots, firstly, selecting soil 15cm below the ground and filtering the soil by a screen, and then, enabling the filtered soil and river sand to be 1 in weight ratio: 3, mixing the components according to the proportion, performing high-temperature sterilization treatment and cooling to obtain a substrate, then filling the substrate with a flowerpot volume of 2/3 into each flowerpot, spreading a layer of arbuscular mycorrhizal fungi composite microbial inoculum on the surface of the substrate, covering the substrate with a thickness of 2cm, then respectively transplanting the Trifolium repens and alfalfa seedlings into each flowerpot, wherein the number of the Trifolium repens or alfalfa seedlings transplanted in each flowerpot is the same and the growth vigor of the Trifolium repens or alfalfa seedlings is the same, finally placing the flowerpot transplanted with the grass seedlings in a greenhouse for cultivation, adopting a random arrangement method, changing positions every two weeks, adopting natural illumination, the daytime temperature is 20-30, the evening temperature is 15-25 ℃, in the process of cultivation in the greenhouse, watering is performed once in the early stage for two days, and is performed once in the later stage for 3 days to ensure sufficient water; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week. Harvesting plants at the end of growing season, separating from the base diameter, deactivating enzymes of the two parts at 105 deg.C for 30min, oven drying at 70 deg.C to constant weight, and measuring aboveground biomass. Carefully washing the plant root system with clear water, and taking care to preserve the integrity of the root system and the root nodule. Carefully picking off root nodules by using tweezers, and recording the number of effective nodules of each plant and the total number of nodules. The contents of total phosphorus and total nitrogen were measured by molybdenum-antimony anti-absorptiometry and kjeldahl method, respectively.
Comparative example 3
Firstly, selecting full-seed white clover (sea method) seeds and alfalfa (WL168) seeds; then, soaking the selected white clover (sea method) seeds and alfalfa (WL168) seeds in 75% ethanol for 1min, washing the seeds with distilled water, soaking the seeds in 5% sodium hypochlorite for 2min, washing the seeds with distilled water, placing the seeds on sterilized vermiculite for germination culture, watering the seeds once every 2 days in the culture process to ensure sufficient water, continuously culturing the seeds for 25 days after the seedlings of the pasture grass emerge to obtain white clover and alfalfa seedlings, then respectively transplanting the white clover and alfalfa seedlings into a plurality of flowerpots, firstly, selecting soil 15cm below the ground and filtering the soil by a screen, and then, enabling the filtered soil and river sand to be 1 in weight ratio: 3, mixing the components according to the proportion, using the components as substrates after high-temperature sterilization treatment and cooling, then filling the substrates with the volume of 2/3 in each flowerpot, respectively transplanting the seedlings of the three-leaf clover and the alfalfa into each flowerpot, wherein the number of the seedlings of the three-leaf clover or the alfalfa transplanted in each flowerpot is the same and the growth vigor of the seedlings of the three-leaf clover or the alfalfa is the same, finally absorbing the rhizobia microbial inoculum by using an injector to inoculate rhizobia according to the amount of 2ml of each seedling plant of the pasture grass, finally placing the flowerpot transplanted with the seedlings of the pasture grass in a greenhouse for cultivation, adopting a random arrangement method, changing the positions once every two weeks, adopting natural illumination, the temperature in the daytime is 20-30, the temperature in the evening is 15-25 ℃, watering is carried out once in two days at the early stage and once in 3 days at the later stage in the greenhouse for ensuring sufficient water; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week. Harvesting plants at the end of growing season, separating from the base diameter, deactivating enzymes of the two parts at 105 deg.C for 30min, oven drying at 70 deg.C to constant weight, and measuring aboveground biomass. Carefully washing the plant root system with clear water, and taking care to preserve the integrity of the root system and the root nodule. Carefully picking off root nodules by using tweezers, and recording the number of effective nodules of each plant and the total number of nodules. The contents of total phosphorus and total nitrogen were measured by molybdenum-antimony anti-absorptiometry and kjeldahl method, respectively.
Example 4
Firstly, selecting full-seed white clover (sea method) seeds and alfalfa (WL168) seeds; then, soaking the selected white clover (sea method) seeds and alfalfa (WL168) seeds in 75% ethanol for 1min, washing the seeds with distilled water, soaking the seeds in 5% sodium hypochlorite for 2min, washing the seeds with distilled water, placing the seeds on sterilized vermiculite for germination culture, watering the seeds once every 2 days in the culture process to ensure sufficient water, continuously culturing the seeds for 25 days after the seedlings of the pasture grass emerge to obtain white clover and alfalfa seedlings, then respectively transplanting the white clover and alfalfa seedlings into a plurality of flowerpots, firstly, selecting soil 15cm below the ground and filtering the soil by a screen, and then, enabling the filtered soil and river sand to be 1 in weight ratio: 3, performing high-temperature sterilization treatment and cooling to obtain a substrate, filling the substrate with the volume of 2/3 in each flowerpot, spreading a layer of arbuscular mycorrhizal fungi complex microbial inoculum on the surface of the substrate, covering the substrate with the thickness of 2cm, transplanting the seedlings of the three-leaf clover and the alfalfa into each flowerpot respectively, wherein the seedlings of the three-leaf clover or the alfalfa transplanted in each flowerpot have the same number and the same growth vigor, finally absorbing the rhizobia inoculant by using an injector to inoculate rhizobia according to the amount of 2ml of each grass seedling plant, finally placing the flowerpot transplanted with the grass seedlings in a greenhouse for culturing, adopting a random arrangement method, changing the positions once every two weeks, adopting natural illumination, the temperature in the daytime is 20-30 ℃, the temperature at night is 15-25 ℃, in the process of culturing in a greenhouse, watering is carried out once in two days at the early stage and once in 3 days at the later stage, so as to ensure sufficient water; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, Hoagland's medium was supplemented 1 time per week. Harvesting plants at the end of growing season, separating from the base diameter, deactivating enzymes of the two parts at 105 deg.C for 30min, oven drying at 70 deg.C to constant weight, and measuring aboveground biomass. Carefully washing the plant root system with clear water, and taking care to preserve the integrity of the root system and the root nodule. Carefully picking off root nodules by using tweezers, and recording the number of effective nodules of each plant and the total number of nodules. The contents of total phosphorus and total nitrogen were measured by molybdenum-antimony anti-absorptiometry and kjeldahl method, respectively.
4 experimental treatments were formed by experimental design whether the addition of arbuscular mycorrhizal fungi (+ AMF, -AMF) and rhizobia (+ R, -R): comparative example 1 was performed without adding AMF and Rhizobium (-AMF/-R), comparative example 2 was performed without adding Rhizobium (+ AMF/-R), comparative example 3 was performed without adding AMF (-AMF/+ R) and example 4 was performed with adding AMF and Rhizobium (+ AMF/+ R). FIG. 1 is a graph of the effect of four examples on aboveground biomass of alfalfa; FIG. 2 is a graph of the effect of four examples on the phosphorous content of alfalfa overground parts; FIG. 3 is a graph showing the effect of four examples on the nitrogen content in the aerial parts of alfalfa, and FIG. 4 is a graph showing the effect of four examples on the number of alfalfa nodules; FIG. 5 shows the effect of four examples on aboveground biomass of white clover and FIG. 6 shows the effect of four examples on the number of nodules of white clover.
As can be seen from fig. 1-6, inoculation with AMF and rhizobia significantly increased the aboveground and underground biomass of alfalfa compared to no inoculation, and inoculation with AMF and rhizobia significantly increased the aboveground nitrogen and phosphorus content, promoting the nodulation of alfalfa, demonstrating that inoculation with AMF and rhizobia has a positive effect on nutrient absorption by legumes. Compared with non-inoculated treatment, the inoculation of AMF and rhizobia creates favorable conditions for growth, nodulation and nitrogen fixation of the trifolium repens, improves the aboveground biomass of the trifolium repens, remarkably promotes the nodulation of the trifolium repens, increases available nitrogen and phosphorus nutrition in soil, can reduce the inter-species nutrient competition strength among plants, thereby improving the diversity and stability of communities, improving the utilization rate of space soil fertilizer, promoting the nitrogen fixation of leguminous plants, and transmitting a large part of nitrogen to adjacent non-nitrogen fixation plants through a hypha network, thereby contributing the nitrogen to the whole plant community, assisting the host plants to absorb soil nutrients, loosening the soil, eliminating soil hardening, improving the soil structure, reducing the using amount of chemical fertilizers and pesticides, preventing the loss of artificially applied chemical nitrogen fertilizers, reducing environmental pollution, protecting ecological safety, and being higher than the chemical fertilizers in utilization rate, Has long action time, is simple and easy to operate, can increase the organic matter and nutrient content of soil, improve the fertility soil and reduce the emission of greenhouse gases, thereby promoting the virtuous cycle of a grassland agricultural ecosystem, further promoting the improvement of the production and quality of the whole grassland and providing excellent pasture for livestock
The above-described examples are merely illustrative of preferred embodiments of the present invention and do not limit the scope of the present invention. Many possible variations and modifications may be made to the invention by one skilled in the art using the above methods and techniques without departing from the spirit and scope of the invention. Therefore, variations and modifications of the above embodiments according to the technical essence of the present invention are within the scope of the present invention, unless they depart from the technical solution of the present invention.
Claims (5)
1. A method for promoting leguminous plant growth by using arbuscular mycorrhizal fungi and rhizobia, characterized by comprising the steps of:
A. selecting seeds; selecting leguminous forage seeds with full seeds; the leguminous forage grass seeds are selected from Trifolium repens sea method seeds and alfalfa WL168 seeds;
B. and (3) sterilization treatment: sterilizing the selected leguminous forage seeds;
C. germination and culture: placing the sterilized leguminous forage seeds on sterilized vermiculite for germination culture until the forage seeds grow seedlings, continuing to culture for 25 days to obtain leguminous forage seedlings, and watering once every 1-3 days in the germination culture process;
D. transplanting: transplanting leguminous forage seedlings into a plurality of flowerpots, wherein the specific method comprises the following steps: firstly, selecting soil 15cm below the ground, filtering the soil by using a screen, wherein the aperture of the screen is 2mm, and mixing the filtered soil and sand according to the weight ratio of 1: 3 as a substrate after high-temperature sterilization treatment and cooling, wherein the temperature of the high-temperature sterilization treatment is 121 ℃ and the time is 2 hours, then the substrate with the volume of 2/3 flower pots is filled into each flower pot, a layer of arbuscular mycorrhizal fungi complex microbial inoculum is spread on the surface of the substrate, the substrate with the thickness of 2cm is covered, then leguminous grass seedlings are transplanted into each flower pot, the number of the leguminous grass seedlings transplanted in each flower pot is the same, the growth vigor of the leguminous grass seedlings is the same, then the rhizobia microbial inoculum is absorbed by an injector according to the amount of 1.5-3ml of each grass seedling plant to inoculate rhizobia, finally the flower pot with the leguminous grass seedlings is placed in a greenhouse for cultivation, the flower pot is randomly arranged, natural illumination is adopted, the daytime temperature is 20-30, the evening temperature is 15-25 ℃, in the greenhouse cultivation process, pouring every 2-3 daysWater; observing the growth condition of the grass seedlings within one week of transplantation, and if the seedlings die, selecting the same grass seedlings with good growth for replacement; after 1 month of culture in the greenhouse, 1 Hoagland's culture medium was supplemented every week; the arbuscular mycorrhizal fungi composite microbial inoculum is obtained by adopting the following method: selecting high-quality sorghum seeds, carrying out aseptic seedling culture in a growth room for 15 days, preparing a sterile plastic basin with the diameter of 10cm, filling the sterile plastic basin into 2/3 substrate, and then adding a layer of composite microbial inoculum with the thickness of 1-2cm, wherein the composite microbial inoculum is a culture substrate with spores and hyphae, and comprises ascosphaera radicata (A), (B), (C) and D), (C) and a) and (C)Glomus intraradices) Morse sacculus mildew (A)Glomus mosseae) And Botrytis nubigena (Glomus etunicatum) Then adding a layer of matrix with the thickness of 2cm, transplanting sorghum seedlings into a sterilized plastic pot to serve as a host plant, wherein 4-6 strains are planted in each pot, and the pot soil cultured for 4 months is the arbuscular mycorrhizal fungi composite microbial inoculum; the rhizobium inoculant is obtained by adopting the following method: selecting rhizobium meliloti ACCC18007 and rhizobium meliloti ACCC17676, inoculating to YMA inclined plane for activation, culturing at 25 deg.C for 3 days, transferring to a triangular flask containing YMA culture solution, shaking at 28 deg.C for 48h with a shaking table 150, and culturing to logarithmic growth phase to obtain liquid rhizobium inoculant with bacterial count of 1 × 109Per ml; the Hoagland culture solution consists of tetrahydrate calcium nitrate, potassium nitrate, ammonium nitrate, anhydrous potassium dihydrogen phosphate, magnesium sulfate heptahydrate, an iron salt solution and a trace element solution, and comprises the following components in percentage by weight: 1180 mg.L-1 of calcium nitrate tetrahydrate, 505 mg.L-1 of potassium nitrate, 80 mg.L-1 of ammonium nitrate, 68 mg.L of anhydrous monopotassium phosphate with pH =6.0-1493 mg. L magnesium sulfate heptahydrate-122.5 mg. L of iron salt solution-15.151 mg.L of trace element liquid-1。
2. A method for promoting the growth of leguminous plants using arbuscular mycorrhizal fungi and rhizobia as claimed in claim 1, wherein: in the step B, the process of sterilizing the selected leguminous grass seeds is as follows: soaking the seeds of the leguminous forage in 75% ethanol for 1min, washing with distilled water, soaking in 5% sodium hypochlorite for 2min, and washing with distilled water.
3. A method for promoting the growth of leguminous plants using arbuscular mycorrhizal fungi and rhizobia as claimed in claim 1, wherein: in step C, water was poured every 2 days during germination cultivation.
4. A method for promoting the growth of leguminous plants using arbuscular mycorrhizal fungi and rhizobia as claimed in claim 1, wherein: in the step D, the number of seedlings of leguminous forage transplanted in each flowerpot is 6.
5. A method for promoting the growth of leguminous plants using arbuscular mycorrhizal fungi and rhizobia as claimed in claim 1, wherein: in the step D, the injector absorbs the rhizobium inoculant to inoculate rhizobium according to the amount of 2ml per pasture seedling plant.
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