CN111748496A - Application of Brevibacillus laterosporus MES818 in tomato cultivation - Google Patents
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Abstract
The invention belongs to the technical field of plant cultivation by utilizing microorganisms, and particularly relates to an application of Brevibacillus laterosporus MES818 in tomato cultivation, wherein the strain is Brevibacillus laterosporus MES818 which is preserved in China general microbiological culture collection center in 2018, 12 and 17 months, and the preservation number is CGMCC NO. 16859. The Brevibacillus laterosporus MES818 can be applied to the aspects of preventing and treating tomato damping-off and increasing tomato yield. The invention has the beneficial effects that: the Brevibacillus laterosporus MES818 has obvious inhibition effect on the tomato damping-off pathogen Rhizoctonia solani, can alleviate the occurrence and harm of tomato damping-off and promote the improvement of tomato yield and quality.
Description
Technical Field
The invention belongs to the technical field of plant cultivation by using microorganisms, and particularly relates to application of Brevibacillus laterosporus MES818 in tomato cultivation.
Background
The tomato is popular with people because of rich nutrition and good taste, China is the biggest export country of tomato products all over the world, and the tomato has important significance for people's life and national economic development. However, in recent years, tomato seedling blight frequently occurs in seedling stage, the tomato seedling often dies in pieces when the tomato seedling is seriously attacked, and the tomato seedling is seriously attacked in years with continuous rainy weather, little light and high temperature in the seedling stage. The disease can be found in both the seedlings just coming out of the ground and the big seedlings. The stem base of the diseased seedling turns brown, the diseased part becomes thin by shrinkage, and the stem leaves wither and die; slightly bigger seedlings wither in the day and recover at night, and when the disease spots surround the stem for a week, the seedlings die gradually but do not fall suddenly; the primary oval dark brown color spot of the diseased part has concentric ring lines and light brown spidromous mildew which is not obvious sometimes, and hypha can form brown sclerotia with different sizes. Generally causes 10 to 20 percent of yield reduction, reaches 40 to 50 percent of yield reduction in severe years, and greatly influences the development of the tomato industry.
The tomato Rhizoctonia solani is mainly caused by the infection of Rhizoctonia solani of fungi imperfecti subphylum. The bacterium does not produce spores and propagates and multiplies mainly as mycelium. The germs live through the winter in the soil as mycelium or sclerotium. Hyphae can directly invade a host and spread through water flow, farm tools, compost with bacteria and the like. The germs are favored by high temperature and high humidity environment, and the optimum temperature for the onset of the germs is about 20 ℃.
Chemical control is mostly adopted for the traditional control of tomato damping-off. The chemical control effect is good, but the use of chemical pesticides can cause serious environmental pollution, destroy the ecological environment of soil, cause the accumulation of toxic substances and finally cause the reduction of the life quality of human beings. Therefore, the pollution problem can be fundamentally solved only by applying biological control.
The green prevention and control method for preventing and controlling plant diseases by utilizing microorganisms has the characteristics of safety, high efficiency and environmental friendliness, accords with the development direction of future agriculture, and is one of the most ideal prevention and control measures at present. At present, a great deal of research is carried out on antagonistic bacteria for preventing and treating tomato damping-off at home and abroad, and researchers screen and find that some microorganisms such as bacillus subtilis, trichoderma and the like have the effect of inhibiting the growth of pathogenic bacteria. At present, no report of using Brevibacillus laterosporus (Bacillus laterosporus) to control tomato damping-off is found.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the application of the brevibacillus laterosporus MES818 in the aspect of tomato cultivation, the brevibacillus laterosporus MES818 has obvious inhibition effect on the tomato damping-off pathogenic bacterium Rhizoctonia solani, can reduce the occurrence and harm of the tomato damping-off and promote the improvement of the tomato yield and quality.
The invention provides an application of Brevibacillus laterosporus MES818 in tomato cultivation, wherein the Brevibacillus laterosporus MES818 is preserved in China general microbiological culture collection center in 2018, 12 and 17 months, and the preservation number is CGMCC NO. 16859.
As a preferred aspect of the present invention, Brevibacillus laterosporus MES818 can be used for controlling tomato damping-off.
As a preferred aspect of the present invention, Brevibacillus laterosporus MES818 can be used for increasing tomato yield.
As a preferable mode of the invention, the Brevibacillus laterosporus MES818 is used for preventing and treating the tomato damping-off by antagonizing pathogenic bacteria of the tomato damping-off.
The invention also provides an application of the brevibacillus laterosporus MES818 in preparation of a tomato cultivation microbial inoculum, wherein the microbial inoculum comprises the brevibacillus laterosporus MES 818.
As the optimization of the invention, the microbial inoculum is applied to the aspect of preventing and controlling the tomato damping-off.
Preferably, the microbial inoculum is applied to the tomato yield increase aspect.
As a preferred aspect of the present invention, theThe effective viable count CFU in the microbial inoculum is more than or equal to 1.0 × 1010Per gram.
Preferably, the microbial inoculum is diluted by 550-650 times by water and is sprayed on leaves or applied in holes.
Preferably, the microbial inoculum is obtained by sequentially carrying out shake flask culture, primary seed culture, fermentation and concentration on the brevibacillus laterosporus MES 818.
Preferably, the fermentation is ended when the sporulation rate is greater than 90%.
Further preferably, the preparation of the microbial inoculum comprises the steps of: (1) and (3) shake flask culture: performing seed shake flask culture on the Brevibacillus laterosporus MES818 strain activated by the inclined plane at the temperature of 28-32 ℃, and performing shake culture for 18-24h under the condition of rotating speed of 180-220 rpm;
(2) first-order seed culture: culturing strain MES818 activated by test tube slant in an ampoule under the conditions: culturing at 28 deg.C for 24 h;
(3) fermentation: at 50m330L of liquid Brevibacillus laterosporus fermentation culture medium is filled in the expansion tank, the temperature is 121 ℃, moist heat sterilization is carried out for 40min, then cooling is carried out to 32-35 ℃, the first-stage seeds are inoculated in the Brevibacillus laterosporus liquid fermentation culture medium, the pressure of the tank is controlled to be 0.02MPa-0.07MPa, and the culture is carried out for 26h at 35 ℃;
(4) when the microscopic spore formation rate is more than 90%, ending the fermentation;
(5) compounding the fermentation liquor and a stabilizer to prepare a liquid preparation;
(6) concentrating and spray-drying in spray-drying tower to obtain powder with effective viable count CFU not less than 1.0 × 1010The strain/g of Brevibacillus laterosporus product.
The culture media used for preparing the microbial inoculum are as follows:
the shake flask culture medium in the step (1) is a beef extract peptone culture medium, and the components and the using amounts are as follows: 8-10 g of soybean peptone, 2-4 g of beef extract, 4-5 g of sodium chloride, 18-20 g of agar and 1000 ml of ultrapure water, wherein the pH value is 7.0-7.5;
the culture medium in the flask in the step (2) is a broth culture medium, and the culture medium comprises the following components in parts by weight: 8-10 g of soybean peptone, 2-4 g of beef extract, 4-5 g of sodium chloride, 18-20 g of agar and 1000 ml of ultrapure water, wherein the pH value is 7.0-7.5;
the fermentation medium in the step (3) comprises the following components in percentage by weight: 3.5 to 4.5 percent of bean cake powder, 5 to 6 percent of corn flour, 0.3 to 1 percent of cane sugar, 0.3 to 1 percent of glucose, 0.05 to 0.2 percent of sodium chloride, 0.01 to 0.03 percent of manganese sulfate, 0.2 to 0.5 percent of disodium hydrogen phosphate, 0.3 to 0.5 percent of calcium carbonate, 0.02 to 0.05 percent of potassium hydroxide and the pH value of the culture medium is adjusted to 6.0 to 7.5.
The invention has the beneficial effects that: the Brevibacillus laterosporus has obvious inhibition effect on tomato damping-off pathogen Rhizoctonia solani, can alleviate the occurrence and harm of tomato damping-off, and promotes the improvement of tomato yield and quality. The bactericide is used for preventing and treating tomato damping-off, has the prevention and treatment effect of over 80 percent, is green and environment-friendly, has low cost and is convenient to use in a large area. In addition, after the brevibacillus laterosporus bacterial manure is applied, the tomato yield increase amplitude can reach 15%, and meanwhile, the fruit type of the tomato can be improved, so that the main roots are strong, and the fibrous roots are increased.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of the inhibition of tomato damping-off by Brevibacillus laterosporus MES818 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention is further described in detail with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
The application strain in the aspect of tomato cultivation provided by the invention is Bacillus laterosporus MES818, the preservation date of the strain is 2018, 12 and 17 months, the preservation unit name is China general microbiological culture Collection center (CGMCC for short), and the preservation number is as follows: CGMCC No. 16859.
It should be noted that the Brevibacillus laterosporus strain of the present invention has been filed a patent application with application No. 201911381797.3 by the present company.
The brevibacillus laterosporus is a gram-positive bacterium, the cell is rod-shaped, the euscaphis laterosporus is short and oval, the cyst is expanded, and the bacterial colony is round, small, grey white, neat in edge, moist and smooth in surface and semitransparent on a nutrient agar culture medium. The compound microbial inoculum can grow under the anaerobic condition, is positive in catalase and oxidase, can reduce nitrate, decompose casein, is positive in gelatin liquefaction, cannot hydrolyze starch, produces acid in D-mannitol, produces acid in glucose, produces acid in sucrose, produces acid in D-xylose, produces acid in L-arabinose, and cannot grow on a culture medium containing 7% of sodium chloride.
Brevibacillus laterosporus MES818 was isolated from the root soil of kiwi fruit by I R.R. 6 in 2018. Weighing 10 g of soil sample, placing the soil sample into a triangular flask containing 100mL of sterile water, shaking for 30 minutes, sucking 1mL of sterile water, adding the sterile water into a 9mL suction pipe for gradient dilution, drawing a flat plate, culturing at 28 ℃ for 24 hours, picking a single colony to prepare a slant strain, and refrigerating at 4 ℃. The single colony is picked for microscopic examination and then identified as Brevibacillus laterosporus by a strain detection mechanism of department of agriculture, and named as MES 818. The method comprises the steps of taking the separated and preserved Rhizoctonia solani of tomato as a test pathogenic bacterial strain, carrying out a plate confrontation experiment on the separated and preserved Rhizoctonia solani by using brevibacillus laterosporus, and culturing the Rhizoctonia solani in a constant-temperature incubator at 28 ℃ for 5 days to find that the brevibacillus laterosporus can obviously inhibit the growth of the Rhizoctonia solani hyphae and generate a wider bacteriostatic strip.
The Brevibacillus laterosporus MES818 can be applied to the aspect of preventing and controlling tomato damping-off.
The Brevibacillus laterosporus MES818 has antagonism to Rhizoctonia solani, so that the pathogenic bacteria of Rhizoctonia solani can be antagonized to prevent and control the tomato damping off.
The Brevibacillus laterosporus MES818 can be applied to the tomato yield increase aspect.
Based on the antagonism of Brevibacillus laterosporus MES818 to Rhizoctonia solani, the Brevibacillus laterosporus MES818 can also be prepared into a tomato Rhizoctonia solani prevention and treatment agent for preventing and treating tomato damping off. Based on the tomato yield increasing effect of brevibacillus laterosporus MES818, the brevibacillus laterosporus MES818 can be prepared into a tomato yield increasing microbial inoculum for increasing the tomato yield. The microbial agent can use Brevibacillus laterosporus MES818 as a main active ingredient, and can also use Brevibacillus laterosporus MES818 as an auxiliary active ingredient to be matched with other microorganisms or functional ingredients, and the microbial agent has the beneficial effects of Brevibacillus laterosporus MES 818.
When Brevibacillus laterosporus MES818 is prepared into a tomato rhizoctonia solani prevention and control agent or a tomato yield increasing agent, the effective viable count CFU in the agent is more than or equal to 1.0 × 1010Per gram.
When in use, the microbial inoculum is diluted by 550-650 times by water and is sprayed on leaves or applied in holes. The dilution factor may be, for example, but not limited to, 550 times, 560 times, 570 times, 580 times, 590 times, or 600 times.
Example 1 preparation of Brevibacillus laterosporus MES818 bacterial agent for controlling tomato damping-off
(1) And (3) shake flask culture: performing seed shake flask culture on the strain subjected to slant activation, wherein a shake flask culture medium is a beef extract peptone culture medium, and the components and the contents are as follows: 8-10 g of soybean peptone, 2-4 g of beef extract, 4-5 g of sodium chloride, 18-20 g of agar and 1000 ml of ultrapure water, wherein the pH value is 7.0-7.5. Performing shake culture for 18-24h at the temperature of 28-32 ℃ and the rotation speed of 180-220 rpm;
(2) first-order seed culture: culturing Brevibacillus laterosporus MES818 strain activated by test tube slant in an ampoule, wherein the ampoule culture medium is broth culture medium, and the formula of the culture medium is as follows: 8-10 g of soybean peptone, 2-4 g of beef extract, 4-5 g of sodium chloride, 18-20 g of agar and 1000 ml of ultrapure water, wherein the pH value is 7.0-7.5. The culture conditions were: culturing at 28 deg.C for 24 h;
(3) fermentation: at 50m330L of liquid Brevibacillus laterosporus fermentation culture medium is filled in the expansion tank, the temperature is 121 ℃, moist heat sterilization is carried out for 40min, then cooling is carried out to 32-35 ℃, the first-stage seeds are inoculated in the Brevibacillus laterosporus liquid fermentation culture medium, the pressure of the tank is controlled to be 0.02MPa-0.07MPa, and the culture is carried out for 26h at 35 ℃; the raw materials and the dosage of the culture medium used for fermentation are as follows: 3.5-4.5% of bean cake powder, 5-6% of corn flour, 0.3-1% of cane sugar, 0.3-1% of glucose, 0.05-0.2% of sodium chloride, 0.01-0.03% of manganese sulfate, 0.2-0.5% of disodium hydrogen phosphate, 0.3-0.5% of calcium carbonate, 0.02-0.05% of potassium hydroxide and the pH value of a culture medium is adjusted to 6.0-7.5;
(4) when the microscopic spore formation rate is more than 90%, ending the fermentation;
(5) compounding the fermentation liquor and a stabilizer to prepare a liquid preparation;
(6) concentrating and spray-drying in spray-drying tower to obtain powder, detecting and counting, wherein the effective viable Count (CFU) of Brevibacillus laterosporus product is not less than 1.0 × 1010Per gram.
The tail gas generated in the preparation process is discharged after being treated by chemical oxidation, plasma irradiation or ultraviolet irradiation for deodorization, deamination and denitrification or hydrogen sulfide removal.
Example 2 inhibition of tomato damping-off by Brevibacillus laterosporus MES818
Test time and place: 2019 in 5 months in a company research and development laboratory.
(II) test method
(1) Sources of tomato damping-off germs to be tested: the tomato Rhizoctonia solani is collected from field disease plants in Wuqing district in Tianjin city, is separated and purified by company researchers, is identified as Rhizoctonia solani, and the pathogenicity determination shows strong pathogenicity.
(2) Experiment of slab confrontation
The method comprises the steps of firstly inoculating the tomato rhizoctonia solani in the center of a plate of a potato agar culture medium, then dipping a small amount of activated Brevibacillus laterosporus MES818 to inoculate at about 2cm positions on two sides of pathogenic bacteria, and meanwhile, setting a plate only inoculated with the tomato rhizoctonia solani as a blank control. And (3) performing constant-temperature culture at 28 ℃, measuring the control growth amount (colony radius) and the treatment growth amount (bacteriostatic growth radius after MES818 is inoculated) of the tomato rhizoctonia solani when a blank control is about to grow over the whole culture dish, and expressing the antagonistic effect by using the bacteriostatic rate:
(3) results
The inhibition effect of the brevibacillus laterosporus MES818 on the tomato damping-off is shown in figure 1, the inhibition bandwidth of the brevibacillus laterosporus MES818 on the tomato damping-off is 7.9mm, and the inhibition rate is 89.38%. The bacillus laterosporus MES818 has obvious inhibiting effect on tomato damping-off and has biocontrol potential for preventing and treating tomato damping-off.
Example 3 control test of Brevibacillus laterosporus MES818 against tomato damping-off
(I) test agent
(1) Microbial agent
MES818 liquid formulation prepared in example 1 was diluted 600-fold with water.
(2) Chemical agent
50% thiram wettable powder (Shandong Libang Nonghua Co., Ltd.) was diluted 500-fold with water.
(3) Blank control
The treatment with only clear water spray was set as a blank control.
(II) test time and location
In 2019, 6 months and 20 days, the method is carried out in a seedling growing room of Tianjin Kun He Biotechnology group, Ltd.
(III) test method
The tomato seeds are sprouted in a seedling tray after pregermination, seedlings with uniform growth vigor are selected and transplanted into plastic seedling pots with the caliber of 26 cm when the height of the seedlings is about 5 cm, 1 plant is planted in each pot, 5 pots are processed in each pot, and the processing is repeated for 3 times. When about 6-7 true leaves grow out from the seedling, the MES818 liquid preparation prepared in example 1 is diluted 600 times with water, sprayed evenly on the leaves and stems of the plant, and the chemical agent treatment and the blank clear water spraying control treatment are carried out by the same spraying method as MES 818. Inoculating Rhizoctonia solani mycelium blocks on the same leaf positions of each tomato seedling after 3 days, beating the cultured fresh pathogenic bacteria mycelium blocks into uniform-sized mycelium blocks by using a puncher with the diameter of 6mm before inoculation, putting the surface with the mycelium downwards on the leaf surface, and covering a preservative film on the inoculated part for moisture preservation. After two weeks, when obvious disease spots appear in the blank control, the occurrence condition of the tomato damping-off is investigated, the disease level and the number of plants are recorded, and the disease index and the prevention and treatment effect are calculated. Wherein:
the tomato damping off disease is classified according to the following standards:
level 0: the base of the stem has no disease spots;
level 1: the lesion at the base of the stem accounts for less than 1/3 of the whole circumference of the stem;
and 3, level: the lesion spots at the base of the stem account for 1/3-1/2 of the whole circumference of the stem;
and 5, stage: the lesion spots at the base of the stem account for 1/2-3/4 of the whole circumference of the stem;
and 7, stage: the lesion at the base of the stem accounts for more than 3/4 of the whole circumference of the stem;
the calculation method of disease index and prevention and treatment effect comprises the following steps:
wherein, CK0 and CK1 are disease indexes before and after drug administration of the blank control respectively; PT0 and PT1 are disease indexes before and after drug administration of the drug treatment respectively.
(IV) results
The test results are shown in table 1, the disease index (8.89) of the tomato plant treated by the brevibacillus laterosporus MES818 preparation is obviously lower than the disease index (89.64) of the tomato plant treated by a blank control, the difference of the disease index (7.16) of the tomato plant treated by the chemical agent is not obvious, the control effect of the brevibacillus laterosporus MES818 on the tomato damping off is 81.75 percent, and the control effect of the brevibacillus laterosporus MES818 and the microbial agent thereof on the tomato damping off is better and has huge application value.
TABLE 1 control of tomato damping-off by Brevibacillus laterosporus MES818
| Treatment of | Index of disease condition | Control effect (%) |
| MES818 preparation | 8.89b | 81.75 |
| Thiram (thiram) | 7.16bc | 90.60 |
| Blank control | 89.64a | 0.00 |
Example 4 yield enhancement of tomato by Brevibacillus laterosporus MES818
(I) test materials
MES818 liquid formulation prepared in example 1 was diluted 600-fold with water.
(II) test method
The experiment has 3 treatment modes, namely a treatment mode 1, namely diluting the MES818 liquid preparation prepared in example 1 by 600 times with water and spraying the MES818 liquid preparation on leaf surfaces, a treatment mode 2, diluting the MES818 liquid preparation prepared in example 1 by 600 times with water and performing hole application, a treatment mode 3, spraying clear water for comparison, 3 times of repetition and random block arrangement are arranged in the experiment, and the area of each treatment cell is 3 × 3m2. Digging holes for watering or spraying in the seedling stage of the tomatoes.
(III) statistical method
Counting the yield of each treated tomato, and calculating the yield increase rate, wherein the formula for calculating the yield increase rate of the tomato is as follows:
(IV) results
The effect of Brevibacillus laterosporus MES818 on the increase in tomato damping-off is shown in Table 2. As can be seen from the results in table 2, the tomato yields of both treatments with the bacillus laterosporus agent (treatment 1 and treatment 2) were higher than the control without the agent (treatment 3), and the hole application effect was better than the spray treatment effect.
TABLE 2 increase in tomato damping-off effect of Brevibacillus laterosporus MES818
| Treatment method | Yield (kg/667 m)2) | Yield increase (%) |
| 1 | 2164 | 10.4 |
| 2 | 2258 | 15.1 |
| 3 | 1961 | - |
The present invention has been described in detail with reference to the examples, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Claims (10)
1. The application of Brevibacillus laterosporus MES818 in tomato cultivation is characterized in that the Brevibacillus laterosporus MES818 is preserved in the China general microbiological culture collection center in 2018, 12 and 17 months, and the preservation number is CGMCC NO. 16859.
2. The use according to claim 1, characterized in that Brevibacillus laterosporus MES818 is used for controlling tomato damping-off.
3. The use according to claim 1, characterized in that Brevibacillus laterosporus MES818 is used in tomato production enhancement.
4. The use of Brevibacillus laterosporus MES818 in the preparation of a tomato cultivation inoculum as set forth in claim 1, wherein the inoculum comprises Brevibacillus laterosporus MES 818.
5. The use of claim 4, wherein the microbial inoculum is used for controlling tomato damping-off.
6. The use according to claim 4, characterized in that the fungicide is used in tomato stimulation.
7. The use of any one of claims 4-6, wherein the effective viable count CFU in the microbial inoculum is more than or equal to 1.0 × 1010Per gram.
8. The use as claimed in claim 7, wherein the microbial inoculum is diluted 550-650 times with water for foliar spraying or hole application.
9. The application of claim 7, wherein the agent is obtained by sequentially performing shake flask culture, first-order seed culture, fermentation and concentration on Brevibacillus laterosporus MES 818.
10. Use according to claim 9, characterized in that the fermentation is ended when the sporulation rate is greater than 90%.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480197A (en) * | 2022-02-09 | 2022-05-13 | 山东农业大学 | Brevibacillus laterosporus, microbial inoculum and application thereof in biological pesticide fertilizer |
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| CN111748496B (en) | 2022-05-13 |
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