Disease-preventing and growth-promoting compound microbial agent and application thereof to vegetables

Abstract

The invention discloses a disease prevention and growth promotion type compound microbial agent and application thereof to vegetables, and belongs to the technical field of microbial agents, and the compound microbial agent comprises burkholderia gladioli, bacillus cereus, antibiotic lysobacter and bacillus velezensis. The fertilizer provides nutrient substances and growth factors required by plants, promotes root growth, improves the soil environment and improves the nutrient absorption efficiency. By using natural microorganisms, the use of chemical pesticides and fertilizers is reduced, the environmental pollution is reduced, the diversity of soil microorganisms is improved, and the soil health is recovered. The biological agent disclosed by the invention can increase the yield and quality of crops, improve the economic benefit, improve the appearance and taste of fruits and improve the commodity value. The disease index of downy mildew is reduced by 34.14% compared with that of a control group, the disease index of powdery mildew is reduced by 25.53% compared with that of the control group, the wilt dead plant rate is reduced by 82.66% compared with that of the control group, the yield is increased by 12.37% compared with that of the control group, and the economic benefit is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial inoculants, and more specifically, to a disease-preventing and growth-promoting composite microbial inoculant and its application in vegetables. Background Art

[0002] In modern agriculture, plant diseases and soil degradation are important issues affecting crop yield and quality. Although traditional chemical pesticides and fertilizers can increase yields in the short term, long-term use can lead to problems such as soil compaction, reduced microbial diversity, and environmental pollution.

[0003] In recent years, microbial inoculants, as a green and environmentally friendly solution, have gradually attracted attention. Through the metabolic activities of microorganisms, these inoculants can inhibit the growth of pathogenic bacteria, promote plant growth, and enhance disease resistance. At the same time, they can improve soil structure, increase soil fertility, reduce the use of chemical substances, and thus achieve sustainable agricultural development.

[0004] However, the effects of single strains are often limited. Therefore, the development of composite microbial inoculants by mixing multiple beneficial strains to exert synergistic effects has become a research hotspot. Such composite inoculants can not only reduce plant diseases but also improve crop yield and quality, with broad application prospects.

[0005] In this context, the present invention has studied a composite inoculant composed of multiple microorganisms such as Burkholderia gladioli mixed in a specific ratio, which has excellent effects of preventing diseases and promoting growth, and is expected to provide a new effective means for agricultural production. Summary of the Invention

[0006] 1. Technical Problems to be Solved

[0007] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a disease-preventing and growth-promoting composite microbial inoculant and its application in vegetables, which can achieve the use of natural microorganisms, reduce the use of chemical pesticides and fertilizers, reduce environmental pollution, increase soil microbial diversity, restore soil health, increase crop yield and quality, improve economic benefits, improve the appearance and taste of fruits, and enhance commercial value.

[0008] 2. Technical Solutions

[0009] To solve the above problems, the present invention adopts the following technical solutions.

[0010] A disease-preventing and growth-promoting composite microbial inoculant is prepared by mixing Burkholderia gladioli, Bacillus cereus, Lysobacter antibioticus, and Bacillus velezensis in a volume ratio of 1:1:1:1.

[0011] The preparation method of the disease-preventing and growth-promoting compound microbial inoculant includes the following steps:

[0012] 1) Inoculate the selected four strains of bacteria into suitable culture media respectively, and place them under suitable temperature and conditions for cultivation, usually for 24 - 48 hours until the bacteria reach the logarithmic growth phase;

[0013] 2) Collect the bacterial cells of each strain by centrifugation or filtration. When centrifuging, select a rotational speed of 3000 - 5000 rpm and centrifuge for 10 - 15 minutes to collect the precipitate;

[0014] 3) Select an ultrasonic device and set the frequency to 20 - 40 kHz. Put the resuspended bacterial cells into the ultrasonic device, use a high-pressure homogenizer, place the bacterial cell suspension in the device, adjust the pressure to 200 - 260 MPa, and the number of homogenization times depends on the characteristics of the bacterial cells to ensure that the outlet temperature does not inactivate the bacterial cells;

[0015] 4) Mix the four resuspended bacterial cells together according to a volume ratio of 1:1:1:1. You can gently stir with a sterile stirrer to ensure uniform mixing;

[0016] 5) Dispense the mixed compound microbial inoculant into sterile containers, seal and store it. Select refrigeration or freezing storage to extend the shelf life of the inoculant.

[0017] Furthermore, the 4 strains of bacteria were deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2024. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0018] Furthermore, the deposit numbers of Burkholderia gladioli, Bacillus cereus, Lysobacter antibioticus, and Bacillus velezensis are respectively: CCTCC NO: M 2024576, CCTCC NO: M 2024575, CCTCC NO: M2024577, CCTCC NO: M 2024578.

[0019] Furthermore, the antagonistic effect of the disease-preventing and growth-promoting compound microbial inoculant against the pathogen of cucumber fusarium wilt.

[0020] Furthermore, in step 5), select a suitable vacuum packaging machine to ensure the sterility of the equipment. Dispense the mixed inoculant into sterile containers, ensure that the container material is suitable for vacuum packaging. During the vacuum packaging process, minimize the exposure time of the inoculant to oxygen. After vacuum sealing, check the integrity and tightness of the packaging. Before packaging, an inert gas can be added to the container to replace oxygen to ensure that the packaging material has good oxygen barrier performance.

[0021] 3. Beneficial effects

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] 1) In the present invention, the compound microbial agent effectively reduces the occurrence of plant diseases by inhibiting the growth of pathogenic bacteria. Each strain acts synergistically to form a strong biological barrier and enhance the disease resistance of plants.

[0024] 2) The present invention provides the nutrients and growth factors required by plants, promotes root growth, improves the soil environment, and enhances the nutrient absorption efficiency. The use of natural microorganisms reduces the use of chemical pesticides and fertilizers, reduces environmental pollution, increases soil microbial diversity, and restores soil health.

[0025] 3) The biological microbial agent of the present invention can increase the yield and quality of crops, improve economic benefits, improve the appearance and taste of fruits, and enhance the commercial value.

[0026] 4) The compound microbial agent formed by mixing Burkholderia gladioli L3-3, Bacillus cereus L3-1, Lysobacter antibioticus 42-3, and Bacillus velezensis L5-4 in the same proportion in the present invention has the best effect on preventing diseases and promoting growth of cucumbers. The seedling vigor index increases by 14.01% compared with the control, the vitamin C content increases by 37.86% compared with the control, the soluble sugar content increases by 20.19% compared with the control, the soluble solid content increases by 22.03% compared with the control, the downy mildew disease index decreases by 34.14% compared with the control, the powdery mildew disease index decreases by 25.53% compared with the control, the withering disease death rate of plants decreases by 82.66% compared with the control, and the yield increases by 12.37% compared with the control. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the 16S rDNA phylogenetic tree of the strain L3-3 of the present invention;

[0028] Figure 2 It is a schematic diagram of the 16S rDNA phylogenetic tree of the strain L3-1 of the present invention;

[0029] Figure 3 It is a schematic diagram of the 16S rDNA phylogenetic tree of the strain 42-3 of the present invention;

[0030] Figure 4 It is a schematic diagram of the 16S rDNA phylogenetic tree of the strain L5-4 of the present invention.

[0031] Figure 5 It is a schematic diagram of the cell morphology of Burkholderia gladioli L3-3 of the present invention.

[0032] Figure 6 It is a schematic diagram of the cell morphology of Bacillus cereus L3-1 of the present invention.

[0033] Figure 7 Schematic diagram of the cell morphology of Lysobacter antibioticus 42-3 of the present invention.

[0034] Figure 8 Schematic diagram of the cell morphology of Bacillus velezensis L5-4 of the present invention.

[0035] Figure 9 Biological preservation diagram of 4 cultures of the present invention. Detailed implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention; obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] Example 1:

[0038] A disease-preventing and growth-promoting composite microbial inoculant is prepared by mixing Burkholderia gladioli, Bacillus cereus, Lysobacter antibioticus, and Bacillus velezensis in a volume ratio of 1:1:1:1.

[0039] The four strains of bacteria were deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2024. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Their deposit numbers are: CCTCC NO: M 2024576, CCTCC NO: M2024575, CCTCC NO: M 2024577, CCTCC NO: M 2024578.

[0040] The preparation method of the disease-preventing and growth-promoting composite microbial inoculant includes the following steps:

[0041] 1) Inoculate the selected four strains of bacteria into appropriate culture media respectively, and place them under appropriate temperature and conditions for cultivation, usually for 24 to 48 hours until the bacteria reach the logarithmic growth phase;

[0042] 2) Collect the bacterial cells of each strain by centrifugation or filtration. When centrifuging, select a rotation speed of 3000 - 5000 rpm and centrifuge for 10 - 15 minutes to collect the precipitate;

[0043] 3) Select an ultrasonic device, set the frequency to 20 - 40 kHz, put the resuspended bacterial cells into the ultrasonic device, use a high-pressure homogenizer, place the bacterial cell suspension in the device, adjust the pressure to 200 - 260 MPa, and the number of homogenization times depends on the characteristics of the bacterial cells to ensure that the outlet temperature does not inactivate the bacterial cells;

[0044] 4) Mix the four resuspended bacterial cells together according to a volume ratio of 1:1:1:1. You can gently stir them using a sterile stirrer to ensure uniform mixing;

[0045] 5) Dispense the mixed composite microbial inoculant into sterile containers, seal them, and choose to store them refrigerated or frozen to extend the shelf life of the inoculant. Select a suitable vacuum packaging machine to ensure the equipment is sterile, dispense the mixed inoculant into sterile containers, ensure that the container material is suitable for vacuum packaging, and minimize the exposure time of the inoculant to oxygen during the vacuum packaging process. After vacuum sealing, check the integrity and tightness of the packaging. Before packaging, an inert gas can be added to the container to replace oxygen and ensure that the packaging material has good oxygen barrier properties.

[0046] Antagonistic effect of the disease-preventing and growth-promoting composite microbial inoculant against the pathogen of cucumber Fusarium wilt.

[0047] I. Isolation, identification, and preservation of strains

[0048] 1. Isolation of strains

[0049] The four strains of the present invention were all isolated from rhizosphere soil samples collected from cucumber experimental fields in Wuwei, Jingyuan, and Lanzhou, Gansu Province, using the soil dilution method.

[0050] 2. Identification of strains

[0051] Identify the strains based on the morphological characteristics of the bacterial cells and the analysis of the gyrB gene sequence.

[0052] 2.1. Morphological characteristics of strains

[0053] Burkholderia gladioli L3-3: The colonies on nutrient agar medium are round, light yellow, smooth and slightly convex on the surface, and the edges are neat. The bacterial cells are short rod-shaped, with blunt ends at both ends, arranged singly or in pairs, and are Gram-negative aerobic bacteria.

[0054] Bacillus cereus L3-1: The colonies on nutrient agar medium are relatively large, round, light yellow, flat, rough and slightly shiny on the surface, and the surface becomes frosted glass-like in the later stage of cultivation. The bacterial cells are rod-shaped, with square ends at the ends, arranged in short or long chains, and are Gram-positive facultative aerobic bacteria.

[0055] Lysobacter antibioticus 42-3: The colonies on nutrient agar medium are round, smooth and shiny, with neat edges, opaque and thin liquid-like, dark yellow in the initial stage of cultivation, and brown in the later stage of cultivation. The bacterial cells are rod-shaped without flagella and are Gram-negative aerobic bacteria.

[0056] Bacillus velezensis L5-4: The colonies on nutrient agar medium are milky white, opaque, round or nearly round, with uneven edges and wrinkled surfaces. The cells are rod-shaped, Gram-positive aerobic bacteria.

[0057] 2.2、Sequence analysis of the gyrB gene of the strains

[0058] The 16S rDNA sequences of the 4 strains were aligned on the Ezbiocloud website and phylogenetic trees were constructed. The results showed that the strains L3-3, L3-1, 42-3, and L5-4 were identified as Burkholderia gladioli, Bacillus cereus, Lysobacter antibioticus, and Bacillus velezensis, respectively.

[0059] 3. Preservation of the strains

[0060] The 4 strains were deposited at the China Center for Type Culture Collection (CCTCC) on March 27, 2024. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit numbers are: CCTCC NO: M2024576, CCTCC NO: M2024575, CCTCC NO: M 2024577, CCTCC NO: M 2024578.

[0061] II. Antagonistic effects of the strains against the pathogen of cucumber fusarium wilt

[0062] The antagonistic effects of the 4 strains against the pathogen of cucumber fusarium wilt were determined by the inhibition zone method. The results showed that all 4 strains had certain antagonistic effects against the pathogen of cucumber fusarium wilt. Among them, the strain L3-3 had the strongest antagonistic effect against the pathogen of cucumber fusarium wilt, with an inhibition zone diameter of 22.56 mm and an inhibition rate of 24.46%, as shown in Table 1:

[0063]

[0064] Note: Inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%

[0065] III. Antagonistic effects of strain L3-3 against 8 vegetable pathogens

[0066] Select the strain with the best antagonistic effect against cucumber Fusarium wilt, and use the inhibition zone method to determine its antagonistic effect against the pathogens of other vegetable diseases. The results show that strain L3-3 has a certain inhibitory effect on 8 kinds of vegetable diseases, with the inhibition zone diameter ranging from 21.12 to 35.32 mm and the inhibition rate ranging from 23.16 to 38.17%. Among them, the antagonistic effect of strain L3-3 against the pathogens of Phytophthora capsici, Botrytis cinerea and Alternaria solani of pepper is significantly higher than that against cucumber Fusarium wilt, and there is no significant difference in the antagonistic effect against Pythium aphanidermatum of pepper, Fusarium wilt of watermelon, Fusarium wilt of cabbage, Fusarium wilt of melon and Rhizoctonia solani of pepper compared with that against cucumber Fusarium wilt. It shows that strain L3-3 can be used to control other vegetable diseases, as shown in Table 2:

[0067]

[0068] IV. Growth-promoting performance of strains

[0069] The acetylene reduction method was used to determine the nitrogenase activity, the plate phosphorus solubilization circle method was used to determine the amount of organic phosphorus solubilized, and the Salkowski colorimetric method was used to determine the IAA secretion amount. The results in Table 3 show that the amount of organic phosphorus solubilized by strain L3-1 is the highest, reaching 20.85 μg / mL. The nitrogenase activity of strain 42-3 is the highest, reaching 780.20 nmol(C2H4) / h·mL. The IAA concentration of strain L5-4 is the highest, reaching 17.56 μg / mL, as shown in Table 3:

[0070]

[0071]

[0072] V. Screening test of compound microbial inoculants

[0073] 1. Materials and methods

[0074] Cucumber varieties for test: Del A10 (Tianjin Kernel Cucumber Research Institute)

[0075] Test strains: Burkholderia gladioli L3-3, Bacillus cereus L3-1, Lysobacter antibioticus 42-3, Bacillus velezensis L5-4 (developed by Vegetable Research Institute, Gansu Academy of Agricultural Sciences).

[0076] Spray on the base of the cucumber stem after the seedlings are established and the slow seedling stage. Six treatments were set up in the test, L3-3:L3-1:42-3:L5-4 = 1:1:1:1 (B1), L3-3:L3-1:42-3:L5-4 = 2:1:1:1 (B2), L3-3:L5-4 = 1:1 (B3), L3-3:L5-4 = 2:1 (B4), and the concentration was 1×10 8CFU / ml, and 5 ml was sprayed on each plant. A 600-fold solution of 30% metalaxyl·hymexazol (B5), 50 ml was applied to each plant by irrigation, and sterile water was sprayed on the base of the stem as the control (CK). There were 3 replicates, arranged in a randomized block design, with a total of 18 plots, and 100 cucumber seedlings in each plot. The ridge width was 80 cm, the furrow width was 60 cm, the plant spacing was 30 cm, and the plot area was 21 m 2 .

[0077] Disease index = ∑(number of plants at disease level × representative level) / (total number of plants surveyed × highest level value) × 100. Vigorous seedling index = stem diameter / plant height × fresh weight of whole plant × 10

[0078] 2. Results and Analysis

[0079] 2.1 Effects of compound microbial inoculant on the growth and development of cucumber

[0080] The application of the compound microbial inoculant had effects on the plant height, stem diameter, and fresh weight of the whole plant of cucumber. The B1 treatment (L3-3:L3-1:42-3:L5-4 = 1:1:1:1) had the best comprehensive performance, with a vigorous seedling index of 10.01, an increase of 14.01% compared with the control. The average plant height increased by 26.67 cm, the average stem diameter increased by 0.09 cm, and the average fresh weight of the whole plant increased by 42.89 g, showing a significant growth-promoting effect, as shown in Table 4:

[0081]

[0082]

[0083] 2.2 Effects of compound microbial inoculant on the quality of cucumber

[0084] The application of the compound microbial inoculant could improve the quality of cucumber. Among them, the B1 treatment was the best, with vitamin C reaching 13.51 mg / 100 g, an increase of 37.86% compared with the control. The soluble sugar content was 2.56 g / 100 g, an increase of 20.19% compared with the control. The soluble solid content reached 4.32%, an increase of 22.03% compared with the control, as shown in Table 5:

[0085]

[0086] 2.3 Effects of compound microbial inoculant on cucumber diseases

[0087] The disease indices of cucumber downy mildew and powdery mildew and the death rate of wilted plants under each treatment were all lower than those of the control. Among them, the B1 treatment had the best comprehensive performance, and the disease indices of downy mildew and powdery mildew were reduced by 34.14% and 25.53% respectively compared with the control. The death rate of wilted plants was reduced by 82.66% compared with the control, as shown in Table 6:

[0088]

[0089] 2.4 Effects of compound microbial inoculum on cucumber yield

[0090] The yields of B1, B2, and B3 were significantly higher than that of the control. Among them, B1 had the highest single fruit weight, number of fruits per plant, and yield, reaching 0.28 Kg, 14.22, and 12648.18 Kg / 667 m2 respectively, with a 12.37% increase in yield compared to the control. The specific data are shown in Table 7:

[0091]

[0092] The test results showed that the compound microbial inoculum composed of Burkholderia gladioli L3-3, Bacillus cereus L3-1, Lysobacter antibioticus 42-3, and Bacillus velezensis L5-4 mixed in the same proportion had the best effect on preventing diseases and promoting growth of cucumbers. The strong seedling index increased by 14.01% compared to the control, the vitamin C content increased by 37.86% compared to the control, the soluble sugar content increased by 20.19% compared to the control, the soluble solid content increased by 22.03% compared to the control, the downy mildew disease index decreased by 34.14% compared to the control, the powdery mildew disease index decreased by 25.53% compared to the control, the dead plant rate of fusarium wilt decreased by 82.66% compared to the control, and the yield increased by 12.37% compared to the control.

[0093] L3-3 sequence: 1450 bp

[0094]

[0095] L3-1 sequence: 1449 bp

[0096]

[0097] 42-3 Sequence: 1458 bp

[0098]

[0099] L5-4 sequence: 1458bp

[0100]

[0101] As described above, it is only a preferred specific embodiment of the present invention; however, the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its improved concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A disease-preventing and growth-promoting composite microbial agent, characterized in that: The preparation method is prepared by mixing gladiolus Burkholderia, Bacillus cereus, antibiotic lysobacter and Bacillus velez in a volume ratio of 1:1:1:

1.

2. The method for preparing a disease-preventing and growth-promoting composite microbial agent according to claim 1, characterized in that The steps include: 1) The four selected bacterial strains are inoculated into appropriate culture media respectively and cultured under appropriate temperature and conditions, usually for 24 to 48 hours, until the bacteria reach the logarithmic growth phase; 2) Collect the cells of each bacterial species by centrifugation or filtration. When centrifuging, select a speed of 3000-5000 rpm for 10-15 minutes and collect the precipitate; 3) Select an ultrasonic device, set the frequency to 20-40kHz, place the resuspended bacteria in the ultrasonic device, use a high-pressure homogenizer, place the bacterial suspension in the device, adjust the pressure to 200-260MPa, and the number of homogenizations depends on the characteristics of the bacteria to ensure that the discharge temperature does not inactivate the bacteria; 4) Mix the four resuspended cells together in a volume ratio of 1:1:1:

1. Use a sterile blender to gently stir to ensure a uniform mix. 5) The mixed composite microbial inoculant is packaged into sterile containers, sealed and stored in a refrigerated or frozen state to extend the shelf life of the inoculant.

3. The disease-preventing and growth-promoting composite microbial agent according to claim 1, characterized in that: The four strains were deposited in the China Center for Type Culture Collection (CCTCC) on March 27, 2024, and the deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

4. The disease-preventing and growth-promoting composite microbial agent according to claim 3, characterized in that: The deposit numbers of the gladiolus Burkholderia, Bacillus cereus, Bacillus antibioticus and Bacillus velez are CCTCC NO: M 2024576, CCTCC NO: M 2024575, CCTCC NO: M2024577 and CCTCC NO: M 2024578, respectively.

5. An antagonistic effect of the disease-preventing and growth-promoting composite microbial agent described in claim 1 on the pathogen of cucumber wilt.

6. The method for preparing a disease-preventing and growth-promoting composite microbial agent according to claim 1, characterized in that: In the step 5), a suitable vacuum packaging machine is selected to ensure that the equipment is sterile, and the mixed bacterial agent is packaged into sterile containers. It is ensured that the container material is suitable for vacuum packaging. During the vacuum packaging process, the time the bacterial agent is exposed to oxygen is minimized. After vacuum sealing, the integrity and sealing of the package are checked. Before packaging, an inert gas can be added to the container to replace oxygen to ensure that the packaging material has good oxygen barrier properties.

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