Preparation method and application of a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops

Abstract

The invention discloses a method for preparing a bioenzyme preparation for inhibiting and promoting growth of crops and its application. The invention separates and screens a new strain of Bacillus velezensis F41‑141 from rhizosphere soil of cotton fields in Hami, Xinjiang, and obtains a bioenzyme preparation for inhibiting and promoting growth by fermenting and metabolizing the strain. The bioenzyme preparation has a control effect on cotton Verticillium wilt, with a control effect of 78.61%. It also has an inhibitory effect on cotton wilt pathogens, tomato root rot pathogens, pear fire blight and red date black spot, and is not easy to produce drug resistance, has good drug efficacy persistence, is safe for humans and animals, and has no environmental pollution problems; at the same time, the bioenzyme preparation for inhibiting and promoting growth can also improve the yield of cotton, and has the potential to promote the growth of crops as an organic fertilizer.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme preparation, and in particular to a method for preparing a crop bacteriostasis and growth promotion biological enzyme preparation and the technical field of application thereof. Background Art

[0002] Antibacterial and growth-promoting bio-enzymes are a type of bio-enzymes. Antibacterial and growth-promoting bio-enzymes can change the growth environment of plants, increase the fresh and dry weight of seedlings, root length, root activity, plant height, and stem thickness, enhance crop growth, and increase leaf area and chlorophyll content to promote photosynthetic rate, transpiration rate, stomatal conductance, intercellular concentration and other photosynthetic indicators, thereby increasing crop yields.

[0003] Antibacterial and growth-promoting bio-enzyme fertilizer is a new type of organic fertilizer. It uses organic matter as raw material and is fermented through the action of biological complex enzymes. The enzyme fertilizer contains rich beneficial microorganisms and a large number of biological nutrient elements. These elements are biologically active substances that cannot be synthesized by artificial and chemical fertilizers. When applied to the soil and in contact with fertilizers or corresponding substrates in the soil, it can reduce the energy required for the biochemical degradation of such substances, rapidly shorten the decomposition cycle of substances such as cellulose, release nutrients, improve fertilizer utilization and the release rate of organic nutrients, and improve the physical and chemical properties of the soil, thereby promoting plant growth. At the same time, it can also reduce the amount of fertilizer used and improve fertilizer utilization.

[0004] With the growth of population, industrial and urban development in my country, the cultivated land area has been decreasing year by year. For a long time, chemical fertilizers have been used in large quantities while the role of microorganisms and organic fertilizers has been ignored, resulting in unbalanced nutrients in crops, and reduced yield and quality. In addition, extreme weather changes often trigger and aggravate the phenomenon of crop lodging, resulting in poor crop maturity, poor cotton fiber quality and reduced wheat flour output, poor taste of fruits and vegetables, reduced nutritional quality, and poor benefits. These problems have led to a serious shortage of comprehensive agricultural production capacity, directly threatening and affecting the safe production of my country's agriculture, and seriously restricting the sustainable development of the agricultural economy. The antibacterial and growth-promoting bioenzyme fertilizer can improve the ability of crops to resist waterlogging and drought, enhance the immunity of crops, help resist insect pests, and thus reduce dependence on pesticides; at the same time, it can adsorb soil heavy metal ions and reduce the harm of heavy metals to soil and plants. The antibacterial and growth-promoting bioenzyme fertilizer is not only beneficial to improving agricultural production, but also beneficial to ecological environmental protection. In the production of green food, the antibacterial and growth-promoting bioenzyme fertilizer is a main fertilizer, which contains rich nutrients and is harmless to plants, and is conducive to improving the quality of green products. Therefore, applying antibacterial and growth-promoting bio-enzyme fertilizer is the most economical and effective way to improve soil microbial activity and soil fertility. Summary of the invention

[0005] Aiming at the technical problem that currently chemical fertilizers are used in large quantities and the role of microorganisms and organic fertilizers is ignored, resulting in unbalanced nutrients in crops and reduced yield and quality, the present invention aims to provide a preparation method and application of a microbial antibacterial and growth-promoting bioenzyme preparation. The present invention isolates and screens a new strain of Bacillus velezensis F41-141 that has been identified through a series of tests from the rhizosphere soil of a cotton field in Hami, Xinjiang. The new strain of Bacillus velezensis F41-141 is used to velezensis) F41-141 fermentation metabolism prepared by the antibacterial and growth-promoting biological enzyme preparation, the disease index of cotton Verticillium wilt after treatment is 17.15, the control effect is 78.61%, indicating that the antibacterial and growth-promoting biological enzyme preparation has a control effect on cotton Verticillium wilt, and the antibacterial and growth-promoting biological enzyme preparation also has a good inhibitory effect on cotton wilt bacteria, tomato root rot bacteria, amylopectin and red date black spot disease; at the same time, it is not easy to produce drug resistance, the drug effect is long-lasting, it is safe for humans and animals, and there is no environmental pollution problem; in terms of growth promotion, the antibacterial and growth-promoting biological enzyme preparation can improve the yield of cotton. When the antibacterial and growth-promoting biological enzyme preparation is used, the seed cotton yield reaches 6206.21kg / hm 2 , the cotton output reached 2602.26kg / hm 2 The difference was significant compared with the control. The seed cotton yield and lint cotton yield of the control group were only 74.78% and 72.45% of those treated with enzyme preparations. It has the potential to be used as an organic fertilizer to promote the growth of crops and has broad market prospects and promotion value.

[0006] In order to achieve the above technical effects, the present invention is implemented through the following technical solutions.

[0007] The invention provides a crop bacteriostatic and growth-promoting biological enzyme. The crop bacteriostatic and growth-promoting biological enzyme is obtained by metabolism of a new bacterial species Bacillus velezensis F41-141. The new bacterial species Bacillus velezensis F41-141 is deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection with a deposit number of CGMCC No.31741, and a 16Sr RNA gene sequence thereof is shown in SEQ ID NO:1.

[0008] The invention provides a crop bacteriostasis and growth promotion biological enzyme preparation, which comprises an enzyme obtained by fermentation metabolism of a new strain of Bacillus velezensis F41-141.

[0009] The antibacterial and growth-promoting bioenzyme preparation of the present invention is a liquid preparation or a powder.

[0010] In the present invention, the culture medium used for purifying Bacillus velezensis F41-141 comprises: 3 g / L beef extract, 7 g / L peptone, 5 g / L NaCl, 17 g / L agar, and a pH value of 7.0.

[0011] The present invention provides a method for preparing a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops, which specifically comprises the following steps:

[0012] (1) Inoculating a new strain of Bacillus velezensis F41-141 at a mass volume ratio of 1% to 5% into a corn flour and soybean flour culture medium with a pH value of 7.0, fermenting and culturing for 28 to 38 hours at 18 to 40° C. and a shaking speed of 120 to 220 rpm to obtain a fermentation liquid of Bacillus velezensis F41-141; centrifuging the fermentation liquid at 8000 r / min for 10 minutes, and absorbing the supernatant to obtain a bio-enzyme preparation for inhibiting and promoting growth of crops;

[0013] (2) Detect the number of bacteria and spores in the fermentation liquid obtained in step (1), and stop the fermentation culture when the mature spores in the fermentation liquid account for 90% of the total number of spores and bacteria, and prepare a liquid enzyme preparation or a powdered enzyme preparation of a bio-enzyme preparation for inhibiting bacterium and promoting growth of crops as needed.

[0014] The above-mentioned corn flour and soybean flour culture medium is: corn flour 2.0g / L, soybean flour 2.5g / L, NaCl 0.5g / L, MnSO4·H2O 0.7g / L, pH value = 7.0.

[0015] The viable bacteria count of the above-mentioned antibacterial and growth-promoting bioenzyme preparation is not less than 10 8 CFU / mL.

[0016] The invention provides an application of a crop bacteriostasis and growth-promoting bioenzyme preparation in cotton growth promotion, disease prevention or stress resistance.

[0017] The invention provides an application of a crop bacteriostatic and growth-promoting bioenzyme preparation in preparing medicines for preventing and treating cotton Verticillium wilt, cotton Fusarium wilt, amylovora and red date black spot.

[0018] At the same time, the present invention provides an application of a crop bacteriostatic and growth-promoting bioenzyme preparation in the preparation of fertilizers.

[0019] Through the above technical scheme, the present invention obtains the following technical effects:

[0020] (1) After the antibacterial and growth-promoting bioenzyme preparation provided by the present invention was used to treat cotton Verticillium wilt, the disease index was 17.15, and the control effect was 78.61%, indicating that the antibacterial and growth-promoting bioenzyme preparation has a control effect on cotton Verticillium wilt, and the antibacterial and growth-promoting bioenzyme preparation also has a good inhibitory effect on cotton wilt bacteria, tomato root rot bacteria, pear fire blight and red date black spot disease; at the same time, it is not easy to produce drug resistance, has good long-lasting efficacy, is safe for humans and animals, and does not cause environmental pollution.

[0021] (2) The antibacterial and growth-promoting bioenzyme preparation provided by the present invention can extend the length of cotton fibers, increase the fiber elongation, and reduce the micronaire value.

[0022] (3) Treatment with the antibacterial and growth-promoting bioenzyme preparation provided by the present invention can improve the yield structure of cotton and is conducive to the high yield of cotton. The weight of a single boll of cotton can reach 5.56 g, the number of bolls per plant can reach 6.05, and the lint percentage can reach a maximum of 41.93% after enzyme treatment. After using the antibacterial and growth-promoting bioenzyme preparation, the seed cotton yield is 6206.21 kg / hm 2 , lint production 2602.26kg / hm 2 . BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is a schematic diagram of the number of strains isolated from cotton field soil and cotton stalks using different culture media.

[0024] Figure 2 The figure shows the plate confrontation effect between the screened antagonistic strains and Verticillium dahliae SMN-1.

[0025] Figure 3 Shown are the colony morphology and Gram staining results of Bacillus velezensis F41-141.

[0026] Figure 4 Shown is the phylogenetic tree of Bacillus velezensis F41-141.

[0027] Figure 5 Shown is a schematic diagram of the results of the growth-promoting performance test of Bacillus velezensis F41-141.

[0028] Figure 6 The graph shows the effect of different growth conditions on the antibacterial activity of enzyme preparation of Bacillus velezensis F41-141.

[0029] Figure 7Schematic diagram showing the dynamic analysis of the number of Bacillus velezensis F41-141 colonizing the roots of cotton seedlings over time

[0030] Figure 8 The figure shows the effect of Bacillus velezensis F41-141 enzyme treatment on cotton plant height, root length, root hair number and leaf number. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0032] The culture medium used in the present invention is potato dextrose agar medium (PDA), NA medium, King's B medium (KB), tryptone soy agar (TSA), R2A agar medium, improved Gao's medium No. 1, cellulase enzyme production medium, protease enzyme production medium, and amylase enzyme production medium.

[0033] King's B medium: peptone 20 g, K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, glycerol 10.0 mL, L-tryptophan 0.1 g.

[0034] Ashby medium (1L): K2HPO4 0.2g, MgSO4·7H2O 0.2g, CaSO4·2H2O 0.1g, NaCl 0.2g, CaCO3 5.0g, mannitol 10g, agar 18g.

[0035] PKO medium (1L): glucose 10 g, (NH4)2SO4 0.5 g, NaCl 0.3 g, KC1 0.3 g, MgSO4 0.3 g, FeSO4 0.03 g, MnSO4 1 g, Ca3(PO4)25 g, agar 15 g.

[0036] The cotton Verticillium dahliae SMN-1 used in the present invention was isolated and identified from Shihezi cotton stalks by our laboratory in the early stage according to a standard screening procedure, and was identified as a conventional strain.

[0037] The test soil of the present invention is collected from cotton field soil and cotton stalks in the eastern Xinjiang-Hami City, southern Xinjiang-Zep County, and northern Xinjiang-Shihezi areas of Xinjiang where cotton Verticillium wilt is seriously infected.

[0038] Example 1: Isolation and screening of strains producing IAA and having antagonistic effects on Verticillium dahliae (I) Isolation and purification

[0039] Cotton field soil samples: 10 g of soil sample was placed in a conical flask filled with 90 mL of sterile saline solution, and cultured at 30°C and 150 r / min for 30 min. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 Dilution was performed in a concentration gradient. 100 μL of soil dilutions of each gradient was taken and spread on NA, KB, TSA, R2A and modified Gao's medium No. 1 plates, inverted and cultured in a 30°C constant temperature incubator for 24 hours, and bacterial single colonies of different morphologies were selected for multiple purifications.

[0040] Cotton stalk sample: Disinfect the surface of the sample by placing the cotton stalk in 75% alcohol for 30 seconds, and in 1% sodium hypochlorite for 60 seconds. Rinse with sterile water three times and dry with sterile filter paper. Take 1g of sample tissue block, cut it into pieces, place it in a sterile mortar and crush it, and add 9mL of sterile saline, dilute it in a gradient of 10 -1 , 10 -2 , 10 -3 100 μL of each gradient was spread on NA, KB, TSA, R2A and modified Gao's medium No. 1 plates, inverted in a 30°C constant temperature incubator and cultured for 24 hours. After the colonies grew out, single colonies of different colors and morphologies were picked for multiple purifications.

[0041] The strains were isolated and purified from the cotton field soil and cotton stalk samples, and a total of 316 strains were obtained. The results of isolation and culture using different culture media showed that the number of strains isolated from cotton field soil samples was higher than that from cotton stalk samples. The number of strains isolated from NA culture medium was the largest, totaling 106 strains, while the number of strains isolated from modified Gao's No. 1 culture medium was relatively small. The number of strains isolated from cotton field soil and cotton stalks can be found in the attached Figure 1 .

[0042] (II) Screening of IAA-producing strains

[0043] The isolated and purified strains were inoculated into King's B medium and cultured. The Salkowski colorimetric method was used for color development. If the color turned pink, it indicated that IAA could be secreted. The positive strains were inoculated into King's B liquid medium at 30°C and 150 r / min. -1 Oscillating culture for 3 days, 12000 r·min -1Centrifuge for 5 minutes, take an equal volume of the supernatant and add Salkowski colorimetric reagent, place at room temperature and away from light for 30 minutes, then measure the OD 530 The King's B liquid medium without bacteria was used as the control, and the IAA (analytical grade) gradient dilution solution was used to draw a standard curve, and the IAA content in the fermentation broth was calculated based on the standard curve.

[0044] Standard curve drawing: Prepare 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 mg·L of IAA stock solution -1 Take 1 mL of the standard IAA gradient solution and add an equal amount of Salkowski reagent to mix. Place it at room temperature and avoid light for 30 minutes before measuring the OD 530 value.

[0045] A total of 45 IAA-producing strains were screened from the 316 isolated and purified strains. The detailed results are shown in Table 1. The IAA secretion of the strains ranged from 0.69 to 54.82 mg·L -1 Among them, strains T46-19, T41-19, and T50-17 had relatively strong IAA production capabilities, with IAA production of 54.82 mg·L -1 , 38.85mg·L -1 , 28.05mg·L -1 .

[0046] Table 1: IAA secretion in the screened strains

[0047] Strainnumber <![CDATA[Concentration / mg·L -1 ]]> Strainnumber <![CDATA[Concentration / mg·L -1 ]]> F31-10 3.86±0.42 N57-14 3.65±0.37 N67-19 2.23±0.36 N48-17 3.46±0.53 T46-12 5.01±0.19 R15-9 6.58±0.67 F41-141 4.15±0.97 R7-19 3.86±0.46 T50-17 28.05±0.51 R9-11 5.70±0.55 F27-15 16.45±0.14 T47-10 7.46±0.72 N69-11 15.27±0.36 N46-2 5.62±0.92 R3-12 11.30±0.53 N11-12 57.57±0.49 N85 3.67±0.52 R7-15 6.85±0.47 N61-8 4.82±0.46 T46-19 54.82±0.98 T4-15 4.82±0.43 N5-16 15.59±0.69 T8-14 3.14±0.32 N111 8.85±0.70 F35-4 3.73±0.75 R7-18 28.37±0.08 T47-18 4.55±0.51 R14-1 2.53±0.30 N110 23.14±0.04 N35-19 5.03±0.28 F39-2 2.74±0.14 T41-18 38.85±0.93 K5-13 0.69±0.16 T15-19 3.99±0.00 N38-20 5.03±0.74 R11-8 23.70±0.32 K11-5 25.94±0.68 R12-19 7.59±0.07 R9-5 4.34±0.53 T3-9 14.10±0.23 N94 0.87±0.99 N6-15 4.47±0.24 R10-1 6.02±0.20 N48-9 4.69±0.05 N57-5 2.39±0.79 - -

[0048] (III) Screening of antagonistic strains

[0049] Antagonistic strains were screened using the plate confrontation method. Preparation of antagonistic strain fermentation broth: The screened strains were inoculated into NB liquid culture medium and cultured at 30°C and 150 r·min -1 The culture was shaken for 48 hours to obtain the fermentation liquid. Preparation of spore suspension of pathogenic fungus Verticillium dahliae SMN-1: 15 spores of Verticillium dahliae SMN-1 were punched out with a 6mm sterile puncher and inoculated into PDB liquid culture medium at 28°C and 150 r·min. -1 After shaking culture for 5 days, the spore suspension was filtered through four layers of sterile gauze. The concentration of the spore suspension was adjusted to 1×10 6Spores / mL. 100μL of Verticillium dahliae SMN-1 spore suspension was applied to the PDA plate and allowed to stand for 30 minutes. Three holes were punched at equal distances at 2 cm from the center of the PDA plate using a sterile hole puncher. 100μL of antagonistic bacteria sterile filtrate was added to each well. The sterilized NB medium was used as a control. The plates were cultured at 28°C for 4 days and the diameter of the inhibition zone was measured.

[0050]

[0051] The plate confrontation method was used to detect the antagonistic function of 45 strains against Verticillium dahliae SMN-1. Among them, 8 strains had good antagonistic effect on Verticillium dahliae SMN-1. The results are shown in Figure 2 The results are shown in Table 2. Among them, the strain numbered F41-141 had the most significant antagonistic effect, with an inhibition zone diameter of 25.33±0.09 mm, an inhibition rate of 76.32%, and an IAA production of 4.15 mg·L against Verticillium dahliae SMN-1. -1 Therefore, the strain numbered F41-141 was selected as the target strain for subsequent research.

[0052] Table 2: Antagonistic effect of antagonistic strains on Verticillium dahliae SMN-1

[0053] Strainnumber Inhibitorycirclediameter / mm Antibacterialrate / % F41-4 25.33±0.09a 76.32±0.08a F39-2 23.94±0.15b 74.94±0.16b R9-5 21.61±0.23c 72.23±0.30c T47-18 20.29±0.24d 70.43±0.35d N67-19 18.52±0.27e 67.59±0.47e N111 18.30±0.33e 67.22±0.59e R7-19 17.11±0.15f 64.93±0.30f N5-16 17.02±0.15f 64.75±0.32f

[0054] Example 2: Identification of morphological and physiological and biochemical characteristics of Bacillus velezensis F41-141

[0055] (I) Morphological identification

[0056] Refer to the Manual of Common Bacterial Systematic Classification for physiological and biochemical identification. Observe the colony morphology, color, ridge shape and transparency, etc., and determine the strain type by Gram staining the strain and observing it with an oil immersion lens. Perform citrate utilization, VP test, enzyme activity and other tests.

[0057] The results are shown in the attached Figure 3As shown in the figure, the strain numbered F41-141 was cultured on the NA plate for 48 hours, and the colony was nearly round, milky white, opaque, dry and wrinkled on the surface, sticky, Gram-staining positive, and rod-shaped under electron microscope. The results of physiological and biochemical identification are shown in Table 3. The strain numbered F41-141 was positive in contact enzyme test, starch hydrolysis, indoleacetic acid, nitrogen fixation test, phosphate solubility, etc., and negative in oxidase, urease, VP reaction, sodium citrate, methyl red, etc. It can be seen that the strain numbered F41-141 has distinct differences in physiological and biochemical characteristics from the common Bacillus velezensis, showing some characteristics of a new strain.

[0058] Table 3: Physiological and biochemical characteristics of strain Bacillus velezensis F41-141

[0059] Test items feature Test items feature Catalase + D-Mannitol + Oxidase - D-Galactose + Urease - D-Pectin - Starch hydrolysis + sucrose + VP - Twain 40 + Sodium Citrate - pH5 + Methyl red - pH6 + Indoleacetic acid + 1% NaCl + Nitrogen fixation - 4% NaCl + Phosphate solubilization + 8% NaCl +

[0060] Note: “+” means positive; “-” means negative.

[0061] (II) Identification of physiological and biochemical characteristics

[0062] The genomic DNA of the strain was extracted using a bacterial genome extraction kit, and the 16S rDNA gene of the strain was amplified using the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). PCR reaction system (25μL): genomic DNA 1μL, Mix 12.5μL, primer 0.5μL, ddH2O 11μL. PCR reaction conditions: 95℃5min; 95℃30s, 58℃15s, 72℃2min, a total of 20 cycles; 72℃10min. The PCR product was sequenced by Shanghai Shenggong Biotechnology Co., Ltd. The sequence length of the strain numbered F41-141 is 1411bp, and its sequence is shown in SEQ ID NO: 1. The obtained sequence results were uploaded to the EzBioCloud database for sequence alignment, and homologous sequences with high similarity were selected to construct a phylogenetic tree using the neighbor-joining method using MEGA 11.0 software. The results are shown in the attached Figure 4As shown, the gene sequence homology of the strain numbered F41-141 and Bacillus velezensis CR-502 (ID: AY603658) is 98.36%; after molecular identification of the strain system well known in the art and systematic test verification combined with the physiological and biochemical characteristics of the strain, it was confirmed that the strain numbered F41-141 belongs to a new species in the Bacillus velezensis branch, and it is tentatively named Bacillus velezensis F41-141. The strain was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on August 26, 2024, and the strain deposit number is: CGMCC No.31741.

[0063] Example 3: Effect of different growth conditions on the activity of Bacillus velezensis F41-141

[0064] Screen the conditions for the best antibacterial effect under different culture time, inoculation amount, temperature and pH, and simultaneously measure the OD under different influencing factors 600 The values ​​were used to judge the growth of the strain. (1) The fermentation time was 24, 48, 72, 96, and 120 h; (2) The inoculation amount was 1%, 2%, 3%, 4%, and 5%; (3) The culture temperature was 26, 28, 30, 32, and 34°C; (4) The initial pH was 5, 6, 7, 8, and 9.

[0065] The results of the effects of culture time on strain growth and antibacterial activity are shown in the Appendix. Figure 6 -a, after 24 to 72 hours of culture, the growth of the strain decreased, but the antibacterial activity of the enzyme preparation continued to increase, reaching the maximum value at 72 hours, and the diameter of the inhibition zone was 24.84±0.51mm. After 72 hours, the antibacterial activity of the enzyme preparation decreased with the extension of the culture time.

[0066] The results of the effect of inoculum size on strain growth and enzyme antibacterial activity are shown in the Appendix. Figure 6 -b, when the inoculation amount was 1-3%, the growth of the strain and the antibacterial activity of the enzyme preparation gradually increased, reaching the maximum value when the inoculation amount was 3%, and the diameter of the inhibition zone was 25.13±0.38mm, OD 600 The value is 1.54.

[0067] The results of the effects of pH on strain growth and enzyme antibacterial activity are shown in the Appendix. Figure 6-c shows that the strains can grow in the pH range of 5-9. When the pH is 5-7, the growth ability of the strains and the antibacterial activity of the enzyme preparations gradually increase; when the pH is 7, it reaches the best, and the diameter of the inhibition zone is 25.05±0.06mm; when the pH>7, the growth rate and the antibacterial activity of the enzyme preparations gradually decrease.

[0068] The results of the effects of temperature on strain growth and enzyme antibacterial activity are shown in the Appendix. Figure 6 -d, as shown in Figure 3, with the increase of temperature, the growth of the strain gradually increased, and the antibacterial activity of the enzyme preparation also increased accordingly, reaching a maximum value at 30°C, with an inhibition zone diameter of 25.49±0.11mm. After that, the growth amount and the antibacterial activity of the enzyme preparation decreased as the temperature continued to rise.

[0069] Example 4: Determination of enzyme production and growth-promoting function of Bacillus velezensis F41-141

[0070] Enzyme production capacity detection: Take 5 μL of the fermentation liquid of the antagonistic strain and inoculate it on the solid culture medium for cellulase production, the solid culture medium for amylase production, and the solid culture medium for protease production. Culture it at 30°C for 3 days, observe whether there is a transparent circle, measure the diameter of the transparent circle (D) and the diameter of the colony (d), and judge the enzyme production capacity of the antagonistic strain by the ratio (D / d).

[0071] Detection of nitrogen fixation ability: The antagonistic strain was inoculated on an Ashby medium plate and cultured at 30°C for 5 days, and then the growth of the colony was observed. The strains that could grow normally on Ashby medium after five consecutive subcultures were considered to have nitrogen fixation activity or nitrogen fixation ability.

[0072] Detection of phosphate solubilization ability: The strain was inoculated onto PKO solid culture medium and cultured at 30℃ for 7 days. The diameter of the phosphate solubilization circle (D) and the diameter of the colony (d) were measured. The phosphate solubilization ability of the strain was preliminarily judged based on whether the phosphate solubilization circle could be produced and the size of the D / d value.

[0073] The test results are shown in the attached Figure 5 As shown, Bacillus velezensis F41-141 has the ability to produce protease, cellulase and amylase. Figure 5 -a, a white transparent circle can be directly seen on the protease qualitative plate, the diameter of the transparent circle (D) is (38.14±0.63) mm, the diameter of the colony (d) is (12.54±0.63) mm, and the ratio of the diameter of the transparent circle (D) to the diameter of the colony (d) is (3.10±0.18) (7-b); Figure 5-b, the cellulase-producing strain will produce a transparent zone after Congo red staining and sodium chloride washing. The diameter of the transparent zone (D) is (27.58±1.30) mm, the colony diameter (d) is (9.69±1.02) mm, and the ratio of the transparent zone diameter (D) to the colony diameter (d) is (2.86±0.18) (7-a); Figure 5 -c The amylase-producing plate also had a transparent zone after being stained with iodine solution. The diameter of the transparent zone (D) was (22.69±0.30) mm, the diameter of the colony (d) was (16.56±0.05) mm, and the ratio of the diameter of the transparent zone (D) to the diameter of the colony (d) was (1.37±0.01) (7-c); Figure 5 -d, Bacillus velezensis F41-141 strain formed a transparent zone on PKO solid culture medium, indicating that it had a dissolving effect on calcium phosphate. The diameter of the transparent zone (D) was (11.35±0.43) mm, the diameter of the colony (d) was (5.99±0.44) mm, and the ratio of the diameter of the phosphate-dissolving zone (D) to the diameter of the colony (d) was (1.90±0.15) (7-d), indicating that Bacillus velezensis F41-141 strain did not grow on Ashby solid culture medium, indicating that it had no nitrogen fixation ability.

[0074] Example 5: A method for preparing a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops

[0075] A method for preparing a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops, comprising the following steps:

[0076] (1) Inoculating a new strain of Bacillus velezensis F41-141 at a mass volume ratio of 1% to 5% into a corn flour and soybean flour culture medium with a pH value of 7.0, fermenting and culturing for 28 to 38 hours at 18 to 40° C. and a shaking speed of 120 to 220 rpm to obtain a fermentation liquid of Bacillus velezensis F41-141; centrifuging the fermentation liquid at 8000 r / min for 10 minutes, and absorbing the supernatant to obtain a bio-enzyme preparation for inhibiting and promoting growth of crops;

[0077] (2) Detect the number of bacteria and spores in the fermentation liquid obtained in step (1), and stop the fermentation culture when the mature spores in the fermentation liquid account for 90% of the total number of spores and bacteria, and prepare a liquid enzyme preparation or a powdered enzyme preparation of a bio-enzyme preparation for inhibiting bacterium and promoting growth of crops as needed.

[0078] The corn flour and soybean flour culture medium comprises: 2.0 g / L corn flour, 2.5 g / L soybean flour, 0.5 g / L NaCl, 0.7 g / L MnSO4·H2O, and a pH value of 7.0.

[0079] The number of viable bacteria in the antibacterial and growth-promoting bioenzyme preparation is not less than 10 8 CFU / mL.

[0080] Example 6: Application of antibacterial and growth-promoting bioenzyme preparations in cotton seedling pot planting

[0081] The soil was sterilized at 160℃ for 2h, and the cotton seed Xinluzao No. 36 was planted. When the cotton seedlings grew to two leaves and one heart, the inoculation concentration was 2×10 7 The fermentation liquid of Verticillium wilt was inoculated with 10 mL of spores / mL of Verticillium wilt, and all were marked as VD. After 6 days, the roots were inoculated with antibacterial and growth-promoting bioenzyme preparations at a concentration of 2×10 8 CFU / mL, the inoculation volume was 10mL / plant, and the control group VD was inoculated with the same volume of sterilized NB medium. Root irrigation was performed every 6 days for a total of 3 times.

[0082] The colonization amount of Bacillus velezensis F41-141 in the root soil was determined. Two days after the third inoculation of Bacillus velezensis F41-141, one row of cotton seedlings was taken from each treatment. The root soil was collected after the seedlings were taken, mixed, air-dried, and 5 g was weighed into a conical bottle filled with 45 mL of sterile saline. The soil was incubated at 25 ° C and 150 r min -1 Oscillate for 2 hours and select 10 -4 , 10 -5 , 10 -6 Dilution gradient, take 100 μL of each and spread it on NA medium plate, culture at 30℃ for 24h, count the number of bacterial colonies, sample once every 5 days, for a total of 5 times.

[0083] For the determination of cotton plant biomass, 4 to 7 cotton seedlings were randomly selected from each treatment group and control group, their roots were rinsed clean, and the plant height, number of root hairs, number of leaves and root length of the cotton seedlings were counted.

[0084] Disease survey was conducted after 60 days of growth of cotton seedlings. Grade 0: healthy plants; Grade 1: 1 to 2 cotyledons were diseased; Grade 2: 1 true leaf was diseased; Grade 3: more than 2 true leaves were diseased or fell off; Grade 4: all leaves fell off or the top of the leaves died. According to the cotton Verticillium wilt disease index grading standard, the cotton Verticillium wilt disease index and control effect were calculated.

[0085]

[0086] Example 7: Application of antibacterial and growth-promoting bioenzyme preparations in cotton seedling pot planting

[0087] (I) Colonization of Bacillus velezensis F41-141 producing antibacterial and growth-promoting enzymes in cotton roots

[0088] The results are shown in the attached Figure 7 As shown in the figure, the colonization number of strain Bacillus velezensis F41-141 decreased within 2 to 7 days after inoculation, gradually increased from 7 to 17 days, and decreased from 17 to 22 days. From the overall colonization analysis, the strain had a high colonization number in the soil and good adaptability. The colonization number was 1.58×10 7 ~4.17×10 7 CFU / mL range.

[0089] (II) The growth-promoting effect of antibacterial and growth-promoting bioenzyme preparations on cotton plants

[0090] The results are shown in the attached Figure 8 As shown in the figure, 2 to 22 days after treatment with antibacterial and growth-promoting bioenzyme preparations, the plant height, number of leaves and number of root hairs in the treatment group increased to a certain extent compared with the control group. During the 2 to 17 day period, the plant height, number of root hairs and number of leaves showed an increasing trend, and began to decline after 17 days. The results showed that the treatment with antibacterial and growth-promoting bioenzyme preparations increased the plant height, number of leaves and number of root hairs of cotton plants, and promoted the growth of cotton plants.

[0091] (III) The effect of antibacterial and growth-promoting bioenzyme preparations on the prevention of cotton Verticillium wilt

[0092] The results of potted plant research showed that the disease index of the VD treatment group was 80.20, and the disease index after treatment with antibacterial and growth-promoting bioenzyme preparations was 17.15, with a control effect of 78.61%, which was significantly different from the VD treatment group. This showed that the enzyme preparation treatment group of strain F41-141 could reduce the disease index of cotton Verticillium wilt and had a control effect on cotton Verticillium wilt.

[0093] (IV) Effects of antibacterial and growth-promoting bioenzyme preparations on cotton fiber quality

[0094] It can be seen from Table 4 that after using the antibacterial and growth-promoting bioenzyme preparation, the average length of the upper half of the cotton fiber reached 32.5 mm, the uniformity reached 86.8%, the elongation reached 12.7%, and the textile parameters (P<0.05) could reach 172, indicating that the antibacterial and growth-promoting bioenzyme preparation extended the fiber length, increased the fiber elongation, and reduced the micronaire value.

[0095] Table 4: Effects of antibacterial and growth-promoting bioenzyme preparations on cotton fiber quality

[0096]

[0097] Note: Data are mean ± standard deviation; Different lowercase letters in the same column indicate significant differences among treatments in the same year at the 0.05 level.

[0098] (V) Effects of antibacterial and growth-promoting bioenzyme preparations on improving fertilizer utilization

[0099] When cotton is harvested, the H2SO4-H2O2 digestion method is used, the Kjeldahl nitrogen analyzer is used to determine the nitrogen content of the plants, the molybdenum antimony colorimetric method is used to determine the phosphorus content, and the flame spectrophotometry is used to determine the potassium content. Nutrient utilization rate = (total amount of nutrients absorbed by the crop - total amount of nutrients absorbed by the crop in the unfertilized area) / total amount of the nutrient in the fertilizer × 100%

[0100] As can be seen from Table 5, the antibacterial and growth-promoting bioenzyme preparations have a significant effect on the utilization rate of nitrogen, phosphorus and potassium fertilizers in cotton. The amount of N absorbed by cotton treated with enzyme preparations in the soil is 1784.95 kg / hm 2 , the absorption of P is 550.17kg / hm 2 The absorption of K is 1695.47 kg / hm 2 , it was concluded that enzyme preparations significantly promoted cotton's absorption of nitrogen, phosphorus and potassium in the soil. The fertilizer utilization rates of cotton were 46.52% for nitrogen fertilizer (N), 17.39% for phosphorus fertilizer (P2O5), 39.67% for potassium fertilizer (K2O), and 52.36% for total fertilizer utilization. The results showed that cotton's fertilizer utilization rate was potassium fertilizer > nitrogen fertilizer > phosphorus fertilizer; the total fertilizer utilization rate was higher than the utilization rate of each fertilizer used alone, indicating that nitrogen, phosphorus and potassium formula fertilization is conducive to improving fertilizer utilization.

[0101] Table 5: Effects of antibacterial and growth-promoting bioenzyme preparations on cotton nutrient absorption

[0102]

[0103] Note: Data are mean ± standard deviation; Different lowercase letters in the same column indicate significant differences among treatments in the same year at the 0.05 level.

[0104] (VI) Effects of antibacterial and growth-promoting bioenzyme preparations on cotton yield

[0105] As can be seen from Table 6, the weight of a single boll of cotton treated with antibacterial and growth-promoting bioenzyme preparations can reach 5.56g, which is significantly higher than that of the control group; the number of bolls per plant reaches 6.05, which is significantly higher than that of the control group; the number of harvested plants is not much different among the treatments; the lint percentage of the enzyme preparation treatment is the highest at 41.93%, which is significantly different from the control group. In terms of final yield, the seed cotton yield after using the antibacterial and growth-promoting bioenzyme preparations is 6206.21kg / hm 2 , lint production 2602.26kg / hm 2The difference was significant compared with the control. The seed cotton yield and lint cotton yield of the control group were only 74.78% and 72.45% of those treated with enzyme preparations. The yield composition of the treatment with antibacterial and growth-promoting bioenzyme preparations was the best, indicating that the treatment with antibacterial and growth-promoting bioenzyme preparations could improve the yield composition of cotton and facilitate high yield formation.

[0106] Table 6: Effects of antibacterial and growth-promoting bioenzyme preparations on cotton fiber quality

[0107]

[0108] Note: Data are mean ± standard deviation; Different lowercase letters in the same column indicate significant differences among treatments in the same year at the 0.05 level.

[0109] Example 8: Application of Bacillus velezensis F41-141 enzyme preparation in the preparation of drugs for preventing and treating cotton wilt, amylovora blight and red date black spot

[0110] The three pathogenic fungi tested in this example are from the Crop Beneficial Microorganism Laboratory of the Institute of Microbial Application of Xinjiang Academy of Agricultural Sciences, namely: Erwinia amylovora Ea-1, Fusarium oxysporium f.sp. vasinfectum F2015 and Alternaria alternata A2015. The inhibitory effect of the determination method of Example 5 on the three plant pathogenic fungi is shown in Table 7 for details.

[0111] Table 7: Inhibitory effects of antibacterial and growth-promoting bioenzyme preparations on three plant pathogenic fungi

[0112] Pathogen name Control growth (mm) Treatment growth (mm) Antibacterial rate (%) Erwinia amylovora 83 8 90.36 Fusarium wilt 82 7 91.46 Black spot pathogen 84 5 94.05

[0113] It can be seen from the data in Table 7 that the average antibacterial rate of the microbial enzyme preparation against the three tested pathogenic fungi is 91.96%, indicating that the microbial enzyme preparation provided by the present invention has a broad spectrum of antibacterial activity.

[0114] The above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A bio-enzyme for inhibiting bacteria and promoting growth of crops, characterized in that: The crop antibacterial and growth-promoting biological enzyme is obtained by metabolism of a new strain of Bacillus velezensis F41-141; the new strain of Bacillus velezensis F41-141 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.31741, and its 16Sr RNA gene sequence is shown in SEQ ID NO:

1.

2. A bio-enzyme preparation for inhibiting bacteria and promoting growth of crops, characterized in that: The crop bacteriostasis and growth-promoting biological enzyme preparation comprises an enzyme obtained by fermentation metabolism of a new bacterial species Bacillus velezensis F41-141.

3. The crop antibacterial and growth-promoting bioenzyme preparation according to claim 2, characterized in that: The antibacterial and growth-promoting bioenzyme preparation is a liquid preparation or a powder.

4. A culture medium for purifying Bacillus velezensis F41-141, characterized in that The culture medium comprises: 3 g / L beef extract, 7 g / L peptone, 5 g / L NaCl, 17 g / L agar, and a pH value of 7.

0.

5. A method for preparing a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops, characterized in that: The specific steps include: (1) Inoculating a new strain of Bacillus velezensis F41-141 at a mass volume ratio of 1% to 5% into a corn flour and soybean flour culture medium with a pH value of 7.0, fermenting and culturing for 28 to 38 hours at 18 to 40° C. and a shaking speed of 120 to 220 rpm to obtain a fermentation liquid of Bacillus velezensis F41-141; centrifuging the fermentation liquid at 8000 r / min for 10 minutes, and absorbing the supernatant to obtain a bio-enzyme for inhibiting and promoting growth of crops; (2) Detect the number of bacteria and spores in the fermentation liquid obtained in step (1), and stop the fermentation culture when the mature spores in the fermentation liquid account for 90% of the total number of spores and bacteria, and prepare a liquid enzyme preparation or a powdered enzyme preparation of a bio-enzyme preparation for inhibiting bacterium and promoting growth of crops as needed.

6. The method for preparing a bioenzyme preparation for inhibiting bacteria and promoting growth of crops according to claim 5, characterized in that: The corn flour and soybean flour culture medium comprises: 2.0 g / L corn flour, 2.5 g / L soybean flour, 0.5 g / L NaCl, 0.7 g / L MnSO4·H2O, and a pH value of 7.

0.

7. The method for preparing a bioenzyme preparation for inhibiting bacteria and promoting growth of crops according to claim 5, characterized in that: The number of viable bacteria in the antibacterial and growth-promoting bioenzyme preparation is not less than 10 8 CFU / mL.

8. Application of a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops in promoting growth, preventing diseases or resisting stress in cotton.

9. Application of a bio-enzyme preparation for inhibiting and promoting growth of crops in the preparation of drugs for preventing and treating cotton Verticillium wilt, cotton Fusarium wilt, apricot fire blight and red date black spot.

10. Application of a bio-enzyme preparation for inhibiting bacteria and promoting growth of crops in the preparation of fertilizers.

Images