CN111454849A - Separation method of soil microorganism bacterium for inhibiting wheat ear rot pathogenic bacteria - Google Patents
Separation method of soil microorganism bacterium for inhibiting wheat ear rot pathogenic bacteria Download PDFInfo
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Abstract
The invention discloses a method for separating soil microorganism bacteria for inhibiting wheat ear rot pathogenic bacteria, which is prepared and diluted to 10 ℃ in advance‑2Multiple, 10‑3Multiple, 10‑4The method comprises the steps of multiplying a soil extracting solution, culturing wheat ear rot pathogenic bacteria by using barley grains, and then effectively screening by using the inhibition effects of the barley grains dyed with the wheat ear rot pathogenic bacteria and microorganisms in the soil extracting solution, wherein the number of the ear rot pathogenic bacteria on the barley grains is very large, the number of the microorganisms in the soil extracting solution diluted by spraying is very small, and the number of the microorganisms in the soil extracting solution is very different, so that the microorganisms capable of living in the gaps of the ear rot pathogenic bacteria are colonies with strong fertility and strong antibacterial effect, invalid microorganisms are effectively eliminated, the purpose is clear, the workload of blindly separating the soil microorganisms can be greatly reduced, the labor and the cost are saved, and the effective microorganisms are not easy to leak, so that the soil extracting solution for inhibiting the wheat ear rot pathogenic bacteria is improvedThe separation efficiency of the soil microorganism bacteria is high, and the practicability is strong.
Description
Technical Field
The invention relates to the technical field of microbiology, in particular to a method for separating soil microorganisms for inhibiting wheat ear rot pathogens.
Background
Common ear rot pathogens harmful to gramineae plants such as wheat, barley, corn, rice and the like include gibberella zeae, asexual stage fusarium graminearum, rhizoctonia solani, corynebacterium tritici and the like. The pathogenic bacteria live through winter or summer in disease residues such as rice stakes or seeds by using mycelia, ascospores are emitted from ascospores formed in the next growing season, primary infection is carried out by air current transmission, and the ascospores are re-infected by rainwater transmission. High temperature, rainy, foggy and humid weather are favorable for the occurrence of the disease.
The method for preventing and treating wheat ear rot pathogenic bacteria comprises physical prevention and treatment, chemical bactericide prevention and treatment, disease-resistant wheat variety breeding and biological prevention and treatment. Wherein the biological control is a new method for effectively controlling crop diseases by utilizing beneficial microorganisms and microbial metabolites. For example, although gibberellic disease bacteria can multiply and grow in soil, spores or sporangia of gibberellic disease bacteria are rarely observed on the surface of soil because the soil contains a large amount of antagonistic bacteria that inhibit the mass propagation of fusarium head blight. Thus, the microorganisms used for biological control include antagonistic bacteria selected from the soil, including bacteria, fungi, a small amount of actinomycetes, and the like.
How to separate antagonistic bacteria capable of inhibiting the growth of wheat ear rot pathogenic bacteria from soil is an important link of biological control. The method for separating antagonistic bacteria from soil generally comprises the steps of separating all floras in the soil, and then screening beneficial bacteria with antagonistic action by using an oxford cup method as an inhibition effect experiment. However, the soil is the major nutrient for the life of microorganisms, and as much as 10 percent exists in 1g of soil6～109If soil microorganism separation is carried out firstly and then an antibacterial test is carried out on each microorganism individual, the large soil separation workload is easily caused, and effective microorganisms with inhibiting effects are easily leaked.
Disclosure of Invention
The invention aims to provide a method for separating soil microorganisms for inhibiting wheat head rot pathogenic bacteria.
The technical scheme for realizing the purpose of the invention is as follows: a method for separating soil microorganism bacteria for inhibiting wheat ear rot pathogenic bacteria comprises the following steps:
1) preparing a soil extracting solution: collecting a soil sample with the depth of 5-20 cm below the surface layer, air-drying the soil sample collected from the field, putting the soil sample into a plastic sprayer filled with sterilized water, and respectively diluting the soil sample solution after full oscillation to 10 DEG-2Multiple, 10-3Multiple, 10-4Doubling, and transferring into a spray bottle respectively;
2) culturing wheat head rot pathogenic bacteria by barley grains: placing the steamed or boiled barley grains into a culture bottle, sterilizing at high temperature, cooling to room temperature, inoculating wheat ear rot pathogenic bacteria into the bottle filled with the barley grains, and culturing at 25-26 ℃ for several days to ensure that the barley grains fully grow with the wheat ear rot pathogenic bacteria;
3) inoculating barley grains carrying wheat head rot pathogenic bacteria on a plate culture medium: respectively inoculating a plurality of barley grains carrying wheat ear rot pathogenic bacteria in a plurality of plate culture media, and uniformly distributing the inoculated barley grains carrying the wheat ear rot pathogenic bacteria on each plate culture medium;
4) preliminary screening of antagonistic microbe strains:
4.1) uniformly spraying the soil extracting solutions with three concentrations prepared in the step 1) on the surfaces of the flat culture mediums inoculated with the barley grains prepared in the step 3) one by one; after spraying, sealing the edge of the plate culture medium inoculated with barley grains carrying wheat ear rot pathogenic bacteria by using a sealing film, and observing the expansion condition of the wheat ear rot pathogenic bacteria in the plate culture medium after culturing for a plurality of days at 25-26 ℃:
4.2) picking out bacterial colonies capable of inhibiting the growth of the wheat ear rot pathogenic bacteria in the plate culture medium cultured in the step 4.1), respectively coating the bacterial colonies in a new plate culture medium, simultaneously hooking part of the wheat ear rot pathogenic bacteria hypha in a shape like Chinese character 'ji', coating the hypha on the culture medium of each primary sieve, then continuously sealing the edge of the plate culture medium by using a sealing film, and culturing for a plurality of days at the temperature of 25-26 ℃; observing the bacteriostasis condition of the microorganisms in the plate culture medium, and screening out bacterial colonies capable of obviously inhibiting the growth of wheat ear rot pathogenic bacteria in the plate culture medium as bacterial colonies with good inhibition effect on the selected wheat ear rot pathogenic bacteria.
Further, the method also comprises the step 5) of secondary screening of the microbe bacteria with antagonism:
5.1) selecting the bacterial colony which is screened in the step 4.2) and has good inhibition effect on the selected wheat head rot pathogenic bacteria, repeating the step 4.2), and purifying and selecting a single bacterial colony; compared with the bacterial colony screened in the step 4.2), the single bacterial colony has the advantages of stability and difficult variation;
5.2) respectively inoculating the single colonies purified in the step 5.1) into a barley grain culture bottle with sterilized treatment, simultaneously putting at least two barley grains inoculated with wheat head rot pathogenic bacteria cultured in the step 2) into the barley grain culture bottle, sealing the barley grain culture bottle, culturing for a plurality of days at 25-26 ℃, shaking the glass bottle every day during the culture period, and finally standing for 1-2 d to observe whether hyphae are inhibited; the step is used for verifying whether the separated beneficial microorganisms can effectively inhibit the growth of wheat ear rot pathogenic bacteria when the beneficial microorganisms and the harmful wheat ear rot pathogenic bacteria simultaneously infect barley grain organisms;
5.3) observing the bacteriostasis effect of the microbial bacteria with bacteriostasis in the step 5.2), and selecting the microbial bacteria in the corresponding culture bottle without obvious expansion of the wheat head rot pathogenic bacteria as target antagonistic bacteria.
Further, it also includes step 6): re-screening the microbe bacteria with antagonism obtained in the step 5.3):
6.1) inoculating the microbial bacteria selected in the step 5.3) into a sterilized barley grain culture bottle for culture to obtain barley grains carrying microbial bacteria with an antibacterial effect;
6.2) respectively placing one barley grain carrying wheat head rot pathogenic bacteria at the centers of a plurality of new plate culture dishes, then evenly placing two to four barley grains carrying microbial bacteria with the antibacterial effect in the step 6.1) at the periphery of the barley grain carrying the wheat head rot pathogenic bacteria in each plate culture dish, observing the antibacterial effect, and selecting the microbial bacteria in the corresponding culture dish as target antagonistic bacteria, wherein the microbial bacteria do not obviously expand and have good antagonistic action on the wheat head rot pathogenic bacteria. The screening method in the step 6) is convenient to operate and not easy to cause pollution, and forms double verification with the secondary screening of the microbial bacteria with antagonistic action in the step 5), so that the screening accuracy is improved.
Further, when four barley grains of the microorganism having the bacteriostatic effect cultured in the step 6.1) are placed on the plate culture dish in the step 6.2), the placing method comprises the following steps: the method comprises the steps of manufacturing a plurality of plastic circular cross-shaped scale plates with white background and black characters, wherein the diameter of each cross-shaped scale plate is equal to the outer diameter of a plate culture dish, aligning one cross-shaped scale plate under a new plate culture dish before inoculation, the plate culture dish is of a transparent structure, a plate culture medium is semitransparent, putting barley grains carrying wheat ear rot pathogenic bacteria in the center of the cross-shaped scale plate, and respectively putting the barley grains carrying the microbial bacteria with the bacteriostatic effect cultured in the four steps 6.1) on a cross image mapped on the plate culture medium by the cross-shaped scale plate.
Furthermore, the wheat head rot pathogenic bacteria are any harmful bacteria capable of being propagated on barley grains from fusarium head blight, botrytis cinerea, smut bacteria, penicillium and aspergillus flavus.
Further, the scheme of the step 5.2) is repeated on the single colony with the bacteriostatic effect obtained in the step 5.1) for two to three times of screening to screen out the microbial bacteria with better bacteriostatic effect and reproductive effect.
Further, the microbial bacteria with the bacteriostatic effect obtained in the step 6.1) are screened for two to three times by repeating the scheme of the step 6.2) to screen out the microbial bacteria with better bacteriostatic effect and reproductive effect.
Further, the soil in the step 1) is soil for continuously planting barley or wheat. For example, fusarium head blight, which has the characteristics of saprophytic and parasitic, can survive and propagate in soil, so that a microorganism bacterium for restraining fusarium head blight can be easily separated from nature if a soil sample is collected from a head blight identification nursery;
further, the high-temperature sterilization process in the step 2) comprises the following steps: bottling the steamed or boiled barley grains, and sterilizing at 120-125 deg.C for 20-25 min.
Further, when barley grains are inoculated in the step 3) and the step 6.2), the forceps heads of the sterile forceps are heated, small holes are formed in the positions, to be inoculated, of the flat culture medium through the heated forceps heads, and then the barley grains carrying the wheat ear rot pathogenic bacteria or the microbial bacteria are placed in the small holes to prevent the barley grains from moving due to the movement of the flat culture medium.
The method comprises the following key steps of preparing a soil extracting solution in advance, culturing wheat ear rot pathogenic bacteria by using barley grains, and effectively screening by using the inhibition effects of the wheat ear rot pathogenic bacteria and soil microorganisms.
Drawings
FIG. 1 is a graph showing the effect of soil microorganisms in the plate medium of step 4.1) on the inhibition of the growth of barley grain hyphae carrying Fusarium graminearum;
FIG. 2 is a graph showing the inhibition of microorganisms in the plate medium in step 5.1);
FIG. 3 is a diagram showing the growth of Fusarium head blight hyphae in two culture flasks in step 5.3);
FIG. 4 is a diagram showing the distribution structure of barley grains on each culture dish with the cross scale plate in step 6.2);
FIG. 5 is a graph showing the antagonistic effect of the microorganisms with good bacteriostatic effect in step 6.2) in the culture dish.
Detailed Description
The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
A method for separating soil microorganism bacteria for inhibiting wheat ear rot pathogen-gibberellic disease comprises the following steps:
1) preparing a soil extracting solution: the soil sample is from a soil sample with the depth of 5-20 cm below the surface layer of the soil of a wheat scab resistance identification test nursery garden of a test base of an agricultural institute of Nanping City, a Jianyang district, Fujian province, a Nanping city, a Yangxi city, a farm city, a field, after being air-dried, 3g of the dried soil sample is weighed and put into a plastic sprayer (the sprayer is sterilized by alcohol and then is cleaned by sterilizing water), an oscillator is adopted to oscillate for 30 min-1 h to shake out microorganisms in the soil, and then soil-2,10-3,10-4Doubling, and transferring into plastic spraying bottles respectively;
2) and (3) culturing fusarium head blight by barley grains: filling the steamed or boiled barley grains into a culture bottle, sterilizing at high temperature, cooling to room temperature, inoculating fusarium head blight into the bottle filled with the barley grains, and culturing at 25-26 ℃ for 5-6 days to ensure that the barley grains are fully full of fusarium head blight;
3) inoculating barley grains carrying fusarium head blight with a plate culture medium: respectively inoculating 9 barley grains carrying fusarium head blight on a plurality of plate culture mediums, and uniformly distributing the inoculated barley grains carrying fusarium head blight on each plate culture medium;
4) preliminary screening of antagonistic microbe strains:
4.1) uniformly spraying the soil extracting solutions with three concentrations prepared in the step 1) on the surfaces of different flat culture mediums inoculated with barley grains prepared in the step 3) one by one, and setting a sterile water blank control; after the cloth is sprayed, sealing the edge of a plate culture medium inoculated with barley grains carrying fusarium head blight by using a sealing film, culturing for 4-5 days at 25-26 ℃, and observing the expansion condition of fusarium head blight hyphae in the plate culture medium; as shown in FIG. 1, the hyphae of Fusarium head blight have a strong aerial growth ability, and when the surface of the plate medium is inhibited, a large amount of aerial hyphae are formed and cover the lid of the plate medium, so that the hyphae expansion can be observed from the back of the culture dish.
4.2) picking out bacterial colonies with the inhibiting effect on the wheat scab germs in the step 4.1), respectively coating the bacterial colonies in plate culture media for primary screening, simultaneously hooking part of the character-shaped patterns of the fusarium head blight hyphae on the plate culture media for each primary screening, independently hooking the character-shaped patterns of the fusarium head blight hyphae on the plate culture media to serve as blank controls, sealing the edge of the plate culture media by using a sealing film, and culturing for 7-8 days at the temperature of 25-26 ℃; observing the bacteriostasis condition of microorganisms in the plate culture medium, and screening bacterial colonies capable of obviously inhibiting the growth of the wheat scab in the plate culture medium as bacterial colonies with good inhibiting effect on the selected wheat scab;
5) secondary screening of microbe bacteria with antagonism:
5.1) selecting the bacterial colony which is screened in the step 4.2) and has good inhibition effect on the selected wheat scab germ, and repeating the step 4.2) to purify a single bacterial colony with good inhibition effect; as shown in FIG. 2, the culture dish with poor bacteriostatic effect shows that red (dark part in the figure) fusarium head blight grows out of the whole culture dish; few or no fusarium head blight hyphae grow in the culture dish with good bacteriostasis effect;
5.2) respectively inoculating the single bacterial colonies purified in the step 5.1) into barley grain culture bottles with sterilized treatment, simultaneously putting at least two barley grains inoculated with fusarium head blight and cultured in the step 2) into the barley grain culture bottles, wherein the distance between the barley grains in the barley grain culture bottles and the bottle openings is 2cm for the upward growth of hyphae, sealing the barley grain culture bottles, culturing at 25-26 ℃ for 5-7 d, shaking the glass bottles every day for 3-5 days in the culture period, and finally standing for 2d to observe whether the hyphae are inhibited; meanwhile, equivalently selecting the barley grains of the fusarium head blight cultured in the step 2) into another culture bottle filled with the sterilized barley grains to serve as a blank control, sealing the blank control, and culturing at 25-26 ℃ for 5-7 d; the step is used for verifying whether the separated beneficial microorganism strain can effectively inhibit the growth of fusarium head blight hyphae when the beneficial microorganism strain and the harmful fusarium head blight simultaneously infect organisms;
5.3) observing the bacteriostatic effect of the microbial bacteria with bacteriostatic action in the step 5.2), and selecting the microbial bacteria in the corresponding culture bottles without obvious expansion of the wheat head rot pathogenic bacteria; as shown in FIG. 3, it was observed that the culture bottles of the blank control group were full of Fusarium head blight hyphae, and that there were almost no Fusarium head blight hyphae in the culture bottles inoculated with the microorganism having a good bacteriostatic effect;
6) re-screening the microbe bacteria with antagonism obtained in the step 5.3):
6.1) inoculating the microbial bacteria selected in the step 5.3) into a sterilized barley grain culture bottle for culture to obtain barley grains carrying microbial bacteria with an antibacterial effect;
6.2) make the circular cross scale plate of plastics of black word in white background of a plurality of, the diameter of every cross scale plate equals with the external diameter of plate culture dish, before the inoculation, align a cross scale plate earlier and place under a 9cm plate culture dish, the plate culture dish is transparent structure, the plate culture medium is semitransparent, place the barley grain that carries bacteriostatic effect microbial bacteria that four grains 6.1) step were cultivateed on the cross image of being mapped on the plate culture medium by the cross scale plate respectively, at this moment, the barley grain that carries gibberellic disease fusarium is placed at every plate culture medium center (as shown in figure 4), observe the bacteriostatic effect. An example of a better suppression effect is shown in fig. 5: in fig. 5, a transparent bacteriostatic zone appears between the central fusarium graminearum colony and the peripheral barley grains carrying the microbes with bacteriostatic effect, which indicates that the microbes expand the central fusarium graminearum colony to effectively inhibit, and then the microbes with the best bacteriostatic effect are selected.
The inventor extracts 22 examples from the step 6.2), each example is repeated 3 times, and the comparison example is set to obtain the bacteriostasis conditions shown in the table 1 by measuring the net growth amount of the fusarium graminearum and the average colony diameter of the fusarium graminearum of each culture dish in the 22 examples and the comparison example:
TABLE 1
Hypha growth inhibition ratio (%) in table 1 = (hyphal growth diameter of CK group-colony diameter of example group)/hyphal growth diameter of CK group [ ] 100%
As can be seen from table 1, the bacteriostatic effects of 5, 10, 11, 15, 18, and 20 are better, and the bacteriostatic rates are respectively 66.4%, 60.87%, 61.63%, 60.87%, 69.15%, and 68.40%.
The bacteriostasis of the antagonistic bacteria in each culture flask in the step 5.3) is shown in the following table 2:
table 2:
in table 2, the hypha growth inhibition rate (%) = (CK group hypha growth height-hypha growth height)/control hypha growth height) (% 100).
It can be seen from table 2 that the numbers 5, 18 and 20 have good bacteriostatic effect, and the bacteriostatic rate reaches 100%, i.e. the gibberellic disease is inhibited in the selected barley kernels of the microorganisms 5, 18 and 20, and can not grow at all, which corresponds to the barley kernels carrying the gibberellic disease in the left culture flask in fig. 3, and no hypha development is seen.
Step 4.1) of the invention is to uniformly spray the soil extract with three concentrations prepared in step 1) on the surface of the flat culture medium inoculated with barley grains prepared in step 3): the barley grains are full of hypha of wheat head rot pathogenic bacteria, the soil extracting solution is diluted microbial bacteria, the base number of the pathogenic bacteria is far higher than that of the soil extracting solution, and therefore the microbial bacteria with the bacteriostatic effect in the soil extracting solution have absolute advantages in bacteriostatic effect and growth speed, and the bacteriostatic effect can be seen on the culture medium. The method has the advantages that the screened antagonistic bacteria has absolute advantages in growth speed and bacteriostatic effect, and the bacteriostatic effect is better; the barley grains with wheat pathogenic ear rot bacteria and the microbial bacteria are cultured together on the barley grains, so that the fact that the target microbial bacteria can be propagated only by means of biological nutrients can be proved, the comparison with the wheat pathogenic ear rot bacteria on the propagation speed can also be verified, compared with the fact that the bacteriostatic effect is verified on a culture medium, the operation is more convenient than a bacterium inoculation test carried out in a greenhouse, the condition is well controlled, a powerful basis is laid for the subsequent field experiment, and the biological control efficiency of the live wheat ears is guaranteed. And 5.1) selecting a microbial colony, purifying and selecting a single colony, wherein the single colony is stable and not easy to change, and the error of an experimental result is small.
The wheat ear rot pathogenic bacteria are not limited to fusarium head blight adopted in the embodiment, but can be any harmful bacteria capable of propagating on barley grains, such as botrytis cinerea, black ear bacteria, penicillium and aspergillus flavus, the culture time of each step is determined according to the growth rate of specific pathogenic bacteria, a culture medium used for screening the inhibiting bacteria of the fusarium head rot in the embodiment is a PDA culture medium, the components of the PDA culture medium are 250g of potato, 15-25g of glucose, 15-20g of agar and 1000m of water L, 180-250g of potato is peeled and cut during the preparation, potato juice is obtained after boiling, 15-25g of glucose or sucrose is added, 15-20g of agar is added, water is added to 1000m L for sterilization, the sterilization is carried out at 122 ℃, the soil in the step 1) is a field block for continuously planting large wheat, a sample collected from a bacteria source is more easily separated from nature, restricted to the bacteria of the wheat ear head rot pathogenic bacteria in a natural field, 5.1) is screened out, the soil is used for realizing the purification of the bacteria of the wheat ear rot pathogenic bacteria, the bacteria is inoculated to the bacteria, the bacteria carried in the high-temperature sterilization process of the high-resistant bacteria, the bacteria carried by the high-temperature sterilization of the high-resistant culture medium, the high-resistant bacteria culture medium, the high-resistant culture of the high-resistant culture medium is tested large-resistant bacteria, the high-resistant bacteria is tested large-resistant bacteria, the high-resistant bacteria of the high-resistant bacteria is tested large-resistant large ear rot-resistant large-sterile large-strain-.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent flow transformations made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (9)
1. A separation method of soil microorganism bacteria for inhibiting wheat ear rot pathogenic bacteria is characterized in that: which comprises the following steps:
1) preparing a soil extracting solution: collecting a soil sample with the depth of 5-20 cm below the surface layer, air-drying the soil sample collected from the field, putting the soil sample into a plastic sprayer filled with sterilized water, and respectively diluting the soil sample solution after full oscillation to 10 DEG-2Multiple, 10-3Multiple, 10-4Doubling, and transferring into a spray bottle respectively;
2) culturing wheat head rot pathogenic bacteria by barley grains: placing the steamed or boiled barley grains into a culture bottle, sterilizing at high temperature, cooling to room temperature, inoculating wheat ear rot pathogenic bacteria into the bottle filled with the barley grains, and culturing at 25-26 ℃ for several days to ensure that the barley grains fully grow with the wheat ear rot pathogenic bacteria;
3) inoculating barley grains carrying wheat head rot pathogenic bacteria on a plate culture medium: respectively inoculating a plurality of barley grains carrying wheat ear rot pathogenic bacteria in a plurality of plate culture media, and uniformly distributing the inoculated barley grains carrying the wheat ear rot pathogenic bacteria on each plate culture medium;
4) preliminary screening of antagonistic microbe strains:
4.1) uniformly spraying the soil extracting solutions with three concentrations prepared in the step 1) on the surfaces of the flat culture mediums inoculated with the barley grains prepared in the step 3) one by one; after spraying, sealing the edge of a plate culture medium inoculated with barley grains carrying wheat ear rot pathogenic bacteria by using a sealing film, culturing for a plurality of days at 25-26 ℃, and observing the expansion condition of the wheat ear rot pathogenic bacteria in the plate culture medium;
4.2) picking out bacterial colonies capable of inhibiting the growth of the wheat ear rot pathogenic bacteria in the plate culture medium cultured in the step 4.1), respectively coating the bacterial colonies in a new plate culture medium, simultaneously hooking part of the wheat ear rot pathogenic bacteria hypha in a shape like Chinese character 'ji', coating the hypha on the culture medium of each primary sieve, then continuously sealing the edge of the plate culture medium by using a sealing film, and culturing for a plurality of days at the temperature of 25-26 ℃; observing the bacteriostasis condition of the microorganisms in the plate culture medium, and screening out bacterial colonies capable of obviously inhibiting the growth of wheat ear rot pathogenic bacteria in the plate culture medium as bacterial colonies with good inhibition effect on the selected wheat ear rot pathogenic bacteria.
2. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 1, characterized in that: it also comprises the step 5) of secondary screening of the microbe bacteria with antagonism:
5.1) selecting the bacterial colony which is screened in the step 4.2) and has good inhibition effect on the selected wheat head rot pathogenic bacteria, repeating the step 4.2), and purifying and selecting a single bacterial colony;
5.2) respectively inoculating the single colonies purified in the step 5.1) into a barley grain culture bottle with sterilized treatment, simultaneously putting at least two barley grains inoculated with wheat head rot pathogenic bacteria cultured in the step 2) into the barley grain culture bottle, sealing the barley grain culture bottle, culturing for a plurality of days at 25-26 ℃, shaking the glass bottle every day during the culture period, and finally standing for 1-2 d to observe whether hyphae are inhibited;
5.3) observing the bacteriostasis effect of the microbial bacteria with bacteriostasis in the step 5.2), and selecting the microbial bacteria in the corresponding culture bottle without obvious expansion of the wheat head rot pathogenic bacteria as target antagonistic bacteria.
3. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 2, characterized in that: it further comprises step 6): re-screening the microbe bacteria with antagonism obtained in the step 5.3):
6.1) inoculating the microbial bacteria selected in the step 5.3) into a sterilized barley grain culture bottle for culture to obtain barley grains carrying microbial bacteria with an antibacterial effect;
6.2) respectively placing one barley grain carrying wheat head rot pathogenic bacteria at the centers of a plurality of new plate culture dishes, then evenly placing two to four barley grains carrying microbial bacteria with antibacterial effect cultured in the step 6.1) at the periphery of the barley grain carrying the wheat head rot pathogenic bacteria in each plate culture dish, observing the antibacterial effect, and selecting the microbial bacteria in the corresponding culture dish as target antagonistic bacteria, wherein the microbial bacteria do not obviously expand and have better antagonistic action on the wheat head rot pathogenic bacteria.
4. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 3, characterized in that: when four barley grains of the microbial bacteria with the bacteriostatic effect cultured in the step 6.1) are placed on the plate culture dish in the step 6.2), the placing method comprises the following steps: the method comprises the steps of manufacturing a plurality of plastic circular cross scale plates with white background and black characters, wherein the diameter of each cross scale plate is equal to the outer diameter of a plate culture dish, before inoculation, a cross scale plate is aligned and placed under a new plate culture dish, the plate culture dish is of a transparent structure, a plate culture medium is semitransparent, barley grains carrying wheat ear rot pathogenic bacteria are placed in the center of the cross scale plate, and the barley grains carrying the microbial bacteria with the bacteriostatic effect in the four steps 6.1) are placed on a cross image mapped on the plate culture medium by the cross scale plate respectively.
5. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 1, characterized in that: the wheat head rot pathogenic bacteria are any one of fusarium head blight, botrytis cinerea, black ear germ, penicillium and aspergillus flavus which can be propagated on barley grains.
6. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 3, characterized in that: and (3) carrying out secondary to tertiary repeated screening on the single colony with the bacteriostatic effect obtained in the step 5.1) by repeating the scheme of the step 5.2), and carrying out secondary to tertiary repeated screening on the microbial strain with the bacteriostatic effect obtained in the step 6.1) by repeating the scheme of the step 6.2).
7. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 1, characterized in that: the soil in the step 1) is a field block for continuously planting barley or wheat.
8. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 1, characterized in that: the high-temperature sterilization process in the step 2) comprises the following steps: bottling the steamed or boiled barley grains, and sterilizing at 120-125 deg.C for 20-25 min.
9. The method for isolating soil microorganisms that repress wheatear rot pathogenic bacteria according to claim 1, characterized in that: when barley grains are inoculated in the step 3) and the step 6.2), the forceps heads of the sterile forceps are heated, small holes are formed in positions, to be inoculated, of the flat culture medium through the heated forceps heads, and then the barley grains carrying the wheat ear rot pathogenic bacteria or the microbial bacteria are placed in the small holes, so that the barley grains can be effectively prevented from being displaced.
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