CN110577902A - Preparation method of deep-color endophytic mycorrhizal fungi agent assembly - Google Patents
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Abstract
The invention discloses a preparation method of a dark color entomogenous mycorrhizal fungi agent assembly, which specifically comprises the following steps: selecting and processing plant roots, preparing a culture system, performing separation culture on strains, performing purification culture on the strains, preparing a strain seed solution, performing fermentation culture on the strain seed solution, preparing a dry powder microbial inoculum, and preparing a dark color endophytic mycorrhizal fungi microbial inoculum assembly. The deep color endophytic mycorrhizal fungi inoculant combination prepared by the method has the advantages of good inoculant combination quality, high infection rate, high colonization rate, long quality guarantee period and stable infection effect, and the preparation method can be widely applied to the fields of scientific research, ecological prevention and control, soil improvement and recovery and the like, realizes large-scale industrial operation and meets the industrial requirements.
Description
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to a deep-color endophytic mycorrhizal fungi agent.
Background
The dark color has an endophytic mycorrhiza which is a symbiont of plant root systems and fungi widely existing in nature. Dark-color Septate endophytic mycorrhiza fungi (DSE) are small soil fungi which can colonize in plant root systems and form mycorrhizal structures with the plant root systems. DSE is mainly distributed in extreme environments such as heavy metal polluted areas, desert areas, alpine areas, polar regions and the like, has the function of mycorrhizal ecology, can enhance the pressure resistance of plants to biological and non-biological factors, helps the plants to maintain water and nutrition transportation in a drought environment, can enhance the stress resistance of the plants by improving the activity of certain enzymes in the plants, promotes the growth of the plants, and has important significance in recovering desertification lands, repairing heavy metal polluted soil and the like.
At present, the process of obtaining high-quality microbial inoculum by adopting a mass and high-efficiency production method is very complicated due to the characteristics of living body nutrition, obligate symbiosis, no sporulation phenomenon in the growth history and the like of DSE. The main research results in the DSE pure culture at the present stage are as follows: the research results of the loess plateau medlar rhizosphere microecological characteristics and the symbiotic fungi regulation host growth and drought tolerance response mechanism of the loess plateau medlar root zone of Zhang Hai culvert and the like show that the optimal carbon source and the optimal nitrogen source for the growth of different DSE strains are different, Phomachrysanticola is glucose and glycine, Cladosporium cladosporioides LBF3 is mannitol and proline, and Cladosporium cladosporioides LBF6 is xylose and glycine; the research result of a method for establishing a DSE-plant symbiotic system based on DSE rapid propagation, which is disclosed in patent No. CN 105918127B of Banyihui et al, shows that the most suitable carbon source, nitrogen source, temperature and pH for G.Cylindrosporus growth are glucose, peptone, 25 ℃ and 5.0 respectively; the research results of Xufengmei et al, the research on root fungi of Chinese pine in the area where wilt disease occurs and the area where wilt disease does not occur, show that the DSE strain Dialocephala fortini is respectively lactose and malt extract powder in solid culture, and is respectively soluble starch and peptone in liquid culture, and the optimal growth temperature and pH are respectively 20 ℃ and 3.0. At the present stage, the main research results in the aspect of the infection rate of the DSE to the host plant are as follows: the research result of the research on the DSE resource distribution and the inoculation effect of the mongolian ammopiptanthus mongolicus by Wang Shaojie and the like shows that the average colonization rate of the DSE in the root system of the mongolian ammopiptanthus mongolicus is 52.02 percent; the research results of Gejiali et al, that is, the colonization of deep color endophytic fungi in desert plant and the correlation between the deep color endophytic fungi and soil factors, show that the average colonization rates of DSE in red sand and Ephedra sinica Stapf root systems are respectively 13% and 66.7%. The production methods do not form a system frame, large-scale factory operation cannot be realized, and the infection effect of the produced microbial inoculum in a plant production system is unstable, so that the industrial requirements cannot be met.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a deep-color endophytic mycorrhizal fungi agent combination, so as to solve the problems that a system framework is not formed in the conventional deep-color endophytic mycorrhizal fungi agent production method, large-scale industrial operation cannot be realized, and the infection effect of the prepared fungi agent is unstable; further achieves the purposes of low preparation cost, no pollution, high utilization rate of raw materials, high infection rate of the microbial inoculum and prolonged shelf life of the microbial inoculum, and realizes the industrial operation of the endophytic mycorrhiza with dark color.
In order to solve the technical problems, the invention provides a preparation method of a dark endophytic mycorrhizal fungi inoculant combination.
A preparation method of a dark color endophytic mycorrhizal fungi agent assembly comprises the following specific steps:
S1: selecting a wild annual fibrous root herbaceous plant, cleaning, cutting and sterilizing the root system of the wild annual fibrous root herbaceous plant;
S2: preparing a potato glucose agar solid culture system, a potato glucose liquid culture system and a potato glucose agar dual-antibody solid culture system;
S3: inoculating the root system treated by the S1 to a potato glucose agar dual-antibody solid culture system for strain isolation culture;
S4: inoculating mature mycelia to a potato glucose agar solid culture system for strain purification culture;
S5: preparing strain seed liquid;
S6: inoculating the seed liquid into a potato glucose liquid culture system for fermentation culture to obtain fermentation liquid and mycelium pellets;
S7: preparing 10-12 parts of dry powder microbial inoculum from the fermentation liquor and the mycelium pellets, adding 70-75 parts of water-retaining agent, 15-20 parts of nutrient and 0.5-1.0 part of slow-release agent into the dry powder microbial inoculum according to the proportion, and fully and uniformly mixing to obtain the deep color septate endophytic mycorrhizal fungi microbial inoculum combination.
Further, the annual fibrous root herbaceous plant of S1 includes quassia pectinata, decumbent bugleweed herb, and potentilla discolor.
Further, the resistance factors added to the potato dextrose agar dual-antibody solid culture system in the step S2 are ampicillin and streptomycin sulfate.
Further, the dosage of the resistance factor is 100 μ L each of ampicillin and streptomycin sulfate added to 1000mL of distilled water at 50 mg/L.
Further, 3-5 root segments are inoculated on the potato dextrose agar double-antibody solid culture system in the S2.
Further, the inoculation amount of the seed solution in S6 is 10%.
Further, the water retaining agent in S7 is sterilized diatomite and porous rock particles.
further, the nutrient in the S7 is sterilized composite humus soil.
Further, the slow release agent in S7 is polyacrylate.
Due to the adoption of the technical scheme, the technical progress of the invention is as follows.
According to the preparation method of the deep color sound endophytic mycorrhizal fungi fungicide assembly, the water-retaining agent, the nutrient and the slow-release agent are added into the components, so that the deep color endophytic mycorrhizal fungi fungicide assembly has the advantages of good quality, high infection rate, high colonization rate, long quality guarantee period and stable infection effect, can be widely applied to the fields of scientific research, ecological prevention and control, soil improvement and recovery and the like, and the system framework of the production method of the deep color endophytic mycorrhizal fungi fungicide assembly can realize large-scale industrialized operation and meet industrial requirements.
Drawings
FIG. 1 is a diagram showing the effect of the deep color entomogenous mycorrhizal fungi agent combination prepared by the present invention on the infection of plantain plant root segment;
FIG. 2 is a graph showing the growth conditions of the activated culture after the dark color entomogenous mycorrhizal fungi agent combination prepared by the present invention is stored for 1 year.
Detailed Description
A preparation method of a dark color endophytic mycorrhizal fungi agent assembly comprises the following specific steps:
S1: collecting well-growing, healthy and disease-free wild annual fibrous root herbaceous plants, such as: cutting off plant root systems of the ctenocarpus haryngodii, the mud-weeds, the upturned potentilla chinensis and the like, washing the plant root systems under running water, and cutting off root sections with the length of about 1 cm. Cutting off two ends of a fresh root segment which contains dark-color endophytic mycorrhizal fungi hyphae in microscopic examination, cutting the root segment into root segments of 0.3-0.5 cm, disinfecting the surface of the root segments with 75% ethanol for 3min, washing the root segments with sterile water for 3 times, disinfecting the root segments with 8% sodium hypochlorite solution for 8min, and washing the root segments with sterile water for 3 times.
S2: peeling fresh potatoes, cleaning, cutting into blocks, weighing three parts of 200g potatoes, adding 1000mL of distilled water into each part of potatoes, boiling for 30min, and filtering with four layers of gauze to obtain three parts of filtrate.
And (3) taking two parts of filtrate, adding 20g of glucose and 20g of agar into the two parts of filtrate, boiling until the two parts of filtrate are dissolved, and then placing the two parts of filtrate at the temperature of 121.3 ℃ under the condition of 103.4KPa for 15-20 min for high-temperature moist heat sterilization to prepare two parts of potato glucose agar solid culture systems.
adding 20g of glucose into the other filtrate, boiling until the glucose is dissolved, and then placing the filtrate under the conditions of 103.4KPa and 121.3 ℃ for 15-20 min to carry out high-temperature moist heat sterilization to prepare the potato glucose liquid culture system.
1000mL of distilled water was measured, and 100. mu.L each of 50mg/L of ampicillin and streptomycin sulfate was added and mixed well.
And pouring one part of the prepared potato glucose agar solid culture system into a plate, and after solidification, uniformly coating the prepared ampicillin and streptomycin sulfate on the surface of the culture system to obtain the potato glucose agar-ampicillin/streptomycin sulfate dual-resistant solid culture system.
S3: and lightly placing the disinfected root segments on the surface of a potato glucose agar-ampicillin/streptomycin sulfate double-antibody solid culture system, and culturing at the constant temperature of 28 ℃ in the dark.
S4: after the root segments grow dark hyphae, mature dark hyphae are selected and inoculated on the surface of the potato glucose agar solid culture system prepared in S2 for purification culture, and 2 times of subculture is carried out.
S5: subpackaging the potato glucose liquid culture system into conical flasks, inoculating mature hyphae obtained in the last subculture in S3 into the culture system, shaking uniformly, culturing at 28 ℃ for 72h in the dark at a constant temperature of 110r/min, and obtaining the seed liquid when the color of the bacterial liquid is dark brown.
s6: inoculating the seed solution into a potato glucose liquid culture system, controlling the inoculation amount of the seed solution to be 10%, carrying out constant-temperature dark culture for 96h under the conditions of 28 ℃ and 110r/min, and collecting fermentation liquor and mycelium pellets.
s7: and (3) putting the fermentation liquor and the mycelium pellets into a 200-target standard inspection sieve for filtering, collecting the mycelium pellets, drying at 28 ℃, and grinding the dried mycelium pellets into powder, thus obtaining the dry powder microbial inoculum.
And adding 70-75 parts of sterilized diatomite and pore rock particles, 15-20 parts of sterilized combined humus and 0.5-1.0 part of polyacrylate into 10-12 parts of dry powder microbial inoculum, and fully and uniformly mixing to obtain the deep color endophytic mycorrhizal fungi microbial inoculum combination.
The invention will be described in further detail below with reference to the figures and specific examples.
Example 1
s1: collecting well-growing, healthy and disease-free wild annual fibrous root herbaceous plant Potentilla tormentosa, cutting off the root system of Potentilla tormentosa, washing under running water, and cutting into root segments with a length of about 1 cm. And (3) microscopic examination is carried out on the root segment, a fresh root segment containing dark endophytic mycorrhizal fungi hyphae in the microscopic examination is selected, two ends of the fresh root segment are cut off, the root segment is cut into root segments of 0.3-0.5 cm, the surfaces of the root segments are disinfected by 75% ethanol for 3min, then the root segments are washed by sterile water for 3 times, then the root segments are disinfected by 8% sodium hypochlorite solution for 8min, and finally the root segments are washed by sterile water for 3 times.
S2: peeling fresh potatoes, cleaning, cutting into blocks, weighing three parts of 200g of fresh potatoes, adding 1000mL of distilled water into each part of potatoes, boiling for 30min, and filtering with four layers of gauze to obtain three parts of filtrate.
and (3) taking two parts of filtrate, adding 20g of agar and 20g of glucose into the two parts of filtrate, boiling until the two parts of filtrate are dissolved, and then placing the two parts of filtrate at the temperature of 121.3 ℃ under the condition of 103.4KPa for 15-20 min for high-temperature moist heat sterilization to prepare two parts of potato glucose agar solid culture systems.
Adding 20g of glucose into the other filtrate, boiling until the glucose is dissolved, and then placing the filtrate under the conditions of 103.4KPa and 121.3 ℃ for 15-20 min to carry out high-temperature moist heat sterilization to prepare the potato glucose liquid culture system.
1000mL of distilled water was measured, and 100. mu.L each of 50mg/L of ampicillin and streptomycin sulfate was added and mixed well.
and pouring one part of the prepared potato glucose agar solid culture system into a plate, and after solidification, uniformly coating the prepared ampicillin and streptomycin sulfate on the surface of the culture system to obtain the potato glucose agar-ampicillin/streptomycin sulfate dual-resistant solid culture system.
S3: and lightly placing the disinfected root segments on the surface of a potato glucose agar-ampicillin/streptomycin sulfate double-antibody solid culture system, and culturing at the constant temperature of 28 ℃ in the dark.
S4: after the root segments grow dark hyphae, selecting mature dark hyphae, inoculating the dark hyphae on the surface of a potato glucose agar solid culture system for purification culture, and carrying out subculture for 2 times.
S5: subpackaging the potato glucose liquid culture system into conical flasks, inoculating mature mycelia obtained in the last subculture in S3 into the culture system, shaking, culturing at 28 deg.C under 110r/min in dark at constant temperature for 72h, and making into seed liquid when the color of the liquid becomes dark brown.
s6: inoculating the seed solution into a potato glucose liquid culture system, wherein the inoculation amount is 10%, culturing for 96h at the constant temperature of 28 ℃ and 110r/min in the dark, and collecting fermentation liquor and mycelium pellets.
S7: and (3) putting the fermentation liquor and the mycelium pellets into a 200-target standard inspection sieve for filtering, collecting the mycelium pellets, drying at 28 ℃, and grinding the dried mycelium pellets into powder, thus obtaining the dry powder microbial inoculum.
And adding 73 parts of sterilized diatomite and pore rock particles, 20 parts of sterilized combined humus and 1.0 part of polyacrylate into 10 parts of dry powder microbial inoculum, and fully and uniformly mixing to obtain the deep color entomogenous mycorrhizal fungi microbial inoculum combination.
Example 2
S1: collecting well-growing, healthy and disease-free wild annual fibrous root herbaceous plant mud-coriander, cutting off the root system of the mud-coriander, washing the root system under running water, and cutting off the root section with the length of about 1 cm. And (3) microscopic examination is carried out on the root segment, a fresh root segment containing dark endophytic mycorrhizal fungi hyphae in the microscopic examination is selected, two ends of the fresh root segment are cut off, the root segment is cut into root segments of 0.3-0.5 cm, the surfaces of the root segments are disinfected by 75% ethanol for 3min, then the root segments are washed by sterile water for 3 times, then the root segments are disinfected by 8% sodium hypochlorite solution for 8min, and finally the root segments are washed by sterile water for 3 times.
S2: peeling fresh potatoes, cleaning, cutting into blocks, weighing three parts of 200g of fresh potatoes, adding 1000mL of distilled water into each part of potatoes, boiling for 30min, and filtering with four layers of gauze to obtain three parts of filtrate.
And (3) taking two parts of filtrate, adding 20g of agar and 20g of glucose into the two parts of filtrate, boiling until the two parts of filtrate are dissolved, and then placing the two parts of filtrate at the temperature of 121.3 ℃ under the condition of 103.4KPa for 15-20 min for high-temperature moist heat sterilization to prepare two parts of potato glucose agar solid culture systems.
Adding 20g of glucose into the other filtrate, boiling until the glucose is dissolved, and then placing the filtrate under the conditions of 103.4KPa and 121.3 ℃ for 15-20 min to carry out high-temperature moist heat sterilization to prepare the potato glucose liquid culture system.
1000mL of distilled water was measured, and 100. mu.L each of 50mg/L of ampicillin and streptomycin sulfate was added and mixed well.
And pouring one part of the prepared potato glucose agar solid culture system into a plate, and after solidification, uniformly coating the prepared ampicillin and streptomycin sulfate on the surface of the culture system to obtain the potato glucose agar-ampicillin/streptomycin sulfate dual-resistant solid culture system.
S3: and lightly placing the disinfected root segments on the surface of a potato glucose agar-ampicillin/streptomycin sulfate double-antibody solid culture system, and culturing at the constant temperature of 28 ℃ in the dark.
S4: after the root segments grow dark hyphae, selecting mature dark hyphae, inoculating the dark hyphae on the surface of a potato glucose agar solid culture system for purification culture, and carrying out subculture for 2 times.
s5: subpackaging the potato glucose liquid culture system into conical flasks, inoculating mature mycelia obtained in the last subculture in S3 into the culture system, shaking, culturing at 28 deg.C under 110r/min in dark at constant temperature for 72h, and making into seed liquid when the color of the liquid becomes dark brown.
S6: inoculating the seed solution into a potato glucose liquid culture system, wherein the inoculation amount is 10%, culturing for 96h at the constant temperature of 28 ℃ and 110r/min in the dark, and collecting fermentation liquor and mycelium pellets.
S7: and (3) putting the fermentation liquor and the mycelium pellets into a 200-target standard inspection sieve for filtering, collecting the mycelium pellets, drying at 28 ℃, and grinding the dried mycelium pellets into powder, thus obtaining the dry powder microbial inoculum.
And adding 75 parts of sterilized diatomite and pore rock particles, 15 parts of sterilized combined humus and 0.5 part of polyacrylate into 11 parts of dry powder microbial inoculum, and fully and uniformly mixing to obtain the deep color entomogenous mycorrhizal fungi microbial inoculum combination.
Example 3
S1: collecting a wild annual fibrous root herbaceous chlamys farreri with good growth, health and no diseases, cutting off the root system of the chlamys farreri, washing the root system under running water, and cutting off a root section with the length of about 1 cm. And (3) microscopic examination is carried out on the root segment, a fresh root segment containing dark endophytic mycorrhizal fungi hyphae in the microscopic examination is selected, two ends of the fresh root segment are cut off, the root segment is cut into root segments of 0.3-0.5 cm, the surfaces of the root segments are disinfected by 75% ethanol for 3min, then the root segments are washed by sterile water for 3 times, then the root segments are disinfected by 8% sodium hypochlorite solution for 8min, and finally the root segments are washed by sterile water for 3 times.
S2: peeling fresh potatoes, cleaning, cutting into blocks, weighing three parts of 200g of fresh potatoes, adding 1000mL of distilled water into each part of potatoes, boiling for 30min, and filtering with four layers of gauze to obtain three parts of filtrate.
And (3) taking two parts of filtrate, adding 20g of agar and 20g of glucose into the two parts of filtrate, boiling until the two parts of filtrate are dissolved, and then placing the two parts of filtrate at the temperature of 121.3 ℃ under the condition of 103.4KPa for 15-20 min for high-temperature moist heat sterilization to prepare two parts of potato glucose agar solid culture systems.
And adding 20g of glucose into the other filtrate, boiling until the glucose is dissolved, and then placing the filtrate under the conditions of 103.4KPa and 121.3 ℃ for maintaining for 15-20 min for high-temperature moist heat sterilization to prepare a potato glucose liquid culture system.
1000mL of distilled water was measured, and 100. mu.L each of 50mg/L of ampicillin and streptomycin sulfate was added and mixed well. And pouring one part of the prepared potato glucose agar solid culture system into a plate, and after solidification, uniformly coating the prepared ampicillin and streptomycin sulfate on the surface of the culture system to obtain the potato glucose agar-ampicillin/streptomycin sulfate dual-resistant solid culture system.
S3: and lightly placing the disinfected root segments on the surface of a potato glucose agar-ampicillin/streptomycin sulfate double-antibody solid culture system, and culturing at the constant temperature of 28 ℃ in the dark.
s4: after the root segments grow dark hyphae, selecting mature dark hyphae, inoculating the dark hyphae on the surface of a potato glucose agar solid culture system for purification culture, and carrying out subculture for 2 times.
S5: subpackaging the potato glucose liquid culture system into conical flasks, inoculating mature mycelia obtained in the last subculture in S3 into the culture system, shaking, culturing at 28 deg.C under 110r/min in dark at constant temperature for 72h, and making into seed liquid when the color of the liquid becomes dark brown.
S6: inoculating the seed solution into a potato glucose liquid culture system, wherein the inoculation amount is 10%, culturing for 96h at the constant temperature of 28 ℃ and 110r/min in the dark, and collecting fermentation liquor and mycelium pellets.
S7: and (3) putting the fermentation liquor and the mycelium pellets into a 200-target standard inspection sieve for filtering, collecting the mycelium pellets, drying at 28 ℃, and grinding the dried mycelium pellets into powder, thus obtaining the dry powder microbial inoculum.
and adding 70 parts of sterilized diatomite and pore rock particles, 17 parts of sterilized combined humus and 0.7 part of polyacrylate into 12 parts of dry powder microbial inoculum, and fully and uniformly mixing to obtain the deep color entomogenous mycorrhizal fungi microbial inoculum combination.
The deep color entomogenous mycorrhizal fungi agent combinations prepared in examples 1 to 3 are subjected to a colonization rate detection experiment and a quality guarantee period detection experiment respectively.
1) Colonization rate detection test:
Respectively infecting tender plantain plants with the deep color endophytic mycorrhizal fungi agent combination prepared in the embodiments 1 to 3 and the common deep color endophytic mycorrhizal fungi agent, collecting the plantain root sections after the plantain root sections are mature, cutting the root sections into 1cm long, putting the root sections into alkali liquor of KOH solution with the mass fraction of 10%, heating the mixture in water bath at 100 ℃ for 30min, filtering the alkali liquor, and rinsing the mixture for 3 times by using distilled water; then, the gel was stained with 0.5% by weight of acid fuchsin solution for 2.5 hours, and after three decolorizations at room temperature with lactic acid glycerol (lactic acid: glycerol is 1: 1), microscopic examination was performed to compare the current average colonization rate.
The formula for calculating the colonization rate is as follows:
the colonization rate (%) is equal to the number of colonization root segments/total number of microscopic root segments multiplied by 100%
the staining results of examples 1-3 are shown in FIG. 1, and the statistical results of colonization rates are shown in Table 1.
Table 1: colonization rate (unit:%)
The experimental results show that the average infection rate of the deep color endophytic mycorrhizal fungi agent combination prepared by the invention reaches 81.33%, and is greatly improved compared with the infection rate of 52.02% of the common deep color endophytic mycorrhizal fungi agent.
2) detection of shelf life:
The dark-colored endomycorrhizal fungi inoculum combination prepared in examples 1 to 3 was stored in a sealed bag at 20 ℃ in the dark for 1 year in a dry state. One year later, 3 parts of the suspension were taken out at random, activated with sterile water, cultured in a potato dextrose agar solid culture system, and observed for the growth of the dark septate endophyte strains, the results are shown in figure 2.
The experimental result shows that after the strain is stored for one year, the strain still grows normally, which shows that the production method of the deep color endophytic mycorrhizal fungi agent combination adopted by the invention can produce the deep color endophytic mycorrhizal fungi agent combination with high infection rate and high stability, and obtains outstanding effect.
Claims (9)
1. A preparation method of a dark color endophytic mycorrhizal fungi agent assembly is characterized by comprising the following steps:
S1: selecting a wild annual fibrous root herbaceous plant, cleaning, cutting and sterilizing the root system of the wild annual fibrous root herbaceous plant;
S2: preparing a potato glucose agar solid culture system, a potato glucose liquid culture system and a potato glucose agar dual-antibody solid culture system;
S3: inoculating the root system treated by the S1 to a potato glucose agar dual-antibody solid culture system for strain isolation culture;
S4: inoculating mature mycelia to a potato glucose agar solid culture system for strain purification culture;
S5: preparing strain seed liquid;
s6: inoculating the seed liquid into a potato glucose liquid culture system for fermentation culture to obtain fermentation liquid and mycelium pellets;
S7: preparing 10-12 parts of dry powder microbial inoculum from the fermentation liquor and the mycelium pellets, adding 70-75 parts of water-retaining agent, 15-20 parts of nutrient and 0.5-1.0 part of slow-release agent into the dry powder microbial inoculum according to the proportion, and fully and uniformly mixing to obtain the deep color septate endophytic mycorrhizal fungi microbial inoculum combination.
2. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: said annual fibrous root herbaceous plant of S1 comprises Carnis Coturnicis Japonicae, rhizoma Seu herba Linderae Caudatae, and herba Potentillae Anserinae.
3. the method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: the resistance factors added into the potato dextrose agar dual-antibody solid culture system in the step S2 are ampicillin and streptomycin sulfate.
4. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 3, wherein the method comprises the following steps: the dosage of the resistance factors is 100 mu L of each of 50mg/L ampicillin and streptomycin sulfate added into each 1000mL of distilled water.
5. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: and 3-5 root segments are inoculated on the potato glucose agar double-antibody solid culture system in the S2.
6. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: the inoculation amount of the seed solution in the S6 is 10%.
7. the method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: the water-retaining agent in the S7 is sterilized diatomite and porous rock particles.
8. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: the nutrient in the S7 is sterilized composite humus soil.
9. The method for preparing the dark endophytic mycorrhizal fungi inoculant combination according to claim 1, wherein the method comprises the following steps: the slow release agent in the S7 is polyacrylate.
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| CN111500469A (en) * | 2020-04-17 | 2020-08-07 | 中国矿业大学(北京) | Method for producing bacterial manure by using DSE root system (section) |
| CN112442451A (en) * | 2020-12-22 | 2021-03-05 | 黑龙江省农业科学院乡村振兴科技研究所 | Endophytic mycorrhizal fungi for promoting growth of blueberries and application thereof |
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