CN111088186A - Bacillus, microbial agent and application thereof - Google Patents
Bacillus, microbial agent and application thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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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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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention provides bacillus CGMCC No.19084 and a microbial agent containing the bacillus, and provides a preparation method of the microbial agent, which can effectively increase the disease resistance of plants by applying the bacillus and the microbial agent into soil for plant growth.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to bacillus, a microbial agent and application thereof.
Background
The alfalfa root rot disease is root rot and stem rot disease caused by soil-borne pathogen infection. The disease is a worldwide disease, commonly occurring in all alfalfa cultivation areas of the world, and particularly occurring in countries such as the united states, canada, australia, russia, japan and argentina. It is estimated that the annual worldwide yield loss due to the disease is around 20%, with severe land development reaching even 40%. Since 1991, China discovered the disease, and has related reports in Xinjiang, Gansu, Qinghai and other places. Because the alfalfa is used for a long time, the root rot becomes an extremely important cause of the yield reduction and the plant decay of the alfalfa.
Most of the existing methods for preventing and treating alfalfa root rot are chemical agent prevention and treatment, but a large amount of pesticide used for a long time can seriously threaten the environment, organisms and human health. Therefore, a safe and effective biological agent is urgently needed to be found to solve the existing alfalfa root rot disease.
Disclosure of Invention
The present disclosure is directed to a safe and effective biological agent to address the existing alfalfa root rot disease.
In order to achieve the above object, the present disclosure provides a bacillus, characterized in that: the Bacillus is alfalfa endophyte, is classified and named as Bacillus (Bacillus sp.) and has a preservation number of CGMCC No. 19084.
The disclosure also provides application of the bacillus in preventing and treating alfalfa root rot.
The disclosure also provides a preparation method of the microbial agent, which is characterized in that the microbial agent is obtained by activated culture and fermentation of bacillus.
Optionally, the activating culturing step comprises: inoculating bacillus CGMCC No.19084 in an activation culture medium, wherein the culture temperature is 30-37 ℃, and the culture time is 48 h.
Optionally, the fermenting step comprises: inoculating the cultured bacillus into a fermentation culture medium according to the inoculation proportion of 5%, and performing shake culture on a shaking table at the culture temperature of 28-37 ℃ for 24-72 h.
Optionally, the activation medium is beef extract peptone medium, nutrient broth medium, or LB medium; the fermentation medium is a beef extract peptone medium, a nutrient broth medium or an LB medium.
Optionally, the method further comprises mixing the material obtained after culturing in the fermentation medium with an inert solid carrier and a binder; the inert solid carrier comprises at least one of clay, silicon dioxide, diatomite, kaolin, attapulgite, montmorillonite, bentonite, talcum powder, white carbon black and calcium carbonate; the binder comprises maltose and/or dextrin.
The present disclosure also provides a microbial agent.
Optionally, in each gram of the microbial agent, the viable count of bacillus with the preservation number of CGMCC No.19084 is 108-1011CFU。
Optionally, the microbial agent is a powder or solution.
The disclosure also provides application of the microbial agent in preventing and treating alfalfa root rot.
Through the technical scheme, the bacillus and the microbial agent provided by the disclosure can effectively prevent and treat alfalfa root rot diseases, and the bacillus CGMCC No.19084 is derived from alfalfa endophytes, can be protected by alfalfa plant tissues, and is easier to play than other biocontrol factors.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Biological material preservation
The Bacillus disclosed by the invention is a pure culture separated from alfalfa roots by the inventor, the preservation number of the pure culture is CGMCC No.19084, the preservation date of the pure culture is 12 months and 5 days in 2019, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, the address of the pure culture is located in the institute of microorganisms of China academy of sciences No. 3 of North Chen West Lu No.1 of the sunward area in Beijing, and the pure culture is classified and named as Bacillus (Bacillus sp.).
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
The present disclosure provides a Bacillus, which is alfalfa endophyte NA NX51R-5, and is classified and named as Bacillus (Bacillus sp.), wherein the preservation number of the Bacillus is CGMCC No. 19084.
The Bacillus is a new strain separated and cultured from alfalfa root samples by the inventor of the present disclosure, and the strain is identified to be Bacillus (Bacillus sp.). The strain is preserved in the general microbiological culture collection center of China microbiological culture collection management Committee of preservation units appointed by the State intellectual Property office, the preservation date is 12 months and 5 days in 2019, and the preservation number is CGMCC No. 19084.
The bacillus of the present disclosure can survive and propagate in conventional bacterial culture media. The bacillus disclosed by the invention is an alfalfa endophyte and has an outstanding effect on preventing and treating alfalfa root rot.
The invention also provides a preparation method of the microbial agent, which is characterized in that the microbial agent is obtained by activating, culturing and fermenting the bacillus CGMCC No. 19084.
Wherein the activating and culturing steps comprise: inoculating bacillus CGMCC No.19084 in an activated culture medium, wherein the culture temperature is 30-37 ℃, and the culture time is 48 h.
Wherein the fermentation step comprises: inoculating the cultured bacillus into a fermentation culture medium according to the inoculation proportion of 5%, and performing shake culture on a shaking table at the culture temperature of 30-37 ℃ for 24-72 h.
Wherein the activation culture medium is a beef extract peptone culture medium, a nutrient broth culture medium or an LB culture medium; the fermentation medium is a beef extract peptone medium, a nutrient broth medium or an LB medium. The above-mentioned medium can be commercially available or prepared according to the description of "microbiological Culture Medium Manual". Furthermore, the above-mentioned various culture media can be sterilized according to conventional sterilization methods, for example, under the conditions of 115 ℃ and 125 ℃ and 1.5-2 standard atmospheric pressures for 10-30 minutes.
Optionally, the method further comprises mixing the material obtained after culturing in the fermentation medium with an inert solid carrier and a binder; the inert solid carrier comprises at least one of clay, silicon dioxide, diatomite, kaolin, attapulgite, montmorillonite, bentonite, talcum powder, white carbon black and calcium carbonate; the binder comprises maltose and/or dextrin.
Preferably, the inert solid carrier and the binder are used in such an amount that the content of the inert solid carrier in the microbial agent is 30 to 90% by weight; the content of the binder is 5-20 wt%. Wherein, the mixed materials can be further dried and granulated into granules.
The present disclosure also provides a microbial agent.
According to the present disclosure, the amount of bacteria contained in the microbial agent can vary widely, for example, the viable count of Bacillus having a accession number of CGMCC No.19084 per gram of microbial agent can be 108-1011The CFU, preferably, per gram of microbial agent, the viable count of the bacillus with the preservation number of CGMCC No.19084 can be 109-1010CFU。
Wherein, the microbial agent is powder or solution. The bacterial liquid after fermentation culture can be directly used as a microbial agent, and preferably, the bacterial liquid is further processed into a microbial agent in a more convenient storage formulation by the steps of sterile filtration, freeze drying and the like.
The disclosure also provides application of the microbial agent in preventing and treating alfalfa root rot.
By applying the bacillus as described above and the microbial agent as described above to soil where plants grow, disease resistance of plants can be effectively increased.
The present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to the following examples.
Example 1
The bacillus CGMCC No.19084 is inoculated into beef extract peptone culture solution, and shaking culture is carried out for 12h at 37 ℃ and 180rpm to prepare seed solution. Adding 100mL of the seed solution into 100L of fermentation medium, culturing at 30 deg.C, sampling during the culture process, and observing by microscope direct counting method until viable count of Bacillus per gram of culture solution is 1010And CFU, adsorbing the fermentation liquor by using diatomite, centrifuging or filtering by using a plate frame, and drying to form wettable powder, wherein the obtained wettable powder is the microbial agent in the embodiment.
Comparative example 1
Trichoderma purchased from China general microbiological culture Collection center with preservation number CGMCC No.11220 is inoculated into a potato glucose agar solid medium (PDA) from a preservation slant, cultured in an incubator at 25 ℃ for 7 days, and 3-4 fungus blocks are taken from the culture medium and added into a seed bottle. Shaking and culturing at 25 deg.C and 180rpm on shaking table for 48 hr to obtain seed solution. Adding the seed liquid into a seed fermentation tank according to the volume ratio of 1:150 for culture, then adding the seed liquid into a fermentation tank of a chlamydospore fermentation medium according to the volume ratio of 1:20, and culturing for 132 hours at 28 ℃ until 90% of hypha forms chlamydospore to obtain chlamydospore fermentation liquid; then using diatomite to adsorb, centrifuge or plate-frame filter and dry the fermentation liquor to form wettable powder until the viable count of trichoderma is 1010CFU/g, the obtained wettable powder is the microbial agent of the comparative example.
Comparative example 2
Inoculating Bacillus subtilis purchased from China general microbiological culture Collection center with the preservation number of CGMCC No.8995 into beef extract peptone culture solution, and carrying out shake culture on a shaker at 37 ℃ and 180rpm for 12h to prepare seed solution. Adding 100mL of the seed solution into 100L of fermentation medium, culturing at 30 deg.C, sampling during the culture process, and observing by microscope direct counting method until viable count of Bacillus per gram of culture solution is 1010And CFU, adsorbing the fermentation liquor by using diatomite, centrifuging or filtering by using a plate frame, and drying to form wettable powder, wherein the obtained wettable powder is the microbial agent in the embodiment.
Comparative examples 3 to 7
Respectively inoculating 5 strains of bacillus separated together with the bacillus with the preservation number of CGMCC No.19084 into beef extract peptone culture solution, carrying out shake cultivation for 12h at 37 ℃ and 180rpm on a shaking table, and respectively marking the obtained bacterium solutions as seed solutions of comparative examples 4-8. Adding 100mL of the seed solution into 100L of fermentation medium, culturing at 30 deg.C, sampling during the culture process, and observing by microscope direct counting method until viable count of Bacillus subtilis per gram of culture solution is 1010CFU, the resulting culture broth isThe microbial agent is the microbial agent of comparative examples 4-8.
Comparative example 8
From Rhizophorane-Xiye, Shandong McKe Biotech, Inc.
Test example 1
The test was conducted in a field trial to determine the effectiveness of the microbial agents of the invention in controlling alfalfa root rot. The test field is located at the test base of the Chinese academy of agricultural sciences, corridor, Hebei province, and the alfalfa variety is alfalfa (Zhongalfalfa No.1, provided by the middle-stage base of Chinese herbage planting, and the catalog number is 00068).
The pathogenic alfalfa is pulled out from the alfalfa field with the root rot, cut into pieces and soaked in clear water with the weight 10 times of that of the alfalfa, and then the root rot disease stock solution is obtained.
The microbial agents of example 1 and comparative examples 1-8 were applied in different planting areas 20 days after sowing as experimental groups, wherein the application amount of the microbial agent of example 1 and comparative examples 1-7 was 0.2kg/m2The commercially available phloretin of comparative example 8 was diluted 500 times in accordance with the instruction for application and sprayed at an application rate of 1.5g/m2(stock solution). And a blank fermentation medium was used as a blank control. After 2 days of applying the microbial inoculum, 100mL of infectious disease stock solution of the root rot is sprayed per square meter. After 30 days, the relative survival ratio (i.e. the number of the experimental group survivors divided by the number of the blank control group survivors), the relative plant height (i.e. the average plant height of the experimental group divided by the average plant height of the blank control group) and the relative aboveground biomass (i.e. the average aboveground fresh weight of the experimental group divided by the average aboveground fresh weight of the blank control group) of the turfgrass were counted, and the results are shown in table 1.
TABLE 1
| Microbial agent | Relative survival ratio | Relative plant height | Relative aboveground biomass |
| Example 1 | 2.1 | 1.98 | 2.89 |
| Comparative example 1 | 1.85 | 1.71 | 1.78 |
| Comparative example 2 | 1.66 | 1.44 | 1.69 |
| Comparative example 3 | 1.74 | 1.56 | 1.76 |
| Comparative example 4 | 1.53 | 1.43 | 1.66 |
| Comparative example 5 | 1.68 | 1.50 | 1.71 |
| Comparative example 6 | 1.71 | 1.45 | 1.73 |
| Comparative example 7 | 1.89 | 1.78 | 1.98 |
| Comparative example 8 | 1.81 | 1.75 | 1.87 |
It can be seen from table 1 that the bacillus with the preservation number of CGMCC No.19084 of the present disclosure can effectively control the root rot of alfalfa.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. In order to avoid unnecessary repetition, various possible combinations will not be separately described in this disclosure.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (10)
1. A bacillus, characterized in that: the Bacillus is alfalfa endophyte, is classified and named as Bacillus (Bacillus sp.) and has a preservation number of CGMCC No. 19084.
2. The application of the bacillus of claim 1 to control alfalfa root rot.
3. A method for producing a microbial agent, characterized in that the microbial agent is obtained by activated culture and fermentation of the Bacillus of claim 1.
4. The method of claim 3, wherein the activating culture step comprises: inoculating bacillus CGMCC No.19084 into an activation culture medium, wherein the culture temperature is 30-37 ℃, and the culture time is 48 h;
the fermentation step comprises: inoculating the cultured bacillus into a fermentation culture medium according to the inoculation proportion of 5%, and performing shake culture on a shaking table at the culture temperature of 30-37 ℃ for 24-72 h.
5. The method of claim 4, wherein the activation medium is a beef extract peptone medium, nutrient broth medium, or LB medium; the fermentation medium is a beef extract peptone medium, a nutrient broth medium or an LB medium.
6. The method of claim 3, further comprising mixing the fermented material with an inert solid carrier and a binder; the inert solid carrier comprises at least one of clay, silicon dioxide, diatomite, kaolin, attapulgite, montmorillonite, bentonite, talcum powder, white carbon black and calcium carbonate; the binder comprises maltose and/or dextrin.
7. A microbial agent, characterized by being prepared according to the method of any one of claims 3 to 6.
8. The microbial agent according to claim 7, wherein the viable count of bacillus with the preservation number of CGMCCNo.19084 is 10 per gram of the microbial agent8-1011CFU。
9. The microbial agent according to claim 7, wherein the microbial agent is a powder or solution.
10. The use of the microbial agent of any one of claims 7-9, for controlling alfalfa root rot.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114717156A (en) * | 2022-05-06 | 2022-07-08 | 南京财经大学 | Biological agent produced by fixing bacillus with diatomite |
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