CN116121123B - Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping - Google Patents
Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping Download PDFInfo
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- CN116121123B CN116121123B CN202211578485.3A CN202211578485A CN116121123B CN 116121123 B CN116121123 B CN 116121123B CN 202211578485 A CN202211578485 A CN 202211578485A CN 116121123 B CN116121123 B CN 116121123B
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- 235000009754 Vitis X bourquina Nutrition 0.000 title claims abstract description 48
- 235000012333 Vitis X labruscana Nutrition 0.000 title claims abstract description 48
- 235000014787 Vitis vinifera Nutrition 0.000 title claims abstract description 48
- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 44
- 241000219095 Vitis Species 0.000 title abstract description 47
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- 239000003899 bactericide agent Substances 0.000 claims abstract description 28
- 239000003814 drug Substances 0.000 claims abstract description 25
- 229940079593 drug Drugs 0.000 claims abstract description 25
- 241000123650 Botrytis cinerea Species 0.000 claims abstract description 20
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- RJGDLRCDCYRQOQ-UHFFFAOYSA-N anthrone Chemical compound C1=CC=C2C(=O)C3=CC=CC=C3CC2=C1 RJGDLRCDCYRQOQ-UHFFFAOYSA-N 0.000 description 1
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- ONUFESLQCSAYKA-UHFFFAOYSA-N iprodione Chemical compound O=C1N(C(=O)NC(C)C)CC(=O)N1C1=CC(Cl)=CC(Cl)=C1 ONUFESLQCSAYKA-UHFFFAOYSA-N 0.000 description 1
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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
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
- A01N43/40—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom six-membered rings
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
- A01N43/54—1,3-Diazines; Hydrogenated 1,3-diazines
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P3/00—Fungicides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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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
-
- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses bacillus belicus and application thereof in grape gray mold and grape fresh-keeping, and belongs to the technical field of microorganisms. The bacillus belicus of the invention is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of No. M20221278 in the year 2022 and 8 and 15. The bacillus bailii disclosed by the invention has stronger tolerance to pH and UV, has a control effect on multi-drug resistant grape gray mold and can realize the fresh-keeping effect of foods. Therefore, the bacillus belicus can be prepared into a microbial preparation, and the microbial preparation can be applied to the fields of germ prevention and control, food preservation and the like, and has wide application prospect. Meanwhile, the bacillus belicus also has strong tolerance to the chemical bactericide, so that the bacillus belicus and the chemical bactericide can be used for preventing and controlling multi-drug-resistant botrytis cinerea together, thereby reducing the pesticide application amount.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus beijerinus and application thereof in grape gray mold and grape fresh-keeping.
Background
Grape (grape vinifera L.) is one of main cash crops in China, the existing variety is 700 or more, and the planting area is more than 7.0X10 5 hm 2 The fresh food and the brewing production of the rice become one of important industries for driving the development of agriculture and rural economy in China. However, the occurrence of grape diseases has been increased year by year, and the characteristic of the grape itself that is not storage-stable has become a central factor for limiting the yield and quality of grapes. Among them, grape gray mold is one of the most common and serious diseases in grape production.
The etiology of Botrytis cinerea (Botrytis cinerea) is that can infect grape inflorescences, leaves and young fruits, and also endanger mature fruits, thereby endangering the whole industrial chain of grapes from planting to fruit storage, causing significant economic losses. In the grape planting stage, chemical pesticides of different structural types have been used as the principal force for grape gray mold control, wherein representative chemical pesticides include carbendazim and iprodione which are widely used in early stages, and boscalid and azoxystrobin which are popularized and used in recent years. Although the chemical pesticide can better prevent and control the grape gray mold, the chemical pesticide is used in excess and the pesticide residue exceeds the standard vicious circle because the action mechanism of the chemical pesticide is relatively single and the genetic variability of the grape gray mold is strong, so that the field resistant strain is continuously appeared. In the grape storage stage, the grape gray mold prevention and control and chemical pesticide use contradiction are more prominent due to the harsher requirements on pesticide residues.
In comparison with chemical bactericides, the search for potential strains from microorganisms for controlling plant diseases has become one of the important directions for the development of future green pesticides. At present, bacteria such as bacillus and streptomyces and partial strains of fungi such as trichoderma and penicillium have been successfully developed into commercial bactericides. The microbial agent has the advantages of good degradability, high safety, difficult generation of drug resistance and the like, and has important significance for developing environment-friendly ecological agriculture.
Disclosure of Invention
The invention focuses on a plurality of 'neck clamping' problems in the grape production and storage process: (1) The problems that chemical bactericides are difficult to control and the risk of pesticide residues is increased due to frequent occurrence of multi-drug resistant grape gray mold; (2) The problem of difficult storage and preservation of the grape caused by double superposition of the storage intolerance characteristic of the grape and the gray mold infection of the multi-drug resistant grape.
Based on the problems, the invention provides the following technical scheme:
the invention provides bacillus beleiensis NH-13-5, which is separated from suaeda salsa rhizosphere soil collected in a national level natural protection area of Shandong yellow river delta, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of No. M20221278 in 8 months and 15 days of 2022.
The bacillus belicus has proved to have the effect of preventing and treating plant diseases, and particularly has remarkable preventing and treating effects on multi-drug resistant grape gray mold and plant diseases caused by the gray mold. Therefore, the invention provides the application of the bacillus belicus in preventing and/or treating the multi-drug resistant botrytis cinerea and/or plant diseases caused by the multi-drug resistant botrytis cinerea; in this application, the purpose of prevention and treatment may be diagnostic or non-diagnostic. In particular, the invention provides application of the bacillus beijerinus in preparing a microbial preparation for preventing and/or treating multi-drug resistant botrytis cinerea and/or plant diseases caused by the multi-drug resistant botrytis cinerea.
The invention further provides application of the bacillus belicus in food fresh-keeping. Such foods include, but are not limited to, fruits, vegetables, and foodstuffs; further preferred is grape.
The bacillus beliae has better chemical bactericide tolerance, and based on the bacillus beliae, the invention provides the application of the bacillus beliae in a chemical bactericide application environment; such applications include, but are not limited to, the following: (1) When the chemical bactericide is applied, the bacillus belicus is applied to improve the microenvironment of the grape planting soil, so that the problems of dysbacteriosis, plant pathogenic bacteria resistance enhancement and the like caused by excessive application of the chemical bactericide are prevented; (2) The bacillus belicus is used for preventing and controlling the multi-drug resistant botrytis cinerea together with a chemical bactericide, namely the bacillus belicus is used for inhibiting the multi-drug resistant botrytis cinerea and/or plant diseases caused by the multi-drug resistant botrytis cinerea together with the chemical bactericide. The invention further provides application of the bacillus belicus in preparing bactericides for inhibiting multi-drug resistant botrytis cinerea and/or plant diseases caused by the multi-drug resistant botrytis cinerea in combination with chemical bactericides.
The chemical bactericide is one or more selected from boscalid and azoxystrobin.
The invention provides a multi-drug resistant grape gray mold combined bactericide, which comprises a chemical bactericide and bacillus bailii; the chemical bactericide is selected from one or more of boscalid and azoxystrobin.
The bacillus beleiensis is proved to have stronger pH and UV tolerance, and based on the bacillus beleiensis, the invention provides application of the bacillus beleiensis in inhibiting multi-drug resistant botrytis cinerea and/or plant diseases caused by the multi-drug resistant botrytis cinerea in combination with UV.
The invention provides a microbial preparation comprising the bacillus bailii. The microbial preparation can be used for preventing and treating multi-drug resistant grape gray mold bacteria and/or plant diseases caused by the same.
The microbial preparation includes, but is not limited to, solid or liquid preparations such as powders or suspensions. If the bacillus subtilis is a suspending agent, the bacterial content of the bacillus subtilis is more than or equal to 10 8 CFU/mL。
The invention provides a fermentation culture of the bacillus beijerinus.
The preparation method of the fermentation culture comprises the following steps:
fermenting the bacillus beljalis by an LB culture medium for 2-3 d to obtain a fermentation culture; the LB culture medium is selected from the following components: 1% tryptone, 0.5% yeast extract, 1% NaCl, distilled water, pH was adjusted to 7.0.
In the fermentation culture, the bacterial content of the bacillus beijerinus is more than or equal to 10 8 CFU/mL。
The beneficial effects of the invention are as follows:
the bacillus bailii disclosed by the invention has stronger tolerance to pH and UV, has a control effect on multi-drug resistant grape gray mold and can realize the fresh-keeping effect of foods. Therefore, the bacillus belicus can be prepared into a microbial preparation, and the microbial preparation can be applied to the fields of germ prevention and control, food preservation and the like, and has wide application prospect. Meanwhile, the bacillus belicus also has strong tolerance to the chemical bactericide, so that the bacillus belicus and the chemical bactericide can be used for preventing and controlling multi-drug-resistant botrytis cinerea together, thereby reducing the pesticide application amount.
Drawings
FIG. 1 is a colony morphology of strain NH-13-5;
FIG. 2 is a phylogenetic tree of strain NH-13-5 constructed based on the 16S rDNA gene;
FIG. 3 shows the resistance of strain NH-13-5 to chemical fungicides;
FIG. 4 shows the pH and UV resistance of strain NH-13-5;
FIG. 5 shows the effect of strain NH-13-5 on the prevention of multi-drug resistant grape gray mold;
FIG. 6 shows the therapeutic effect of strain NH-13-5 on multidrug-resistant grape gray mold;
FIG. 7 shows the preservation effect of strain NH-13-5 on grape fruits;
FIG. 8 shows the safety evaluation results of strain NH-13-5.
Detailed Description
Separating and identifying strains:
the strain is separated from suaeda salsa rhizosphere soil collected in a national level natural protection area of Shandong yellow river delta, and is obtained by separating and purifying by a dilution flat plate coating and a flat plate dilution scribing method, and the specific steps are as follows: placing suaeda salsa rhizosphere soil in an oven, and drying residual moisture at 45 ℃; taking 10g of soil sample to a 250mL Erlenmeyer flask, adding 90mL of sterile water, and shake culturing at 28 ℃ and 180rpm for 30min to obtain a soil suspension mother solution, and sequentially diluting by 10 times and 100 times; respectively sucking 200 mu L of the diluent, uniformly coating the diluent on the surface of an LB plate prepared from artificial seawater (3% sea salt aqueous solution), and arranging three parallel gradients; standing and culturing the flat plate at 28 ℃, picking out bacterial colonies with different forms on the surface of a culture medium, scribing and separating on an LB flat plate prepared from artificial seawater, and observing the growth condition of the bacterial colonies at regular time; and (3) separating and purifying the strain by adopting a flat dilution streaking method, and preserving for standby, wherein the strain number is NH-13-5.
Strains NH-13-5 were streaked onto LB plates and incubated at 28℃for 2d, with colony morphology as shown in FIG. 1. The surface of the colony is rough, the center of the colony is raised, and the edge of the colony is wrinkled and yellowish.
The 16S rDNA gene sequence of the strain NH-13-5 is determined as follows:
the sequence information was compared with the corresponding sequence information of known strains in the Genbank database, and a phylogenetic tree was constructed, as shown in FIG. 2, in which the strain was the most homologous (99.65%) to strain Bacillus velezensis CBMB (Accession number: NR 116240.1) and was in the same clade, thus identifying Bacillus bailii (Bacillus velezensis). The strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20221278 at 8 and 15 of 2022.
Other terms used in the present invention, unless otherwise indicated, generally have meanings commonly understood by those of ordinary skill in the art. The invention will be described in further detail below in connection with specific embodiments and with reference to the data. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
Chemical sterilant resistance test:
commercial boscalid and azoxystrobin stock (98%) were selected as test chemicals and purchased from Shanghai leaf Biotechnology Co. The specific test steps are as follows:
the boscalid and azoxystrobin raw materials with certain mass are weighed and mixed with a certain volume of PDA culture medium at 45 ℃ to obtain mixed culture media with the concentration of 800mg/L, 400mg/L and 200mg/L respectively. The mixed culture media with different concentrations are respectively poured into a quarter-cell culture dish (with the diameter of 9 cm), and PDA culture media are used as a control. The strain NH-13-5 is respectively streaked on a plate, cultured for 48 hours at 28 ℃, and the growth condition of the strain is observed.
The test results are shown in FIG. 3:
even on the 800mg/L boscalid and azoxystrobin plates, strain NH-13-5 was able to grow normally, indicating that it has a strong resistance to chemical bactericides.
Example 2
pH and UV resistance test:
single colony of strain NH-13-5 is selected, inoculated into a 250mL triangular flask filled with 50mL LB culture medium, and shake-cultured at 28 ℃ for 12 hours at 180rpm to obtain seed liquid.
pH tolerance test: according to the inoculum size of 5%, LB medium with pH=2, 3, 4, 5, 6, 8, 9, 10 and 11 was inoculated respectively, and pH=7 was used as a control. After shaking culture at 28℃and 180rpm for 12 hours, the strain survival rate was calculated by dilution and coating.
UV resistance test: and (3) placing the seed liquid under a 254nm ultraviolet lamp, respectively irradiating for 20min, 40min and 60min, and diluting and coating to calculate the survival rate of the strain by taking normal light irradiation as a contrast.
The test results are shown in FIG. 4:
the survival rate of the strain NH-13-5 is over 90 percent within the pH value range of 5 to 9; even at pH values as low as 2 or as high as 11, survival rates still exceeded 55%. The survival rate of the strain NH-13-5 after 20min of irradiation of strong ultraviolet rays is more than 80%, and the survival rate can still be more than 40% after 40 min. This indicates that the strain NH-13-5 has a strong environmental tolerance.
Example 3
Grape gray mold control test:
(1) Test plant pathogenic fungi
The multi-drug resistant gray mold fungus is isolated from a grape picking garden of Qingdao Laiyitose, and is a common chemical bactericide for gray mold: boscalid and azoxystrobin have high drug resistance, and EC 50 Value of>150mg/L; in contrast, non-EC of drug-resistant Botrytis cinerea against both 50 Value of<40mg/L。
(2) Preparation of Botrytis cinerea spore suspension
The multidrug resistant gray mold bacteria were inoculated into PDA plate, and cultured at 25℃for 7d. Picking up mycelia with conidia to 0.5% glucose aqueous solution, shaking thoroughly to disperse conidia uniformly, and diluting to about 1×10 5 And (5) conidium/mL for later use.
(3) Grape fruit pretreatment
The test grape fruit is selected from a representative variety 'Daze mountain Yi No.', which is widely planted in Qingdao. Grape fruits with uniform size and no mechanical damage or infection are selected, and the surface is washed for 20min by running water to remove surface stains. Sterilizing the surface of the 2% sodium hypochlorite solution for 2min, rinsing with sterile water for 3-4 times to remove residual sodium hypochlorite, and naturally airing.
(4) Fermentation culture of test strain NH-13-5
Single colony of strain NH-13-5 is selected, inoculated into a 250mL triangular flask filled with 50mL LB culture medium, and shake-cultured at 28 ℃ for 12 hours at 180rpm to obtain seed liquid. Inoculating a certain amount of seed solution (5% of inoculating amount) into LB culture medium, shake culturing at 28deg.C at 180rpm for 48 hr, and diluting the fermentation broth until the bacterial concentration is 1×10 8 CFU/mL, and further diluted to 1%, 5% and 10% of the above bacteria concentration for use.
(5) Preventive and therapeutic effects
Prevention effect experiment: surface wounds (2 mm. Times.2 mm) were made in the middle of grape fruits with sterile blades, which were soaked in strain fermentation cultures of different concentrations for 10s with LB medium as negative control. mu.L of spore suspension was aspirated and inoculated into grape fruit lesions, with 3 replicates per treatment, 12 grape fruits per replicate. Sealing grape fruits with a preservative film, placing the grape fruits in a climatic chamber for incubation at 25 ℃ and 80% humidity, observing the disease condition of the grape fruits every 12 hours, and counting the diameter of the disease spots when the diameter of the disease spots of the control is close to the diameter of the grape.
Treatment effect test: firstly, sucking 5 mu L of spore suspension liquid, inoculating the spore suspension liquid to a grape fruit wound, and placing the grape fruit wound in a climatic chamber for incubation for 24 hours at 25 ℃ and 80% humidity; next, it was immersed in strain fermentation cultures of different concentrations for 10s with LB medium as negative control.
The treatment and prevention effect tests all use 1000mg/L carbendazim as a positive control.
The test results are shown in fig. 5 and 6:
as the tested plant pathogenic fungi are multi-drug resistant grape gray mold, the positive control carbendazim has a common control effect on the grape gray mold; the strain NH-13-5 has obvious prevention and treatment effects on grape gray mold with multi-drug resistance caused by grape fruit infection, wherein the prevention effect is obviously better than treatment. This also suggests that the bacterial strain NH-13-5 can be used in time to significantly reduce the incidence of grape gray mold and the impact on grape fruit quality before the grape is exposed to multi-resistant grape gray mold infection.
Example 4
Grape fruit fresh-keeping test:
(1) Sample processing
The grape fruits were pretreated and fermentation cultures of strain NH-13-5 were prepared to 1X 10 according to the method of example 3, respectively 8 CFU/mL and further diluted to 10% for use.
(2) Fresh-keeping effect
Uniformly spraying the fermentation culture of the strain NH-13-5 with the concentration of 10% on the surface of grape fruits, and naturally airing. LB medium is used as a negative control, and commercial bacillus subtilis preservative is used as a positive control. The grape fruits were placed in a refrigerator freezer and stored at 4℃for 28d. The appearance of grape fruits was observed every 7d, and representative evaluation indexes such as hardness, VC, titratable acid and soluble sugar were measured. Hardness was measured using a texture analyzer (Bole Fei China), VC was titrated with 2, 6-dichloroindophenol (fruit tree theory, 2020,37 (4): 520-532; northern gardening, 2022,4: 39-44), titratable acid was titrated with acid-base (fruit tree theory, 2020,37 (4): 520-532), and soluble sugars were colorimetrized with anthrone (fruit tree theory, 2020,37 (4): 520-532; northern gardening, 2022,4: 39-44).
The test results are shown in fig. 7:
the evaluation indexes of grape fruit appearance, hardness, VC, titratable acid, soluble sugar and the like of the strain NH-13-5 treated group are obviously superior to those of a control group. Wherein, the VC and titratable acid content of grape fruits treated by the strain NH-13-5 are also obviously superior to those of a positive control group after 28d.
Example 5
Safety evaluation of strain NH-13-5:
safety of strain NH-13-5 was evaluated using the mouse oral toxicity test (Postharvest Biology and Technology,2021,174,111439) as follows:
(1) Fermentation culture of test strain NH-13-5
A fermentation culture of strain NH-13-5 was prepared in accordance with the method of example 3, and the concentrations were adjusted to 1X 10, respectively 8 CFU/mL and 1X 10 10 CFU/mL, ready for use.
(2) Test mice
ICR mice with ages of 3-4 months and weights of 18-22 g are selected, and male and female mice are half-divided. The raising temperature is 20-22 ℃ and the relative humidity is 45-55%.
(3) Test method
The mice were randomly divided into 3 groups of 30 mice each, each half of which was male and female. Each mouse was gavaged with different concentrations of fermentation cultures (average about 0.4 mL) of strain NH-13-5 at a dose of 20mL/kg, with the same dose of physiological saline as a negative control. The mice to be tested are fasted for 16 hours before being irrigated with stomach, and are not forbidden to water; after gastric lavage, the patients are fasted for 12 hours without water. The mice were continuously observed for 28d, were observed for poisoning and mortality, daily food intake was recorded and weighed every 7d.
The test results are shown in fig. 8:
the daily intake of mice treated with strain NH-13-5 was significantly higher than that of the control group, possibly related to its ability to promote gastrointestinal digestion and absorption in mice, while the average body weight of mice in the treated group was also higher than that of the control group. The mice were not poisoned or killed in both the treated and control groups, as observed for 28d continuously.
In conclusion, the bacillus belicus has low toxicity to human and livestock, can be prepared into a microbial preparation, and is used for preventing and controlling and preserving grape gray mold in the whole grape production process; and can be used together with chemical bactericides to prevent and treat grape gray mold, thereby obviously reducing the pesticide application amount.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the invention in any way, and any person skilled in the art may make modifications or alterations to the disclosed technical content to the equivalent embodiments. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (9)
1. Bacillus belicus (Bacillus velezensis), characterized in that the strain is preserved in the China Center for Type Culture Collection (CCTCC) No. m20221278 at 8 and 15 of 2022.
2. Use of bacillus belgium according to claim 1 for the prevention and/or treatment of multidrug-resistant botrytis cinerea and/or plant diseases caused thereby.
3. The application of bacillus belgium in grape fresh-keeping according to claim 1.
4. Use of bacillus belgium according to claim 1 in a chemical sterilant application environment; the chemical bactericide is boscalid or azoxystrobin.
5. A multi-drug resistant botrytis cinerea combined bactericide which is characterized by comprising a chemical bactericide and bacillus bailii according to claim 1; the chemical bactericide is boscalid or azoxystrobin.
6. The combination bactericide according to claim 5, wherein the chemical bactericide is one or more selected from boscalid and azoxystrobin.
7. Use of bacillus belgium according to claim 1 for inhibiting multidrug-resistant botrytis cinerea and/or a plant lesion caused thereby in combination with UV.
8. A microbial preparation comprising the Bacillus bailii according to claim 1.
9. A method for preparing a fermentation culture, comprising the steps of: fermenting bacillus belgium according to claim 1 by an LB culture medium shaking table for 2-3 d to obtain a fermentation culture; the LB culture medium is selected from the following components: 1% tryptone, 0.5% yeast extract, 1% NaCl, distilled water, pH was adjusted to 7.0.
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