CN111602676B - Application of bacillus subtilis MES810 in prevention and treatment of grape southern blight root rot - Google Patents
Application of bacillus subtilis MES810 in prevention and treatment of grape southern blight root rot Download PDFInfo
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Abstract
The invention belongs to the technical field of prevention and treatment of soil-borne plant diseases by using microorganisms, and particularly relates to application of Bacillus subtilis MES810 in prevention and treatment of grape southern blight, wherein the Bacillus subtilis MES810 is a strain which is preserved in China general microbiological culture collection center in 8, 10 and 2017 and has a preservation number of CGMCC No. 14514. The invention also provides application of the bacillus subtilis MES810 in grape southern blight root rot. The invention has the beneficial effects that: the microbial inoculum has obvious inhibiting effect on pathogenic bacteria of the grape southern blight, has obvious preventing and treating effect when used for preventing and treating the grape southern blight, is green and environment-friendly, has low cost and is convenient to use in a large area.
Description
Technical Field
The invention belongs to the technical field of prevention and treatment of soil-borne plant diseases by using microorganisms, and particularly relates to application of bacillus subtilis MES810 in prevention and treatment of grape southern blight.
Background
The grape root rot is a serious disease of the whole plant, can cause death of the whole plant, and seriously hinders the development of the grape industry. The grape root rot disease can be caused by a plurality of diseases, mainly comprising white streak feather, purple streak feather, southern blight, root rot, round spot root rot and the like. Wherein the Sclerotium vitis is caused by Sclerotium rolfsii Sacc which is a fungus of Deuteromycotina class. The majority of pathogenic bacteria are weak parasitic bacteria, and the time from a few roots getting sick to the main root rotting to the overground part of the plant showing symptoms is one year to several years. The grape southern blight mainly occurs at the root neck of seedlings and young trees, and can also damage leaves and fruits. The disease occurs on the overground part, leaves are small and yellow, brown disease spots are formed on the overground part, the internodes of the branch tips are shortened, and fruits are large and small. When the disease is serious, branches and leaves are withered, and when the disease spot encircles tendrils for one week, the branches and leaves die, and sclerotium grows out. Root and neck affection, brown necrosis of the cortex, severe rot, and overflow of brown sap. In the later period, a plurality of rape seed-like sclerotia which is white at first, brown gradually and black at later period are generated in the diseased part and the surrounding soil.
The prevention and control of the grape southern blight are usually realized by traditional methods such as increasing organic fertilizer application, adjusting soil acidity and alkalinity, sterilizing seedlings, strengthening cultivation management and preventing and controlling medicaments such as amobam, dike pine and shrub roots. The traditional method has complex operation, the chemical agent takes effect quickly, but the drug effect is not lasting, the environment is polluted, and the method does not accord with the development direction of green prevention and control and ecological agriculture.
The microbial control of plant soil-borne diseases is one of the most effective control measures, the microbial control of plant diseases has the advantages of safety, effectiveness and no environmental pollution, accords with the development direction of green control of agriculture in the future, and no report of using bacillus subtilis (B.subtilis) to control grape southern blight is found through retrieval at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the application of the bacillus subtilis MES810 in the prevention and treatment of the southern blight of grapes, the bacillus subtilis MES810 has an obvious inhibiting effect on pathogenic bacteria of the southern blight of grapes, the microbial inoculum is used for preventing and treating the southern blight of grapes, the prevention and treatment effect is obvious, and the microbial inoculum is green, environment-friendly, low in cost and convenient to use in a large area.
In order to achieve the purpose, the invention provides application of Bacillus subtilis MES810 in prevention and control of grape southern blight, wherein the Bacillus subtilis MES810 is preserved in the China general microbiological culture collection center in 2017, 8, 10 and has a preservation number of CGMCC No. 14514.
Preferably, the bacillus subtilis MES810 is used for preventing and treating the grape southern blight by antagonizing pathogenic bacteria of the grape southern blight.
Further preferably, the bacillus subtilis MES810 controls the grape southern blight by antagonizing Sclerotium rolfsii Sacc (Sclerotium rolfsii Sacc).
The invention also provides an application of the bacillus subtilis MES810 in preparation of a microbial inoculum for preventing and treating the grape southern blight, wherein the microbial inoculum comprises the bacillus subtilis MES 810.
Preferably, the effective viable count CFU in the microbial inoculum is more than or equal to 2.0 multiplied by 1010Per gram.
Preferably, the dosage of the microbial inoculum is 2-5 kg/mu.
Further preferably, the microbial inoculum is directly applied in a liquid form by drip irrigation, micro-spraying and flushing, or is applied in a mode of diluting the microbial inoculum by water by 800 times through furrow application, hole application and root irrigation, or is directly applied in a solid form by furrow application, hole application and fertilizer base application.
As a preferable selection of the invention, the bacillus subtilis MES810 is subjected to shake flask culture, first-level seed culture and fermentation in sequence to obtain the microbial inoculum.
Further preferably, the fermentation is terminated when the sporulation rate is greater than 90%.
Further preferably, the preparation method of the bacillus subtilis microbial inoculum comprises the following steps:
(1) and (3) shake flask culture: carrying out seed shake flask culture on the strain activated by the inclined plane, wherein the culture conditions are as follows: the rotation speed is 200-;
(2) first-order seed culture: 100mL of first-level seed culture medium is filled in a 500mL eggplant bottle, moist heat sterilization is carried out for 20-35min at the temperature of 116-121 ℃, 10mL of bacillus subtilis viable bacteria freeze-drying tube strain is inoculated in the first-level seed culture medium, and the first-level seed culture medium is cultured for 10-12h at the temperature of 30-37 ℃;
(3) secondary seed culture: 150L-200L of secondary seed culture medium is filled in a 300L fermentation tank, moist heat sterilization is carried out for 20-40min at the temperature of 115-122 ℃, and 100-300mL of primary seeds are inoculated in the secondary seed culture medium; culturing at 30-38 deg.C for 8-12 h;
(4) fermentation: at 5m3Or 30m3The expansion fermentation tank is filled with 4000L or 20m3Sterilizing the live bacillus subtilis fermentation culture medium at 115-122 deg.C for 20-40min, inoculating 150L or 200L of secondary seeds into the live bacillus subtilis fermentation culture medium, controlling the pressure of the tank to 0.02-0.07 Mpa, and culturing at 30-38 deg.C for 24-36 h;
(5) ending fermentation when the spore formation rate is more than or equal to 90%;
(6) compounding the fermentation liquid with stabilizer to obtain liquid preparation containing Bacillus subtilis product cFu not less than 2.0 × 1010Per gram.
The culture medium applicable to the preparation method comprises the following steps:
the shake flask culture medium used in the step (1) is a beef extract peptone culture medium, and the raw materials and the using amount are as follows: 0.5-3% of beef extract, 0.5-3% of peptone, 0.2-1% of sodium chloride and 6.0-7.5% of culture medium pH;
the raw materials and the using amount of the culture medium used for the first-stage seed culture in the step (2) are as follows: 0.5-3% of beef extract, 0.5-3% of peptone, 0.2-1% of sodium chloride, 1.5-2.0% of nutrient agar and 6.0-7.5% of culture medium pH;
the culture medium used for the secondary seed culture in the step (3) comprises the following raw materials in parts by weight: 0.3-2% of glucose, 1-5% of corn flour, 1-5% of soybean meal powder, 0.1-0.5% of disodium hydrogen phosphate, 0.5-5% of sodium dihydrogen phosphate, 0.2-2% of defoaming agent and the pH value of the culture medium is adjusted to 6.0-7.5;
the raw materials and the dosage of the culture medium used for fermentation in the step (4) are as follows: 0.3 to 2 percent of glucose, 1 to 5 percent of corn flour, 1 to 5 percent of soybean meal, 0.1 to 0.5 per mill of disodium hydrogen phosphate, 0.5 to 5 per mill of sodium dihydrogen phosphate, 0.2 to 2 per mill of antifoaming agent, and the pH value of the culture medium is adjusted to 6.0 to 7.5.
The invention has the beneficial effects that: the bacillus subtilis has strong inhibition effect on pathogenic bacteria of sclerotinia rolfsii (Sacc) which is a fungi of deuteromycotina, can prevent the occurrence of grape southern blight, and promotes the yield and income of grapes.
The bacillus subtilis is prepared into a microbial inoculum for preventing and treating grape southern blight, has remarkable prevention and treatment effect, achieves the prevention effect of more than 60 percent, is green and environment-friendly, has low cost and is convenient to use in a large area. The liquid microbial inoculum meets the integrated requirements of water and fertilizer, is convenient for drip irrigation, micro-spraying and flushing application, and can also be diluted by 800 times and then applied in furrow, hole and root, and the dosage per mu is 2-5 kg. The solid microbial inoculum can be applied in furrow or hole, mixed with fertilizer and applied on base, and the dosage per mu is 2-5 kg.
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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 diagram showing the bacteriostatic effect of Bacillus subtilis MES810 on southern blight of grapes.
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 technical scheme adopted by the invention for solving the technical problems is as follows:
the invention provides an application of Bacillus subtilis MES810 in grape southern blight root rot, the strain is Bacillus subtilis MES810, the preservation date of the strain is 2017, 8 and 10 days, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC No. 14514.
It should be noted that the Bacillus subtilis strain of the present invention has been filed in patent application No. 201710862006.3 by the present company.
The Bacillus subtilis is a gram-positive aerobic bacterium, has a single cell size of 0.7-0.8 × 2-3 μm, and is uniformly colored. Without capsule, the perigenic flagellum can move. The spore is 0.6-0.9 × 1.0-1.5 μm, oval to columnar, located in the center or slightly off-center of the thallus, and the thallus does not expand after spore formation. The colony surface is rough and opaque, and is dirty white or yellowish, and when the colony grows in a liquid culture medium, the skin becomes always formed. Tryptophan can be decomposed to form indole by using protein, various sugars and starch.
The strain of bacillus subtilis MES810 is separated from Zhang Jiakou grape field by developers in 6 months 2015. After plate streaking, culturing at 32 deg.c for 24 hr, picking single colony to form slant strain, and cold storing at 4 deg.c. The separated sclerotium rolfsii is taken as a target, a flat plate confrontation experiment is carried out on the sclerotium rolfsii by using the sclerotium rolfsii, the sclerotium rolfsii is cultured at the temperature of 30 ℃, after 4 days, the sclerotium rolfsii is found to be capable of obviously inhibiting the growth of pathogenic bacteria, a wider antibacterial zone appears and is named as MES810, and then the MES810 is identified by a strain detection mechanism of the department of agriculture, and is the sclerotium rolfsii.
The bacillus subtilis MES810 disclosed by the invention can be used for preventing and treating the grape southern blight by antagonizing pathogenic bacteria of the grape southern blight.
The pathogenic bacteria of the grape Sclerotium rolfsii root rot comprise Sclerotium rolfsii Sacc, and the bacillus subtilis MES810 disclosed by the invention can prevent and treat the grape Sclerotium rolfsii root rot by antagonizing the Sclerotium rolfsii Sacc.
Because the bacillus subtilis MES810 aligns the Sclerotium rolfsii Sacc and has antagonistic action, the bacillus subtilis MES810 can be prepared into a microbial inoculum for preventing and treating the grape southern blight, and the microbial inoculum comprises the bacillus subtilis MES 810. The bacillus subtilis MES810 can be used as a main active ingredient, and the bacillus subtilis MES810 can also be used as an auxiliary active ingredient to be matched with other microorganisms or functional ingredients, so that the bacillus subtilis MES810 has the efficacy.
In the microbial inoculum containing the bacillus subtilis MES810 for preventing and treating the grape southern blight, the effective viable count CFU in the microbial inoculum is required to be more than or equal to 2.0 multiplied by 1010Per gram.
When the microbial inoculum is used, the microbial inoculum is directly applied in a liquid form by drip irrigation, micro-spraying and flushing, or the microbial inoculum is applied in a mode of diluting the microbial inoculum by water by 800 times and applying the microbial inoculum by furrow application, hole application and root irrigation, or the microbial inoculum is concentrated and sprayed to dryness and is directly applied in a solid form by furrow application, hole application and fertilizer mixing base application, wherein the dosage of the microbial inoculum is 2-5 kg/mu.
The bacillus subtilis MES810 is subjected to shake flask culture, primary seed culture, fermentation and concentration in sequence to obtain the microbial inoculum. Preferably, the fermentation is ended when the sporulation rate is greater than 90%.
Example 1 preparation of Bacillus subtilis MES810 microbial inoculum
The preparation method of the bacillus subtilis MES810 microbial agent comprises the following steps:
(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 formula of the beef extract peptone culture medium is as follows: 0.5-3% of beef extract, 0.5-3% of peptone, 0.2-1% of sodium chloride and 6.0-7.5% of culture medium pH. The culture conditions were: the rotation speed is 200-;
(2) first-order seed culture: 100mL of first-level seed culture medium is filled in a 500mL eggplant bottle, moist heat sterilization is carried out for 20-35min at the temperature of 116-121 ℃, 10mL of bacillus subtilis viable bacteria freeze-drying tube strain is inoculated in the first-level seed culture medium, and the first-level seed culture medium is cultured for 10-12h at the temperature of 30-37 ℃; the raw materials and the dosage of the culture medium used for the first-stage seed culture are as follows: 0.5-3% of beef extract, 0.5-3% of peptone, 0.2-1% of sodium chloride, 1.5-2.0% of nutrient agar and 6.0-7.5% of culture medium pH;
(3) secondary seed culture: 150L-200L of secondary seed culture medium is filled in a 300L fermentation tank, moist heat sterilization is carried out for 20-40min at the temperature of 115-122 ℃, and 100-300mL of primary seeds are inoculated in the secondary seed culture medium; culturing at 30-38 deg.C for 8-12 h; the raw materials and dosage of the culture medium used for secondary seed culture are 0.3-2% of glucose, 1-5% of corn flour, 1-5% of soybean meal, 0.1-0.5 per mill of disodium hydrogen phosphate, 0.5-5 per mill of sodium dihydrogen phosphate, 0.2-2 per mill of antifoaming agent, and the pH value of the culture medium is adjusted to 6.0-7.5;
(4) fermentation: at 5m3Or 30m3The expansion fermentation tank is filled with 4000L or 20m3Sterilizing the live bacillus subtilis fermentation culture medium at 115-122 deg.C for 20-40min, inoculating 150L or 200L of secondary seeds into the live bacillus subtilis fermentation culture medium, controlling the pressure of the tank to 0.02-0.07 Mpa, and culturing at 30-38 deg.C for 24-36 h; the raw materials and the dosage of the culture medium used for fermentation are as follows: 0.3-2% of glucose, 1-5% of corn flour, 1-5% of soybean meal powder, 0.1-0.5% of disodium hydrogen phosphate, 0.5-5% of sodium dihydrogen phosphate, 0.2-2% of defoaming agent and the pH value of the culture medium is adjusted to 6.0-7.5;
(5) ending fermentation when the spore formation rate is more than or equal to 90%;
(6) compounding the fermentation liquor and a stabilizer to prepare a liquid preparation; the obtained Bacillus subtilis product cfu is not less than 2.0 × 1010And (4) respectively.
If necessary, the obtained liquid preparation can be concentrated and sprayed to obtain a solid microbial inoculum product.
The tail gas generated in the preparation process is treated by chemical oxidation, plasma irradiation or ultraviolet irradiation for removing odor, deamination and denitrification or hydrogen sulfide removal and then discharged.
Example 2 antagonistic action test of Bacillus subtilis MES810 on southern grape disease
Test time and place: 2019, month 2, in a laboratory in a company research and development center.
(II) test method:
(1) the tested grape southern blight pathogen sources: the grape southern blight is collected from grape southern blight strain in Wuqing district of Tianjin, and is separated and purified by company researchers, and the strain is identified as Sclerotium rolfsii Sacc through 16 SrDNA. The pathogenicity assay is manifested as weak pathogenicity.
(2) Plate confrontation experiment:
firstly, grape germs are in the center of an LB flat plate, then activated bacillus subtilis MES810 is spotted at a position 2.0 cm away from an indicator fungus piece, and a blank control is arranged. And (3) carrying out constant-temperature culture at 37 ℃, measuring the control growth amount (colony radius) and the treatment growth amount (growth inhibition radius after MES810 inoculation) of the sclerotium rolfsii when a blank control is about to grow over the whole culture dish, and expressing the antagonistic action bacteriostasis rate as follows: bacteriostatic ratio (%) (control growth amount-treated growth amount)/control growth amount × 100.
(3) Results the bacteriostatic effect of bacillus subtilis MES810 on grapevine southern blight is shown in fig. 1, and the antagonistic results are shown in table 1. As can be seen from FIG. 1 and Table 1, the inhibition rate of Bacillus subtilis MES810 on southern grape blight reaches 89.4%; the transparent bacteriostatic bandwidth is 8.0 mm. The bacillus subtilis MES810 has obvious inhibition effect on grape southern blight and biological control potential for preventing and treating the grape southern blight.
TABLE 1 antagonistic results of Bacillus subtilis MES810 on southern grape disease
| Strain name | Control growth volume (mm) | Treatment of growth (mm) | Bacteriostatic ratio (%) |
| MES810 | 38.0 | 4.0 | 89.4 |
Example 3 test of controlling Effect of Bacillus subtilis MES810 on southern blight of grape
(I) test agent
(1) And (3) microbial agent: MES810 liquid preparation prepared in example 1 was diluted 300 times, 500 times and 800 times with water, respectively;
(2) chemical agents: 50% amobam (chemical industry of Jinan Yongtai) diluted 500 times with water.
(3) Blank control: and (4) clear water.
(II) test time and place: the method is carried out in the Wuqing grape planting base in 2019 and 4 months;
(III) test method: the number of the plants with the grape southern blight and the disease index of each plant are counted before the test, an annular groove with the width of 15 centimeters and the depth of 20 centimeters is dug at the periphery of the position 20 centimeters away from the root of each grape with the grape southern blight, and each treatment is carried out on 5 plants, and the steps are repeated three times. Selecting plants by a random block arrangement method. 3L of MES810 liquid preparations diluted 300 times, 500 times and 800 times in example 1 are respectively and uniformly poured into annular grooves at the roots of grapes, chemical agent control and blank control are treated by clear water in the same way, and soil is sealed after the treatment is finished. And observing the disease condition of the grape plants after 20 days, and calculating the disease index and the prevention and treatment effect.
Grading the disease condition standard:
level 0: the diseased leaves of the withered and yellow diseased branches are not seen;
level 1: the proportion of infected withered and yellow branches and leaves accounts for 25 percent;
and 3, level: the proportion of infected withered and yellow branches and leaves accounts for 25-50%;
and 5, stage: the ratio of infected withered branches and leaves is more than 50 percent, but the plants are not withered;
and 7, stage: the plants died.
The results of the comparative test results of the bacillus subtilis MES810 on the control effect of the southern blight of the grapes are shown in the table 2, and it can be seen from the table 2 that after the grape plants are treated by the bacillus subtilis MES810 liquid preparation, the disease index (9.18-9.43) is obviously lower than the disease index (89.82) after blank control treatment, and is not significantly different from the disease index (7.46) after chemical agent treatment, and the control effect of the M ES810 on the southern blight of the grapes is more than 89%, which shows that the bacillus subtilis MES810 and the microbial agent thereof have good control effect on the southern blight of the grapes and have the potential of large-area popularization and application.
TABLE 2 control of the control of southern blight by Bacillus subtilis MES810
| Treatment of | Index of disease condition | Control effect (%) |
| 300 times of MES810 liquid preparation | 9.18b | 89.78 |
| 500 times of MES810 liquid preparation | 9.25b | 89.69 |
| 800 times of MES810 liquid preparation | 9.43b | 89.50 |
| Chemical agent | 7.46bc | 91.69 |
| Blank control | 89.82a | 0.00 |
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 (7)
1. Application of bacillus subtilis MES810 in prevention and treatment of grape southern blight, which is characterized in that the bacillus subtilis MES810 prevents and treats the grape southern blight by antagonizing pathogenic bacteria of the grape southern blight, and the pathogenic bacteria of the grape southern blight are sclerotium rolfsii (Sclerotinia sclerotiorum (C.))Sclerotium rolfsii Sacc）；
The Bacillus subtilis (A), (B) and (C)Bacillus subtilis) MES810 has been deposited in the China general microbiological culture Collection center in 2017, 8, 10 and has a preservation number of CGMCC No. 14514.
2. The application of bacillus subtilis MES810 in preparing a microbial inoculum for preventing and treating grape southern blight is characterized in that the microbial inoculum comprises bacillus subtilis MES 810;
the bacillus subtilis MES810 is used for preventing and treating grape southern blight by antagonizing pathogenic bacteria of grape southern blight, and the pathogenic bacteria of the grape southern blight are sclerotium rolfsii (sclerotium rolfsii)Sclerotium rolfsii Sacc）；
The Bacillus subtilis (A), (B) and (C)Bacillus subtilis) MES810 has been deposited in the China general microbiological culture Collection center in 2017, 8, 10 and has a preservation number of CGMCC No. 14514.
3. The use of claim 2, wherein the effective viable count CFU in the microbial inoculum is more than or equal to 2.0 x 1010Per gram.
4. The use according to claim 3, characterized in that the amount of the microbial inoculum is 2-5 kg/acre.
5. The use according to claim 4, characterized in that the microbial inoculum is applied in liquid form by direct drip irrigation, micro-spraying and flushing, or is applied in a mode of diluting the microbial inoculum with water by 800 times through furrow application, hole application and root irrigation, or is applied in a mode of directly mixing the microbial inoculum in solid form through furrow application and hole application with fertilizer base application.
6. The use of any one of claims 2-5, wherein the Bacillus subtilis MES810 is subjected to shake flask culture, primary seed culture, and fermentation sequentially to obtain the microbial inoculum.
7. Use according to claim 6, characterized in that the fermentation is ended when the sporulation rate is greater than 90%.
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