CN111448947B - Preparation and sowing method of ectomycorrhizal fungi granule strain - Google Patents
Preparation and sowing method of ectomycorrhizal fungi granule strain Download PDFInfo
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Abstract
The invention discloses a preparation method of an ectomycorrhizal fungi granule strain, which comprises the following steps: (1) stirring the ectomycorrhizal fungi and smashing mycelia to obtain ectomycorrhizal fungi liquid; (2) adding the nutrient solution into the ectomycorrhizal bacteria liquid obtained in the step (1) to obtain a mixed solution; (3) slowly adding the mixed solution obtained in the step (2) into polyacrylamide, continuously stirring until the mixed solution is completely absorbed, then adding wheat bran powder, and continuously stirring to obtain the ectomycorrhizal fungi granule strain; the seeding method is carried out by cutting roots on a slope or cutting roots in a cave shape. The application of the ectomycorrhizal fungi granule strain improves the problems that the prior art is limited by the distribution of target mycorrhizal fungi, the labor intensity of seeding is high, and the weather influence is large, and the used raw materials are simple and easy to obtain, the preparation method is simple, the operation is simple and convenient, the production cost is reduced, the economic benefit is improved, and the ectomycorrhizal fungi granule strain is suitable for large-area popularization and application.
Description
Technical Field
The invention belongs to the technical field of edible fungus strain production, and particularly relates to a preparation and sowing method of an ectomycorrhizal fungi granule strain.
Background
Many macrofungi and the roots of higher plants form a symbiotic relationship, which we refer to as ectomycorrhiza. There are very abundant ectomycorrhizal fungi in China, and there are known over 600 species of fungi, such as russula, lactarius, amanita, boletus, scleroderma puffball of ascites, pouzolzia and ascomycete tuber. The research and application of the ectomycorrhizal fungi have important significance for developing seedling growth in forestry, promoting the growth and development of forest trees and greening barren mountains and waste lands of mining plants. The ectomycorrhizal fungi is a mycorrhizal complex formed by fungi and roots of higher plants, and metabolites of hosts and mycorrhizal fungi perform bidirectional operation through a network of a Hastella network. The main trees with ectotrophic mycorrhiza in China include oak, pine, willow, basswood, maple, walnut, betulinaceae and the like; many of the mycorrhizal fungi are precious edible fungi, such as: bolete, matsutake mushroom, lactarius deliciosus and the like are frequently found in pine forests and spruce forests. The mycorrhizal fungi with the function of co-growth and co-habitation has obvious economic benefit and environmental benefit. Not only the yield of wood is improved by 40%, but also a large amount of delicious mushrooms can be provided. A large number of research reports prove that the ectomycorrhizal fungi is beneficial to growing luxuriant forests in barren soil, and the infectivity of the ectomycorrhizal fungi is still stronger in a low-nutrition soil environment, so that most plants have common pioneers. While obtaining a large amount of fast-growing trees, the fungi with the same or higher value as the main products are also obtained.
However, the ectomycorrhizal edible and medicinal fungi can not be propagated under pure artificial conditions, the ectomycorrhizal fungi is applied to the host rhizosphere by spraying the ectomycorrhizal fungi liquid by a sprayer, and the mode is not suitable for large-area popularization and application and mainly has the following two factors: (1) the labor intensity is high, and the input-output ratio is not suitable for large-area popularization and application; (2) if drought occurs, the activity of the strains is greatly reduced. The conventional research data are limited in a small range, and a plurality of factors such as strain release, management and the like are not considered, and the two factors are a big bottleneck for large-area popularization and application of the ectomycorrhizal edible and medicinal fungi.
Disclosure of Invention
The invention provides a preparation and sowing method of an ectomycorrhizal fungi granule strain for solving the technical problems. The application prepares the ectomycorrhizal bacteria liquid and the nutrient solution into granules by mixing, and then the granules are applied to soil, so that the new roots can be formed by promoting the callus healing of the root of a host while providing sufficient nutrients and water for the growth of hyphae, and a new mycorrhizal complex is formed, thereby providing a convenient and feasible means for the formation of target mycorrhizal bacteria, further adopting different sowing methods according to the distribution condition of the target mycorrhizal bacteria, improving the problems that the prior art is limited by the distribution of the target mycorrhizal bacteria, the labor intensity of sowing is high, and the influence of weather is large, and the used raw materials are simple and easy to obtain, the preparation method is simple, the operation is simple and convenient, the production cost is reduced, the economic benefit is improved, and the method is suitable for large-area popularization and application.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of an ectomycorrhizal fungi granule strain comprises the following steps: smashing mycelium of ectomycorrhizal fungi by stirring, adding nutrient solution, uniformly mixing, slowly adding the mixed solution into polyacrylamide, stirring until the mixed solution is completely adsorbed, then adding wheat bran powder, and continuously stirring to obtain the ectomycorrhizal fungi granule strain; the ectomycorrhizal fungi are Lactarius deliciosus, bolete and Tricholoma giganteum; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
Further, the preparation method of the ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution;
(3) and (3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain.
Further, in the step (1), the stirring speed is 250-300 r/min, and the time is 3-5 min.
Further, in the step (2), the nutrient solution and the ectomycorrhizal liquid are mixed according to the equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 20-50 parts of raw corn flour, 10-40 parts of glucose, 0.3-1 part of calcium chloride, 0.3-1 part of sodium chloride, 1-3.5 parts of magnesium sulfate, 2-5 parts of monopotassium phosphate, 10.05-0.15 part of VB and 0.5-2 parts of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 60-100 ℃, stirring to dissolve the raw corn flour into corn liquid, cooling to room temperature, then adding other raw materials, and stirring and mixing uniformly to obtain the corn liquid.
Further, in the step (3), the stirring speed of adding the mixed solution into polyacrylamide is 250-300 r/min; the stirring speed of the wheat bran powder is 500-800 r/min.
Further, in the step (3), the volume ratio of the wheat bran powder to the polyacrylamide is 1-2: 1.
Further, in the step (3), the polyacrylamide and the mixed solution are mixed according to the volume ratio of 16-19: 10, and the speed of adding the polyacrylamide into the mixed solution is 10-15 cm3/s。
Further, the diameter of the strain of the ectomycorrhizal fungi granule is 0.2 cm-0.5 cm, and the water content is 30% -45%.
Furthermore, the seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole root-cutting seeding method to perform seeding.
Further, the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer on one side, stripping 5-8 cm of surface soil, digging an annular inclined plane 2-4 m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 200-400 g per square, then covering 5-8 cm of humus soil or surface soil and compacting, and then covering dry branches and fallen leaves; the slope and the depth of the annular inclined plane are respectively 15 degrees and 28-32 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected to be fixed 2-4 m away from the hosts around the four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.1-1.3 m and the middle depth of 0.2-0.4 m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, exogenously mycorrhizal fungi granule strains are uniformly scattered on the pot-shaped slope and are compressed according to 200-400 g per square, humus soil or surface soil with the thickness of 5-8 cm is covered and is compressed, and then the dead branch fallen leaves are covered.
According to the method, the nutrient solution is added into the ectomycorrhizal bacteria liquid according to the carbon source, the nitrogen source and the inorganic salt required by the growth of most fungi, and the pH of the nutrient solution is natural.
The slope root-cutting and hole-shaped root-cutting sowing method is characterized in that the fine roots and fibrous roots of most plants are mainly distributed within the range of 0-30 cm (particularly, the pine with the lactarius deliciosus host, masson pine is the most typical), so that the method can provide sufficient nutrients and water for hypha growth and propagation, and simultaneously can promote the callus of the root of the host to heal to form new roots, so that a new mycorrhizal complex is formed, and a convenient and feasible means is provided for the formation of target mycorrhizal fungi.
Selection of mono, polyacrylamide
The inventor discovers that the method comprises the following steps in the research process of host 'popularization and application of lactarius deliciosus yield-promoting technology under masson pine forest': the traditional method for applying liquid strains conventionally causes that: firstly, a large amount of manpower, material resources and financial resources are consumed; secondly, due to the limitation of natural conditions, if the strain is dry or rainless, the strain can lose activity quickly; the strain of the order replaces the original ectomycorrhizal fungi to improve the yield, and the effect is very little. In view of this, the inventor creatively adopts the characteristic that the high molecular polymer (polyacrylamide) can lock water and nutrients and simultaneously can keep the activity of the strain, solves the problems encountered in practical popularization and application, and further proves which type of polyacrylamide is selected through design experiments, and the specific experiments are as follows:
(A) Material
1. Raw materials: 250-800 million units of anionic polyacrylamide, 250-800 million units of cationic polyacrylamide, 250-800 million units of nonionic polyacrylamide and 10-50 meshes of wheat bran powder;
2. the main reagents are as follows: glucose;
3. liquid spawn: adopting an optimized liquid culture medium (the liquid culture medium is prepared from the following raw materials, by weight, 20-50 parts of raw corn flour and 10-40 parts of glucose), and fermenting for 9 days to obtain a strain (80-120 spores in a 100-fold microscopic view);
4. the main equipment is as follows: a stirrer, a triangular flask, a high-temperature high-pressure steam sterilization pot, a constant-temperature constant-humidity incubator, a vacuum drying oven, a ten-thousandth electronic balance, a culture dish, filter paper and the like.
(II) method
1. Reagent preparation
Firstly, diluting a solution: sterilizing a preferable liquid culture medium (the liquid culture medium is prepared from the following raw materials, by weight, 20-50 parts of raw corn flour and 10-40 parts of glucose) at high temperature and high pressure for later use;
secondly, diluting strains: diluting according to the ratio of 1:1, measuring 5mL of liquid strains (80-120 spores in 100-fold microscope vision), and then adding 5mL of diluent and stirring uniformly;
measuring 17mL of anionic, cationic and nonionic polyacrylamide respectively, and pouring into three triangular flasks respectively;
and fourthly, measuring 120mL of wheat bran powder with the mesh size of 10-50 meshes for later use.
2. Strain production method
Measuring 5mL of liquid strain, adding 5mL of equivalent diluent, fully and uniformly stirring to obtain strain diluent, slowly pouring the strain diluent into a triangular flask filled with polyacrylamide, slowly pouring the strain diluent into the triangular flask by using a stirrer at 250-300 r/min while stirring until the liquid is uniformly and fully absorbed, adjusting the rotating speed of the stirrer to 500-800 r/min, slowly pouring 17mL of wheat bran powder, and fully stirring to obtain granules of 0.2-0.5 cm;
preparing a control group strain: the difference from the experimental group is that: the volume ratio of the strain diluted solution to the wheat bran powder is 1:2 without adding polyacrylamide.
(III) Strain Activity experiments and results
1. The method comprises the following steps: cutting sponge into 5 × 0.5cm, spreading in a culture dish, covering with filter paper with the same size as sponge, arranging 3 granules on the filter paper at equal intervals, adding 5mL of water into the culture dish, covering with a cover, and culturing in an incubator at 28 deg.C and relative humidity of 80%.
2. And (3) effect measurement: the hyphal growth was judged by qualitative description and the results are shown in Table 1 below.
TABLE 1 influence of different ionic polyacrylamides on the activity of the strains
As can be seen from the experimental data in Table 1, the nonionic polyacrylamide and the anionic polyacrylamide have the characteristic of locking water and nutrient and can keep the activity of the strain for a long time when being used for preparing the strain, so the nonionic polyacrylamide and the anionic polyacrylamide can be used as the material for preparing the strain, and the nonionic property is slightly better than the anionic property according to the experimental result, so the nonionic polyacrylamide is finally preferred as the material for the strain; the cationic polyacrylamide granules are strongly acidic and are not suitable for hypha growth; the control group is not added with polyacrylamide, has no water retention capacity, and easily loses water in a natural state, so that the activity of the strain is greatly influenced.
This application utilizes polyacrylamide to have extremely strong absorption and flocculation adsorption characteristic, let it lock the mycelium and nutrient substance absorption surface when of moisture, in case meet suitable condition mycelium just can utilize the moisture content and the nutrient growth of granule fungus agent self to spread rapidly in soil or other medium environment, and can slowly release nutrient composition and moisture, continuously provide the required material of growth for the bacterial, thereby can make the bacterial activity hold time longer.
Second, inorganic salt is tested on hypha germination characteristics and activity validity period
The inorganic salt is prepared from the following raw materials in parts by weight: 1-3.5 parts of MgSO42-5 parts of KH2PO40.3-1 part of NaCl and 0.3-1 part of CaCl2。
(II) preparation method of strain
1. Preparing a salt-containing granule strain: weighing 10-35 mg of MgSO420-50 mg KH2PO43-10 mg of NaCl and 3-10 mg of CaCl2Pouring 5mL of diluent (the diluent is prepared from the following raw materials, by weight, 20-50 parts of raw corn flour and 10-40 parts of glucose) into the mixture, heating, stirring and dissolving; after cooling to room temperature, pouring the mixture into 5mL of liquid strain and stirring uniformly;
2. preparation of salt-free granular strains: the preparation method of the salt-containing granule strains is adopted, but inorganic salt is not added into the raw materials to prepare the salt-free granule strains.
(III) measurement method
1. Experiment design: evenly dividing the prepared salt-containing granule strains and salt-free granule strains into six parts at random respectively, carrying out hypha germination experiments in batches, carrying out experiments simultaneously in each batch, carrying out experiments in a batch at intervals of 6 days, and observing and recording hypha growth states when each batch is cultured to 2 days, 3 days and 5 days respectively, wherein 6 batches of experiments are carried out.
2. The experimental method comprises the following steps: cutting the sponge into 5 × 0.5 soaking water, spreading in a culture dish, covering with filter paper with the same size as the sponge, equidistantly arranging 3 granules of microbial inoculum on the filter paper, adding 5mL of water into the culture dish, covering with a cover, and culturing in a constant temperature and humidity incubator with the temperature of 28 ℃ and the relative humidity of 80%.
3. Data processing
The experiment adopts qualitative description on the growth condition of hypha, and the experimental result is shown in the following table 2; then determining hypha growth index assignment by using two factors of hypha compactness and hypha length through weight assignment, wherein the experimental result is shown in the following table 3; according to the hypha growth index assignment result, the strain validity period is judged in a vertical graph mode, and the result is shown in figure 1.
TABLE 2
TABLE 3 assignment of hyphal growth index
(IV) results of the experiment
1. As can be seen from Table 2, the salt-containing granule strains can promote hypha germination more quickly, are beneficial to hypha growth, can maintain the activity for a longer time, and gradually reduce the activity after 23 days, while the salt-free granule strains continuously reduce the activity; therefore, the activity of the salt-containing granule strains is optimally within 20 days, and the produced salt-containing granule strains need to be applied within 20 days, and the latest period of time is not more than 30 days.
2. As is evident from fig. 1 (vertical plot of strain validity period): the salt-containing granule strains can promote hypha germination more quickly, are beneficial to hypha growth, can keep the activity for a longer time, and gradually reduce the activity after 23 days, while the salt-free granule strains continuously reduce the activity; therefore, the activity of the salt-containing granule strains is optimally within 20 days, the produced salt-containing granule strains must be applied within 20 days, and the latest period of time must not exceed 30 days; and the growth state of hyphae under the condition of the laboratory is more vigorous with the time, and the 5 th day is better than the 3 rd day.
Third, experimental observation of plant root regeneration and ectomycorrhizal fungi parasitizing by adding growth hormone in strain
(I) Experimental method
1. Preparation of granules containing growth hormone
Weighing 10-35 mg of MgSO420-50 mg KH2PO43-10 mg of NaCl and 3-10 mg of CaCl2Pouring 5mL of diluent (the diluent is prepared from the following raw materials, by weight, 20-50 parts of raw corn flour and 10-40 parts of glucose) into the mixture, heating, stirring and dissolving;
cooling to room temperature, pouring the mixture into 5mL of liquid strain, and uniformly stirring; then, 3 growth hormone (potassium indolebutyrate) inoculants with the concentrations of 500ppm (experiment 1), 1000ppm (experiment 2) and 2000ppm (experiment 3) are respectively prepared.
(II) plant root regeneration and ectomycorrhizal fungi parasitism experiment (seedling root cutting method)
1. Nutrition bag seedling root cutting treatment experiment:
cutting seedling stem in 1cm, spreading granular agent homogeneously on the cross section, covering and pressing with nylon gauze, and covering the other half of the cut nutritious bag with mud;
② the concentration of each growth hormone is respectively 3, repeated for 1 time, totally 18;
③ contrasting: cutting root by the same method, and treating 3 plants without adding granules;
and fourthly, placing the treated nutrition bag seedlings in an incubator with the temperature of 24 ℃, the relative humidity of 80 percent and the illumination level of 1 at an inclination angle of 30-45 degrees for culture, and spraying water once every other day.
2. And (4) microscopic observation: and (5) culturing for 20d, taking the survival nursery stock, putting the survival nursery stock into clear water, soaking for 3-5 h, lightly shaking off soil, putting the root system into a culture dish, filling water into the culture dish to submerge the root system, and observing under a 10-time microscope.
(II) results of the experiment
1. The root-cutting experiment started at 2/15 and ended at 6/3 in 2018, and the results are shown in Table 4.
2. Root 10-fold microscopic observation: the periphery of the root system is covered with a film material with a large number of mycelia, the film material is a high molecular polymer attached to the surface of the root hair, and new roots are obvious at the same time, as shown in figure 2.
TABLE 4 Nutrition bag seedling root cutting treatment experiment results
As can be seen from the experimental data in Table 4, the strain has strong water-retaining property, the requirement of the plant root system on water can be fully ensured, and meanwhile, the water content of the strain provides guarantee for the growth of the ectomycorrhizal fungi. As can be seen from FIG. 2, the bacterial strains have strong adhesiveness around the root hairs after absorbing water, and the hyphae grow vigorously, so that the added plant growth hormone has a promoting effect on the growth of the roots. Therefore, the granular strains have good water retention and moisture retention functions, can promote and guarantee the growth of plant roots and are beneficial to the growth and propagation of fungal mycelia carried by the strains.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
(1) the application mixes the ectomycorrhizal bacteria liquid and the nutrient solution to prepare granules, and then the granules are applied to soil, so that sufficient nutrients and water can be provided for hypha growth, simultaneously, callus at the root of a host can be promoted to heal to form new roots, and then new mycorrhizal complex is formed, a convenient and feasible means is provided for the formation of target mycorrhizal bacteria, and then different sowing methods can be adopted according to the distribution condition of the target mycorrhizal bacteria, so that the problems that the prior art is limited by the distribution of the target mycorrhizal bacteria, the sowing labor intensity is high, and the influence of weather is large are solved.
(2) The activity of the strain of the exogenously mycorrhizal fungi granule prepared by the method is measured by a sponge and filter paper culture method, the activity is measured once every 6d, the growth condition of hyphae is observed in an experiment, and the hyphae grow in the strain preparation 30d, which indicates that the strain of the exogenously mycorrhizal fungi granule can well cope with the natural environment and cannot cause the great reduction of the strain activity due to drought.
(3) The ectomycorrhizal fungi granule strain can be sown by adopting a slope root-cutting method and a hole-shaped root-cutting method. The method is simple in manufacturing and sowing, reduces labor intensity, has good water-retaining property, does not cause great reduction of strain activity due to drought, and provides a convenient and feasible means for forming new mycorrhizal bodies for immersion and replacement of target strains.
Drawings
In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some examples of the present invention, and it is also possible for a person skilled in the art to obtain other drawings without inventive step on the basis of these drawings:
FIG. 1 is a graph of the activity of the strain of the ectomycorrhizal fungi granule of the present application;
FIG. 2 shows a 10-fold microscope of the root of the granule strain of ectomycorrhizal fungi, which is added with growth hormone for plant root regeneration and parasitism of ectomycorrhizal fungi.
Detailed Description
The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
A preparation method of an ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid; the stirring speed is 250r/min, and the stirring time is 3 min;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution; mixing the nutrient solution and the ectomycorrhizal liquid according to equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 20 parts of raw corn flour, 10 parts of glucose, 0.3 part of calcium chloride, 0.3 part of sodium chloride, 1 part of magnesium sulfate, 2 parts of monopotassium phosphate, 10.05 parts of VB and 0.5 part of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 60 deg.C, stirring to dissolve raw corn flour into corn liquid, cooling to room temperature, adding other raw materials, stirring, and mixing;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain; the stirring speed of adding the mixed solution into polyacrylamide is 250 r/min; the stirring speed of the wheat bran powder is 500 r/min; the volume ratio of the wheat bran powder to the polyacrylamide is 1: 1; the polyacrylamide and the mixed solution are mixed according to the volume ratio of 16:10, and the speed of adding the polyacrylamide into the mixed solution is 10cm3/s；
The ectomycorrhizal fungi is lactarius deliciosus; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
The ectomycorrhizal fungi granule strain prepared by the preparation method has the particle diameter of 0.2cm and the water content of 30 percent.
A seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole-shaped root-cutting seeding method to perform seeding; the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer on one side, stripping surface soil by 5cm, digging an annular inclined plane 2m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 200g per square, then covering humus soil or surface soil with the thickness of 5cm and compacting, and then covering dry branch and fallen leaves; the slope degree of the annular inclined plane is 15 degrees, and the depth of the annular inclined plane is 28 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected and fixed 2m away from the hosts at four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.1m and the middle depth of 0.2m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, exogenetic mycorrhizal fungi granule strains are uniformly scattered on the pot-shaped slope and are compressed according to 200g per square, humus soil or surface soil with the thickness of 5cm is covered and is compressed, and then the dead branch fallen leaves are covered.
Example 2
A preparation method of an ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid; the stirring speed is 300r/min, and the stirring time is 5 min;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution; mixing the nutrient solution and the ectomycorrhizal liquid according to equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 50 parts of raw corn flour, 40 parts of glucose, 1 part of calcium chloride, 1 part of sodium chloride, 1-3.5 parts of magnesium sulfate, 5 parts of monopotassium phosphate, 10.15 parts of VB and 2 parts of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 100 deg.C, stirring to dissolve raw corn flour into corn liquid, cooling to room temperature, adding other raw materials, stirring, and mixing;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain; the stirring speed of adding the mixed solution into polyacrylamide is 300 r/min; stirring during wheat bran powder additionThe stirring speed is 800 r/min; the volume ratio of the wheat bran powder to the polyacrylamide is 2: 1; the polyacrylamide and the mixed solution are mixed according to the volume ratio of 19:10, and the speed of adding the polyacrylamide into the mixed solution is 15cm3/s；
The ectomycorrhizal fungi is bolete; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
The ectomycorrhizal fungi granule strain prepared by the preparation method has the particle diameter of 0.5cm and the water content of 45 percent.
A seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole-shaped root-cutting seeding method to perform seeding; the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer, stripping 8cm of surface soil, digging an annular inclined plane 4m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 400g per square, then covering 8cm of humus soil or surface soil and compacting, and then covering the dry branch and fallen leaves; the slope of the annular inclined plane is 15 degrees and the depth is 32 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected and fixed at a distance of 4m from the hosts at four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.3m and the middle depth of 0.4m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, exogenetic mycorrhizal fungi granule strains are uniformly scattered on the pot-shaped slope and compressed according to 400g per square, humus soil or surface soil with the thickness of 8cm is covered and compressed, and then the dead branch fallen leaves are covered.
Example 3
A preparation method of an ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid; the stirring speed is 260r/min, and the time is 3.5 min;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution; mixing the nutrient solution and the ectomycorrhizal liquid according to equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 30 parts of raw corn flour, 20 parts of glucose, 0.5 part of calcium chloride, 0.5 part of sodium chloride, 1.5 parts of magnesium sulfate, 3 parts of monopotassium phosphate, 10.08 parts of VB and 1.0 part of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 70 deg.C, stirring to dissolve raw corn flour into corn liquid, cooling to room temperature, adding other raw materials, stirring, and mixing;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain; the stirring speed of adding the mixed solution into polyacrylamide is 260 r/min; the stirring speed of the wheat bran powder is 600 r/min; the volume ratio of the wheat bran powder to the polyacrylamide is 1.2: 1; the polyacrylamide and the mixed solution are mixed according to the volume ratio of 17:10, and the speed of adding the polyacrylamide into the mixed solution is 11cm3/s；
The ectomycorrhizal fungi is tricholoma giganteum; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
The ectomycorrhizal fungi granule strain prepared by the preparation method has the particle diameter of 0.3cm and the water content of 35 percent.
A seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole-shaped root-cutting seeding method to perform seeding; the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer on one side, stripping surface soil by 6cm, digging an annular inclined plane 2.5m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 250g per square, then covering humus soil or surface soil with the thickness of 6cm and compacting, and then covering dry branches and fallen leaves; the slope and the depth of the annular inclined plane are respectively 15 degrees and 29 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected to be fixed 2.5m away from the hosts at four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.15m and the middle depth of 0.25m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, 250g of exogenous mycorrhizal fungi granular strains per square are uniformly scattered on the pot-shaped inclined plane and are compressed, humus soil or surface soil with the thickness of 6cm is covered and is compressed, and then the dead branch fallen leaves are covered.
Example 4
A preparation method of an ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid; the stirring speed is 290r/min, and the time is 4.5 min;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution; mixing the nutrient solution and the ectomycorrhizal liquid according to equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 40 parts of raw corn flour, 30 parts of glucose, 0.0 part of calcium chloride, 0.9 part of sodium chloride, 3 parts of magnesium sulfate, 4 parts of monopotassium phosphate, 10.12 parts of VB and 1.5 parts of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 90 deg.C, stirring to dissolve raw corn flour into corn liquid, cooling to room temperature, adding other raw materials, stirring, and mixing;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain; the stirring speed of adding the mixed solution into polyacrylamide is 290 r/min; the stirring speed when the wheat bran powder is added is 700 r/min; the volume ratio of the wheat bran powder to the polyacrylamide is 1.9: 1; the polyacrylamide and the mixed solution are mixed according to the volume ratio of 18:10, and the speed of adding the polyacrylamide into the mixed solution is 14cm3/s；
The ectomycorrhizal fungi is bolete; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
The ectomycorrhizal fungi granule strain prepared by the preparation method has the particle diameter of 0.4cm and the water content of 40 percent.
A seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole-shaped root-cutting seeding method to perform seeding; the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer, stripping surface soil by 7cm, digging an annular inclined plane at a distance of 3.5m from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 350g per square, then covering and compacting humus soil or surface soil with the thickness of 7cm, and then covering dry branch and fallen leaves; the slope and the depth of the annular inclined plane are respectively 15 degrees and 31 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected and fixed 3.5m away from the hosts at four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.25m and the middle depth of 0.25m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, exogenous mycorrhizal fungi granule strains are uniformly scattered on the pot-shaped inclined plane and are compressed according to 350g per square, humus soil or surface soil with the thickness of 7cm is covered and is compressed, and then the dead branch fallen leaves are covered.
Example 5
A preparation method of an ectomycorrhizal fungi granule strain comprises the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and smashing mycelia to obtain ectomycorrhizal fungi liquid; the stirring speed is 275r/min, and the time is 4 min;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution; mixing the nutrient solution and the ectomycorrhizal liquid according to equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 35 parts of raw corn flour, 25 parts of glucose, 0.7 part of calcium chloride, 0.6 part of sodium chloride, 2 parts of magnesium sulfate, 4 parts of monopotassium phosphate, 10.10 parts of VB and 1.2 parts of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 80 deg.C, stirring to dissolve raw corn flour into corn liquid, cooling to room temperature, adding other raw materials, stirring, and mixing;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain; the stirring speed of adding the mixed solution into polyacrylamide is 275 r/min; the stirring speed when the wheat bran powder is added is 650 r/min; the volume ratio of the wheat bran powder to the polyacrylamide is 1.5: 1; the polyacrylamide and the mixed solution are mixed according to the volume ratio of 18:10, and the speed of adding the polyacrylamide into the mixed solution is 13cm3/s；
The ectomycorrhizal fungi is lactarius deliciosus; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide.
The ectomycorrhizal fungi granule strain prepared by the preparation method has the particle diameter of 0.4cm and the water content of 37%.
A seeding method of the ectomycorrhizal fungi granule strains adopts a slope root-cutting or hole-shaped root-cutting seeding method to perform seeding; the slope root-cutting seeding method comprises the following steps: in rainy seasons, targeting a dry branch and fallen leaf layer on one side, stripping surface soil by 6cm, digging an annular inclined plane 3m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting exogenous mycorrhizal fungi granule strains on the inclined plane according to 300g per square, then covering and compacting humus soil or surface soil with the thickness of 7cm, and then covering dry branch and fallen leaves; the slope of the annular inclined plane is 15 degrees and the depth of the annular inclined plane is 30 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected and fixed 3m away from the hosts at four weeks, a dead branch fallen leaf layer is firstly targeted at one side along a hole by taking the point as the center of circle, a surface soil layer is stripped at the edge, then a pot-shaped hole with the radius of 1.2m and the middle depth of 0.3m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, the exophytic rhizopus granular strains are uniformly scattered on the pot-shaped slope and are compressed according to 300g per square, humus soil or surface soil with the thickness of 6cm is covered and is compressed, and then the dead branch fallen leaves are covered.
To sum up, this application makes the granule through mixing ectomycorrhizal fungus liquid and nutrient solution, puts in soil again, can provide sufficient nutrient and moisture for the hypha growth and can also promote host root callus to heal and form new root when can promoting, then form new mycorrhizal complex, for the formation of purpose mycorrhizal fungus provides convenient feasible means, and then can adopt different seeding methods according to the distribution situation of purpose mycorrhizal fungus, improved prior art and received the restriction of purpose mycorrhizal fungus distribution and sow intensity of labour big, receive the great problem of weather effect, and the raw materials that uses are simple easily obtained, preparation method is simple, easy and simple to handle, reduced manufacturing cost, improved economic benefits, suitable large tracts of land and popularize and apply. The activity of the exogenously-grown mycorrhizal fungi granule strain prepared by the method is measured by adopting a filter paper culture method and a PDA liquid culture method, the growth condition of hyphae is observed by performing an experiment every 7 days, and the hyphae grow in 30 days of the strain preparation, so that the exogenously-grown mycorrhizal fungi granule strain can well cope with the natural environment, and the strain activity cannot be greatly reduced due to drought.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (4)
1. A preparation method of an ectomycorrhizal fungi granule strain is characterized by comprising the following steps:
(1) putting the ectomycorrhizal fungi into a container, stirring and breaking mycelia to obtain ectomycorrhizal fungi liquid;
(2) adding the nutrient solution into the ectomycorrhizal bacteria solution obtained in the step (1) and uniformly stirring to obtain a mixed solution;
(3) placing polyacrylamide in a container, slowly adding the mixed solution obtained in the step (2) into the polyacrylamide under the stirring state, continuously stirring until the mixed solution is completely adsorbed, adding wheat bran powder, and continuously stirring to obtain the ectomycorrhiza fungi granule strain;
the ectomycorrhizal fungi are Lactarius deliciosus, bolete and Tricholoma giganteum; the polyacrylamide is nonionic polyacrylamide or anionic polyacrylamide;
the diameter of the ectomycorrhizal fungi granule strain is 0.2 cm-0.5 cm, and the water content is 30% -45%;
in the step (3), the mixed solution is added into polyacrylamide, and the stirring speed is 250-300 r/min; the stirring speed of the wheat bran powder is 500-800 r/min;
in the step (3), the volume ratio of the wheat bran powder to the polyacrylamide is 1-2: 1;
in the step (3), the polyacrylamide and the mixed solution are mixed according to the volume ratio of 16-19: 10, and the speed of adding the polyacrylamide into the mixed solution is 10-15 cm3/s。
2. The method of claim 1, wherein the method comprises the steps of: in the step (1), the stirring speed is 250-300 r/min, and the time is 3-5 min.
3. The method of claim 1, wherein the method comprises the steps of: in the step (2), the nutrient solution and the ectomycorrhizal liquid are mixed according to the equal volume; the nutrient solution is prepared from the following raw materials in parts by weight: 20-50 parts of raw corn flour, 10-40 parts of glucose, 0.3-1 part of calcium chloride, 0.3-1 part of sodium chloride, 1-3.5 parts of magnesium sulfate, 2-5 parts of monopotassium phosphate, 10.05-0.15 part of VB and 0.5-2 parts of potassium indolebutyrate; the preparation method comprises the following steps: adding raw corn flour into water, heating to 60-100 ℃, stirring to dissolve the raw corn flour into corn liquid, cooling to room temperature, then adding other raw materials, and stirring and mixing uniformly to obtain the corn liquid.
4. A sowing method of the ectomycorrhizal fungi granule strain prepared by the preparation method of any one of claims 1 to 3, which is characterized in that: seeding by adopting a slope root-cutting or hole-shaped root-cutting seeding method; the slope root-cutting seeding method comprises the following steps: in rainy seasons, raking a dry branch and fallen leaf layer, stripping 5-8 cm of topsoil, digging an annular inclined plane 2-4 m away from a host trunk, taking the trunk as a circle center, uniformly spreading and compacting the exogenous mycorrhizal fungi granule strains on the inclined plane according to 200-400 g per square, then covering 5-8 cm of humus soil or topsoil and compacting, and then covering the dry branch and fallen leaves; the slope and the depth of the annular inclined plane are respectively 15 degrees and 28-32 cm;
the hole-shaped broken root sowing method comprises the following steps: in a forest with evenly distributed hosts, a point is selected to be fixed 2-4 m away from the surrounding hosts, a dead branch fallen leaf layer is firstly raked on one side along an cave by taking the point as the center of circle, a surface soil layer is stripped on the edge, then a pot-shaped cave with the radius of 1.1-1.3 m and the middle depth of 0.2-0.4 m is dug by taking the point as the center of circle, the slope of the edge is 15 degrees, the exogenous mycorrhizal fungi granule strains are uniformly scattered on the pot-shaped slope and are compressed according to 200-400 g per square, humus soil or surface soil with the thickness of 5-8 cm is covered and compressed, and then the dead branch fallen leaves are covered.
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