CN111394259B - Preparation method of DSE dry microbial inoculum capable of promoting plant growth and easy to store and transport - Google Patents

Preparation method of DSE dry microbial inoculum capable of promoting plant growth and easy to store and transport Download PDF

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CN111394259B
CN111394259B CN202010305355.7A CN202010305355A CN111394259B CN 111394259 B CN111394259 B CN 111394259B CN 202010305355 A CN202010305355 A CN 202010305355A CN 111394259 B CN111394259 B CN 111394259B
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needle
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microbial inoculum
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毕银丽
王淑惠
全文智
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Beijing Heshengyuan Ecological Environment Engineering Technology Co ltd
China University of Mining and Technology Beijing CUMTB
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China University of Mining and Technology Beijing CUMTB
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Abstract

The invention discloses a preparation method of a DSE dry microbial inoculum which promotes plant growth and is easy to store and transport. The preparation method of the DSE dry microbial inoculum comprises the following steps: adopting a liquid culture medium culture needle A2-7 (at 25-28 ℃, 150-; collecting solids in the bacterial liquid; drying at 20-45 deg.C to obtain DSE dry bacteria agent. The DSE dry bacterium agent prepared by the preparation method provided by the invention is inoculated in soil, and then alfalfa seeds are planted and normally cultured. The result shows that the DSE dry bacterium agent prepared by the preparation method provided by the invention can obviously promote the growth of alfalfa. The preparation method of the DSE dry microbial inoculum provided by the invention has the advantages of short preparation time (only 6 days) and low cost, and the prepared DSE dry microbial inoculum is easy to store and transport and has good effect of promoting plant growth. The invention has great application and popularization value for plant planting, and is convenient for large-scale application in different places.

Description

Preparation method of DSE dry microbial inoculum capable of promoting plant growth and easy to store and transport
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a DSE dry microbial inoculum which can promote plant growth and is easy to store and transport.
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 research shows that the DSE has the ecological functions of promoting the mineral nutrition absorption of the 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.
Currently, DSE agents are mainly achieved by inoculating a DSE mass (obtained by solid culture) or a DSE bacterial solution (obtained by liquid culture) to the roots of plants. However, the DSE bacterium blocks and the DSE bacterium liquid are not easy to store and are not suitable for mass transportation. Therefore, the DSE bacterium block and the DSE bacterium liquid are only suitable for laboratory culture or small-batch and near-to-near application, and are not practical for large-scale application in different places.
Disclosure of Invention
The invention aims to prepare a microbial inoculum which can promote the growth of plants and is easy to store and/or transport.
The invention firstly protects a preparation method of a DSE dry microbial inoculum, which sequentially comprises the following steps:
(1) adopting a liquid culture medium culture needle A2-7 to obtain a bacterial liquid; the culture time may be 6 days;
(2) collecting solids in the bacterial liquid;
(3) drying to obtain DSE dry bacterium agent;
the DSE dry bacterium agent can promote plant growth.
In the step (1), the culturing condition can be 25-28 deg.C (such as 25-26 deg.C, 26-27 deg.C, 27-28 deg.C, 25 deg.C, 26 deg.C, 27 deg.C or 28 deg.C), 150-200r/min (such as 150-180r/min, 180-200r/min, 150r/min, 180r/min or 200r/min), shaking culture.
In the step (1), the needle A2-7 can be added in the form of a mushroom tablet or mushroom cake. The strain pieces or strain cakes are obtained by inoculating needle A2-7 on solid culture medium (such as PDA solid culture medium), culturing at 25-28 deg.C (such as 25-27 deg.C, 27-28 deg.C, 25 deg.C, 26 deg.C, 27 deg.C or 28 deg.C) for 7-10 days (such as 7-8 days, 8-9 days, 9-10 days, 7 days, 8 days, 9 days or 10 days), and perforating at the colony edge. The diameter of the mushroom pieces or mushroom cakes can be 4-6mm (e.g., 4-5mm, 5-6mm, 4mm, 5mm, or 6 mm).
In the step (1), the ratio of the bacterial slices or bacterial cakes to the liquid culture medium can be 1 bacterial slice or bacterial cake: (100-200mL) liquid medium, such as "1 bacterial pellet or cake: (100-150mL), liquid medium "," 1 pellet or cake: (150-: 100mL of liquid medium "," 1 pellet or cake: 150mL of liquid medium "or" 1 pellet or cake: 200mL of liquid medium ".
Any of the above liquid media can be liquid MMN media. The preparation method of the liquid MMN culture medium can be as follows: adding CaCl2 0.05g、MgSO4 0.15g、NaCl 0.025g、FeCl3 0.01g、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.
And (3) in the step (2), collecting the solid in the bacterial liquid by filtering or centrifuging. Qualitative filter paper was used for the filtration. The qualitative filter paper can be medium-speed qualitative filter paper, namely qualitative filter paper with the aperture of 30-50 microns (such as 30-40 microns, 40-50 microns, 30 microns, 40 microns or 50 microns).
In the step (3), the drying temperature may be 20-45 deg.C (such as 20-25 deg.C, 25-30 deg.C, 30-35 deg.C, 35-40 deg.C, 40-45 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C or 45 deg.C). From the economical point of view, the cost is lowest when the drying temperature is 20-25 ℃ (such as 20-23 ℃, 23-25 ℃, 20 ℃, 23 ℃ or 25 ℃).
In the step (3), the drying may be natural air drying or forced air drying.
In any of the above-mentioned methods, a carrier may be added before step (2) and/or before step (3). The carrier may be a solid carrier or a liquid carrier. The solid carrier may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of bran, soybean meal, corn flour, bean flour and starch. The high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.
In any of the above-mentioned preparation methods, before performing step (2) and/or before performing step (3), a surfactant (e.g., tween 20, tween 80, etc.), a binder, a stabilizer (e.g., an antioxidant), a pH adjuster, etc. may be added as necessary.
In any of the above methods, the moisture content of the DSE dry microbial inoculum may be 1-5% (e.g., 1-2%, 2-5%, 1%, 2%, or 5%).
In any of the above preparation methods, the promoting plant growth is specifically promoting plant fresh weight increase.
In any of the above-mentioned preparation methods, the needle A2-7 is called dark septate endophytic fungus (Darksiidea sp.) needle A2-7, which has been deposited in the China general microbiological culture Collection center on 19 th 11 th 2019 with the deposit number of CGMCC No. 18811.
The DSE dry bacterium agent prepared by any one of the preparation methods is easy to store and/or transport.
Any of the above plants may be a Papilionoideae plant.
Any of the plants described above may be a medicago plant.
Any of the plants described above may be alfalfa, for example alfalfa. The variety of the alfalfa can be specifically Athena alfalfa.
The DSE dry microbial inoculum (such as needle A2-7 dry microbial inoculum c, needle A2-7 dry microbial inoculum 2, needle A2-7 dry microbial inoculum d, needle A2-7 dry microbial inoculum e, needle A2-7 dry microbial inoculum f and needle A2-7 dry microbial inoculum g) prepared by the preparation method provided by the invention is inoculated in soil, and then the seeds of the alfalfa of Athena are planted and cultured normally. The result shows that the DSE dry bacterium agent prepared by the preparation method provided by the invention can obviously promote the fresh weight increase of the Athena alfalfa, namely the growth of the Athena alfalfa. Therefore, the preparation method of the DSE dry bacterium agent provided by the invention has the advantages of short preparation time (only 6 days) and low cost, and the prepared DSE dry bacterium agent is easy to store and transport and has a good effect of promoting plant growth. The invention has great application and popularization value for plant planting, and is convenient for large-scale application in different places.
Drawings
FIG. 1 is a photograph of the morphology of needle A2-7 within the root system.
FIG. 2 is a photograph of the morphology of needle A2-8 within the root system.
FIG. 3 shows the form of hypha and microsclerotia in alfalfa roots of the dry inoculant 1 of inoculating needle A2-7.
Biological material preservation instructions
Classification nomenclature of biological materials: darksidea sp.
Strain number of biological material: needle A2-7
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.18811
Biological material preservation instructions
Classification nomenclature of biological materials: pleosporales 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
Biological material preservation instructions
Classification nomenclature of biological materials: darksidea sp.
Strain number of biological material: needle A2-1
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.17465
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures in the following examples are conventional unless otherwise specified.
The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
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 acquisition and preservation of strains
The three strains (needle A2-7, needle A2-8 and needle A2-1) are all strains separated and purified from the root system of the needle cogongrass in the northern electric winning mine area of the union of the autonomous region of the inner Mongolia, Siling.
The ITS sequence of detection probe A2-7 is shown as SEQ ID No: 1, identified as Darksideasp. The needle A2-7 has been preserved in China general microbiological culture Collection center (CGMCC) No.18811 in 2019 at 19.11. Morphological characteristics of dark septate endophytes are as follows: the hyphae are dark brown in the root system and have obvious transverse septa. A photograph of the morphology of needle A2-7 in the root system is shown in FIG. 1.
The ITS sequence of detection probe A2-8 is shown as SEQ ID No: 2, identified as a strain of gelidium Pleospora sp. The needle A2-8 has been preserved in China general microbiological culture Collection center (CGMCC) No.18812 in 2019, 11 and 19 months. The morphological characteristics of needle A2-8 are as follows: has typical dark hypha and microsclerotia structure formed by the close packing of cells with enlarged and thickened cell walls. A photograph of the morphology of needle A2-8 in the root system is shown in FIG. 2.
The ITS sequence of detection probe A2-1 is shown as SEQ ID No: 3, identified as Darksidesasp. The needle A2-1 has been preserved in China general microbiological culture Collection center (CGMCC) at 2019, 4 and 8, with the preservation number of CGMCC No. 17465.
Example 2 Effect of inoculation of DSE Wet inoculant and DSE Dry inoculant on the infection Rate and growth of Medicago sativa
The temperature for natural air drying in this example was 25 ℃.
The test strains were needle A2-7, needle A2-8 and needle A2-1, respectively.
The alfalfa is specifically Athena alfalfa.
Preparation of needle A2-7 wet bacterial agent 1-needle A2-7 wet bacterial agent 4
1. Inoculating activated needle A2-7 to PDA solid culture medium, and performing inverted culture at 28 deg.C for 7 days; then, holes are punched at the edges of the colonies to obtain bacterial slices with the diameter of 5 mm.
2. After the step 1 is finished, inoculating the bacterial slices into 150mL of liquid MMN culture medium, and carrying out shaking culture at 28 ℃ and 180r/min for 4 days to obtain a needle A2-7 bacterial liquid.
3. And (3) after the step 2 is finished, filtering the bacterial liquid of the needle A2-7 by using sterilized qualitative filter paper (the aperture is 30-50 microns, and the medium speed) under the aseptic condition, and collecting the solid on the qualitative filter paper. The solid is needle A2-7 wet bacterial agent 1.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 6 days, and obtaining the needle A2-7 wet bacterial agent 2 without changing other steps.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 8 days, and keeping the other steps unchanged to obtain the needle A2-7 wet bacterial agent 3.
According to the above steps, the shaking culture was changed from 4 days to 10 days, and the other steps were not changed, to obtain needle A2-7 wet bacterial agent 4.
Secondly, preparation of needle A2-7 dry bacterial agent 1-needle A2-7 dry bacterial agent 5
1. Inoculating activated needle A2-7 to PDA solid culture medium, and performing inverted culture at 28 deg.C for 7 days; then, holes are punched at the edges of the colonies to obtain bacterial slices with the diameter of 5 mm.
2. After the step 1 is finished, inoculating the bacterial slices into 150mL of liquid MMN culture medium, and carrying out shaking culture at 28 ℃ and 180r/min for 4 days to obtain a needle A2-7 bacterial liquid.
3. And (3) after the step 2 is finished, filtering the bacterial liquid of the needle A2-7 by using sterilized qualitative filter paper (the aperture is 30-50 microns, and the medium speed) under the aseptic condition, and collecting the solid on the qualitative filter paper. The solid is needle A2-7 wet bacterial agent a.
4. And (3) after the step (3) is finished, taking the needle A2-7 wet microbial inoculum a, and naturally drying in the air under an aseptic condition to obtain the needle A2-7 dry microbial inoculum 1 with the water content of 2%.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 6 days, and keeping the other steps unchanged to obtain a needle A2-7 wet bacterial agent b and finally obtain a needle A2-7 dry bacterial agent 2.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 7 days, and keeping the other steps unchanged to obtain a needle A2-7 wet bacterial agent c and finally obtain a needle A2-7 dry bacterial agent 3.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 8 days, and keeping the other steps unchanged to obtain a needle A2-7 wet bacterial agent d, and finally obtaining a needle A2-7 dry bacterial agent 4.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 10 days, and keeping the other steps unchanged to obtain a needle A2-7 wet microbial inoculum e, and finally obtaining a needle A2-7 dry microbial inoculum 5.
0.06g of needle A2-7 dry microbial inoculum 1 can be prepared from every 3g of needle A2-7 wet microbial inoculum a.
0.06g of needle A2-7 dry microbial inoculum 2 can be prepared from every 3g of needle A2-7 wet microbial inoculum b.
0.06g of needle A2-7 dry microbial inoculum 3 can be prepared from every 3g of needle A2-7 wet microbial inoculum c.
0.06g of needle A2-7 dry microbial inoculum 4 can be prepared from every 3g of needle A2-7 wet microbial inoculum d.
0.06g of needle A2-7 dry microbial inoculum 5 can be prepared from every 3g of needle A2-7 wet microbial inoculum e.
Preparation of needle A2-8 dry bacterial agent 1-needle A2-8 dry bacterial agent 4
1. Inoculating activated needle A2-8 into PDA solid culture medium, and performing inverted culture at 28 deg.C for 7 days; then, holes are punched at the edges of the colonies to obtain bacterial slices with the diameter of 5 mm.
2. After the step 1 is finished, inoculating the bacterial slices into 150mL of liquid MMN culture medium, and carrying out shaking culture at 28 ℃ and 180r/min for 4 days to obtain the bacterial liquid of the needle A2-8.
3. And (3) after the step 2 is finished, filtering the bacterial liquid of the needle A2-8 by using sterilized qualitative filter paper (the aperture is 30-50 microns, and the medium speed) under the aseptic condition, and collecting the solid on the qualitative filter paper. The solid is needle A2-8 wet bacterial agent 1.
4. And (3) after the step (3) is finished, taking the wet microbial inoculum 1A 2-8, and naturally drying in the air under an aseptic condition to obtain the dry microbial inoculum 1A 2-8 with the water content of 2%.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 6 days, and keeping the other steps unchanged to obtain the needle A2-8 dry microbial inoculum 2.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 8 days, and keeping the other steps unchanged to obtain the needle A2-8 dry microbial inoculum 3.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 10 days, and keeping the other steps unchanged to obtain the needle A2-8 dry microbial inoculum 4.
0.06g of needle A2-8 dry bacterial agent 1 can be prepared from every 3g A2-8 wet bacterial agent 1.
0.06g of needle A2-8 dry microbial inoculum 2 can be prepared from every 3g A2-8 wet microbial inoculum 2.
0.06g of needle A2-8 dry microbial inoculum 3 can be prepared from every 3g A2-8 wet microbial inoculum 3.
0.06g of needle A2-8 dry microbial inoculum 4 can be prepared from every 3g A2-8 wet microbial inoculum 4.
Preparation of needle A2-1 wet bacterial agent 1-needle A2-1 wet bacterial agent 4
1. Inoculating activated needle A2-1 into PDA solid culture medium, and performing inverted culture at 28 deg.C for 7 days; then, holes are punched at the edges of the colonies to obtain bacterial slices with the diameter of 5 mm.
2. After the step 1 is finished, inoculating the bacterial slices into 150mL of liquid MMN culture medium, and carrying out shaking culture at 28 ℃ and 180r/min for 4 days to obtain the bacterial liquid of the needle A2-1.
3. And (3) after the step 2 is finished, filtering the bacterial liquid of the needle A2-1 by using sterilized qualitative filter paper (the aperture is 30-50 microns) under an aseptic condition, and collecting solids on the qualitative filter paper. The solid is needle A2-1 wet bacterial agent 1.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 6 days, and obtaining the needle A2-1 wet bacterial agent 2 without changing other steps.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 8 days, and obtaining the needle A2-1 wet bacterial agent 3 without changing other steps.
According to the steps, replacing the shaking culture for 4 days with the shaking culture for 10 days, and obtaining the needle A2-1 wet bacterial agent 4 without changing other steps.
Fifthly, inoculating DSE wet microbial inoculum or DSE dry microbial inoculum to alfalfa
The DSE wet microbial inoculum is needle A2-7 wet microbial inoculum 1-4 and needle A2-1 wet microbial inoculum 1-4 respectively.
The DSE dry microbial inoculum is needle A2-7 dry microbial inoculum 1-5 and needle A2-8 dry microbial inoculum 1-4 respectively.
Culture medium: collecting sand from north beach of Beijing, sieving (mesh number is 2mm), sterilizing with high temperature and high pressure steam at 121 deg.C for 2 hr, and air drying.
Test pots: taking disposable paper cups (specification is 270mL), sterilizing 75% (v/v) alcohol water solution, filling 250g of culture medium into each paper cup, and watering until the water capacity of the culture medium is 70%.
1. Selecting alfalfa seeds with similar size and full grains, and using H with the concentration of 10%2O2Soaking in the solution for 10min (for disinfection), and cleaning with sterile water for 5-6 times to obtain sterile herba Medicaginis seed.
2. After step 1 was completed, sterile alfalfa seeds were taken and set into 18 groups of three replicates each for a total of 54 test pots. Each group was treated as follows:
a first group: digging holes on the culture substrate by using a sterile iron spoon; then inoculating 3g of needle A2-7 wet microbial inoculum 1 into the inoculation hole by using sterile forceps, and covering a little sandy soil; then inoculating the sterile alfalfa seeds into a culture medium, and covering soil.
Second group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-7 wet bacterial agent 2, and the rest are not changed.
Third group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-7 wet bacterial agent 3, and the rest are not changed.
And a fourth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-7 wet bacterial agent 4, and the rest are not changed.
And a fifth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-7 dry bacterial agent 1, and the rest are not changed.
A sixth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-7 dry bacterial agent 2, and the rest are not changed.
A seventh group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-7 dry bacterial agent 3, and the rest are not changed.
And an eighth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-7 dry bacterial agent 4, and the rest are not changed.
Ninth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-7 dry bacterial agent 5, and the rest are not changed.
The tenth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-8 dry bacterial agent 1, and the rest are not changed.
Eleventh group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-8 dry bacterial agent 2, and the rest are not changed.
A twelfth group: the wet bacterial agent 1 of the needle A2-7 of 3g in the first group is replaced by the dry bacterial agent 3 of the needle A2-8 of 0.06g, and the rest are not changed.
Group thirteen: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 0.06g of needle A2-8 dry bacterial agent 4, and the rest are not changed.
A fourteenth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by the 3g of needle A2-1 wet bacterial agent 1, and the rest are not changed.
A fifteenth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-1 wet bacterial agent 2, and the rest are not changed.
Sixteenth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-1 wet bacterial agent 3, and the rest are not changed.
Seventeenth group: the 3g of needle A2-7 wet bacterial agent 1 in the first group is replaced by 3g of needle A2-1 wet bacterial agent 4, and the rest are not changed.
Eighteenth group (blank control group): digging holes on the culture substrate by using a sterile iron spoon; then inoculating the sterile alfalfa seeds into a culture medium, and covering soil.
Each test pot was inoculated evenly with 10 sterile alfalfa seeds.
The inoculation depths of the DSE wet microbial inoculum or the DSE dry microbial inoculum in each test basin are basically consistent.
The depth of inoculation of sterile alfalfa seeds in each test pot was substantially consistent.
3. After step 2, 54 test pots were placed in an artificial climate box and cultured alternately in light and dark at 23-25 deg.C (16h light/8 h dark, light intensity in light culture was 2000-. During the culture period, the water holding capacity of the culture substrate is maintained between 60% and 80%.
Sixthly, infection condition
After the fifth step is completed, detecting the DSE colonization rate, hypha colonization rate and microsclerotia colonization rate (colonization rate is also called infection rate) of each plant, and averaging according to groups.
The method for detecting the colonization rate comprises the following steps: taking the roots of the plants, cutting the roots into root segments with the length of about 1cm, randomly taking 30 root segments from each plant, processing the root segments by adopting an acid fuchsin staining method, performing microscopic examination under a microscope, and calculating the total colonization rate of DSE (the colonization rate is the number of colonized root segments/the number of microscopic root segments multiplied by 100 percent).
The statistical results are shown in Table 1.
TABLE 1
Figure BDA0002455590110000091
Figure BDA0002455590110000101
The shapes of hypha and microsclerotia in alfalfa roots of the inoculating needle A2-7 dry inoculant 1 are shown in figure 3(A is hypha, B is microsclerotia).
Results show that a DSE-alfalfa symbiotic system is established by inoculating the DSE wet microbial inoculum and the DSE dry microbial inoculum, and the activity (infection rate) of the DSE dry microbial inoculum is improved to a certain extent or has no obvious difference compared with that of the DSE wet microbial inoculum (namely, the activity of the DSE dry microbial inoculum is not lower than that of the DSE wet microbial inoculum); the activity of needle A2-8 prepared as a dry microbial inoculum is inferior to that of needle A2-7, although it is also a DSE bacterium.
Seventh, influence on the growth of alfalfa
1. And after the fifth step is finished, detecting the fresh weight of each plant, and taking an average value according to groups.
The results are shown in Table 2. The results show that fresh weight average of alfalfa treated in the sixth, seventh and eighth groups was significantly increased and there was no significant difference between the three groups compared to the blank control group.
TABLE 2
Figure BDA0002455590110000102
2. The plants treated with the blank control group (eighteenth group) were used as reference plants, and the fungicide contribution rates of the first to seventeenth groups were calculated. For fresh weight, the microbial inoculum contribution rate is (fresh weight of treated plants of each group-fresh weight of reference plant)/fresh weight of reference plant x 100%.
The results of the microbial inoculum contribution rate are shown in Table 3 (mean of 3 plants).
TABLE 3
Inoculated DSE wet bacterial agent or DSE dry bacterial agent Contribution rate to fresh weight
First group 3g needle A2-7 Wet bacterial agent 1 5.67％
Second group 3g needle A2-7 Wet bacterial agent 2 19.90％
Third group 3g needle A2-7 Wet bacterial agent 3 11.58％
Fourth group 3g needle A2-7 Wet bacterial 4 0.80％
Fifth group 0.06g needle A2-7 Dry microbial inoculum 1 25.94％
Sixth group 0.06g needle A2-7 dry bacterial agent 2 30.31％
Seventh group 0.06g needle A2-7 Dry microbial inoculum 3 35.37％
Eighth group 0.06g needle A2-7 is dryBacterial agent 4 38.39％
Ninth group 0.06g needle A2-7 dry bacterial agent 5 -4.62％
Ninth group 0.06g needle A2-8 Dry microbial inoculum 1 3.51％
Tenth group 0.06g needle A2-8 dry bacterial agent 2 22.12％
Eleventh group 0.06g needle A2-8 dry bacterial agent 3 5.48％
Twelfth group 0.06g needle A2-8 dry bacterial agent 4 2.77％
Group thirteen 3g needle A2-1 Wet bacterial agent 1 9.74％
Fourteenth group 3g needle A2-1 Wet bacterial agent 2 21.75％
Fifth group 3g needle A2-1 Wet bacterial agent 3 12.82％
Sixteenth group 3g needle A2-1 Wet bacterial 4 0.31％
Seventeenth group Is free of 0.00％
The results show that the contribution rate of the sixth group, the seventh group and the eighth group to the fresh weight of the alfalfa is obviously higher than that of other groups.
Therefore, the needle A2-7 dry microbial inoculum 2, the needle A2-7 dry microbial inoculum 3 and the needle A2-7 dry microbial inoculum 4 can effectively promote the growth of alfalfa and are convenient to store and transport. Wherein, the preparation time of the needle A2-7 dry microbial inoculum 2 is shortest; from the viewpoint of saving (e.g., time, cost, effort), needle a2-7 dry microbial inoculum 2 is the best microbial inoculum for promoting plant growth.
Example 3 Effect of drying temperature on promoting growth of alfalfa A2-7 Dry Mushroom
Firstly, preparation of needle A2-7 dry bacterial agent a-needle A2-7 dry bacterial agent h
According to the method for preparing the needle A2-7 dry microbial inoculum 2 in the second step of the embodiment 2, natural air drying at 25 ℃ is replaced by natural air drying at 10 ℃, natural air drying at 15 ℃, natural air drying at 20 ℃, natural air drying at 30 ℃, forced air drying at 35 ℃, forced air drying at 40 ℃, forced air drying at 45 ℃ or forced air drying at 50 ℃, and other steps are not changed, so that the needle A2-7 dry microbial inoculum a, the needle A2-7 dry microbial inoculum b, the needle A2-7 dry microbial inoculum c, the needle A2-7 dry microbial inoculum d, the needle A2-7 dry microbial inoculum e, the needle A2-7 dry microbial inoculum f, the needle A2-7 dry microbial inoculum g and the needle A2-7 dry microbial inoculum h with the water content of 2% are sequentially obtained.
Secondly, inoculating a needle A2-7 dry microbial inoculum a-needle A2-7 dry microbial inoculum h to the alfalfa
According to the method of the fifth step in the embodiment 2, the alfalfa is respectively inoculated with the needle A2-7 dry microbial inoculum a-needle A2-7 dry microbial inoculum h, the needle A2-7 dry microbial inoculum 2 and water.
Influence on growth of alfalfa
1. And after the second step is finished, detecting the fresh weight of each plant, and taking an average value according to the group.
The results are shown in Table 4. The results show that compared with a blank control group, the fresh weights of alfalfa processed by the needle A2-7 dry microbial inoculum c, the needle A2-7 dry microbial inoculum 2, the needle A2-7 dry microbial inoculum d, the needle A2-7 dry microbial inoculum e, the needle A2-7 dry microbial inoculum f and the needle A2-7 dry microbial inoculum g are all remarkably increased and have no remarkable difference.
TABLE 4
Inoculated needle A2-7 dry bacterium agent Fresh weight (g)
Needle A2-7 Dry microbial inoculum a 0.1839±0.0374
Needle A2-7 Dry microbial inoculum b 0.1909±0.0285
Needle A2-7 Dry microbial inoculum c 0.2206±0.0147
Needle A2-7 Dry microbial inoculum 2 0.2115±0.0120
Needle A2-7 Dry microbial inoculum d 0.2173±0.0238
Needle A2-7 Dry microbial inoculum e 0.2111±0.0156
Needle A2-7 Dry microbial inoculum f 0.2272±0.0108
Needle A2-7 Dry microbial inoculum g 0.2143±0.0217
Needle A2-7 Dry microbial inoculum h 0.1880±0.0131
None (i.e. blank control group) 0.1623±0.0119
2. And taking the plants treated by the blank control group as reference plants, and calculating the contribution rate of the microbial inoculum of other groups. For fresh weight, the microbial inoculum contribution rate is (fresh weight of treated plants of each group-fresh weight of reference plant)/fresh weight of reference plant x 100%.
The results of the microbial inoculum contribution rate are shown in Table 5 (mean of 3 plants).
TABLE 5
Figure BDA0002455590110000121
Figure BDA0002455590110000131
The results show that the contribution rate of needle A2-7 dry bacterial agent c, needle A2-7 dry bacterial agent 2, needle A2-7 dry bacterial agent d, needle A2-7 dry bacterial agent e, needle A2-7 dry bacterial agent f and needle A2-7 dry bacterial agent g to the fresh weight of alfalfa is obviously higher than that of other groups.
Therefore, the effect of the needle A2-7 dry bacterial agent prepared at the drying temperature of more than 45 ℃ or less than 20 ℃ on promoting the growth of plants is not as good as that of the needle A2-7 dry bacterial agent prepared at the drying temperature of between 20 and 45 ℃. The needle A2-7 dry microbial inoculum c, the needle A2-7 dry microbial inoculum 2, the needle A2-7 dry microbial inoculum d, the needle A2-7 dry microbial inoculum e, the needle A2-7 dry microbial inoculum f and the needle A2-7 dry microbial inoculum g can effectively promote the growth of the alfalfa and are convenient to store and transport. Wherein, the needle A2-7 dry bacterial agent c, the needle A2-7 dry bacterial agent 2 and the needle A2-7 dry bacterial agent d can be realized under natural conditions, and can be realized by a device (such as an air blower) if the natural conditions are not satisfied; from the aspect of saving (such as energy sources; the higher the temperature is, the more energy sources are consumed), the needle A2-7 dry microbial inoculum c, the needle A2-7 dry microbial inoculum 2 and the needle A2-7 dry microbial inoculum d are all the microbial inocula which can optimally promote the growth of plants.
<110> Beijing Synbiotic ecological engineering technology Co., Ltd, China university of mining industry (Beijing)
<120> preparation method of DSE dry microbial inoculum capable of promoting plant growth and easy to store and transport
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 589
<212> DNA
<213> Darksidea sp.
<400> 1
cttccgtagg tgaacctgcg gaaggatcat tacctggcct tgggccgctc gcgggagcta 60
gtcgcttgcg acgacgctac cgagggcgct tagcccttga ctatcacctt gactacgtgc 120
accttttgtt gtttcctcgg caggtcacct gccgccagga accccctaaa ccttttgtaa 180
tagcatccaa acttctgaaa acaaaccaaa ttatttacaa cttttaacaa tggatctctt 240
ggttctggca tcgatgaaga acgcagcgaa atgcgataag tagtgtgaat tgcagaattc 300
agtgaatcat cgaatctttg aacgcacatt gcgccccatg gtattccgtg gggcatgcct 360
gttcgagcgt catttacccc ctcaagctcc gcttggtgtt gggcgtctgt cccgcttcgc 420
gcgcggactc gccccaaagg tattggcagc ggtcgtgcca gcttctcgcg cagcacattg 480
cgcttctcga ggcaccggcg ggcccgcgtc catcaagctc accccccagt ttgacctcgg 540
atcaggtagg gatacccgct gaacttaagc atatcaataa gcggaggaa 589
<210> 2
<211> 593
<212> DNA
<213> Pleosporales sp.
<400> 2
cttccgtagg tgaacctgcg gaaggatcat tacctggcct tgggccgctc gggggagcca 60
gtcgctcgcg acggcgctgc cttgggcgct tagcccttga ctatcacctt gactacgtgc 120
accttttgtt gtttcctcgg caggtcctct gccgccagga accccctaaa cctttttgca 180
atagcatcca aacttctgaa aacaaaccaa atcatttaca acttttaaca atggatctct 240
tggttctggc atcgatgaag aacgcagcga aatgcgatat gtagtgtgaa ttgcagaatt 300
cagtgaatca tcgaatcttt gaacgcacat tgcgccccat ggtattccgt ggggcatgcc 360
tgttcgagcg tcatttaccc cctcaagctc cgcttggtgt tgggcgtctg tcccgcttcg 420
cgcgcggact cgccccaaag gtattggcag cggtcatgcc agcttctcgc gcagcacatt 480
gcgcttctcg aggcaccggc gggcccgcgt ccatcaagct ctcacccccc cagtttgacc 540
tcggatcagg tagggatacc cgctgaactt aagcatatca ataagcggag gaa 593
<210> 3
<211> 589
<212> DNA
<213> Darksidea sp.
<400> 3
cttccgtagg tgaacctgcg gaaggatcat tacctggcct tgggccgctc gcgggagcta 60
gtcgcttgcg acgacgctac cgagggcgct tagcccttga ctatcacctt gactacgtgc 120
accttttgtt gtttcctcgg caggtcacct gccgccagga accccctaaa ccttttgtaa 180
tagcatccaa acttctgaaa acaaaccaaa ttatttacaa cttttaacaa tggatctctt 240
ggttctggca tcgatgaaga acgcagcgaa atgcgataag tagtgtgaat tgcagaattc 300
agtgaatcat cgaatctttg aacgcacatt gcgccccatg gtattccgtg gggcatgcct 360
gttcgagcgt catttacccc ctcaagctcc gcttggtgtt gggcgtctgt cccgcttcgc 420
gcgcggactc gccccaaagg tattggcagc ggtcgtgcca gcttctcgcg cagcacattg 480
cgcttctcga ggcaccggcg ggcccgcgtc catcaagctc accccccagt ttgacctcgg 540
atcaggtagg gatacccgct gaacttaagc atatcaataa gcggaggaa 589

Claims (10)

1. A preparation method of a DSE dry microbial inoculum sequentially comprises the following steps:
(1) culturing dark septate endophytic fungi by adopting liquid culture mediumDarksidea sp.Needle A2-7 to obtain bacterial liquid; dark-colored septate endophytic fungiDarksidea sp.The preservation number of the needle A2-7 is CGMCC No. 18811; the culture time is 6 days;
(2) collecting solids in the bacterial liquid;
(3) drying to obtain DSE dry bacterium agent;
the DSE dry bacterium agent can promote plant growth;
the plant is the alfalfa of Athena.
2. The method of claim 1, wherein: in the step (1), the culture conditions are 25-28 ℃ and 150-.
3. The method of claim 1, wherein: in the step (1), the needle A2-7 can be added in the form of a mushroom slice or a mushroom cake; the diameter of the fungus pieces or fungus cakes is 4-6 mm; the ratio of the bacterial tablets or bacterial cakes to the liquid culture medium is 1 bacterial tablet or bacterial cake: (100-200mL) liquid medium.
4. The method of claim 1, wherein: the liquid culture medium is a liquid MMN culture medium.
5. The method of claim 1, wherein: and (3) in the step (2), collecting the solid in the bacterial liquid by filtering or centrifuging.
6. The method of claim 5, wherein: qualitative filter paper was used for the filtration.
7. The method of claim 1, wherein: in the step (3), the drying temperature is 20-45 ℃.
8. The method of claim 1, wherein: in the step (3), the drying is natural air drying or forced air drying.
9. The method of claim 1, wherein: before step (2) and/or before step (3), a carrier may be added.
10. The method of any of claims 1 to 9, wherein: the water content of the DSE dry microbial inoculum is 1-5%.
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