CN110616172B - Bacillus with crucifer clubroot prevention and treatment function and preparation method and application thereof - Google Patents
Bacillus with crucifer clubroot prevention and treatment function and preparation method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
Abstract
The invention discloses a bacillus with the function of preventing and treating cruciferous clubroot and a preparation method and application thereof, the bacillus strain is bacillus methylotrophicus (WSWFJ-29, preservation unit: china general microbiological culture Collection center (CGMCC), preservation number: the microbial inoculum of the bacillus methylotrophicus WSWFJ-29 is a microbial inoculum obtained by performing strain activation, strain purification, seed solution preparation and fermentation on the bacillus methylotrophicus WSWFJ-29, and the preparation method of the microbial inoculum of the bacillus methylotrophicus WSWFJ-29 comprises the steps of strain activation, strain purification, seed solution preparation and fermentation, and the preparation with the bacillus methylotrophicus WSWFJ-29 as an active ingredient has a remarkable prevention and treatment effect on microbial bacteria and clubroot of cruciferae.
Description
Technical Field
The invention belongs to the technical field of microorganism separation culture, and particularly relates to bacillus with a function of preventing and treating clubroot of cruciferae, a preparation method and application thereof.
Background
The cruciferous clubroot is a disease seriously harmful to the yield of vegetables and is a soil-borne disease popular in cruciferous vegetables at present. The disease is widely distributed in China, particularly has serious disease in vegetable planting places in southern provinces such as Yunnan province (more than 40 counties) and Sichuan province in China, and specially harms Chinese cabbages, green vegetables, mustard, cabbage, radishes, cabbages, cauliflowers and the like of cruciferae. After the cruciferous vegetables are infected, the roots and stems are prevented from absorbing and transmitting nutrients, poor growth, wilting and dwarfing are caused, the yield and the quality are damaged, the yield is reduced, the failure is caused seriously, and the serious economic loss is caused. At present, the disease incidence area of the clubroot of cruciferae is gradually enlarged, and because the prevention and treatment effect of the conventional medicament on the clubroot of cruciferae is not ideal, the prevention and treatment are difficult, the cancer in the main vegetable production area such as Tonghai in Yunnan province and the like, even the cancer is called as the vegetable disease by vegetable growers, the cancer becomes the first disease of the cruciferae vegetables, and the production and development of the local vegetables are severely restricted.
The prevention and control of the clubroot of cruciferae is mainly carried out in the seedling stage, and because chemical agents and physical prevention and control have poor prevention and control effects on diseases, the prevention and control of the clubroot are carried out by adopting agricultural microbial agents based on the rapid development of the biological industry, so that the crops can be prevented from generating drug resistance, the green environmental protection can be realized, and no residue is left, and the development of a functional strain for efficiently preventing and controlling the clubroot and a prevention and control product prepared by the functional strain are particularly important for the healthy continuous development of the vegetable industry.
Disclosure of Invention
The first purpose of the invention is to provide a Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29 microbial inoculum which is efficient, nontoxic, safe, residue-free, environment-friendly and convenient to use and can be used for preventing and treating the clubroot disease of cruciferae; the second purpose is to provide a preparation method of the Bacillus methylotrophicus WSWFJ-29 microbial inoculum.
The first purpose of the invention is realized by that the Bacillus strain for preventing and treating the crucifer clubroot disease is Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC, address: No. 3 Xilu-1 of Beijing, Chaoyang, Chengxiang, institute of microbiology, China academy of sciences, postal code 100101), preservation date: 22/07/2019, accession number: CGMCC No.18269, said Bacillus methylotrophicus belongs to the kingdom of bacteria, Bacteromycotina, class Bacteromycetales, order Bacillaceae, genus Bacillus.
The Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29 is separated from a Yunan Songming vegetable planting base, and is determined by morphology, culture properties, conventional physiological biochemistry and a Biolog full-automatic identification system, so that the strain is a new strain (Bacillus methylotrophicus) WSWFJ-29 of the Bacillus methylotrophicus. The strain has the following characteristics:
1) the colony on the PDA culture medium is milky round, the surface is wet, the surface of the colony is in a crater shape, the edge is smooth, no diaphragm is arranged, and the colony is semitransparent and thick.
2) The strain has strong colonization ability and bacteriostasis ability, has the functions of sterilization, disease prevention and yield increase, and can also colonize in the rhizosphere soil of cruciferae.
The first purpose of the invention is realized by that the Bacillus methylotrophicus strain (Bacillus methylotrophicus) WSWFJ-29 is prepared by strain activation, strain purification, seed liquid preparation and fermentation.
The second purpose of the invention is realized by the steps of strain activation, strain purification, seed liquid preparation and fermentation, and specifically comprises the following steps:
A. activating strains: selecting strain preservation solution of Bacillus methylotrophicus (WSWFJ-29), inoculating the strain preservation solution into a strain culture medium, and performing activated culture for 24-48 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 150-180 rpm to obtain activated culture solution of the strain;
B. and (3) strain purification: inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 1-3 days in an incubator at the temperature of 35-38 ℃, and selecting a single colony with better growth vigor and colony characteristics conforming to the colony characteristics of the functional strains as an activated seed colony;
C. preparing a seed solution: b, scraping the activated seed bacterial colony in the first ring step B by using an aseptic inoculating ring, inoculating the seed bacterial colony into 50ml of strain culture medium, and performing activated culture for 48-72 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 160rpm to obtain seed liquid;
D. fermentation: inoculating the strain seed liquid into a fermentation culture solution in a fermentation tank according to the proportion of 1-5 per mill, and culturing for 48-60 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min to obtain fermentation liquid, namely the methylotrophic bacillus fungicide with the function of preventing and treating the clubroot of cruciferous vegetables.
The preparation taking the bacillus methylotrophicus as the active ingredient can be applied to the prevention and treatment of the clubroot of cruciferae, can be used as a microbial bactericide, and can be added into a fertilizer and a seedling substrate to prepare a fertilizer product and a biological seedling substrate with the function of preventing and treating the clubroot of cruciferae for use, and has the following advantages:
1. bacillus methylotrophicus WSWFJ-29 is a high-efficiency biocontrol bacterium, and a preparation taking the Bacillus methylotrophicus WSWFJ-29 as an active ingredient has remarkable control effects on clubroot of cruciferae and microbial bacteria.
2. The preparation method of the preparation is simple, feasible, reasonable, effective and low in cost, and is beneficial to industrial production, application and popularization of the preparation.
3. The Bacillus methylotrophicus WSWFJ-29 and the preparation taking the Bacillus methylotrophicus WSWFJ-29 as an active ingredient are nontoxic to human and livestock and have strong environmental adaptability and environmental friendliness.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting in any way, and any variations or modifications which are based on the teachings of the present invention are intended to fall within the scope of the invention.
The Bacillus strain with the function of preventing and treating the clubroot of cruciferae is a Bacillus methylotrophicus strain (Bacillus methylotrophicus) WSWFJ-29, and is screened and preserved by a microorganism fermentation engineering research center limited company in Yunnan province. The preservation unit: china general microbiological culture Collection center (CGMCC), preservation date: 22/07/2019, accession number: CGMCC No.18269, said Bacillus methylotrophicus belongs to the kingdom of bacteria, Bacteromycotina, class Bacteromycetales, order Bacillaceae, genus Bacillus.
The microbial inoculum of the Bacillus methylotrophicus WSWFJ-29 is obtained by performing strain activation, strain purification, seed solution preparation and fermentation on the Bacillus methylotrophicus WSWFJ-29.
The preparation method of the Bacillus methylotrophicus WSWFJ-29 microbial inoculum comprises the steps of strain activation, strain purification, seed solution preparation and fermentation, and specifically comprises the steps of
A. Activating strains: selecting strain preservation solution of Bacillus methylotrophicus (WSWFJ-29), inoculating the strain preservation solution into a strain culture medium, and performing activated culture for 24-48 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 150-180 rpm to obtain activated culture solution of the strain;
B. and (3) strain purification: inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 1-3 days in an incubator at the temperature of 35-38 ℃, and selecting a single colony with better growth vigor and colony characteristics conforming to the colony characteristics of the functional strains as an activated seed colony;
C. preparing a seed solution: b, scraping the activated seed bacterial colony in the first ring step B by using an aseptic inoculating ring, inoculating the seed bacterial colony into 50ml of strain culture medium, and performing activated culture for 48-72 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 160rpm to obtain seed liquid;
D. fermentation: inoculating the strain seed liquid into a fermentation culture solution in a fermentation tank according to the proportion of 1-5 per mill, and culturing for 48-60 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min to obtain fermentation liquid, namely the methylotrophic bacillus fungicide with the function of preventing and treating the clubroot of cruciferous vegetables.
The bacterial strain in the step A is obtained by conventional liquid and solid fermentation culture, is a milky round bacterial colony, has smooth surface and edge, no diaphragm and semitransparent and thick bacterial colony, and is preserved at the temperature of minus 80 ℃ by a glycerol preservation method.
And A, B, C, wherein the strain culture medium is a beef extract peptone liquid culture medium.
The A, B, C step comprises the following components in parts by weight: 2-5 parts of beef extract, 8-12 parts of peptone, 4-7 parts of sodium chloride and 800-1200 parts of water, and adjusting the pH to 6-8 by adopting hydrochloric acid and sodium hydroxide
The A, B, C step comprises the following components in parts by weight: 2-5 parts of beef extract, 8-12 parts of peptone, 4-7 parts of sodium chloride, 15-25 parts of agar and 800-1200 parts of water, and the pH is adjusted to be 6-8 by hydrochloric acid and sodium hydroxide.
And the fermentation culture solution in the step D comprises the following components in parts by weight: 2-4 parts of soybean meal, 0.5-1 part of cane sugar, 1-3 parts of beef extract, 2-3 parts of corn flour, 0.8-1.5 parts of calcium carbonate, 1-2 parts of fish meal, 0.05-0.1 part of potassium dihydrogen phosphate, 0.05-0.1 part of dipotassium hydrogen phosphate, 1-5 parts of sodium chloride and 0.1-0.3 part of defoaming agent, and the pH is adjusted to 6.8-7.5 by adopting hydrochloric acid and sodium hydroxide.
The effective viable count of the bacillus methylotrophicus prepared in the step D is more than 2.0 multiplied by 1010cfu/mL。
The microbial inoculum of Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29 is applied to preparing a microbial bactericide for preventing and treating clubroot of cruciferae.
When the microbial inoculum of the bacillus methylotrophicus WSWFJ-29 is used as a microbial bactericide, the method is to dilute the microbial inoculum into 1800-2200 times of solution by using clean water, so that the effective viable count is controlled to be 1 multiplied by 107cfu/mL, irrigated to the roots of the plants.
The invention is further illustrated by the following specific examples:
example 1
The invention provides a preparation method of a bacillus methylotrophicus WSWFJ-29 microbial inoculum.
Firstly, inoculating 0.1ml of strain preservation solution of Bacillus methylotrophicus (WSWFJ-29) which is preserved by adopting glycerol at the temperature of-80 ℃ into 10ml of strain culture medium, and carrying out activated culture for 24 hours at the temperature of 35-38 ℃ and the rotating speed of 150-180 rpm to obtain activated culture solution of the strain;
secondly, inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 1 day in an incubator at the temperature of 35-38, and selecting a single colony with better growth vigor and colony characteristics which accord with the colony characteristics of the functional strains as an activated seed colony;
thirdly, scraping the activated seed colonies in the first step by using an aseptic inoculating ring, inoculating the seed colonies into 50ml of strain culture medium, and performing activated culture for 48 hours under the conditions that the temperature is 35 ℃ and the rotating speed is 160rpm to obtain seed liquid;
fourthly, inoculating the strain seed liquid into the fermentation culture liquid in a fermentation tank according to the proportion of 1 per mill, and culturing for 48 hours under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min, so as to obtain the effective viable count which is more than 2.0 multiplied by 1010cfu/mL fermentation liquor, namely the methylotrophic bacillus microbial inoculum with the function of preventing and treating the clubroot of cruciferae, can be directly used as a microbial bactericide.
Example 2
The invention provides a preparation method of a bacillus methylotrophicus WSWFJ-29 microbial inoculum.
Firstly, inoculating 0.1ml of strain preservation solution of Bacillus methylotrophicus (WSWFJ-29) which is preserved by adopting glycerol at the temperature of-80 ℃ into 10ml of strain culture medium, and carrying out activated culture for 36h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 150-180 rpm to obtain activated culture solution of the strain;
secondly, inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 2 days in an incubator at the temperature of 35-38, and selecting a single colony with better growth vigor, the colony characteristics of which accord with the colony characteristics of the functional strains, as an activated seed colony;
thirdly, scraping the activated seed colonies in the first step by using an aseptic inoculating ring, inoculating the seed colonies into 50ml of strain culture medium, and performing activated culture for 60 hours under the conditions that the temperature is 35-38 and the rotating speed is 160rpm to obtain seed liquid;
fourthly, inoculating the strain seed liquid into the fermentation culture liquid in a fermentation tank according to the proportion of 3 per mill, and culturing for 54 hours under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min to obtain the effective viable count which is more than 2.0 multiplied by 1010cfu/mL fermentation liquor, namely the methylotrophic bacillus microbial inoculum with the function of preventing and treating the clubroot of cruciferae, can be directly used as a microbial bactericide.
Example 3
The invention provides a preparation method of a bacillus methylotrophicus WSWFJ-29 microbial inoculum.
Firstly, inoculating 0.1ml of strain preservation solution of Bacillus methylotrophicus (WSWFJ-29) which is preserved by adopting glycerol at the temperature of-80 ℃ into 10ml of strain culture medium, and carrying out activated culture for 48h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 150-180 rpm to obtain activated culture solution of the strain;
secondly, inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 3 days in an incubator at the temperature of 35-38, and selecting a single colony with better growth vigor, the colony characteristics of which accord with the colony characteristics of the functional strains, as an activated seed colony;
thirdly, scraping the activated seed colonies in the first step by using an aseptic inoculating ring, inoculating the seed colonies into 50ml of strain culture medium, and performing activated culture for 72 hours under the conditions that the temperature is 35-38 and the rotating speed is 160rpm to obtain seed liquid;
fourthly, inoculating the strain seed liquid into the fermentation culture liquid in a fermentation tank according to the proportion of 5 per mill, and culturing for 60 hours under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min, so as to obtain the effective viable count which is more than 2.0 multiplied by 1010cfu/mL fermentation liquor, namely the methylotrophic bacillus microbial inoculum with the function of preventing and treating the clubroot of cruciferae, can be directly used as a microbial bactericide.
Example 4
Optionally, the Bacillus methylotrophicus WSWFJ-29 microbial agent preparation prepared in the embodiments 1-3 has an inhibitory effect on clubroot of cruciferae.
4.1 preparation before experiment
Performing experiments in Qinglong town region of Anning city of Kunming, Yunnan province, selecting 4 seedling raising ponds with the volume size of 1m multiplied by 0.1m, wherein the cabbage seeds and the seedling raising substrates are conventional vegetable seeds and seedling raising substrates purchased in the market; regulating the moisture of the seedling raising substrate to 40-50%, namely kneading the seedling raising substrate into a cluster by hand and naturally dispersing the seedling raising substrate when the seedling raising substrate falls to the ground; the seeding depth of the Chinese cabbage seeds in the seedling culture substrate is 0.2-0.3 cm, 1 seed per hole is not covered with the substrate after seeding is finished, the tray is lightly placed into 4 seedling culture pools, and a proper amount of clear water is injected into the seedling culture pools in advance; the number of selected effective viable bacteria is 2 multiplied by 1010WSWFJ-29 microbial inoculum of cfu/mL and active spore concentration of 5 multiplied by 1012cfu/mL of plasmodiophora root pathogenic bacteria spore liquid for later use.
4.2 specific experimental mode:
1, seedling raising pool: in the seedling raising day, 5000mL of WSWFJ-29 microbial inoculum preparation is added into a seedling raising pool 1;
and (3) seedling raising pond 2: in the seedling raising day, 5000mL of WSWFJ-29 microbial inoculum preparation is added into the seedling raising pool 2, and after 10 days of seedling raising, 0.2mL of plasmodiophora radical disease pathogenic bacteria spore liquid is added into the seedling raising pool 2;
and 3, seedling raising pond 3: after 10 days of seedling culture, 0.2mL of plasmodiophora root pathogen spore liquid is added into the seedling culture pond 3;
and (4) seedling raising pond: no microbial inoculum is added into the seedling raising pond 4 to directly raise seedlings as a blank control.
The effective viable count of the WSWFJ-29 microbial inoculum for the experiment is 1 multiplied by 10 in the final concentration of water in the nursery ponds 1-37cfu/mL; the final concentration of active spores of plasmodiophora radical pathogenic bacteria spore liquid in water in the seedling raising ponds 1-3 for the experiment is 105cfu/mL. No fertilizer is applied in the process of seedling culture, so that the water supply of the seedling culture pond is normal, the temperature of the seedling culture shed is proper, the environment is suitable for crop growth, and no plant diseases and insect pests exist; the results of observing the emergence condition, growth condition and disease occurrence condition of crops and measuring the contents of the colonized functional strains in the root, overground part and matrix of the Chinese cabbage are as follows.
4.2.1 statistics of emergence, see Table 1
TABLE 1
| Pond of growing seedlings | Rate of emergence | Condition of seedling formation |
| Pond of growing seedlings 1 | 92.05％ | 90.31％ |
| Seedling raising pond 2 | 92.14％ | 90.22％ |
| Pond of growing seedlings 3 | 90.32％ | 88.14％ |
| Pond of growing seedlings 4 | 90.51％ | 89.02％ |
4.2.2 statistics of disease incidence, see Table 2
TABLE 2
| Pond of growing seedlings | Incidence of disease |
| Pond of growing seedlings 1 | 5.3％ |
| Seedling raising pond 2 | 5.2％ |
| Pond of growing seedlings 3 | 8.34％ |
| Pond of growing seedlings 4 | 23.17％ |
4.2.3 determination of the colonized bacteria content of functional strains in crops, see Table 3
TABLE 3
| Pond of growing seedlings | Root of herbaceous plant | Aerial parts | Substrate |
| Pond of growing seedlings 1 | 2.85E+05 | 8.00E+04 | 5.30E+06 |
| Seedling raising pond 2 | 1.85E+06 | 3.60E+05 | 6.80E+06 |
| Pond of growing seedlings 3 | 0 | 0 | 0 |
| Pond of growing seedlings 4 | 0 | 0 | 0 |
4.3 conclusion of the test:
the clubroot prevention and control functional microbial inoculum used in the test can play a certain promoting role in seedling raising of the Chinese cabbage, has a growth promoting function, is added in the early stage of seedling raising to inhibit the growth of pathogenic bacteria, can effectively reduce the infection of the pathogenic bacteria in the seedling raising process, and can well protect the Chinese cabbage seedlings. The functional strain can be colonized in Chinese cabbage and seedling raising matrix, and has the functions of fixing nitrogen, dissolving phosphorus, dissolving potassium, improving crop disease resistance, secreting auxin, secreting bacteriostat, etc., so that it has the effects of promoting growth and preventing diseases.
Example 5
The bacillus methylotrophicus WSWFJ-29 microbial inoculum preparation prepared in the embodiment 1-3 has the effect of preventing and controlling the clubroot of cruciferae.
5.1, early preparation:
in the Liu factory town area of Xiangyun county, Dali, four vegetable planting greenhouses with serious disease incidence are randomly selected, one vegetable is planted in each vegetable planting greenhouse, namely Shanghai green, cauliflower, leaf lettuce and lettuce, and each planting greenhouse is divided into two parts along the axis, so that one side of each planting greenhouse is a test group and the other side of each planting greenhouse is a control group; the test group of each vegetable planting greenhouse adopts a biological organic fertilizer added with the bacillus methylotrophicus WSWFJ-29 microbial inoculum, the control group adopts a conventional biological organic fertilizer as a control, and the cultivation management modes of the four groups are the same; and applying base fertilizer before sowing, wherein the application amount is 100 kg/mu, and simultaneously ploughing the greenhouse soil to enable the fertilizer to be ploughed into the soil.
5.2, experimental mode:
5.2.1 field disease
Transplanting four vegetable seedlings in 3 months and 5 days, harvesting in 5 months and 1 day, planting a greenhouse in the total growth period of 57 days for each vegetable seedling, dividing the greenhouse into a left area and a right area from the center of the greenhouse, wherein the left area and the right area are respectively a test group and a control group, the area of each greenhouse is 0.48 mu, and the greenhouse is serious in disease occurrence. The onset of disease is shown in Table 4.
TABLE 4
According to the statistics in the table 1, the bio-organic fertilizer added with the functional microbial inoculum is used as the base fertilizer, so that clubroot is effectively reduced, the cruciferous vegetables are effectively protected before being infected by pathogenic bacteria, and the disease prevention effect is achieved.
5.2.2, statistics of agronomic traits are shown in Table 5
TABLE 5
As can be seen from Table 2, the addition of the functional microbial inoculum can promote the growth of vegetables, indicating that the microbial inoculum has the effect of promoting the growth of crops.
5.2.3 statistics of the yield of four vegetable plants, see Table 6
TABLE 6
| Treatment of | Shanghai Qing tea | Cauliflower | Leaf of Chinese lettuce | Lettuce |
| Test group | 3125 kg/mu | 3250 kg/mu | 3033 kg/mu | 2994 kg/mu |
| Control group | 2396 kg/mu | 2415 kg/mu | 2298 kg/mu | 2138 kg/mu |
The control group clubroot disease condition has certain influence on the vegetable yield, and the disease prevention and growth promotion effects of the functional microbial inoculum promote the yield of the test group to be greatly improved compared with the control group.
5.3 summary of the test
The bacillus methylotrophicus WSWFJ-29 microbial inoculum with the clubroot disease prevention function is added into an organic fertilizer and is applied as a base fertilizer before sowing, so that the infection of clubroot bacteria on vegetables can be effectively blocked, the incidence rate of clubroot diseases of cruciferae can be effectively reduced, the growth of crops can be promoted, and the yield and the quality of the crops can be improved.
Claims (9)
1. A bacillus strain with the function of preventing and treating clubroot of cruciferae is characterized in that: the Bacillus strain is Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29, and the preservation unit is as follows: china general microbiological culture Collection center (CGMCC), preservation date: 22/07/2019, accession number: CGMCC No.18269, said Bacillus methylotrophicus belongs to the kingdom of bacteria, Bacteromycotina, class Bacteromycetales, order Bacillaceae, genus Bacillus.
2. A bacterial agent of Bacillus methylotrophicus (WSWFJ-29) according to claim 1, wherein: the Bacillus methylotrophicus strain (Bacillus methylotrophicus) WSWFJ-29 is obtained by strain activation, strain purification, seed liquid preparation and fermentation.
3. A method for preparing a Bacillus methylotrophicus (Bacillus methylotrophicus) WSWFJ-29 bacterial agent of claim 2, wherein the method comprises the following steps: comprises the steps of strain activation, strain purification, seed liquid preparation and fermentation, and specifically comprises
A. Activating strains: selecting strain preservation solution of Bacillus methylotrophicus (WSWFJ-29), inoculating the strain preservation solution into a strain culture medium, and performing activated culture for 24-48 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 150-180 rpm to obtain activated culture solution of the strain;
B. and (3) strain purification: inoculating strains in the activated culture solution to a strain culture medium flat plate by using a sterile inoculating loop, performing purification streak culture, culturing for 1-3 days in an incubator at the temperature of 35-38 ℃, and selecting a single well-grown bacterial colony with bacterial colony characteristics conforming to the bacterial colony characteristics of the functional strains as an activated seed bacterial colony;
C. preparing a seed solution: b, scraping the activated seed bacterial colony in the first ring step B by using an aseptic inoculating ring, inoculating the seed bacterial colony into 50ml of strain culture medium, and performing activated culture for 48-72 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 160rpm to obtain seed liquid;
D. fermentation: inoculating the strain seed liquid into a fermentation culture solution in a fermentation tank according to the proportion of 1-5 per mill, and culturing for 48-60 h under the conditions that the temperature is 35-38 ℃ and the rotating speed is 100-140 rpm/min to obtain fermentation liquid, namely the methylotrophic bacillus fungicide with the function of preventing and treating the clubroot of cruciferous vegetables.
4. The production method according to claim 3, characterized in that: the bacterial strain in the step A is obtained by conventional liquid and solid fermentation culture, is a milky round bacterial colony, has smooth surface and edge, no diaphragm and semitransparent and thick bacterial colony, and is preserved at the temperature of minus 80 ℃ by a glycerol preservation method.
5. The production method according to claim 3, characterized in that: and A, B, C, wherein the strain culture medium is a beef extract peptone liquid culture medium.
6. The production method according to claim 3 or 5, characterized in that: the A, B, C step comprises the following components in parts by weight: 2-5 parts of beef extract, 8-12 parts of peptone, 4-7 parts of sodium chloride and 800-1200 parts of water, and adjusting the pH value to be 6-8 by adopting hydrochloric acid and sodium hydroxide.
7. The production method according to claim 3 or 5, characterized in that: the A, B, C step comprises the following components in parts by weight: 2-5 parts of beef extract, 8-12 parts of peptone, 4-7 parts of sodium chloride, 15-25 parts of agar and 800-1200 parts of water, and the pH is adjusted to be 6-8 by hydrochloric acid and sodium hydroxide.
8. The production method according to claim 3, characterized in that: and the fermentation culture solution in the step D comprises the following components in parts by weight: 2-4 parts of soybean meal, 0.5-1 part of cane sugar, 1-3 parts of beef extract, 2-3 parts of corn flour, 0.8-1.5 parts of calcium carbonate, 1-2 parts of fish meal, 0.05-0.1 part of potassium dihydrogen phosphate, 0.05-0.1 part of dipotassium hydrogen phosphate, 1-5 parts of sodium chloride and 0.1-0.3 part of defoaming agent, and the pH is adjusted to 6.8-7.5 by adopting hydrochloric acid and sodium hydroxide.
9. The production method according to claim 3, characterized in that: the effective viable count of the bacillus methylotrophicus prepared in the step D is more than 2.0 multiplied by 1010cfu/mL。
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Denomination of invention: A Bacillus subtilis with the function of preventing and treating root nodules in the Brassicaceae family, its preparation method and application Effective date of registration: 20231205 Granted publication date: 20210817 Pledgee: Kunming Dongfeng Sub branch of Bank of China Ltd. Pledgor: YUNNAN YUNZHIYE BIOTECHNOLOGY CO.,LTD.|YUNNAN YUNYE FERTILIZER Co.,Ltd.|MICROBIAL FERMENTATION ENGINEERING RESEARCH CENTER Co.,Ltd. OF YUNNAN PROVINCE Registration number: Y2023980069266 |