CN111500469B - Method for producing bacterial manure by using DSE root system (section) - Google Patents
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- CN111500469B CN111500469B CN202010304460.9A CN202010304460A CN111500469B CN 111500469 B CN111500469 B CN 111500469B CN 202010304460 A CN202010304460 A CN 202010304460A CN 111500469 B CN111500469 B CN 111500469B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
The invention discloses a method for producing bacterial manure by using DSE root system (section). The invention provides a method for preparing a DSE solid microbial inoculum, which comprises the following steps: plant seeds are infected by using the gelidium Pleospora sp needle A2-8 CGMCC No.18812, and the root system or rhizosphere soil of the infected plant is collected to obtain the solid microbial inoculum. According to the invention, DSE fungi rapidly invade the plant root system, and then the infected plant root segment is used as the solid microbial inoculum, so that various defects of the traditional liquid microbial inoculum in preparation, transportation and application are overcome, and a foundation is provided for the preparation of green biological bacterial fertilizers, granular bacterial fertilizers and the like.
Description
Technical Field
The invention belongs to the technical field of bioengineering, relates to a method for producing bacterial manure by using a DSE root system (segment), and more particularly relates to application of dark-color septate endophytic fungi in producing green microbial manure by invading a plant root system.
Background
Dark Septate Endophytes (DSEs) have biological functions similar to root fungi, DSEs are ecologically widespread, colonization among different habitat plants indicates that they have little or no host specificity and can establish a reciprocal mutual benefit and a mutually regulated physiological whole with plants, but each has its morphological characteristics.
Researches show that the dark-color endophytic fungi plays a more important role in the stress environment, the absorption area of the plant root system is enlarged through the growth of hyphae, and the absorption capacity of nutrient elements outside the absorption range of the original root system is improved.
The conventional microbial agents require strict aseptic environment in the whole production process so as to prevent pollution from damaging the microbial activity, and the activity of the microbial agents is limited in time, and in addition, the microbial agents also need strict protection in the transportation and application processes. Although the DSE can be purely cultured, in actual production practice, the microbial inoculum cannot achieve ideal effects from purification, production, application and the like, and meanwhile, the liquid microbial inoculum is inconvenient to carry and easy to pollute.
Disclosure of Invention
The invention aims to provide a method for preparing a solid microbial inoculum.
The method for preparing the DSE solid microbial inoculum provided by the invention comprises the following steps: plant seeds are infected by using the gelidium Pleospora sp needle A2-8 CGMCC No.18812, and the root system or rhizosphere soil of the infected plant is collected to obtain the solid microbial inoculum.
In the method, the step of infecting the plant seeds by using the gelidium Pleosporus sp. needle A2-8 CGMCC No.18812 is to sow the plant seeds in a culture medium containing 10-40% of the gelidium Pleosporus sp. needle A2-8 bacterial liquid for infection culture;
the 10% -40% of the bacillus gregarius Pleospora sp needle A2-8 bacterial liquid is prepared according to the following method:
1) inoculating the mycelium of the gelidium Pleospora sp needle A2-8 into a liquid culture medium for liquid culture to obtain a strain stock solution;
2) diluting the strain stock solution by 2.5-10 times to obtain 10% -40% of the bacillus gregarius Pleospora sp. needle A2-8 strain solution.
In the above method, the liquid medium is a liquid MMN medium.
In the method, the infection culture is carried out until the infection rate of the needle A2-8 bacteria infecting the plant is more than or equal to 80%.
In the method, the bacterium concentration in the 10-40% gram spore bacterium Pleospora sp. needle A2-8 bacterium liquid is 0.18-0.98 mg/ml;
or the bacterium concentration in the 10-40% of the bacterial liquid of the gelidium Pleosporus sp. needle A2-8 is 0.18-0.25 mg/ml;
or the bacterial concentration in the 10% of the bacterial liquid of the gelidium Pleospora sp needle A2-8 is 0.23 mg/ml.
In the embodiment of the invention, the adopted plant seeds are corn seeds, and in an infection culture system, the mass ratio of the fungus of the needle A2-8 to the corn seeds is 4.6-18.4 mg: 0.8-1 g; preferably 4.6 mg: 0.8g or 4.6 mg: 1g of a compound;
the infection culture conditions are as follows: culturing at 22 deg.C in dark for 1-3 days; and on 4-13 days of culture, alternately culturing under 16h illumination and 8h dark illumination, wherein the illumination intensity is 1800Lux, the temperature during illumination culture is 25 ℃, and the temperature during dark culture is 22 ℃.
The conditions of the liquid culture are as follows: carrying out dark shaking culture at the temperature of 28 ℃ at 180r/min for 1 day, and then carrying out dark shaking culture at 160r/min for 8 days;
in the method, the method also comprises the following steps after the infected plant root system is collected: the root system is prepared into a root section with the length of 0.8-1.5cm and used as a solid microbial inoculum.
In the method, the method also comprises the following steps after the infected plant root system is collected: preparing the root system into a root section with the length of 0.8-1.5cm, and drying the root section to be used as a solid microbial inoculum.
In the examples of the present invention, the drying is air drying to a moisture content of 4% to 8%.
The solid microbial inoculum prepared by the method is also protected by the invention.
The dry root section and rhizosphere soil in the solid microbial inoculum can be stored at low temperature of a 4 ℃ refrigerator for later use.
The application of the solid microbial inoculum in improving the storage and/or transportation efficiency of the bacillus cereus Pleosporales sp. needle A2-8 as bacterial fertilizer is also within the protection range of the invention;
or, the application of the solid microbial inoculum in the preparation of bacterial manure is also the protection scope of the invention;
or, the application of the solid microbial inoculum in promoting the growth of plants is also the protection scope of the invention.
The application of the gelidium Pleospora sp. needle A2-8 or the bacterial liquid thereof or the root system or rhizosphere soil obtained by infecting plants with the gelidium Pleospora sp. needle in promoting the growth of plants is also within the protection scope of the invention; the gram-spore bacteria Pleospora sp. needle A2-8 has a preservation number of CGMCC No. 18812.
The culture solution of the Geospora Pleosporus sp. needle A2-8 is 10% -40% of the culture solution of the Geospora Pleosporus sp. needle A2-8 in the first purpose.
According to the invention, based on the characteristic that DSE fungi can rapidly invade plant root systems and host specificity is absent, on the basis of a DSE liquid microbial inoculum, corn root systems are infected by the DSE fungi, then plants are infected again by using the infected corn root samples and rhizosphere soil as inoculants, and the capability of the DSE of secondary infection is detected, so that the possibility of rapid production of green bacterial manure is provided, namely, the plant root systems are rapidly invaded by the DSE fungi, and then the infected plant root sections are used as the inoculants to produce green organic fresh bacterial manure, so that a foundation is provided for future production of granular bacterial manure.
The invention perfectly solves various defects of the traditional liquid microbial inoculum in preparation, transportation and application, and simultaneously provides a foundation for preparing green biological bacterial manure, granular bacterial manure and the like, and the invention has the following advantages:
firstly, on the premise of fully considering economic benefits, the economic benefit is the highest by taking 10% concentration bacterial liquid as the first stage inoculation, and the root system and root system soil are collected as solid microbial inoculum; secondly, when the solid microbial inoculum is used as an inoculant in the second stage, the infection efficiency is good and higher than that of a liquid microbial inoculum, and especially, the effect is more remarkable along with the lengthening of the storage time; the infection effect of the root system as the solid microbial inoculum is higher than that of rhizosphere soil as the solid microbial inoculum; thirdly, both the fresh sample and the air-dried sample of the root system can be used as solid microbial inoculum, the infection rate has no obvious difference, and the air-dried sample can be selected in consideration of the transportation and storage time; air-drying the root section: the inoculation amount of 1.5-2% can reach an infection rate of more than 50% on the 10 th day, the potential of the air-dried root section as a microbial inoculum is shown, and the air-dried root section has the main advantages of portability, small volume, convenience in carrying and storage and suitability for large-scale field application of long-distance transportation. Therefore, the invention can obviously improve the storage time, storage mode and application limit of the microbial agent, can use the solid microbial agent as a bacterial fertilizer to promote plant growth, can also be used together with other microbial fertilizers and chemical fertilizers, can greatly promote the application of the microbial fertilizer, provides possibility for the large-scale application of the microbial fertilizer in fields such as ecological reconstruction in mining areas, soil remediation, agricultural biological fertility and the like, and particularly provides a basis for the reasonable application and large-scale production of future microbial fertilizers.
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 graph of the colonization of the root system by needle A2-8.
FIG. 2 shows the infection rate of the second inoculation of the strain A2-8 at different times.
FIG. 3 shows the infection rate of bacterium 001 at different times of the second inoculation.
FIG. 4 shows the infection rate of the second inoculation of the strain A1-3 at different times.
FIG. 5 shows the infection ability of the needle A2-8 cultured with the bacterial liquid for different times
FIG. 6 shows the plant heights of the needle A2-8 after being inoculated with solid and liquid inocula for different times
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.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The quantitative tests in the following examples, all set up three replicates and the results averaged.
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 15 min.
Example 1 isolation and identification of DSE strains and preservation
Selecting fresh root samples of needle fescue collected from north electric power victory mining areas of the union of the autonomous region of inner Mongolia, Silingguo, cutting the root segments into small segments of about 1cm, placing the small segments into 75% ethanol aqueous solution for disinfection for 5min, washing the small segments with sterile water for 3 times, placing 10% sodium hypochlorite aqueous solution for disinfection for 5min, washing the small segments with sterile water for 3 times, and sucking water on the surface of the root samples by using filter paper. PDA medium with antibiotics (50 mg/L ampicilin and 50mg/L streptomycin sulfate) was placed in each dish with 2-3 root segments. 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 No. 5 bacterium is detected, and the fungus is identified as dark septate endophytic fungus (DSE) as shown in 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 preparation of needle A2-8 solid inoculum
The test strains are: the DSE strain needle A2-8 is classified and named as Geospora Pleospora sp, and the preservation number is CGMCC No. 18812.
The corn variety is Zhongnuo No.1 (also called Zhongnuo No. 1; authentication No. 0103006-.
Preparation of bacterial liquid
1. Preparation of needle A2-8 bacterial solution
1) Activation of
Activating the strain of the gelidium Pleospora sp.CGMCC No.18812 from a slant test tube strain preserved at low temperature, and transferring the strain to a PDA culture medium; culturing at 28 deg.C in dark for 7 days, and collecting fungus cake when mycelia are covered on the plate;
2) preparation of bacterial liquid
Inoculating the fungus cake with diameter of 6mm prepared in the step 1) into 100mL of liquid MMN culture medium, performing dark shaking culture at 28 deg.C for 1 day at 180r/min, and performing dark shaking culture at 160r/min for 8 days to obtain needle A2-8 bacterial liquid (calculated concentration of 2.31 mg/mL).
The mg/mL of the above bacterial suspension is the dry weight of the bacterial cells per mL of the bacterial suspension.
2. Dilution of bacterial liquid
The bacterial solution of needle A2-8 obtained in step 1 after 8 days of culture was diluted with sterile water to obtain the following 3 concentrations of bacterial solution:
10% (volume percentage content) of bacterial liquid (diluted by 10 times): taking 10mL of the bacterial liquid A2-8 of the step 1, and diluting the bacterial liquid with sterile water to 100mL to obtain 10% bacterial liquid A2-8 (the concentration is 0.23 mg/mL);
20% (volume percentage content) of bacterial liquid (diluted 5 times): taking 20mL of the bacterial liquid A2-8 of the step 1, and diluting the bacterial liquid A2-8 to 100mL by using sterile water to obtain 20% of bacterial liquid A2-8 (the concentration is 0.46 mg/mL);
40% (volume percentage content) of bacterial liquid (diluted 2.5 times): taking 40mL of the bacterial liquid A2-8 of the step 1, and diluting the bacterial liquid with sterile water to 100mL to obtain 40% bacterial liquid A2-8 (the concentration is 0.92 mg/mL).
The preparation methods of the bacterial liquid A1-3 and 001 of No. 2 and 5A 2-8 are the same, and the difference is only the strain, namely 10% of bacterial liquid A1-3, 20% of bacterial liquid A1-3, 40% of bacterial liquid A1-3, 10% of bacterial liquid 001, 20% of bacterial liquid 001 and 40% of bacterial liquid 001 are obtained respectively.
Preparation of solid microbial inoculum of second, third needle A2-8
The needle A2-8 solid DSE microbial inoculum is obtained through a first-stage infection test of a bacterial solution.
The method comprises the following specific steps:
1. sterilization of materials
Sand and soil: sieving with 2mm sieve, removing root residue, sterilizing at 120 deg.C for 90min under high temperature and high pressure to obtain sterilized soil sample;
selecting corn seeds with similar size and full grains, soaking the corn seeds in 30% sodium hypochlorite aqueous solution for 10min, washing the corn seeds with sterile water for 5 times, then soaking the corn seeds in 70% ethanol solution for 5min, and washing the corn seeds with sterile water for 5 times to obtain sterilized corn seeds for later use.
2. Corn infected by bacterial liquid
Taking 125mL of small cups, putting the 225g of sterilized soil sample into each small cup, and setting 3 culture system groups for each strain, wherein each group is 9 small cups as follows:
first group (10% bacterial suspension group): 20mL of the prepared 10% bacterial liquid is poured into each small cup;
second group (20% bacterial suspension group): pouring 20mL of the bacterial liquid with the concentration of 20% prepared in the first step into each small cup;
third group (40% bacterial suspension group): pouring 20mL of the bacterial liquid into each small cup, wherein the bacterial liquid is 40% of the bacterial liquid prepared in the previous step;
the sterilized corn seeds were sown into small cups (3 seeds were sown in each plastic cup, the weight of the seeds was 0.8g) with tweezers, and then cultured.
The 10% bacterial liquid is respectively 10% bacterial liquid needle A2-8, 10% bacterial liquid needle A1-3 and 10% bacterial liquid 001;
the 20% bacterial liquid is respectively 20% bacterial liquid needle A2-8, 20% bacterial liquid needle A1-3 and 20% bacterial liquid 001;
the 40% bacterial liquid is respectively 30% bacterial liquid needle A2-8, 30% bacterial liquid needle A1-3 and 40% bacterial liquid 001;
in the first group of culture systems, the mass ratio of the needle A2-8 bacteria to the seeds is 4.6 mg: 0.8 g;
in the second group of culture systems, the mass ratio of the needle A2-8 bacteria to the seeds is 9.2 mg: 0.8 g;
in the third group of culture system, the mass ratio of the needle A2-8 bacteria to the seeds is 18.4 mg: 0.8 g;
the culture process comprises the following steps: day 1-3 (the day when seeds are put is taken as the first day), culturing at 22 deg.C in dark, and air relative humidity is 70%; on day 4-13, culturing alternately in dark and light (16h light/8 h dark, light intensity is 1800Lux during light culture, temperature is 25 ℃ during light culture, temperature is 22 ℃ during dark culture), and relative air humidity is 70%.
And (3) detecting the colonization condition of various test strains in each group of culture systems at the root of the plant on the 13 th day of culture, wherein the specific method comprises the following steps:
from each group of culture systems, 90 small root segments of 1cm were selected (randomly selected), stained with acid magenta staining, the number of roots colonized by DSE was microscopically detected, and the colonization rate of A2-8 strain (also referred to as infestation rate; colonization rate: number of roots colonized by DSE/total number of roots) was calculated.
As shown in Table 1, it was found that the colonization rates of the A2-8 bacteria solutions inoculated at the respective concentrations were 80% or more compared with the 001 bacteria solution and the A1-3 bacteria solution after the culture on day 13; from the economical point of view, 10% A2-8 bacterial liquid can be selected to prepare the solid microbial inoculum.
TABLE 1 colonization rate of DSE bacteria liquid with different concentrations
3. Obtaining of solid microbial inoculum
Needle a2-8 solid inoculum (root system): collecting the roots of the infected plants A2-8 in each group of culture systems cultured for 12 days (collected on the 13 th day of culture) in the step 2, cutting the roots into small root sections of about 1cm, and cleaning the small root sections to obtain a solid microbial inoculum (fresh root sample) prepared by 10% of the bacteria liquid needle A2-8, a solid microbial inoculum (fresh root sample) prepared by 20% of the bacteria liquid needle A2-8 and a solid microbial inoculum (fresh root sample) prepared by 40% of the bacteria liquid needle A2-8.
Needle a2-8 solid bacterial agent (root system dried sample): and (3) air-drying the solid microbial inoculum (fresh root sample) prepared by the 10% bacterial liquid needle A2-8, the solid microbial inoculum (fresh root sample) prepared by the 20% bacterial liquid needle A2-8 and the solid microbial inoculum (fresh root sample) prepared by the 40% bacterial liquid needle A2-8 for 5 days at a ventilated and cool place at room temperature to ensure that the water content of the solid microbial inoculum (dry root sample) is 4% -8%, so as to obtain the solid microbial inoculum (dry root sample) prepared by the 10% bacterial liquid needle A2-8, the solid microbial inoculum (dry root sample) prepared by the 20% bacterial liquid needle A2-8 and the solid microbial inoculum (dry root sample) prepared by the 40% bacterial liquid needle A2-8. Storing in a refrigerator at 4 deg.C.
Needle a2-8 solid inoculum (rhizosphere soil): collecting root system soil after infection of A2-8 in each group of culture systems cultured for 12 days (collected on the 13 th day of culture) in the step 2, sieving the root system soil by a 1mm sieve, completely removing the plant root system, and fully mixing all the root system soil to obtain a solid microbial inoculum (rhizosphere soil) prepared by 10% of bacteria liquid needle A2-8, a solid microbial inoculum (rhizosphere soil) prepared by 20% of bacteria liquid needle A2-8 and a solid microbial inoculum (rhizosphere soil) prepared by 40% of bacteria liquid needle A2-8. Storing in a refrigerator at 4 deg.C.
Various solid microbial inocula of No. 001 and needle A2-3 were prepared according to the preparation method of the solid microbial inoculum of needle A2-8, and the difference was only bacterial strains.
The solid microbial inoculum of the dry sample of the root system and the solid microbial inoculum of the rhizosphere soil are stored in a refrigerator at 4 ℃.
Thirdly, the infection capacity of different bacterial strains of the comparison needle A2-8 solid microbial inoculum
And (3) carrying out the following second-stage infection test on various prepared solid microbial agents to verify the infection capacity of the microbial agents:
1. material preparation
Sand and soil: sieving with 2mm sieve, removing root residue, sterilizing at 120 deg.C for 90min under high temperature and high pressure to obtain sterilized soil sample;
corn seeds: selecting corn seeds with similar size and full grains, soaking the corn seeds in 30% sodium hypochlorite aqueous solution for 10min, washing the corn seeds with sterile water for 5 times, then soaking the corn seeds in 70% ethanol solution for 5min, and washing the corn seeds with sterile water for 5 times to obtain sterilized corn seeds for later use.
2. Infection in the second stage
Taking 125mL small cups, putting the 225g sterilized soil sample into each small cup, and setting 18 culture system groups for each strain, wherein each group comprises 6 small cups;
first to sixth groups: infecting plant seeds with the root system fresh-sample solid microbial inoculum and the rhizosphere soil solid microbial inoculum obtained in the second step, and recording as inoculation on day 1;
seventh to twelfth groups: infecting plant seeds with the root system dry sample solid microbial inoculum stored for 11 days and the rhizosphere soil solid microbial inoculum stored for 11 days obtained in the second step, and recording as inoculation on day 11;
thirteenth to eighteenth groups: infecting plant seeds with the root system dry sample solid microbial inoculum stored for 21 days and the rhizosphere soil solid microbial inoculum stored for 21 days, and recording as inoculation on day 21;
the method comprises the following specific steps:
a first group: weighing 4g of solid microbial inoculum (root system fresh sample) prepared by 10% bacteria liquid needle A2-8;
second group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 10 percent of bacteria liquid needle A2-8 is weighed;
third group: weighing 4g of solid microbial inoculum (root system fresh sample) prepared by 20% of fungal liquid needle A2-8;
and a fourth group: weighing 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 20% of fungal liquid needle A2-8;
and a fifth group: 4g of solid microbial inoculum (root system fresh sample) prepared by 40% bacteria liquid needle A2-8;
a sixth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 40% bacteria liquid needle A2-8 is weighed;
a seventh group: 3g of solid microbial inoculum (root system dry sample) prepared by 10 percent of bacteria liquid needle A2-8 and stored for 11 days is weighed;
and an eighth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 10 percent of bacteria liquid needle A2-8 and stored for 11 days is weighed;
ninth group: 3g of solid microbial inoculum (root system dry sample) prepared by 20 percent of fungal liquid needle A2-8 which is preserved for 11 days is weighed;
the tenth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 20 percent of fungal liquid needle A2-8 which is preserved for 11 days is weighed;
eleventh group: 3g of solid microbial inoculum (root system dry sample) prepared by 40% bacteria liquid needle A2-8 and stored for 11 days;
a twelfth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 40% bacteria liquid needle A2-8 and stored for 11 days is weighed;
group thirteen: 3g of solid microbial inoculum (root system dry sample) prepared by 10 percent of fungal liquid needle A2-8 and stored for 21 days;
a fourteenth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 10 percent of fungal liquid needle A2-8 and stored for 21 days is weighed;
a fifteenth group: 3g of solid microbial inoculum (root system dry sample) prepared by 20 percent of fungal liquid needle A2-8 which is preserved for 21 days;
sixteenth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 20 percent of fungal liquid needle A2-8 and stored for 21 days is weighed;
seventeenth group: 3g of solid microbial inoculum (root system dry sample) prepared by a 40% bacteria liquid needle A2-8 and stored for 21 days;
eighteenth group: 0.5g of solid microbial inoculum (rhizosphere soil) prepared by 40% bacteria liquid needle A2-8 and stored for 21 days is weighed;
the corn seeds were sown into small cups (3 seeds were sown per small cup, the weight of the seeds was 1.0g) with tweezers (70% alcohol sterilization), and then secondary infection culture was performed.
The secondary infection culture process comprises the following steps: culturing in dark at 22 deg.C for 1-3 days with air relative humidity of 70%; on day 4-13, culturing alternately in dark and light (16h light/8 h dark, light intensity is 1800Lux during light culture, temperature is 25 ℃ during light culture, temperature is 22 ℃ during dark culture), and relative air humidity is 70%.
And (5) detecting the corn root system infection condition every 3 days from the 7 th day of the secondary infection culture.
The results are shown in FIG. 2, where A represents the treatment of the inoculation on day 1, B represents the treatment of the inoculation on day 11, and C represents the treatment of the inoculation on day 21; it can be seen that the root system solid microbial inoculum and the rhizosphere soil solid microbial inoculum prepared in the second step can both infect roots, and the infection effect of the root system of the solid microbial inoculum is higher than that of the rhizosphere soil of the solid microbial inoculum. In addition, the air-dried sample and the fresh root sample have the same infection capacity, and the infection capacity is similar.
The same method is adopted to detect the secondary infection rate of different solid-state inocula of the 001 strain and the A1-3 strain, and the method is consistent with the method except that the strains are different.
As shown in fig. 3 and 4, it can be seen that the infection effect of the different solid-state inocula of the 001 strain and the different solid-state inoculas of the needle a1-3 strain is smaller than that of the different solid-state inoculas of the needle a2-8 strain; particularly, on the 13 th day of secondary infection culture, the infection rate of different solid bacteria of the needle A2-8 is obviously higher than that of the solid bacteria of the 001 # bacteria and that of the needle A1-3 # bacteria, and after the bacteria are stored for 21 days, the infection rate of the solid bacteria of the needle A2-8 # bacteria (root system dry sample) is obviously higher than that of the solid bacteria of the 001 # bacteria and that of the needle A1-3 # bacteria.
The results show that the solid microbial inoculum prepared by the needle A2-8 has long storage time and the most obvious infection effect.
Infection capacity of liquid microbial inoculum of needle A2-8 with different preservation times
1. Preparation of needle A2-8 liquid microbial inoculum
The 8-day-old needle culture solution A2-8 obtained in the above step 1 was diluted by the method of 2 to obtain 10% of needle A2-8, 20% of needle A2-8 and 40% of needle A2-8 as needle A2-8 liquid preparations.
Continuously carrying out dark shaking culture on the liquid microbial inoculum of the needle A2-8 at 28 ℃ and 160r/min, and collecting culture solution at different time after continuous dark shaking culture as the preserved liquid microbial inoculum for carrying out the following infection experiments.
2. Infection by infection
1) Material preparation
The same as 1 of the second;
2) corn infected by bacterial liquid
Taking 125mL of small cups, putting the 225g of sterilized soil sample into each small cup, and setting 3 culture system groups for each strain, wherein each group comprises 6 small cups as follows:
a first group: each cup was filled with 20mL of a 10% liquid inoculum prepared from needle A2-8 and cultured for 8 days (the first day to inoculate mycelia, referred to as the first day of storage);
second group: pouring 20% liquid microbial inoculum prepared from 20mL needle A2-8 cultured for 8 days into each cup;
third group: pouring 40% liquid microbial inoculum prepared from 20mL needle A2-8 cultured for 8 days into each cup;
and a fourth group: 10% liquid microbial inoculum prepared by 20mL needle A2-8 cultured for 18 days (equivalent to 10 days of liquid microbial inoculum preserved) is poured into each small cup;
and a fifth group: 20 percent of liquid microbial inoculum prepared by 20mL of needle A2-8 for culturing for 18 days is poured into each small cup;
a sixth group: 20 percent of liquid microbial inoculum prepared by 20mL of needle A2-8 for culturing for 18 days is poured into each small cup;
a seventh group: pouring 10% liquid microbial inoculum prepared by 20mL needle A2-8 cultured for 28 days (equivalent to preserving the liquid microbial inoculum for 20 days) into each small cup;
and an eighth group: pouring 20% liquid microbial inoculum prepared from 20mL needle A2-8 for culturing for 28 days into each cup;
ninth group: pouring 20% liquid microbial inoculum prepared from 20mL needle A2-8 for culturing for 28 days into each cup;
the sterilized corn seeds were sown into small cups (3 seeds were sown per small cup, the weight of the seeds was 0.8g) with tweezers, and then cultured.
In the first group of culture systems, the mass ratio of the needle A2-8 bacteria to the seeds is 4.6 mg: 0.8 g;
in the second group of culture systems, the mass ratio of the needle A2-8 bacteria to the seeds is 9.2 mg: 0.8 g;
in the third group of culture system, the mass ratio of the needle A2-8 bacteria to the seeds is 18.4 mg: 0.8 g;
the culture process comprises the following steps: day 1-3 (the day when seeds are put is taken as the first day), culturing at 22 deg.C in dark, and air relative humidity is 70%; on day 4-13, culturing alternately in dark and light (16h light/8 h dark, light intensity is 1800Lux during light culture, temperature is 25 ℃ during light culture, temperature is 22 ℃ during dark culture), and relative air humidity is 70%.
And (3) detecting the colonization condition of various test strains in each group of culture systems at the root of the plant on the 13 th day of culture, wherein the specific method comprises the following steps:
from each group of culture systems, 90 small root segments of 1cm were selected (randomly selected), stained with acid magenta staining, the number of roots colonized by DSE was microscopically detected, and the colonization rate of A2-8 strain (also referred to as infestation rate; colonization rate: number of roots colonized by DSE/total number of roots) was calculated.
The needle A2-8 is cultured for 8, 18 and 28 days (the proportion is similar to the increase of the preservation time of the solid microbial inoculum, and the statistics is carried out once by increasing 10 days), and the infection capacity of the liquid microbial inoculum is respectively detected.
As shown in FIG. 5, A represents the inoculation treatment on the 8 th day of the culture of the liquid microbial inoculum, B represents the 18 th day of the culture of the liquid microbial inoculum, and C represents the 28 th day of the culture of the liquid microbial inoculum; the result shows that the infection capacity of the liquid microbial inoculum is greatly reduced along with the prolonging of the storage time of the liquid microbial inoculum, the activity of the DSE is obviously reduced, and the storage advantage of the solid microbial inoculum is shown from the side.
Example 3 application of solid microbial inoculum of needle A2-8 as bacterial fertilizer
Detecting the height of a 13 th-day corn strain cultured after the liquid microbial inoculum of the needle A2-8 in the third step of the embodiment 2 is treated;
detecting the height of a corn plant cultured on day 13 after the liquid microbial inoculum of needle A2-8 in the fourth needle of example 2 is treated;
as shown in FIG. 6, A in FIG. 6 indicates the height of the corn plants inoculated with the solid microbial inoculum (fresh root system) prepared by needle A2-8 and the solid microbial inoculum (rhizosphere soil) prepared by needle A2-8, B indicates the height of the corn plants inoculated with the solid microbial inoculum (dry root system) prepared by needle A2-8 and the solid microbial inoculum (rhizosphere soil) prepared by needle A2-8, which are preserved for 11 days, C indicates the height of the corn plants inoculated with the solid microbial inoculum (dry root system) prepared by needle A2-8 and the solid microbial inoculum (rhizosphere soil) prepared by needle A2-8, which are preserved for 21 days, D indicates the height of the corn plants inoculated after 8 days of culture of the liquid microbial inoculum, E indicates the height of the corn plants inoculated after 18 days of culture of the liquid microbial inoculum, and F indicates the height of the corn plants inoculated after 28 days of culture of the liquid microbial inoculum. The result shows that the plant strain treated by the solid microbial inoculum is higher than that treated by the liquid microbial inoculum, and the promotion effect of the root system in the solid microbial inoculum on the plant height is more obvious along with the increase of the standing time.
SEQUENCE LISTING
<110> university of mineral industry (Beijing) Xian university of science and technology
<120> method for producing bacterial manure by using DSE root system (section)
<160> 3
<170> PatentIn version 3.5
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cccttccgta gggtgaacct gcggagggat cattacacaa tatgaaagcg ggctggatac 60
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Claims (10)
1. A method for preparing a solid microbial inoculum comprises the following steps: adopting the gelidium Pleospora sp needle A2-8 CGMCC No.18812 to infect corn seeds, and collecting the root system of the infected corn to obtain the solid microbial inoculum.
2. The method of claim 1, wherein:
the method for infecting corn seeds by using the gelidium Pleosporus sp-needle A2-8 CGMCC No.18812 is characterized in that the corn seeds are sowed in a culture medium containing 10-40% of the gelidium Pleosporus sp-needle A2-8 bacterial liquid for infection culture;
the 10% -40% of the bacillus gregarius Pleospora sp needle A2-8 bacterial liquid is prepared according to the following method:
1) inoculating the mycelium of the gelidium Pleospora sp needle A2-8 into a liquid culture medium for liquid culture to obtain a strain stock solution;
2) diluting the strain stock solution by 2.5-10 times to obtain 10% -40% of the bacillus gregarius Pleospora sp. needle A2-8 strain solution.
3. The method of claim 2, wherein: the liquid culture medium is a liquid MMN culture medium.
4. A method according to claim 2 or 3, characterized in that: the infection culture is carried out until the infection rate of the needle A2-8 bacteria infecting the corn is more than or equal to 80%.
5. The method according to any one of claims 2-4, wherein: the bacterium concentration of the 10-40% of the bacterial liquid of the gelidium Pleospora sp needle A2-8 is 0.18-0.98 mg/ml;
or the bacterium concentration in the 10-40% of the bacterial liquid of the gelidium Pleosporus sp. needle A2-8 is 0.18-0.25 mg/ml;
or the bacterial concentration in the 10% of the bacterial liquid of the gelidium Pleospora sp needle A2-8 is 0.23 mg/ml.
6. The method according to any one of claims 1-5, wherein:
the method also comprises the following steps after the infected corn root system is collected: the root system is prepared into a root section with the length of 0.8-1.5cm and used as a solid microbial inoculum.
7. The method according to any one of claims 1-6, wherein:
the method also comprises the following steps after the infected corn root system is collected: preparing the root system into a root section with the length of 0.8-1.5cm, and drying the root section to be used as a solid microbial inoculum.
8. A solid microbial preparation produced by the method according to any one of claims 1 to 7.
9. Use of the solid microbial inoculum of claim 8 for increasing the efficiency of storage and/or transport of said strain of gelidium Pleosporales sp. needle a2-8 as a bacterial fertilizer;
or, the application of the solid microbial inoculum of claim 8 in preparing bacterial manure;
or, the use of the solid microbial inoculum according to claim 8 for promoting the growth of corn plants.
10. The application of a solid microbial inoculum obtained by infecting corn roots with a gram spore bacterium Pleosporales sp needle A2-8 in promoting the growth of corn plants; the gram-spore bacteria Pleospora sp. needle A2-8 has a preservation number of CGMCC No. 18812.
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