CN111471597B - Preparation and application method of DSE (Deuteroxylin-N-acetylneuraminidase) fungicide and symbiotic effect sensitive period monitoring - Google Patents
Preparation and application method of DSE (Deuteroxylin-N-acetylneuraminidase) fungicide and symbiotic effect sensitive period monitoring Download PDFInfo
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Abstract
The invention provides a preparation method and an application method of a DSE microbial inoculum and monitoring of a symbiotic effect sensitive period. The invention provides a gelidium Pleosporus sp needle A2-8, the preservation number is CGMCC No. 18812. The invention also provides a solid microbial inoculum, which is prepared by the method comprising the following steps: 1) inoculating the gelidium into a liquid culture medium for culture to obtain a culture solution; 2) filtering the culture solution, and collecting solid substances to obtain the microbial inoculum. Experiments prove that the invention provides a novel strain and a solid microbial inoculum prepared from the same, which are applied to plants to promote the bacteria and the plants to achieve symbiotic relationship, so that the growth promoting effect of the bacteria on the plants is maximized, the process of forming an infection symbiont of the bacteria and the plants is found, and the symbiotic sensitive period is monitored.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to preparation and application methods of a DSE (Deuteronitrobacter xylinum) fungicide and monitoring of a symbiotic effect sensitive period.
Background
Dark Septate endophytic fungi (DSE) are soil fungi which can form a mutual-benefit symbiotic relationship with most plant roots, and hyphae of the soil fungi are Dark in color and have obvious transverse septation. The prior research shows that the DSE has the ecological functions of promoting the mineral nutrition absorption of host plants, promoting the organic nutrient absorption of the host plants, improving the stress resistance of the host plants and improving the disease resistance of the host plants, and plays an important role in a plant-microorganism-soil three-in-one ecological system. At present, DSE microbial inoculum is limited to laboratory production, and has large consumption of manpower and material resources and high production cost; in order to realize reasonable application and maximize the effect and benefit of the DSE microbial inoculum on plants, the preparation method of the solid fresh microbial inoculum of the DSE microbial inoculum is researched.
In addition, the DSE fungicide is inoculated into the root system of the soil plant to play the role, establishes a symbiotic relationship with the plant to promote the growth of the plant, determines the sensitive period for promoting the growth of the plant after the DSE fungicide forms the symbiotic relationship with the plant, and has important significance for selecting the optimal time for field monitoring.
Disclosure of Invention
An object of the present invention is to provide a novel strain.
The invention provides a gelidium Pleosporus sp needle A2-8, the preservation number is CGMCC No. 18812.
The second purpose of the invention is to provide a microbial inoculum.
The microbial inoculum provided by the invention is a solid microbial inoculum, and is prepared by the method comprising the following steps:
1) inoculating the gelidium into a liquid culture medium for culture to obtain a culture solution;
2) filtering the culture solution, and collecting solid substances (sticky substances in the form of lumps) to obtain the microbial inoculum.
In the microbial inoculum, the culture time is 4-6 days.
In the microbial inoculum, the culture condition is shaking culture at 25-28 ℃ and 160-.
In the microbial inoculum, medium-speed filter paper is adopted for filtering;
or, the liquid culture medium is a liquid MMN culture medium.
In the microbial inoculum, the step of inoculating the gelidium into a liquid culture medium is to inoculate mycelium of the gelidium into the liquid culture medium;
the mycelium of the gelidium is prepared according to the following method:
a) culturing the gelidium in a solid culture medium until the gelidium is covered with mycelia;
b) then taking the fungus slices from the culture medium full of hyphae and inoculating the fungus slices into the liquid culture medium for culture;
in the microbial inoculum, the ratio of the diameter of the bacterial sheet to the liquid culture medium is 1 mm: 25-35 ml; specifically, the ratio of the diameter of the bacterial tablet to the liquid culture medium is 1 mm: 30 ml;
in the embodiment of the invention, the diameter of the mushroom slice is 5 mm; the volume of the liquid medium was 150 ml.
In the microbial inoculum, the concentration of the obtained culture solution is 4.5-8.7mg of DSE dry matter contained in each ml of culture solution.
In the embodiment of the invention, the condition of culturing in the solid culture medium is that the culture is carried out for 7 to 10 days in an inverted way at the constant temperature of between 25 and 28 ℃; the solid culture medium adopted is PDA culture medium.
The application of the Geospora above or the microbial inoculum above in at least one of the following 1) to 5) is also within the protection scope of the invention:
1) promoting the growth of plants;
2) promoting plant growth in lean soil;
3) treating or restoring field ecology;
4) monitoring the symbiotic effect of DES bacteria and plants;
5) land reclamation and/or ecological reconstruction in the field.
It is still another object of the present invention to provide a method for monitoring the symbiotic effect of DES bacteria on plants.
The method provided by the invention comprises the following steps: applying the microbial inoculum prepared by the second purpose to the plants, culturing, and monitoring symbiotic effect in a period of 15-20 days after the selective culture.
The above culture is growth in lean soil.
Compared with the control without inoculating the microbial inoculum, the symbiotic effect is realized by inoculating the microbial inoculum to promote the increase of the fresh weight of the plants.
The culture conditions adopted by the invention are 25 ℃, 3000lx light intensity, illumination time of 16h day/8 h night, and the water holding capacity of the culture substrate is maintained between 60% and 80%.
The 4 th object of the present invention is to provide a method for promoting plant growth.
The method provided by the invention comprises the following steps: the microbial inoculum is applied to plants and cultured to realize the growth promotion of the plants.
In the method, the step of applying the microbial inoculum prepared for the second purpose to the plant is to inoculate the microbial inoculum into a culture medium in which seeds of the plant are located in a manner of inoculating 6g of microbial inoculum to every 2-3g of seeds;
in an embodiment of the present invention, the step of applying the microbial inoculum prepared for the second purpose to the plant is specifically to inoculate 6g of microbial inoculum per 2.3g of seeds (0.06 g of microbial inoculum per 10 seeds), and the microbial inoculum is inoculated into a culture medium in which the seeds of the plant are located.
The culture conditions adopted by the invention are 25 ℃, 3000lx light intensity, illumination time of 16h day/8 h night, and the water holding capacity of the culture substrate is maintained between 60% and 80%.
In the examples of the present invention, the plant used is alfalfa, in particular alfalfa.
Experiments prove that the novel strain and the solid microbial inoculum prepared by the novel strain are applied to plants, the symbiotic relationship between the strain and the plants is promoted, the growth promoting effect of the strain on the plants is maximized, the process that the strain and the plants infect the symbiont is found, the monitoring of symbiotic sensitive time intervals can better reveal the DSE ecological restoration effect, the cost is saved for improving the land reclamation efficiency by using the microbial technology, the large-scale economic benefit is achieved, and a microbial technology support is provided for field land reclamation and ecological reconstruction. The method is more easy to exert the effect efficiency maximization of the DSE microbial inoculum, and provides an economic and efficient scientific method for the scale application of the DSE microbial inoculum.
Biological material preservation instructions
Classification nomenclature of biological materials: alternaria sp.
Strain number of biological material: 001
Deposit name of biological material: china general microbiological culture Collection center
The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)
Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101
Preservation date of biological material: 4 and 8 months in 2019
Accession number to the collection of biological materials: CGMCC No.17463
Biological material preservation instructions
Classification nomenclature of biological materials: darksidea zeta
Strain number of biological material: needle A1-3
Deposit name of biological material: china general microbiological culture Collection center
The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)
Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101
Preservation date of biological material: 4 and 8 months in 2019
Accession number to the collection of biological materials: CGMCC No.17464
Biological material preservation instructions
Classification nomenclature of biological materials: the G.sp.Pleospora sp.
Strain number of biological material: needle A2-8
Deposit name of biological material: china general microbiological culture Collection center
The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)
Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101
Preservation date of biological material: 11/19/2019
Accession number to the collection of biological materials: CGMCC No.18812
Drawings
FIG. 1 is a photograph showing the configuration of the needle A2-8.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
PDA culture medium: mixing potato extract powder 3.0g, glucose 20.0g and agar 14.0g, adding distilled water 1000ml, boiling to dissolve, packaging, and autoclaving at 121 deg.C for 15 min.
Liquid MMN medium (ph 5.5): adding CaCl2 0.05g、MgSO4 0.15g、NaCl 0.025g、FeCl30.01g、KH2PO40.5g, 0.0001g of Vitamin B1 (thiamine), (NH)4)2HPO4Mixing 0.25g, glucose 10g, citric acid 0.2g and Malt extract 10g to obtain mixture, adding distilled water to a constant volume of 1L, packaging, and autoclaving at 121 deg.C for 30 min.
Example 1 isolation and identification of DSE strains and preservation
Selecting fresh root samples of needle cogongrass collected from north electric power victory mine area of Netmori autonomous region, washing with sterile water for multiple times, sterilizing with 75% ethanol water solution for 5min, washing with sterile water for 3 times, sterilizing with 10% sodium hypochlorite water solution for 5min, washing with sterile water for 3 times, and removing water on the surface of the root samples with filter paper. Cutting the root segments into small segments of about 1cm, placing PDA culture medium containing 50mg/L ampicilin and 50mg/L streptomycin sulfate, and placing 2-3 root segments in each culture dish. Isolated and cultured at 28 ℃ in the dark and purified.
Separating and purifying to obtain bacteria No.1, bacteria No. 2 and bacteria No. 5.
The ITS sequence of the No.1 bacterium is detected, and the fungus is identified as dark-color septate endophytic fungus (DSE) as shown in a sequence 1 in a sequence table and is named as 001. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 8.4.2019, the preservation number is CGMCC No.17463, and the strain is classified and named as Alternaria sp.
The ITS sequence of the No. 2 bacterium is detected, and the fungus is identified as dark septate endophytic fungus (DSE) as shown in a sequence 2 in a sequence table, and is named as a needle A1-3. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 8.4.2019, the preservation number is CGMCC No.17464, and the strain is classified and named as Darksidea zeta.
The ITS sequence of the strain No. 5 is detected, and the strain is identified as the strain of Geospora Ploss sp, which is shown as a sequence 3 in a sequence table and is named as a needle A2-8. The strain is preserved in China general microbiological culture Collection center (CGMCC) No.18812 in 19 th 11 th 2019, and is classified and named as Grifola frondosa sp.
The morphology of needle A2-8 is shown in FIG. 1, and has typical Dark septal hyphae with a microsclerotic core structure formed by close packing of cells with enlarged and thickened cell walls, which belong to the Dark Septal Endophytes (DSE).
Example 2 growth promoting Effect of DSE inoculum on alfalfa with different days of cultivation
Preparation of DSE microbial inoculum with different culture days
1. Preparation of needle A2-8 microbial inoculum
1) Activation of
Activating the strain of the gelidium Pleospora sp.CGMCC No.18812 from a slant test tube preserved at low temperature, and transferring the activated strain to a plate of a PDA culture medium; culturing at 28 deg.C under constant temperature in the dark for 7 days, and perforating the edges of bacterial colony to obtain bacterial sheet when the plate is covered with mycelia;
2) preparation of solid microbial inoculum
Inoculating 5 mm-diameter fungus slices (hyphae) into 150ml of liquid MMN culture medium, and performing shake culture at 28 deg.C and 180r/min in dark for 4, 6, 8, and 10 days to obtain culture solution A2-8 (4.6mg/ml) of needle A2-8 (7.9mg/ml) after 4 days of culture, culture solution A2-8 (8.0mg/ml) after 8 days of culture, and culture solution A2-8 (8.7mg/ml) after 10 days of culture; filtering each culture solution with sterilized medium-speed qualitative filter paper (30-50 micron aperture) in a sterile operating platform, collecting solid (sticky mass) in the qualitative filter paper, and obtaining needle A2-8 microbial inoculum (0.6911g) after 4 days of culture, needle A2-8 microbial inoculum (1.1859g) after 6 days of culture, needle A2-8 microbial inoculum (1.1999g) after 8 days of culture and needle A2-8 microbial inoculum (1.3031g) after 10 days of culture for later use.
The concentration of the culture solution is the amount of DSE bacteria dry matter in each ml of culture solution.
The amount of the microbial inoculum is the amount of dry matter.
2. Preparation of needle A1-3 bacterial agent
1) Activation of
Activating Darksidea zeta CGMCC No.17464 strain from low-temperature preserved slant tube, and transferring to PDA culture medium plate; culturing at 28 deg.C under constant temperature in the dark for 7 days, and perforating the edges of bacterial colony to obtain bacterial sheet when the plate is covered with mycelia;
2) preparation of microbial inoculum
Inoculating 5 mm-diameter fungus slices into 150ml of liquid MMN culture medium, performing shake culture at 28 ℃ and 180r/min in the dark for 4, 6, 8 and 10 days respectively, filtering the DSE fungus liquid by using sterilized qualitative filter paper with the aperture of 30-50 microns in an aseptic operation table, and collecting solids (in the form of a massive viscous substance) in the qualitative filter paper to obtain a needle A1-3 microbial inoculum after 4-day culture, a needle A1-3 microbial inoculum after 6-day culture, a needle A1-3 microbial inoculum after 8-day culture and a needle A1-3 microbial inoculum after 10-day culture for later use.
3. 001 preparation of microbial inoculum
1) Activation of
Activating Alternaria sp.CGMCC No.17463 from the slant tube strain preserved at low temperature, and transferring to a plate of PDA culture medium; culturing in dark at 28 deg.C for 7 days, and perforating to obtain bacterial sheet when hyphae are covered on the plate;
2) preparation of microbial inoculum
Inoculating a bacterial sheet with the diameter of 5mm into a 150ml liquid MMN culture medium, culturing for 4, 6, 8 and 10 days at 28 ℃ and 180r/min in dark shaking respectively, filtering the DSE bacterial liquid by sterilized qualitative filter paper with the aperture of 30-50 microns in an aseptic operation table, collecting solids (in the form of a massive viscous substance) in the qualitative filter paper to obtain a 001 bacterial agent after culturing for 4 days, a 001 bacterial agent after culturing for 6 days, a 001 bacterial agent after culturing for 8 days and a 001 bacterial agent after culturing for 10 days for later use.
Secondly, the growth promoting effect of the DSE microbial inoculum with different culture days on the alfalfa
Culture medium: sand (single nutrient content and simulated barren land) collected from north beach of Beijing is sieved (mesh number is 2mm), sterilized by high-temperature high-pressure steam at 121 ℃ for 2h, and naturally dried for later use.
The test plants: alfalfaThe alfalfa variety is Athena alfalfa (available from agricultural Co., Ltd., Shuo county, New county), and the alfalfa seeds with full grains are treated with H with a concentration of 10% (mass fraction)2O2Soaking in water solution for 10min for surface sterilization, and washing with sterile water for 5-6 times.
Test pots: the test basin is a 270ml disposable paper cup sterilized by 75% ethanol solution by volume, and each cup contains 250g of culture medium.
Test protocol: each fungicide is designed with the following 5 treatments:
treatment 1: inoculating a DSE microbial inoculum cultured for 4 days;
and (3) treatment 2: inoculating a DSE microbial inoculum cultured for 6 days;
and (3) treatment: inoculating DSE microbial inoculum cultured for 8 days;
and (4) treatment: inoculating DSE microbial inoculum cultured for 10 days;
control group: no microbial inoculum is inoculated;
each of the above treatments was repeated 3 times for a total of 15 cups.
The specific experiment is as follows:
watering a paper cup until the water capacity of the culture medium is 70%, digging holes on the culture medium by using a sterile iron spoon, respectively inoculating 0.06g of corresponding DSE microbial inoculum into the holes by using sterile tweezers according to the 5 treatment groups of each microbial inoculum, and covering a little sandy soil; then 10 (about 0.023g) alfalfa seeds are inoculated into a culture medium, and are subjected to symbiotic culture by covering the culture medium, wherein the culture conditions are as follows: symbiotic culture is carried out in an artificial climate box, the temperature is 25 ℃, the light intensity is 3000lx, the illumination time is 16h day/8 h night, and the water holding capacity of the matrix is maintained between 60% and 80%. And harvesting all alfalfa plants inoculated with 5 treatment groups corresponding to each microbial inoculum after 30 days, and weighing the fresh weight of all alfalfa plants. 3 pots were received for each treatment group (experiment was repeated 1 time), and the results were averaged.
The 5 treatment groups corresponding to each DSE microbial inoculum are respectively inoculated with the prepared A2-8 microbial inoculum obtained in different culture days, A1-3 microbial inoculum obtained in different culture days and 001 microbial inoculum obtained in different culture days.
The results are as follows:
inoculating A2-8 microbial inoculum to alfalfa for symbiotic culture for 30 days after 4 days of inoculation culture, wherein the fresh weight of the alfalfa is 0.2680 g;
inoculating the needle A2-8 microbial inoculum after 6 days of inoculation culture to the alfalfa, and carrying out symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.2916 g;
inoculating the needle A2-8 microbial inoculum after 8 days of inoculation culture to the alfalfa, and carrying out symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.1924 g;
inoculating A2-8 microbial inoculum to alfalfa for symbiotic culture for 10 days, and then culturing for 30 days to obtain 0.1826g alfalfa fresh weight;
inoculating the needle A1-3 microbial inoculum after 4 days of inoculation culture to the alfalfa, and carrying out symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.2448 g;
inoculating the needle A1-3 microbial inoculum after 6 days of inoculation culture to the alfalfa, and carrying out symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.2254 g;
inoculating the needle A1-3 microbial inoculum after 8 days of inoculation culture to the alfalfa, and carrying out symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.2122 g;
after 10 days of inoculation and culture, inoculating A1-3 microbial inoculum to alfalfa, and performing symbiotic culture for 30 days, wherein the fresh weight of the alfalfa is 0.1560 g.
After 4 days of inoculation culture, the fresh weight of the alfalfa is 0.2382g after the 001 microbial inoculum is inoculated with the alfalfa for 30 days of symbiotic culture;
after 6 days of inoculation and culture, the fresh weight of the alfalfa is 0.1806g after the 001 microbial inoculum is inoculated with the alfalfa for 30 days of symbiotic culture;
after 8 days of inoculation and culture, the fresh weight of the alfalfa is 0.1801g after the 001 microbial inoculum is inoculated with the alfalfa for 30 days of symbiotic culture;
after 10 days of inoculation and culture, the fresh weight of the alfalfa is 0.1491g after the 001 microbial inoculum is inoculated with the alfalfa for 30 days of symbiotic culture.
Control group: the fresh weight of the alfalfa is 0.1630g after 30 days of single culture without inoculating the bacterial agent.
The following percentages in this example are (fresh weight of alfalfa in each group-fresh weight of alfalfa in the control group)/fresh weight of alfalfa in the control group.
Compared with a control group, the growth amount of the needle A2-8 microbial inoculum is improved by 64.41%, 78.84%, 18.04% and 12.02% after 4, 6, 8 and 10 days of inoculation and culture.
The growth amount of the needle A1-3 microbial inoculum after 4, 6, 8 and 10 days of inoculation and culture is improved by 50.16 percent, 38.27 percent, 30.15 percent and-4.29 percent compared with that of a control group.
The growth amount of the 001 microbial inoculum after 4, 6, 8 and 10 days of inoculation culture to the alfalfa is improved by 46.16 percent, 10.82 percent, 10.48 percent and 8.53 percent compared with that of a control group.
The results show that compared with the A1-3 microbial inoculum and the 001 microbial inoculum, the A2-8 microbial inoculum of the inoculating needle obviously promotes the growth amount of the alfalfa, and is embodied in fresh weight. The needle A2-8 microbial inoculum cultured for 4-6 days in the inoculation needle A2-8 microbial inoculum has more obvious effect on promoting the growth of alfalfa, and has shorter culture time and more economical efficiency. Particularly, the A2-8 strain has the most remarkable effect on promoting the growth of the alfalfa after 6 days of culture (the percentage of the increase is the largest compared with that of a control).
Example 3 growth Effect of inoculated DSE inoculum on alfalfa at different time periods
Preparation of DSE microbial inoculum with different culture days
Same as one of embodiment 2;
secondly, the growth effect of the inoculated DSE microbial inoculum on the alfalfa in different time periods
Culture medium: sand (single nutrient content and simulated barren land) collected from north beach of Beijing is sieved (mesh number is 2mm), sterilized by high-temperature high-pressure steam at 121 ℃ for 2h, and naturally dried for later use.
The test plants: the alfalfa is Athena alfalfa, and the plump alfalfa seeds are treated with 10% (mass fraction) H2O2Soaking in water solution for 10min for surface sterilization, and washing with sterile water for 5-6 times.
Test pots: the test basin is a 270ml disposable paper cup sterilized by 75% alcohol by volume and filled with 250g of culture medium per cup.
Test protocol: the A2-8 microbial inoculum is respectively designed into the following 5 treatments:
treatment 1: inoculating DSE microbial inoculum needle A2-8 cultured for 4 days;
and (3) treatment 2: inoculating DSE microbial inoculum needle A2-8 after culturing for 6 days;
and (3) treatment: inoculating DSE microbial inoculum needle A2-8 after 8 days of culture;
and (4) treatment: inoculating DSE inoculum needle A2-8 after culturing for 10 days;
control group: a microbial inoculum needle A2-8 is not inoculated;
each of the above treatments was repeated 3 times for a total of 15 cups.
The specific experiment is as follows:
watering to 70% of the water capacity of the matrix, digging holes on the matrix by using a sterile iron spoon, respectively inoculating 0.06g of corresponding DSE (Dermatophagoides pteronyssinus) fungicide needles A2-8 into the holes by using sterile tweezers according to the 5 treatment groups of each fungicide, and covering a little sandy soil; then 10 (about 0.023g) alfalfa seeds are inoculated into a culture medium, and are subjected to symbiotic culture by covering the culture medium, wherein the culture conditions are as follows: symbiotic culture is carried out in an artificial climate box, the temperature is 25 ℃, the light intensity is 3000lx, the illumination time is 16h day/8 h night, and the water holding capacity of the matrix is maintained between 60% and 80%. And harvesting all alfalfa plants of 5 treatment groups corresponding to the inoculation needle A2-8 microbial inoculum after 10, 15, 20, 25 and 30 days of symbiotic culture, and weighing the fresh weight of all alfalfa plants. Each treatment group received 3 tubs and the results were averaged.
The 5 treatment groups corresponding to the A2-8DSE microbial inoculum are respectively inoculated with the A2-8 microbial inoculum prepared by the method and obtained on different culture days.
The results are shown in Table 1.
TABLE 1 alfalfa fresh weights at different growth times under different treatments with A2-8 microbial inoculum
Calculating the plant growth rate of symbiotic culture in different time periods after inoculation of the A2-8 fungicide according to table 1, and searching the time period of most sensitive symbiosis of DSE and plants; the calculation formula is that the increase rate of the fresh weight of the alfalfa in a time period is (the fresh weight at the later time point-the fresh weight at the previous time point)/the fresh weight at the previous time point is multiplied by 100%, and the result is shown in table 2.
Table 2 shows the increase rate (%)'s of the fresh weight of alfalfa in different growth periods under different treatments with A2-8 microbial inoculum
Test results show that compared with a control group, compared with alfalfa inoculated with the DSE fungal needle A2-8 microbial inoculum, the DSE microbial inoculum cultured for 4-8 days has the advantages that the DSE microbial inoculum and the alfalfa grow faster in a symbiotic way for 15-20 days without obvious difference, the period is the most sensitive period for monitoring the inoculation effect of the DSE, and the monitoring of the inoculation effect at the period can more sensitively reflect the action effect of the DSE; this is substantially consistent with the results of promoting alfalfa growth.
SEQUENCE LISTING
<110> university of mineral industry (Beijing) Xian university of science and technology
<120> preparation and application methods of DSE microbial inoculum and symbiotic effect sensitive period monitoring
<160> 3
<170> PatentIn version 3.5
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ttcacccgtg tcttttgcgt acttcttgtt tccttggtgg gctcgcccgc cacaaggaca 180
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aacaacggat ctcttggttc tggcatcgat gaagaacgca gcgaaatgcg atacgtagtg 300
tgaattgcag aattcagtga atcatcgaat ctttgaacgc acattgcgcc ctttggtatt 360
ccaaagggca tgcctgttcg agcgtcattt gtaccctcaa gctttgcttg gtgttgggcg 420
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Claims (13)
1. The gram-positive spore bacterium Pleospora sp. needle A2-8 has a preservation number of CGMCC No. 18812.
2. The microbial inoculum is prepared by the method comprising the following steps:
1) inoculating the Grifola frondosa of claim 1 into a liquid culture medium for culture to obtain a culture solution;
2) filtering the culture solution, and collecting solid substances to obtain the microbial inoculum.
3. The microbial inoculum of claim 2, wherein: the culture time is 4-6 days.
4. The microbial inoculum of claim 3, wherein:
the culture condition is shaking culture at 25-28 ℃ and 180 r/min.
5. The microbial inoculum according to any one of claims 2 to 4, wherein:
the liquid culture medium is a liquid MMN culture medium;
or, medium-speed filter paper is adopted for filtering.
6. The microbial inoculum according to any one of claims 2 to 4, wherein:
the step of inoculating the Grifola frondosa into the liquid culture medium is to inoculate the mycelium of the Grifola frondosa into the liquid culture medium according to claim 1;
the mycelium of the gelidium is prepared according to the following method:
a) culturing the Grifola frondosa of claim 1 in a solid medium until the mycelia are covered;
b) then taking the fungus slices from the culture medium full of hyphae and inoculating the fungus slices into the liquid culture medium for culture;
the diameter of the bacterial tablet and the ratio of the liquid culture medium are 1 mm: 25-35 ml.
7. The microbial inoculum of claim 6, wherein: the diameter of the bacterial tablet and the ratio of the liquid culture medium are 1 mm: 30 ml.
8. Use of the gelidium sp as defined in claim 1 or the microbial preparation as defined in any one of claims 2 to 7 in at least one of the following 1) to 5):
1) promoting the growth of the Athena alfalfa;
2) promoting the growth of the athena alfalfa in the barren soil;
3) treating or restoring field ecology;
4) monitoring the symbiotic effect of DES bacteria and plants;
5) land reclamation and/or ecological reconstruction in the field.
9. A method for monitoring the symbiotic effect of DES bacteria and Athena alfalfa comprises the following steps: applying the microbial preparation of any one of claims 2 to 7 to alfalfa, culturing, and monitoring symbiotic effects at a period of 15 to 20 days after the selection.
10. A method for promoting the growth of the alfalfa from Athena comprises the following steps: the microbial inoculum of any one of claims 2 to 7 is applied to plants and cultured to promote the growth of the alfalfa of Athena.
11. The method according to claim 9 or 10, characterized in that:
the application of the microbial inoculum according to any one of claims 2 to 7 to the alfalfa of Athena is to inoculate the microbial inoculum into a culture medium containing the seeds of the alfalfa of Athena in a manner that 6g of the microbial inoculum is inoculated into every 2 to 3g of the seeds.
12. The method according to claim 9 or 10, characterized in that:
the application of the microbial inoculum according to any one of claims 2 to 7 to the alfalfa of Athena is to inoculate the microbial inoculum into a culture medium containing the seeds of the alfalfa of Athena in a manner that 6g of the microbial inoculum is inoculated per 2.3g of the seeds.
13. The method according to claim 9 or 10, characterized in that:
the application of the microbial inoculum according to any one of claims 2 to 7 to the alfalfa of Athena is to inoculate the microbial inoculum into a culture medium containing the seeds of the alfalfa of Athena in a manner that 0.06g of the microbial inoculum is inoculated into every 10 seeds.
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