CN117801987A - Bacillus subtilis S1 and application thereof - Google Patents
Bacillus subtilis S1 and application thereof Download PDFInfo
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- CN117801987A CN117801987A CN202311693658.0A CN202311693658A CN117801987A CN 117801987 A CN117801987 A CN 117801987A CN 202311693658 A CN202311693658 A CN 202311693658A CN 117801987 A CN117801987 A CN 117801987A
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Abstract
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis S1 and application thereof. The bacillus subtilis (Bacillus subtilis) S1 has the capability of resisting pathogenic bacteria of corn small spot and pathogenic bacteria of cucumber powdery mildew; producing a plurality of important antibacterial and bactericidal secondary metabolites by themselves; the disease resistance of a cucumber plant system can be induced, and the leaf defensive enzyme activity is obviously improved; can obviously promote the growth of cucumber plants; the method can obviously promote the cellulose decomposition of cucumber root soil and the conversion and utilization of nitrogen, and eliminate the eutrophication of water and the damage to cucumber growth and product quality caused by nitrate accumulation in the soil; the characteristics of the thick extracellular capsule and the spore production enable the bacillus subtilis (Bacillus subtilis) S1 to have better stress resistance and to survive in adverse environments such as drought, ultraviolet irradiation and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bacillus subtilis S1 and application thereof.
Background
Cucumber (cucumber sativus l.) is a favorite fruit and vegetable for the public and has a long cultivation history. Due to perennial large-area planting and continuous cropping, some main diseases occur seriously in cucumber production, and 50 kinds of cucumber diseases are not completely counted. Powdery Mildew (PM) of cucumber commonly known as white hair disease is a fungal disease mainly caused by powdery mildew infection of melon single cyst genus (Podosphaera xanthii) and powdery mildew of sporopouenin genus (Golovinomyces cichoracearum). The cucumber seed extract has the characteristics of wide distribution, short incubation period, strong popularity and quick spread, and is a common disease in cucumber production. Powdery mildew of cucumber occurs throughout the year and has considerable hazard, and the cucumber can be reduced by more than 40% when the disease is serious. Generally, the control effect of a new bactericide on powdery mildew of cucumber can be quickly reduced after three to four years of application. The occurrence of powdery mildew of cucumber in production is frequent, so that the yield and fruit quality of cucumber are seriously affected. It is reported that powdery mildew of cucumber is liable to occur in greenhouse and greenhouse planting, and then the open field cultivation is carried out in spring sowing.
Powdery mildew can be infected in the whole growing period of the cucumber, mainly leaves are damaged, and then petioles and stems are damaged less. Round white powdery spots are generated on the leaves at the initial stage of disease occurrence, the round white powdery spots are expanded into round large spots with insignificant edges, when the round white powdery spots are severe, the whole leaves are fully covered with white powder, yellow brown to black small particles can be scattered at the later stage, namely the capsule shells of germs, and finally the leaves can dry and turn yellow. The mycelium of powdery mildew does not invade into the cells of the host tissue, is generally attached to the surface of the host tissue, and directly absorbs nutrition from the host tissue after forming a sucker which can penetrate into the epidermal cells of the host. The distribution of white powdery mildew layers is generally more in the lower blade than in the upper blade, with more blade back than front. The mildew spots are dispersed individually at early stage. The plant is combined into a large mildew spot which can even cover Quan She, so that photosynthesis is seriously affected, normal metabolism is disturbed, and premature senility is caused.
The control strategies of cucumber powdery mildew mainly include disease-resistant breeding, medicament control and biological control. In practice, powdery mildew resistance breeding Fei Shichang of cucumber has heavy tasks, and plant disease resistance performance is a series of performance of interaction of a host and a pathogen under certain environmental conditions, one of the three is changed, and the original disease resistance can be lost, so that the disease cannot be completely prevented and controlled by only depending on disease-resistant varieties. The prevention and the treatment of the medicament can cause environmental pollution, and germs easily generate drug resistance to the medicament. The biological control, especially the biological control of the bacterial control of the powdery mildew and other diseases of the cucumber has the advantages of environmental protection, low cost, good comprehensive benefit and the like, and is a research hotspot in agricultural production. The excavation and development of efficient biocontrol bacteria resources are important working points in the field.
The cucumber facility cultivation area is large in production, and the chemical fertilizer is excessively applied for a long time, so that the soil fertility is reduced, the soil quality is degraded, the ecological function is reduced, and the nitrate accumulation in the soil can cause eutrophication of surface water bodies and other series of problems. Therefore, the microbial agent capable of improving the soil structure, improving the soil fertility and the biological activity of the soil microbial community is applied, which is beneficial to protecting the environment and promoting the sustainable development of agriculture.
Disclosure of Invention
In order to solve the problems, the invention aims to provide bacillus subtilis S1 and application thereof.
In the invention, the rhizosphere growth-promoting and bacteria-preventing bacillus subtilis (Bacillus subtilis) S1 can synthesize a plurality of important antibacterial secondary metabolites, including polyene antibiotics bacillus, antifungal cyclic lipopeptides (Fengyin), catechol type ferrite bacillus, antibiotics bacillus, surfactants and bacteriocin A, and the antibacterial substances can inhibit bacteria by inhibiting the synthesis of pathogenic bacteria proteins, competing for iron ions, dissolving bacteria and other mechanisms, and can play an antagonistic role on pathogenic bacteria and induce systemic resistance of plants when applied to cucumber rhizosphere and leaf surfaces. According to the measurement, the relative prevention effect of the Bacillus subtilis S strain on the powdery mildew of the cucumber is 63.11%, the Bacillus subtilis S strain has good growth promoting effect on the cucumber plant, and the plant height, the plant fresh weight and the root weight of the cucumber seedling are obviously higher than those of the CK group after Bacillus subtilis S strain 1 is applied; the cucumber seedlings in the Bacillus subtilis S1 treatment group have good growth vigor, large leaf area and more fibrous roots and lateral roots, and are beneficial to improving photosynthesis and transpiration of plants, so that plant growth is promoted; within 30 days after Bacillus subtilis S1 is applied, the activity of soil cellulase is greatly improved, so that the decomposition of cellulose in soil is quickened, sufficient carbon nutrition is ensured to be absorbed and utilized by cucumber seedlings, and the nutrient growth of the cucumber seedlings is promoted; after Bacillus subtilis S is applied, the activity of soil nitrite reductase in cucumber rhizosphere soil is obviously improved, the nitrogen conversion efficiency in the soil is obviously improved, the growth of cucumber plants is promoted, the excessive accumulation of nitrite nitrogen in the soil environment is relieved, and the toxic action of nitrite excess on plants is relieved; bacillus subtilis S1 has thick capsule, and is capable of producing spores, resisting adverse external environmental conditions such as ultraviolet rays and drought, and can be used for preparing microbial fertilizers and biocontrol bactericides, reducing the application amount of pesticides and fertilizers, realizing disease resistance and yield increase of cucumbers and protecting farmland ecological environment.
The plant rhizosphere growth-promoting biocontrol bacteria are separated from rhizosphere soil, have good affinity with host plants, and can be more suitable for different soil environments. The bacillus rhizosphere growth-promoting biocontrol bacillus not only can improve plant biomass and induce plants to produce systemic disease resistance, but also can improve and promote carbon and nitrogen nutrition metabolism in soil and improve soil fertility by regulating soil enzyme activity. Meanwhile, the strain itself can inhibit the growth of plant pathogenic bacteria and even kill the pathogenic bacteria through mechanisms such as nutrition competition, antibiotic production, various antibacterial active substances and the like, and can be used as a main component of a biocontrol microbial inoculum for preventing and controlling plant diseases. For the biocontrol fungus of bacillus, the strain has better adaptability to adverse environmental conditions such as drought, ultraviolet irradiation and the like due to the characteristics of capsule and spore production, so the biocontrol fungus has good application prospect.
The aim of the invention can be achieved by the following technical scheme:
the first object of the present invention is to provide a bacillus subtilis (Bacillus subtilis) S1 with a preservation number of CGMCC No.29146, which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 11 and 27 days of 2023, and with a preservation address of the national institute of microbiology, national institute of sciences No. 3, national institute of sciences, national center 1, which is the region of the morning sun, beijing city.
A second object of the present invention is to provide a gene sequence of 16S rRNA of Bacillus subtilis (Bacillus subtilis) S1, which contains a nucleotide sequence shown in SEQ ID NO. 3.
In one embodiment of the invention, the bacillus subtilis (Bacillus subtilis) S1 has a thick capsule and flagella.
The invention provides a method for culturing bacillus subtilis (Bacillus subtilis) S1, which comprises the following steps:
the activated bacillus subtilis (Bacillus subtilis) S1 is placed in LB culture for anaerobic culture for 18 to 30 hours at 37 ℃.
The third object of the invention is to provide an application of bacillus subtilis (Bacillus subtilis) S1 in preparing a product for treating crop leaf spot.
In one embodiment of the invention, the crop is corn.
The fourth object of the invention is to provide an application of bacillus subtilis (Bacillus subtilis) S1 in preparing products for treating powdery mildew of crops.
In one embodiment of the invention, the crop is melon, vegetable or strawberry.
In one embodiment of the invention, the crop is melon.
In one embodiment of the invention, the crop is cucumber.
A fifth object of the invention is to provide the use of bacillus subtilis (Bacillus subtilis) S1 for the preparation of a product for promoting crop growth.
In one embodiment of the invention, the product is a product that promotes soil enzyme activity associated with carbon nitrogen metabolism in the rhizosphere soil of a crop plant.
In one embodiment of the present invention, the soil enzyme is selected from one or more of soil nitrite reductase, soil cellulase or soil dehydrogenase.
In one embodiment of the invention, the product is a product that promotes nitrogen levels in the rhizosphere soil of a crop.
In one embodiment of the invention, the product is a product that promotes defensive enzymatic activity of a crop leaf.
In one embodiment of the invention, the defensive enzyme is selected from one of POP enzyme, POD enzyme or SOD enzyme.
According to the invention, the microbial fertilizer and microbial agent containing bacillus subtilis (Bacillus subtilis) S1 can reduce the application amount of pesticides and fertilizers, promote growth and disease resistance, improve yield, prevent water eutrophication, protect environment and the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) The bacillus subtilis (Bacillus subtilis) S1 has the capability of resisting corn small spot pathogenic bacteria and cucumber powdery mildew pathogenic bacteria, and can comprehensively induce the systemic disease resistance of cucumber plants. The results of the fermentation liquor plate bacteriostasis experiment show that Bacillus subtilis S1 sterile fermentation liquor can obviously inhibit the growth of the bacterial hyphae of the alternaria corn, and the bacteriostasis rate reaches 47.03%; the spore germination inhibition experimental result shows that the germination inhibition rate of the Bacillus subtilis S1 sterile fermentation liquid to the conidium of the corn small spot germ reaches 42.16% in 6 hours, and the inhibition rate of the control bacillus pumilus 103925 sterile fermentation liquid to the fungus spore is only 35% after 6 hours; the experimental result of the potted cucumber control effect shows that the relative control effect of Bacillus subtilis S1 bacterial liquid seed soaking and leaf surface spraying treatment on cucumber powdery mildew reaches 63.11%; furthermore, the Bacillus subtilis S strain is separated from the rhizosphere soil of the cucumber, can adapt to the cultivation environment of the cucumber better than other biocontrol bacteria, and has better colonization affinity with a host cucumber.
(2) The bacillus subtilis (Bacillus subtilis) S1 of the present invention itself produces a variety of important bacteriostatic and bacteriocidal secondary metabolites. Through whole genome sequencing and bioinformatics analysis of the Bacillus subtilis S strain, the strain can be used for biosynthesizing various antibacterial/bactericidal active substances, including novel polyene antibiotics such as bacteriene, antifungal cyclic lipopeptides (Fengyin), catechol type basellipticin, antibiotics such as Bacilysin, surfactants and bacteriocin A, and the antibacterial/bactericidal substances can play a role in inhibiting or killing bacteria through various mechanisms such as inhibiting the synthesis of pathogenic bacteria proteins, competing iron ions, dissolving bacteria and the like, and can play a remarkable role in inhibiting the growth of cucumber pathogenic bacteria when being applied to the rhizosphere of cucumbers or sprayed on leaf surfaces.
(3) The bacillus subtilis (Bacillus subtilis) S1 can induce cucumber plants to generate systemic disease resistance, and obviously improve leaf defensive enzyme activity. The foliar spray Bacillus subtilis S1 can induce systemic disease resistance of the cucumber and obviously improve the defensive enzyme activity of the cucumber leaf; after the powdery mildew is inoculated artificially to the cucumber, the leaf surface is sprayed with Bacillus subtilis S1 for treatment, and on the 9 th day, the superoxide dismutase (SOD) activity of the detected leaf is found that the SOD activity of the leaf of a Bacillus subtilis S1 treatment group is 5.81 times that of an untreated control group, and the statistical difference is extremely remarkable (p < 0.01); on day 6, the activity of cucumber leaf Peroxidase (POD) in Bacillus subtilis S1 treated group was 1.21 times that in untreated control group, the difference reached extremely significant (p < 0.01); on day 9, the polyphenol oxidase (PPO) activity of the cucumber leaves of the S1 treatment group was 1.81 times that of the control group, and the difference reached extremely significant (p < 0.01).
(4) The bacillus subtilis (Bacillus subtilis) S1 can obviously promote the growth of cucumber plants, and shows good growth promoting effect. The growth promotion experiments of the sterile vermiculite system and the nutrient medium potting system show that the bacillus subtilis S1 strain has obvious growth promotion effect on the cucumber; after Bacillus subtilis S bacterial liquid is applied to the root surrounding soil, the fresh weight and the plant height of the cucumber plant are obviously increased compared with those of a control group, the cucumber plant grows more vigorously, and the growing state is better.
(5) The bacillus subtilis (Bacillus subtilis) S1 can obviously promote cellulose decomposition of cucumber root surrounding soil and conversion and utilization of nitrogen, and eliminates nitrite accumulation in the soil and harm to plant growth. After Bacillus subtilis S1 is applied, the activity of important soil enzymes related to carbon and nitrogen metabolic circulation of cucumber rhizosphere is obviously increased, the decomposition of cellulose and the conversion and utilization of nitrogen in soil are accelerated, and more carbon and nitrogen nutrition is provided for plant growth; the soil cellulase activity of the Bacillus subtilis S1 treatment group is obviously enhanced on the 30 th day, the dehydrogenase activity is obviously enhanced on the 15 th day compared with the CK group, the nitrite reductase activity is obviously enhanced in the whole growth period of the cucumbers, the urease activity is greatly enhanced on the 30 th day, and the differences are obvious compared with the CK group; bacillus subtilis S1 after the application treatment, the nitrous acid reductase activity in the soil surrounding the cucumber root is detected, and the result shows that the nitrous acid reductase activity is obviously enhanced on the 15 th day and the 30 th day compared with the control group; meanwhile, the content of nitrate nitrogen in the soil is extremely lower than that of a control group, and nitrite reductase can degrade nitrite into NO or NH 3 Thereby reducing the accumulation of nitrite nitrogen in the soil and reducing the toxic effect on crops.
(6) The bacillus subtilis (Bacillus subtilis) S1 has the characteristics of thick extracellular capsules and sporulation, so that the bacillus subtilis has better stress resistance and can survive better in adverse environments such as drought, ultraviolet irradiation and the like; the above characteristics are very advantageous for developing the strain into an excellent microbial biocontrol growth-promoting agent.
Drawings
FIG. 1 shows growth-promoting biocontrol bacteria for cucumber rhizosphere: electron microscope and colony morphology photographs of Bacillus subtilis S strain;
FIG. 2 shows growth-promoting biocontrol bacteria for cucumber rhizosphere: bacillus subtilis S1 genome loop map;
FIG. 3 shows the growth-promoting biocontrol bacteria of the rhizosphere of cucumber: bacillus subtilis S1 gene cluster map in seq id no;
fig. 4 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1 inhibition of corn leaf spot pathogen growth by fermentation broth;
FIG. 5 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1 inoculating the whole plant comparison chart (the culture medium is sterile vermiculite) after the cucumber seedlings are inoculated with the Xintaimi thorn;
FIG. 6 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1 cucumber seedling biomass comparison chart (nutrient soil culture medium) after inoculating "Xintaimi thorns";
FIG. 7 shows growth-promoting biocontrol bacteria for cucumber rhizosphere: bacillus subtilis S1A graph of the activity change of soil nitrite reductase after inoculating 'Xintaimi thorn' cucumber;
FIG. 8 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1A soil cellulase activity change graph after inoculating 'Xintaimi thorn' cucumber;
FIG. 9 shows growth-promoting biocontrol bacteria for cucumber rhizosphere: bacillus subtilis S1 inoculating a soil dehydrogenase activity change chart of the cucumber with Xintaimi thorns;
FIG. 10 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1A, inoculating a nitrate nitrogen content change chart in rhizosphere soil after the cucumber seedlings are planted with Xintaimi thorns;
FIG. 11 is a cucumber rhizosphere growth-promoting biocontrol bacterium: bacillus subtilis S1 spraying an activity change graph of leaf defensive enzyme (PPO) after cucumber seedlings of Xintai Mici;
FIG. 12 is a schematic diagram showing growth-promoting biocontrol bacteria for cucumber rhizosphere: bacillus subtilis S1 spraying an activity change pattern of leaf defensive enzymes (PODs) after cucumber seedlings of Xintai Mici;
FIG. 13 shows the growth-promoting biocontrol bacteria of the rhizosphere of cucumber: bacillus subtilis S1A graph of the activity change of leaf defensive enzymes (SOD) after seedling of cucumber is sprayed with Xintai Mici thorn.
Detailed Description
The strain preservation information of the invention is as follows:
strain name: bacillus subtilis S1;
deposit number: CGMCC No.29146;
preservation date: 2023, 11, 27;
preservation unit name: china general microbiological culture Collection center (China Committee for culture Collection);
Deposit unit address: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The invention will now be described in detail with reference to the drawings and specific examples.
In the examples below, unless otherwise specified, all reagents used were commercially available, and all detection means and methods used were conventional in the art.
Example 1
This example provides for the screening of strains.
(S1) collecting rhizosphere soil samples from which antagonistic bacteria are isolated
The experimental samples are collected from Shanghai university experiment farms, 10 parts of rhizosphere soil (plant rhizosphere 3-5 cm) of different crops (cucumber, capsicum, eggplant, tomato and alfalfa corn land) are collected, and the rhizosphere soil is respectively filled into sealing bags and stored in a refrigerator at 4 ℃.
(S101) isolation of bacterial Strain in rhizosphere soil
Preparation of soil suspension: weighing 10g of soil attached to plant rhizosphere with balance, drying in the shade, grinding into powder with grinder, placing into a triangular flask containing 90mL of sterile water (90 mL of deionized water is placed into a triangular flask with 250mL of capacity, sealing and sterilizing for 20 min), oscillating for 30min on a shaker at 180r/min and 37 ℃ and standing at room temperature for 10min to obtain supernatant (with concentration of 10) -1 Is a suspension of soil).
Dilution of soil suspension: 6 sterilized test tubes (20 mL) were prepared, 9mL of sterilized water was added to each test tube, and 1mL of 10 was pipetted with a pipette -1 The concentrated soil suspension is put into a test tube, and is sucked and beaten uniformly to obtain the concentration of 10 -2 The soil suspension is repeatedly subjected to the steps, and the soil suspension is respectively diluted to 10 in sequence -3 、10 -4 、10 -5 、10 -6 Concentration, mixing well for use.
Isolation of soil bacteria: from 10 with a pipette gun -4 ，10 -5 ，10 -6 Three gradient tubes were each coated with 200. Mu.L of soil suspension onto LB plates, 3 plates per gradient. After sealing with sealing film, the flat plate is inverted and fine at 37 DEG CCulturing in a constant temperature incubator for 24 hours, and observing colony growth. Single colonies of different morphological features were picked from plates and individually loaded into 1.5mL centrifuge tubes containing 1mL of liquid LB, labeled, and placed in a 180rpm shaker at 37℃overnight.
Purification of soil bacteria: and (3) respectively streaking and culturing the bacterial liquid cultured overnight on LB plates for 24 hours by using an inoculating loop, then picking single bacterial colonies from each plate, repeating the step of shaking, obtaining bacterial liquid which is separated from soil and subjected to purification culture, and storing all the separated bacterial strains in a refrigerator at the temperature of-4 ℃ to obtain 121 bacterial strains.
(S102) screening of antagonistic bacteria: the method adopts a plate colony diameter method, and uses the pathogenic bacteria of the corn small spot as the fungi to be tested for preliminary screening.
All strains were grown in LB medium, and the resulting bacterial solutions were placed in 50mL centrifuge tubes, and centrifuged at 8000rpm for 5min. Sucking the supernatant with a disposable syringe with a capacity of 5mL, filtering with a filter membrane with a pore diameter of 0.25 μm to obtain fermentation liquor of each separated strain, and loading into a 1.5mL centrifuge tube for marking. Sucking 150 μl of sterile fermentation broth, spreading on PDA plate (1L contains 200g peeled potato decoction, filtering, glucose 20g, sterile deionized water to 1L, pH naturally, adding solid culture medium into 20g agar, sterilizing at 121deg.C for 20 min), and drying in shade to completely absorb. After the corn small spot bacteria are inoculated to the PDA flat plate in advance and the lawn grows to be full of the flat plate, a sterile puncher with the diameter of 5mm is used for punching corn small spot bacteria cakes in a mode of equal diameter from the center point of the flat plate, 1 bacterial cake is hooked by an inoculating needle, the inoculating needle is inverted to the center of the PDA flat plate coated with sterile fermentation liquor, 3 flat plates are arranged in each treatment, and the flat plate coated with sterile water is used as a blank control. After inoculation, all plates were sealed with sealing film and light cultured in a fungus incubator at 28 ℃. In the 2 nd to 5 th days, the diameter of the bacterial lawn was recorded every day by the crisscross method, and photographed. And calculating the growth inhibition rate of germ hyphae on each plate at the 5 d. The calculation formula is as follows: inhibition (%) = (control lawn diameter-treated lawn diameter)/control lawn diameter 100. And (3) through 1 round of primary screening, secondary screening and final screening, selecting a strain with high antibacterial activity and always good antagonistic performance, and carrying out spore germination inhibition rate test secondary screening.
(S2) spore germination inhibition experiment (re-screening) of fermentation broth and antagonistic confirmation by taking powdery mildew pathogenic bacteria of cucumber as test fungi
And re-screening the candidate antagonistic bacteria by adopting a sterile fermentation liquor to carry out a fungal spore germination inhibition method. The filtered aseptic fermentation liquor is filled into a 1.5mL aseptic Eppendorf centrifuge tube, conidium of the corn small spot germ is scraped into the aseptic Eppendorf centrifuge tube by a germ-collecting ring, and the aseptic fermentation liquor is placed on a shaking table at 28 ℃ for shaking after being evenly mixed, and a control group is cultivated by liquid LB. 20 mu L of spore suspension is sucked every 2 hours, placed in the center of a glass slide, covered with a cover slip, the spore germination quantity is observed under an optical microscope, and the spore germination rate and the inhibition rate are calculated. More than 200 conidia are counted in the visual field, the number of germinated spores is recorded, and the germination inhibition rate is calculated, wherein the formula is as follows: spore germination rate (%) =number of spores germinated/total number of survey spores×100.
Spore germination inhibition (%) = (control group spore germination rate-treatment group spore germination rate)/control group spore germination rate×100.
And finally, testing the control effect of the screened candidate antagonistic bacteria, wherein the pathogenic bacteria to be tested are pathogenic bacteria of powdery mildew of cucumber. The specific method comprises the following steps: cucumber seeds are subjected to surface disinfection treatment and then are divided into two groups, one group is placed in a disposable culture dish, the culture dish is soaked in a shallow layer by using a bacterial suspension of Bacillus subtilis S1, the bacterial suspension of Bacillus subtilis S is replaced once every other day until buds come out, and the other group is a control group and is soaked in sterile water. Sowing the germinated seeds into flowerpots filled with nutrient matrixes, planting 10 pots in each of an S1 treatment group and a sterile water control group, sowing 4 seeds in each pot, watering potting soil thoroughly, and keeping water management and illumination humidity management during the period. After a whole leaf grows out of the cucumber seedling, uniformly spraying the prepared cucumber powdery mildew pathogenic bacteria spore suspension on the leaf, and preserving moisture for one night. And spraying Bacillus subtilis S1 bacterial suspension on the leaf surfaces of 10 pots of cucumbers in a treatment group after 24 hours, and spraying clear water on 10 pots of a control group. And when the fourth true leaf grows out of the cucumber, investigation of disease indexes of all disease leaves in the pot plant is carried out, and the disease indexes and the relative control effect of the Bacillus subtilis S strain are calculated. Disease grading standard refers to GB/T17980.30-2000 pesticide field efficacy test criterion (I). The correlation calculation formula is:
Disease index = Σ (number of disease stages x number of leaves of the stage) ×100/(investigation of total number of leaves x highest number of disease stages)
Relative control = (1-treatment group disease index/control group disease index) ×100%
(S3) microbiological morphological observation and scanning electron microscope identification of antagonistic bacteria
(S301) conventional morphological observation
The antagonistic bacteria are inoculated on an LB solid plate, cultured for 12-24 hours at the constant temperature of 37 ℃, then a Bacillus subtilis S single colony is picked up by an inoculating loop under the aseptic condition, coated on a glass slide, flaked and gram-stained, and the size, shape, surface, edge, color, bulge shape and transparency of the colony are observed under a microscope, and the surrounding of the colony, the color of a culture medium and the like are observed (as shown in figure 1).
(S302) scanning electron microscope authentication
Antagonistic bacteria are inoculated on LB liquid medium for overnight culture, 1mL of bacterial liquid is sucked into a sterilized 1.5mL centrifuge tube, the centrifugation is carried out at 8 000rpm for 5min, the supernatant is discarded, the liquid is washed twice with equal amount of sterile water, finally, the liquid is resuspended with equal amount of sterile water, 10 mu L of resuspended bacterial liquid is dripped on a copper mesh, and the liquid is dried in the shade overnight. The dried and immobilized samples were observed for bacterial morphology by a biological transmission electron microscope (Tecnai G2 spiral Biotwin,120kV Bio-TEM; FIG. 1A: 30000X; FIG. 1B: 11000X) (as shown in FIG. 1).
As can be seen from FIG. 1, the gram staining of the thallus is positive, the thallus can form endophytic stress-resistant spores, the thallus is in a rod shape, has thick capsules and periphyton flagella (the structural characteristics endow the thallus with the capability of resisting adverse environmental conditions such as drought, ultraviolet rays and the like), and is easy to fall off; observing cells under an electron microscope generally in a linear arrangement; the form of the colonies on the LB solid medium is milky white and is irregularly round at the edge.
The surface of the single colony at the initial stage is smooth, transparent mucus is contained in the single colony, the colony is dried and shrunken after being placed for a period of time, and the surface is wrinkled and easy to scrape off; the size of the single cell is about (0.6-0.8) mu m x (2.2-2.6) mu m; can utilize various carbon sources such as sucrose, glucose and the like, and has high growth speed.
(S4) PCR amplification of 16S rRNA
(S401) primers used for PCR amplification were 27F and 1492R:
27F(SEQ ID NO.1：5'-ATGCCATTCGTGCGGAGGTTG-3')；
1492R(SEQ ID NO.2：5'-CGTCTCTGCTGTCATCACTTCGTAT-3')；
the primers were synthesized by Shanghai Bioengineering Co.
(S402) the PCR reaction system comprises: 2 XTaq Master Mix 12.5. Mu.L, forward primer 27F (10. Mu. Mol/L) 1.0. Mu.L, reverse primer 1492R (10. Mu. Mol/L) 1.0. Mu.L, dd H 2 O9.5. Mu.L, template DNA 1.0. Mu.L, total volume 25. Mu.L. Wherein 2×Taq Master mix is a product of Shanghai Shaoxin Biotechnology Co., ltd. The composition is as follows: 0.1U Taq polymerase/. Mu.L, 500. Mu. Mol/L dNTP each, 20mmol/L Tris-HCl (pH 8.3), 100mmol/L KCl, 3mmol/L MgCl 2 Other stabilizers and enhancers.
(S403) PCR reaction conditions are: pre-denaturation at 94℃for 10min; denaturation at 94℃for 30s, annealing at 54℃for 30s, elongation at 72℃for 1min,35 cycles; finally, the mixture was extended at 72℃for 7min. The PCR amplified product was about 1.5kb, and the target band was sequenced after recovery and purification by electrophoresis on a 1.2% agarose gel: SEQ ID NO.3 (16S rRNAPCR amplification sequence).
(S5) Whole genome sequencing of Bacillus subtilis S1 bacteria
A Bacillus subtilis S single colony was picked, inoculated into 200mL LB liquid medium, and placed on a 180rpm shaker at 37℃overnight for 12h. After taking out, the bacterial liquid is subpackaged into 4 sterile 50mL centrifuge tubes, and the bacterial liquid is centrifuged at 8000rpm and 25 ℃ for 10min. Pouring out the supernatant, reserving bacterial precipitate, adding an equal amount of 50mL of sterile water, sucking and beating the precipitated bacterial precipitate, mixing uniformly, re-suspending, centrifuging for 10min again, and repeating the steps to wash Bacillus subtilis S bacterial cells twice with the sterile water. Finally, the thalli in the four separation tubes are resuspended by about 10mL of sterile water respectively, transferred into the same centrifuge tube for centrifugation, the supernatant is poured out, the thalli sediment at the bottom of the tube is reserved, and the thalli are sent to a sequencing company for whole genome sequencing of bacteria. Sequencing result analysis included the following steps: and (1) quality control of original data. Filtering reads of low quality and too short a length; (2) genome assembly. De novo assembly of the filtered reads and error correction of the assembled draft genome; (3) genome component analysis. Mainly comprises the following steps: analysis of repetitive sequences, coding genes, non-coding RNAs, prophages, gene islands, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), and the like; (4) functional annotation. Mainly comprises various general databases, such as: nr, eggNOG, KEGG, etc., and CAZyme, PHI, CARD, etc.
As shown in FIG. 2, the genome of the strain is 4083451bp in full length and is free of endoplasmid. CDS is coding sequence; PC is a procoto cluster; CC is cand cluster. Finally, the strain is confirmed to belong to Bacillus subtilis strains, and the strain is named Bacillus subtilis S1; the microorganism culture of the culture medium is preserved in China general microbiological culture Collection center (CGMCC) of 11.27.2023, the national institute of microbiology of national academy of sciences of China, no. 1, no. 3, the Korean region of Beijing, and the preservation number is CGMCC No.29146.
Bacillus subtilis S1 the whole genome sequence is submitted to an antisMASH website for secondary metabolite synthesis prediction analysis, and the sequence comparison result shows that Bacillus subtilis S has complete synthetic polyene antibiotics Bacillaene, antifungal cyclic lipopeptid (Fengycin), catechol type basophilic acid, antibiotic Bacilysin, surfactant Surfactin and bacteriocin A gene cluster (figure 3) on the genome, so that the products can play a role in antagonizing or killing plant pathogenic bacteria by inhibiting the synthesis of pathogenic bacterial proteins, competing iron ions, dissolving bacteria and other mechanisms in the self growth process, and Bacillus subtilis S1 can play a role in disease resistance through root irrigation or leaf surface spraying.
Example 2: identification of bacteriostatic ability of strain fermentation liquor
(1) Determination of inhibition of growth of pathogenic bacteria of corn leaf spot by bacterial fermentation broth (shown in FIG. 4)
The Bacillus subtilis S strain was grown in LB medium, and the resulting broth was placed in a 50mL centrifuge tube and centrifuged at 8000rpm for 5min. The supernatant after centrifugation was aspirated by a disposable syringe having a capacity of 5mL, a filter head having a bacterial filter membrane with a pore size of 0.25 μm was fitted over the needle, and the resulting strain broth (sterile) was filtered and placed into a 1.5mL centrifuge tube. mu.L of the broth was pipetted onto the PDA plate and allowed to stand until complete absorption. Taking out the cultured corn small spot germ cake with a sterile puncher with the diameter of 5mm from the center of the PDA flat plate in an equal diameter manner, hooking 1 germ cake with an inoculating needle, reversely inoculating the corn small spot germ cake to the center of the PDA flat plate coated with fermentation liquor, and inoculating 1 germ cake on each flat plate (S1 treatment group); at least 3 duplicate plates were set for each treatment, with sterile water-coated plates as blank (CK). After inoculation of the bacterial cake, all the plates are sealed by sealing films and placed in a fungus incubator for illumination culture at 28 ℃. In the 2 nd to 5d, measuring the growth diameter of the bacterial lawn after inoculation by a crisscross method every day, and stopping measuring when the blank control group is about to grow up the whole culture dish. And calculating the growth inhibition rate of germ hyphae on each plate at the 5 d. The formula is as follows:
Inhibition (%) = (control lawn diameter-treated lawn diameter)/control lawn diameter 100 ×
According to the figure 4, the Bacillus subtilis S1 sterile fermentation liquid can obviously inhibit the growth of the mycelium of the alternaria alternate, and the bacteriostasis rate can reach 47.03%.
(2) Determination of inhibition of bacterial strain fermentation liquor on germination of pathogenic bacteria spores of corn small spot disease
The strain fermentation broth obtained by filtration is filled into a centrifuge tube with the volume of 1.5mL, conidium of the alternaria alternate is scraped into the centrifuge tube by a fungus inoculation ring, and the fermentation broth is placed on a shaking table with the temperature of 28 ℃ for fungus shaking after being evenly shaken, and a control group is cultivated by liquid LB. Taking 2h as a measurement interval, sucking 20 mu L of spore suspension each time, placing the spore suspension in the center of a glass slide, covering the glass slide, and observing the spore germination quantity under an optical microscope to analyze the inhibition condition of the fermentation broth of the Bacillus subtilis S strain on the germination of the conidium. At least 200 spores were counted in the field of view, the number of spores germinated was recorded, and the germination inhibition was calculated as follows:
spore germination rate (%) =number of spores germinated/total number of survey spores×100
Spore germination inhibition (%) = (control group spore germination rate-treatment group spore germination rate)/control group spore germination rate×100
Wherein the control group is Bacillus pumilus103925.
TABLE 1 inhibition of germination of conidia of pathogenic bacteria of maize small spot disease
Note that: single asterisks indicate that statistically significant differences were achieved (p < 0.05); double asterisks are shown in statistics
The difference in these was extremely pronounced (p < 0.01).
Example 3: prevention and identification of powdery mildew of greenhouse potted cucumber by strain fermentation liquor
The cucumber seeds are divided into two groups after surface disinfection treatment, one group is placed in a disposable sterile culture dish, shallow layer soaking is carried out on the cucumber seeds by using Bacillus subtilis S bacterial suspension, and Bacillus subtilis S bacterial suspension is changed once every day until germination; the other group was a control group, which was immersed in sterile water. After the cucumber seeds germinate, sowing the cucumber seeds into a pot plant filled with a nutrient medium, planting 10 pots in each of an S1 treatment group and a sterile water control group, sowing 4 seeds in each pot, watering potting soil thoroughly, and keeping water, illumination and humidity management during the period. After a piece of true leaves grow on cucumber seedlings, uniformly spraying the prepared cucumber powdery mildew pathogenic bacteria spore suspension on the leaves, and preserving moisture for one night. And spraying Bacillus subtilis S bacterial suspension on leaf surfaces of 10 pots of cucumbers in a treatment group after 24 hours, and spraying clear water on 10 pots of cucumbers in a control group. And when the fourth true leaf grows out of the cucumber, investigation of disease indexes of all disease leaves in the pot plant is carried out, and the disease indexes and the relative control effect of the Bacillus subtilis S strain are calculated. Disease classification refers to the disease classification standard specified in the GB/T17980.30-2000 pesticide field efficacy test criterion (one).
Disease index = Σ (number of disease stages x number of leaves of the stage) ×100/(investigation of total number of leaves x highest number of disease stages)
Relative control = (1-treatment group disease index/control group disease index) ×100%
Table 2 seed of cucumber powdery mildew control effect of strains in seedling stage
Note that: single asterisks indicate that statistically significant differences were achieved (p < 0.05); double asterisks are shown in statistics
The difference in these was extremely pronounced (p < 0.01).
Example 4: promoting effect of strain on cucumber growth
(1) Disinfection and germination accelerating of cucumber seeds
The cucumber seeds are soaked in 75% ethanol and stirred for 30s for surface sterilization, after being washed 3-4 times by sterile water, soaked in 2% NaClO solution for 8-10 min, intermittently stirred, and finally washed 3-4 times by sterile water. Placing two sterilized filter papers into a disposable sterile culture dish, adding sterile water, soaking, spreading the seeds on the sterile culture dish, replacing water for 2 days, and placing the sterile culture dish into a 37 ℃ incubator for germination until buds emerge.
(2) Cultivation and management of cucumber seedlings
Vermiculite soilless culture system (sterile vermiculite as culture substrate): the germinated cucumber seeds were sown in a beaker containing vermiculite. 80g of vermiculite was first loaded into a 1L volume beaker having a thickness of about 3-5 cm. Sealing the beakers with a sealing film of the Erlenmeyer flask, sterilizing at high temperature in a sterilizing pot, cooling, seeding the radicle of the cucumber seed downwards, seeding 5 seedlings in each beaker, watering thoroughly, culturing in a ventilated illumination place, and maintaining moisture management.
Nutrient medium cultivation system (nutrient soil as cultivation medium): uniformly stirring vegetable garden soil and a nutrient matrix, filling the vegetable garden soil with the flowerpot, sowing germinated seeds (radicle length is about 0.4-0.6 cm) on a shallow layer, and watering 3 seeds in each pot thoroughly. After seedling emergence, water is poured once a day, and after cotyledons are fully flattened, water is poured once every two days, so that the surface soil is kept moderately moist.
(3) Bacillus subtilis S1 treatment of cucumber root soil
The Bacillus subtilis S strain was used as a strain at 1% (V/V)The inoculum sizes were inoculated into 100mL of LB medium, respectively, and shaken overnight. The bacterial liquid was centrifuged at 8000rpm for 5min, the supernatant was decanted, and then the bacterial cells were resuspended in sterile water and centrifuged again. The above procedure was repeated twice, and finally the cells were suspended in an equal amount of sterile water. After the first true leaves of the cucumber seedlings are completely grown, the resuspended bacterial liquid is applied as a treatment group into the root soil of the cucumber seedlings, 20mL of bacterial liquid per seedling (10 8 ～10 9 CFU) to administer an equal amount of sterile water as a control group. The above treatments were performed every 3 days for a total of 5 treatments, and 3 pots were set up for each of the treatment group and the control group.
A suspension of Bacillus subtilis S strain was prepared in the same manner as described above. After two true leaves of the cucumber seedlings in vermiculite grow and are fully stretched (about 1 week), the resuspended bacterial liquid is applied to the vicinity of the roots of the cucumber seedlings as a treatment group, and 10mL of bacterial liquid is applied to each seedling, so that an equal amount of sterile water is applied as a control group. The above treatment was performed once every 3 days, and the total treatment was 3 times, and 5 replicates were set for each of the treatment group and the control group.
(4) Cucumber seedling biomass determination
Vermiculite system seedling sampling (as shown in fig. 5): sampling is started 1 week after 3 times of growth promotion treatment, and samples are taken every 7d for 3 times. After the cucumber seedlings are gently taken out together with vermiculite, the vermiculite at the root of the seedling is rinsed by gentle water flow, the wound root systems are separated, and the root water is wiped by paper towels. Then, four biomass indexes of a plant fresh weight (SFW), a plant height (cutting height), a Root Fresh Weight (RFW) and a Root length (Root length) of the cucumber seedlings were measured.
Potted system seedlings were sampled (as shown in fig. 6): sampling is started after 2 weeks after 3 times of growth promotion treatment, and samples are taken every 15d for 3 times. After pouring out the whole pot soil, holding cucumber seedlings and roots by hands, gently shaking off the soil, soaking and washing the soil adhered with the roots of the seedlings with clear water for multiple times, carefully separating the wound root systems of the cucumber seedlings, and wiping off excessive water by using paper towels. Seedling plants are cut at the stem base and divided into overground parts and roots, and biomass indexes such as plant fresh weight (SFW), plant height (cutting height), root Fresh Weight (RFW), root length (Root length) and the like are measured.
As can be seen from FIG. 5, the Bacillus subtilis S strain has a remarkable effect on promoting the growth of cucumber seedlings in vermiculite matrix cultivation. At week 1 after 5 growth-promoting treatments, the cucumber seedlings of the S1-treated group and the CK group began to show a more pronounced growth gap. And, this gap can be maintained to weeks 2 and 3. After Bacillus subtilis S, 3 indexes of the plant height, the plant fresh weight and the root fresh weight of cucumber seedlings are obviously higher than those of a CK group. The cucumber seedlings treated by the S1 have good true leaf growth vigor, large leaf area and more fibrous roots and lateral roots of the roots, and are beneficial to improving photosynthesis and transpiration of plants, so that plant growth is promoted.
As can be seen from fig. 6, the fresh weight of cucumber seedling plants after application of Bacillus subtilis S for 15 days was significantly higher than that of the sterile water treatment group in the nutrient soil potting system. On day 45, the cucumber starts to enter a reproductive growth stage, and the cucumber seedlings treated by S1 show obvious differences from the aseptic water treatment group in plant height, plant fresh weight and root length, so that the growth of the cucumber is obviously promoted by the rhizosphere growth-promoting biocontrol bacillus Bacillus subtilis S1.
Example 5: strain application to increase in soil enzymatic Activity associated with carbon Nitrogen metabolism in cucumber rhizosphere soil
And taking about 200g of rhizosphere soil of the potted cucumber after 15d, 30d and 45d of growth promotion, filling the rhizosphere soil into a sealed bag, and putting the sealed bag into a laboratory for freezing at the temperature of minus 20 ℃. After the soil sample is air-dried at room temperature, grinding the soil sample into powder by a grinder, weighing the soil sample with corresponding weight according to the requirements of soil enzyme kits produced by the Rhinocerotis biological company, adding the soil sample into a centrifuge tube, and carrying out enzymatic reaction by adding all reagents according to the steps of description. After the completion of the reaction, the absorbance was measured at a specific wavelength by an ultraviolet spectrophotometer to determine the activity of each soil enzyme. The activity of each soil enzyme was calculated as follows:
soil nitrite reductase (S-Nir) Activity assay (FIG. 7, S1: strain treated group; CK: control group): the absorbance was measured at 540nm for samples, operated in sequence, with the instructions in the kit as reference. The calculation formula is as follows: S-Nir Activity (. Mu. Mol/d/g soil sample) = 30.84 × [ A blank tube- (A assay tube-A control tube) -0.0088]
Soil cellulase (S-CL) Activity assay (FIG. 8, S1: strain treated group; CK: control group): the absorbance was measured at 620nm for samples, operated in sequence, with the instructions in the kit as reference. The calculation formula is as follows: S-CL viability (mg/d/g soil sample) =19.1× (Δa+ 0.0462), Δa=assay tube absorbance-control tube absorbance.
Determination of soil dehydrogenase (S-DHA) Activity (FIG. 9, S1: strain treated group; CK: control group): the absorbance was measured at 485nm for samples, operated in sequence, with the instructions in the kit as reference. The calculation formula is as follows: s-DHA viability (μg/d/g soil sample) = 473.9 × (Δa+0.0312), Δa=measured tube absorbance-blank tube absorbance.
According to the results shown in figures 7-9, the application of the Bacillus subtilis S strain to the rhizosphere of the cucumber can obviously improve the activities of soil cellulase and soil nitrite reductase, and enhance the conversion efficiency of carbon and nitrogen in the soil, so that the growth of cucumber plants is promoted, the accumulation of nitrite in the soil is effectively prevented, the damage of plant growth and the soil property deterioration are avoided, the eutrophication of water is prevented, and the ecological environment of farmlands is protected.
Example 6: bacillus subtilis S1 application of control of Nitrogen levels in cucumber rhizosphere soil
The soil sampling method comprises the following steps: and taking 200g of rhizosphere soil of the potted cucumber at 15d, 30d and 45d after 5 times of growth promotion, filling the rhizosphere soil into a sealed bag, and putting the sealed bag into a laboratory for freezing at the temperature of minus 20 ℃. After each group of soil samples is air-dried, grinding into powder by a grinder. Each soil sample was weighed into a 5g to 50mL centrifuge tube, 25mL of 1mol/L KCl solution was added, and the mixture was placed on a shaker at 37℃and 180rpm to oscillate for 1h. Taking out, centrifuging at 5000rpm for 10min, and filtering the supernatant by filter paper to obtain 5-8 mL of filtrate to be measured.
Preparation of NH 3 A: buffer solution. Respectively weighing 5.2g of sodium hydroxide, 11g of potassium sodium tartrate and 7.46g of potassium chloride, dissolving in 1L of distilled water together, and uniformly mixing for later use.
Preparation of NH 3 B: sodium salicylate solution. 150g of sodium salicylate and 0.30g of sodium nitroprusside are weighed, dissolved in reagent water and fixed to 1000mL. And reversing and mixing for 5 times. The sodium salicylate solution obtained after filtration through rapid filter paper was stored in dark glass bottles protected from light. The solution needs to be reconstituted every weekAnd (5) preparing once.
Preparation of NH 3 C: active chlorine solution. The reagent needs to be prepared at present, 0.2g of sodium dichloroisocyanurate is weighed before each use, dissolved in 100mL of reagent water, and then added with 0.1mL of needle cleaning liquid for mixing for later use.
NH per 100mL 3 Liquid A and NH 3 Before the solution B is used, 0.1mL of needle washing liquid is added, and the solution B is uniformly shaken for use. The nitrogen content is measured by a full-automatic intermittent chemical analyzer. Loading according to the operation steps of the instrument, and setting corresponding analysis parameters including sample quantity, measurement method, measurement range and the like. Wait for photometer to read and output the measurement result table.
As shown in the results of FIG. 10 (S1: strain treatment group; CK: control group), the application of Bacillus subtilis S strain extremely reduced the nitrate nitrogen content in rhizosphere soil on days 15 and 30 compared with the control, and as Bacillus subtilis S application can promote the growth of cucumber plants and increase their biomass, it is shown that Bacillus subtilis S application can significantly promote the absorption and utilization of nitrate nitrogen in cucumber and reduce the risk of nitrate nitrogen accumulation which is often found under the condition of facility cultivation.
Example 7: bacillus subtilis S1 application induces increased cucumber leaf defensive enzyme activity
30 pots of potted plant cucumber were prepared in the greenhouse. Selecting 10 cucumber seedlings without any treatment, namely a CK control group which is not affected all the time; selecting 10 cucumber seedlings, inoculating the spore suspension, and then performing no foliar spraying Bacillus subtilis S1 treatment, namely an NS untreated group after the disease; the rest 10 pots of cucumber seedlings are sprayed with Bacillus subtilis S bacterial liquid (the concentration is about 10) on the leaf surfaces after inoculating spore suspension 8 CFU/mL), i.e., TS treatment group. When two true leaves of cucumber seedlings grow out in the potted plant, spraying cucumber powdery mildew spore suspension, and preserving moisture overnight. When cucumber seedlings grow to a position requiring shelf insertion, the cucumber leaves at the upper, middle and lower parts of the CK group, the NS group and the TS group are randomly sampled, and the activities of three leaf defensive enzymes are measured. The TS group needs to be sampled and measured for three times on days 3, 6 and 9 after treatment.
( 1) PPO enzyme activity assay (fig. 11, ck: cucumber leaves not inoculated with powdery mildew; NS: after powdery mildew inoculation, the disease is developed but Bacillus subtilis S1 is not sprayed; TS-3/TS-6/TS-9: post inoculation onset and leaf blade at day 3, day 6 and day 9 after Bacillus subtilis S1 spraying )
0.5g of leaf blade to be measured is weighed, placed in a mortar, poured into a proper amount of liquid nitrogen for grinding to powder, added with 5mL of precooled 0.1mol/L phosphate buffer solution (containing 1% PVP and having pH of 6.5) and further ground into homogenate. Transferring the homogenate into a 15mL centrifuge tube, freezing and centrifuging at 5 000rpm for 10min at 4 ℃, and collecting supernatant to obtain crude enzyme extract. The enzyme activity measurement reaction system is shown in the following table:
TABLE 3 enzyme Activity assay reaction System
After the enzyme solution is added into the reaction system, the reaction system is put into a water bath kettle at 37 ℃ for water bath for 10min. Immediately after the completion of the reaction, 2mL of 20% trichloroacetic acid was added to each test tube to terminate the reaction, and after shaking uniformly, the reaction mixture was collected in a 1mL glass cuvette and subjected to color comparison at 525nm.
Before measurement, the preheating machine of the ultraviolet spectrophotometer is started in advance, then Simple Reads software is clicked, and after connection is successful, the default wavelength of 500nm is adjusted to 525nm. The glass dish is rinsed once by distilled water, then rinsed once by the reaction liquid, then 800 mu L of the reaction liquid is added into a dish groove, the reading is clicked, the corresponding absorbance value of the reaction liquid in each test tube is recorded, and the change of OD525 value in each minute is calculated as L enzyme activity units. The formula is as follows:
PPO[U·(g·min) -1 ]=Δaχv inverse total/[ 0.01 (W/vj x V sample) ×t ]＝122×ΔA/W
Wherein Δa: variation of absorbance values over the reaction time; v inverse total: 6.1mL of total volume of the reaction system; w: sampling fresh weight of the leaf blade, 0.5g; v, carrying out: the volume of the added extracting solution is 5mL; v sample: the added sample volume, 0.1mL; t: the reaction time is 10min;
( 2) POD enzyme Activity assay (FIG. 12, CK: cucumber leaves not inoculated with powdery mildew; NS: after powdery mildew inoculation, the disease is developed but Bacillus subtilis S1 is not sprayed; TS-3/TS-6/TS-9: post inoculation onset and leaf blade at day 3, day 6 and day 9 after Bacillus subtilis S1 spraying )
Weighing 0.5g of blade to be measured, placing in a mortar, pouring a proper amount of liquid nitrogen, grinding to powder, adding 5mL of precooled 50 mmol.L -1 Is further ground to a homogenate. Transferring the homogenate into a 15mL centrifuge tube, centrifuging at 10 rpm for 10min at 4 ℃, and collecting supernatant to obtain crude enzyme extract. The enzyme activity measurement reaction system is shown in the following table:
TABLE 4 enzyme Activity measurement reaction System
Adding 0.5mL of crude enzyme extract into a 15mL centrifuge tube, adding l mL of 0.1% guaiacol, mixing uniformly, adding 5mL of deionized water, and finally adding 1mL of 0.2% H 2 O 2 Immediately timing after shaking, and placing in a water bath kettle at 37 ℃ for water bath for 15min. After the completion of the reaction, 0.1mL of 5% metaphosphoric acid was added to each tube to terminate the reaction. After shaking uniformly, the reaction solution was collected by a 1mL glass cuvette and subjected to color comparison at 470nm, and was zeroed with a blank tube. The wavelength of the ultraviolet spectrophotometer was adjusted to 470nm at OD per minute 470 The value change of 0.01 was calculated as 1 enzyme activity unit. The formula is as follows:
POD[U·(g·min) -1 ]=Δaχvj/[ 0.01 (w×v sample×t)]＝333.3×ΔA/W
Wherein Δa: variation of absorbance values over the reaction time; v, carrying out: extracting the total volume of enzyme solution, 5mL; w: sampling fresh weight of the leaf blade, 0.5g; v sample: measuring the volume of enzyme solution used in the measurement, and 0.1mL; t: reaction time, 15min;
( 3) SOD enzyme activity assay (fig. 13, ck: cucumber leaves not inoculated with powdery mildew; NS: after powdery mildew inoculation, the disease is developed but Bacillus subtilis S1 is not sprayed; TS-3/TS-6/TS-9: post inoculation onset and leaf blade at day 3, day 6 and day 9 after Bacillus subtilis S1 spraying )
0.5g of leaf blade to be measured is weighed, placed in a mortar, poured into a proper amount of liquid nitrogen, ground into powder, added with 5mL of precooled 50mmol/L phosphate buffer solution (containing 1% PVP, pH 7.8) and further ground into homogenate. Transferring the homogenate into a 15mL centrifuge tube, centrifuging at 10000rpm and 4 ℃ for 10min, and collecting supernatant to obtain crude enzyme extract. The enzyme activity measurement reaction system is shown in the following table:
TABLE 5 enzyme Activity assay reaction System
After mixing, the control tube is covered with a double-layer black shading cloth, and the control tube, the measuring tube and the maximum light reduction tube are placed in an illumination incubator for culture, the temperature is adjusted to 25 ℃, and the light intensity is adjusted to 4000lx. After 0.5h, the assay tube and the maximum photoreduction tube were covered with black cloth and the reaction was terminated. The reaction mixture was collected in a 1mL glass cuvette, and the mixture was subjected to color comparison at 560nm, and was zeroed with an unattended control tube. The wavelength of the ultraviolet spectrophotometer was adjusted to 560nm, and the enzyme amount for inhibiting photooxidation of 50% NBT was 1 enzyme activity unit. The formula is as follows:
SOD[U·g -1 ]＝[(Ack-Ae)×V]/(0.5×Ack×W×Vt)＝200×(1-Ae/Ack)
Wherein Ack: maximum photoreduction absorbance; ae: measuring the absorbance value of the tube; v: extracting the total volume of enzyme solution, 5mL; w: sampling fresh weight of the leaf blade, 0.5g; vt: the volume of enzyme solution used for measurement was 0.1mL.
From fig. 10 to 12, it can be found that systemic disease resistance of cucumber can be induced by foliar spraying Bacillus subtilis S1, and defensive enzyme activity of cucumber leaves can be significantly improved. After the powdery mildew is inoculated artificially to the cucumber, the leaf surface is sprayed with Bacillus subtilis S1 for treatment, and on the 9 th day, the superoxide dismutase (SOD) activity of the leaf is detected to find that the SOD enzyme activity of the leaf of the S1 treatment group is 5.81 times that of the untreated control group, and the statistical difference is extremely remarkable (p < 0.01). On day 6, the activity of cucumber leaf Peroxidase (POD) in the S1 treated group was 1.21 times that in the untreated control group, and the difference reached a very significant level (p < 0.01). On day 9, the polyphenol oxidase (PPO) activity of the cucumber leaves of the S1 treatment group was 1.81 times that of the control group, and the difference reached extremely significant (p < 0.01).
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the explanation of the present invention, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The bacillus subtilis (Bacillus subtilis) S1 is characterized by being preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation number of CGMCC No.29146 at the date of 2023 and 11 month and having the preservation address of China academy of sciences of China, national institute of microbiology, national institute No.3, north Chen West Lu No. 1, the Korean area of Beijing.
2. The 16S rRNA gene sequence of bacillus subtilis (Bacillus subtilis) S1 according to claim 1, comprising a nucleotide sequence according to SEQ ID No. 3.
3. Use of bacillus subtilis (Bacillus subtilis) S1 according to claim 1 for the preparation of a product for the treatment of maize small spot.
4. Use of bacillus subtilis (Bacillus subtilis) S1 according to claim 1 for the preparation of a product for the treatment of powdery mildew in crops.
5. Use of bacillus subtilis (Bacillus subtilis) S1 according to claim 1 for the preparation of a product for promoting the growth of crops.
6. The use according to claim 5, wherein the product is a product that promotes soil enzyme activity associated with carbon nitrogen metabolism in the rhizosphere soil of a crop plant.
7. The use according to claim 6, wherein the soil enzyme is selected from one or more of soil nitrite reductase, soil cellulase or soil dehydrogenase.
8. The use according to claim 5, wherein the product is a product that promotes nitrogen levels in the rhizosphere soil of crops.
9. The use according to claim 5, wherein the product is a product that promotes the defensive enzymatic activity of a crop leaf.
10. The use according to claim 9, wherein the defensive enzyme is selected from one or more of POP enzyme, POD enzyme or SOD enzyme.
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