Biocontrol bacterium composition and application thereof in prevention and treatment of corn diseases

Abstract

The invention discloses a biocontrol bacterium composition and application thereof in prevention and treatment of corn diseases, and relates to the technical field of biology. The biocontrol bacterium composition comprises bacillus velezensis (Bacillus velezensis) HPP-0-TS7 and bacillus subtilis (Bacillus subtilis) HPP-0-GZL7, and the biocontrol bacterium composition comprises bacillus velezensis (Bacillus velezensis) HPP-0-TS7 and bacillus subtilis (Bacillus subtilis) HPP-0-GZL7. The biocontrol bacterium composition provided by the invention shows excellent technical effects in the aspects of preventing and treating corn diseases and promoting plant growth, and the two strains have a synergistic effect, so that not only is the prevention and control capability on specific diseases enhanced, but also the antibacterial spectrum is expanded, and a reliable guarantee is provided for comprehensive prevention and treatment; a good interaction relationship exists between the two strains, and the two strains jointly play a role through different action mechanisms, so that the stress resistance and yield potential of corn are comprehensively improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a biocontrol bacteria combination and its application in preventing and treating corn diseases. BACKGROUND

[0002] Corn (Zea mays L.) as China's key food and feed crops, plays an indispensable role in ensuring national food security and food safety. However, in recent years, due to the growth of planting area, climate warming and changes in farming system and other factors, the occurrence frequency of corn diseases has increased significantly, which poses a serious threat to the yield and quality of corn.

[0003] Chemical agents are widely used in agricultural production to control diseases and have obvious effects, but long-term single use not only may pollute the environment, but also may lead to the development of drug resistance in pathogenic bacteria, thereby reducing the prevention and treatment effect. Therefore, exploring more environmentally friendly, safe and efficient alternative solutions has become an inevitable trend. Under this background, biological control methods have gradually attracted attention, which is a recognized environmentally friendly disease management strategy and shows great potential in dealing with corn disease challenges.

[0004] In particular, the method of using microbial agents to inhibit pathogenic bacteria is gaining more and more attention. Such methods are not only harmless to human and animal health, but also have little negative impact on other beneficial microorganisms compared to traditional chemical agents. More importantly, these microorganisms can establish a mutually beneficial symbiotic relationship with host plants, promote plant growth and enhance disease resistance, thereby improving agricultural productivity, promoting environmental protection and sustainable development, and ultimately helping to improve global food security.

[0005] Therefore, the development of biological control agents with excellent disease resistance and growth-promoting functions for corn is crucial for improving corn yield and quality. This is not only one of the effective ways to solve the current problems faced by agricultural production, but also a key step to achieve green development of agriculture. SUMMARY

[0006] The purpose of the present application is to provide a biocontrol bacteria combination and its application in preventing and treating corn diseases to solve the problems existing in the prior art. The strain has antagonistic effects on multiple corn pathogenic bacteria and growth-promoting functions, and has good development and application prospects.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] The application provides a biocontrol agent combination applied in corn planting, and the biocontrol agent combination comprises Bacillus velezensis HPP-0-TS7 and Bacillus subtilis HPP-0-GZL7.

[0009] The Bacillus velezensis HPP-0-TS7 is preserved in the China General Microbiological Culture Collection Center on August 8, 2024, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31638.

[0010] The Bacillus subtilis HPP-0-GZL7 is preserved in the China General Microbiological Culture Collection Center on August 8, 2024, and the preservation address is No. 3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31639.

[0011] The application also provides application of the biocontrol agent combination in preparation of a biological agent with the functions of preventing and treating corn diseases and / or promoting growth.

[0012] Further, the corn diseases are corn large spot, corn small spot, wheat stem rot, corn sheath rot, corn ear rot and / or corn root rot.

[0013] Further, the biological agent is a liquid agent, a coating agent or a granular agent.

[0014] The application also provides a biological agent with the functions of preventing and treating corn diseases and / or promoting growth, and the active ingredient comprises the biocontrol agent combination.

[0015] Further, the corn diseases are corn large spot, corn small spot, wheat stem rot, corn sheath rot, corn ear rot and / or corn root rot.

[0016] Further, the biological agent further comprises an auxiliary material.

[0017] Further, the dosage form of the biological agent is a liquid agent, a coating agent or a granular agent.

[0018] Further, when the dosage form of the biological agent is a coating agent, the auxiliary material is a film-forming agent.

[0019] When the dosage form of the biological agent is a granular agent, the auxiliary material is wheat bran.

[0020] The application also provides application of the biocontrol agent combination or the biological agent in preventing and treating corn diseases and / or improving corn yield.

[0021] The application discloses the following technical effects:

[0022] The present application provides a high-efficiency biocontrol bacteria combination, which is composed of Bacillus velezensis HPP-0-TS7 and Bacillus subtilis HPP-0-GZL7. This combination exhibits significant technical effects in biological control and plant growth promotion, specifically in the following aspects:

[0023] 1. Broad-spectrum antibacterial effect:

[0024] Through experimental verification, Bacillus velezensis HPP-0-TS7 has a significant inhibitory effect on corn stalk rot, corn ear rot, corn large spot disease, and small spot disease, and other common diseases. Bacillus subtilis HPP-0-GZL7 also shows strong antagonistic ability and can effectively inhibit various corn fungal pathogens. The synergistic effect of the two strains not only enhances the control ability of specific diseases, but also expands the antibacterial spectrum, providing a reliable guarantee for comprehensive prevention and control.

[0025] 2. Promoting function:

[0026] There is a good interaction between the two strains, and they work together through different mechanisms to comprehensively improve the resistance and yield potential of corn. Experimental results show that after using the biocontrol bacteria combination, the growth rate of corn is accelerated, the plant is more robust, and the final yield is significantly increased.

[0027] 3. Easy to apply and industrialized production:

[0028] The two strains in the biocontrol bacteria combination provided by the present application can be artificially cultured, the culture conditions are simple, the preservation is convenient, and it is suitable for large-scale industrialized production, which has broad market application prospects. In addition, the combination is simple to apply and can be directly applied in the field, which is easy to operate and convenient to use.

[0029] In summary, the biocontrol bacteria combination provided by the present application exhibits excellent technical effects in preventing and controlling corn diseases and promoting plant growth, and is expected to become an important tool for promoting sustainable agricultural development, making positive contributions to food security and environmental protection. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor based on these drawings.

[0031] Figure 1 Phylogenetic tree diagram constructed based on 16S rDNA(A) and gyrB(B) sequences for strain HPP-0-TS7;

[0032] Figure 2 A phylogenetic tree constructed based on 16S rDNA (A) and gyrB (B) sequences for strain HPP-0-GZL7;

[0033] Figure 3 A result graph for compatibility determination of strain HPP-0-TS7 and strain HPP-0-GZL7;

[0034] Figure 4 A graph of antagonistic effect of strain compound treatment on grains;

[0035] Figure 5 A graph of antagonistic effect of compound strain on corn stem rot;

[0036] Figure 6 A graph of indoor growth-promoting effect of different dosage forms;

[0037] Figure 7 A result graph of the effect of strain HPP-0-TS7 on the growth of corn under greenhouse saline-alkali stress conditions; wherein CK (no saline-alkali) is a positive control of sterile water treatment in normal soil; CK (saline-alkali) is a negative control of sterile water treatment in saline-alkali soil; HPP-0-TS7 represents corn seedlings treated with HPP-0-TS7 strain in saline-alkali soil. DETAILED DESCRIPTION

[0038] The following detailed description is provided to understand certain aspects, features and embodiments of the present application. It should be understood that the detailed description and specific examples, while indicating certain embodiments of the application, are given by way of illustration only, and are not by way of limitation of the application.

[0039] It should be understood that the terms used in the specification are for the purpose of describing particular embodiments only and are not intended to limit the present application. In addition, for numerical ranges recited in the specification, it is contemplated that each intervening value, to the upper and lower limits of the ranges is also specifically disclosed. Each smaller range that falls within the integer ranges is also specifically disclosed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0041] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the principles of the application, and such variations are to be considered within the scope of the application as it is set forth in the appended claims. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed material or list of materials.

[0043] Isolation, identification and preservation of strains of Example 1

[0044] 1. Isolation and identification of strains

[0045] The present application isolates a strain from the corn rhizosphere soil in Tangshan City, Hebei Province, named HPP-0-TS7; and a strain from the corn rhizosphere soil in Gongzhuling, Jilin Province, named HPP-0-GZL7.

[0046] According to the Handbook of Systematic Identification of Common Bacteria (Dong Xiuzhu, Cai Miaoying), physiological and biochemical characteristics were identified. Strain HPP-0-TS7 was positive in indole production, citrate utilization, nitrate reduction, starch hydrolysis, and lactose decomposition, but negative in methyl red test, VP determination, and glucose decomposition. Strain HPP-0-GZL7 was positive in indole production, citrate utilization, nitrate reduction, starch hydrolysis, lactose decomposition, and methyl red test, but negative in VP determination and glucose decomposition.

[0047] The 16S rDNA and gyrB gene sequences of strains HPP-0-TS7 and HPP-0-GZL7 were identified, sequenced, and compared using Blastn. The phylogenetic tree of the target strains was constructed using MEGA11. As shown in Figure 1 and Figure 2 Strain HPP-0-TS7 is most closely related to Bacillus velezensis, and strain HPP-0-GZL7 is most closely related to Bacillus subtilis.

[0048] Based on the physiological and biochemical characteristics and molecular identification results, it can be seen that strain HPP-0-TS7 is Bacillus velezensis, and strain HPP-0-GZL7 is Bacillus subtilis.

[0049] 2. Biological preservation

[0050] Bacillus velezensis HPP-0-TS7 was preserved in China General Microbiological Culture Collection Center (CGMCC) on August 8, 2024, the address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31638.

[0051] Bacillus subtilis HPP-0-GZL7 was preserved in China General Microbiological Culture Collection Center (CGMCC) on August 8, 2024, the address of the preservation center is No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 31639.

[0052] Activity detection of the strains in Example 2

[0053] 1. Inhibition activity detection

[0054] The antagonistic effect of strains HPP-0-TS7 and HPP-0-GZL7 on common pathogenic fungi of corn was detected by plate confrontation method. After the activation of the pathogenic fungi of corn, i.e. Fusarium graminearum, Fusarium proliferatum, Fusarium verticillioides, Fusarium oxysporum, Setosphaeria turcica and Cochliobolus heterostrophus, 5mm fungal cakes were inoculated in the center of PDA medium, and Bacillus velezensis HPP-0-TS7 was activated in LB medium, then 0.25μL of bacterial solution was inoculated about 2.5cm away from the fungal cake, and only the indicator bacteria were inoculated as a control. After incubation at 25℃ in the dark, the inhibition radius of each bacterial inoculation on fungal growth and the radius of the control group of fungi were measured when the control group of indicator bacteria grew, and the inhibition rate was calculated. The higher the inhibition rate, the stronger the antagonistic effect of the strain on fungi.

[0055] Inhibition rate calculation formula: inhibition rate = (control group colony growth radius - treatment group colony growth radius) / control group colony growth radius × 100%.

[0056] Table 1 Inhibition rate of strains HPP-0-TS7 and HPP-0-GZL7 on various pathogenic fungi

[0057]

[0058] As shown in the results of Table 1, the strains HPP-0-TS7 and HPP-0-GZL7 have certain inhibitory effects on the pathogenic fungi of F. graminearum, F. sambucinum, F. pseudocatenatum, F. oxysporum, C. turcica and C. zonata, especially on C. turcica and C. zonata. The strains HPP-0-TS7 and HPP-0-GZL7 significantly inhibit the growth of mycelium of the pathogenic fungi under confrontation culture.

[0059] 2. Biological property test

[0060] 2.1 Biological property test of the strain HPP-0-TS7

[0061] (1) Determination of the ability of the strain HPP-0-TS7 to degrade organic phosphorus

[0062] The strain HPP-0-TS7 was inoculated into the organic phosphorus degradation medium, and the growth of the strain was observed to see whether a degradation ring appeared around the strain. If a degradation ring appeared, it indicated that the strain had the ability to degrade organic phosphorus.

[0063] (2) Determination of the ability of the strain HPP-0-TS7 to degrade inorganic phosphorus

[0064] The strain HPP-0-TS7 was inoculated into the inorganic phosphorus degradation medium, and the growth of the strain was observed to see whether a degradation ring appeared around the strain. If a degradation ring appeared, it indicated that the strain had the ability to degrade inorganic phosphorus.

[0065] (3) Determination of the ability of the strain HPP-0-TS7 to degrade potassium

[0066] The strain HPP-0-TS7 was inoculated into the potassium degradation medium, and the growth of the strain was observed to see whether a degradation ring appeared around the strain. If a degradation ring appeared, it indicated that the strain had the ability to degrade potassium.

[0067] (4) Determination of the ability of the strain HPP-0-TS7 to degrade cellulose

[0068] The strain HPP-0-TS7 was inoculated into the cellulose degradation medium, and the growth of the strain was observed to see whether a degradation ring appeared around the strain. If a degradation ring appeared, it indicated that the strain had the ability to degrade cellulose.

[0069] (5) Determination of the ability of the strain HPP-0-TS7 to produce siderophore

[0070] The strain HPP-0-TS7 was inoculated onto the surface of the CAS solid medium, and was placed in a 37°C dark incubator for 48 h. The appearance of an orange halo around the strain was observed. If an orange halo appeared, it indicated that the strain had the ability to secrete siderophore.

[0071] (6) Determination of the ability of the strain HPP-0-TS7 to produce organic acid

[0072] After adding bromocresol purple into NBRIP medium, pour the medium into plates, and then inoculate strain HPP-0-TS7 into the plates. After incubation at 37°C for 3 days, observe whether the strain turns yellow on the plate. If the strain turns yellow, it indicates that the strain can produce organic acid.

[0073] The results show that Bacillus velezensis HPP-0-TS7 has the abilities of degrading organic phosphorus, degrading inorganic phosphorus, degrading potassium, degrading cellulose, producing siderophore and producing organic acid.

[0074] 2.2 Test of biological characteristics of strain HPP-0-GZL7

[0075] (1) Phosphorus and potassium degrading ability

[0076] Inoculate HPP-0-GZL7, which has been activated and adjusted to OD 600 value of 1, into phosphorus and potassium degrading medium, and incubate at 37°C in the dark for 5 days. Observe the growth of the strain and whether degradation rings are produced around the strain, and record the test data. The results show that strain HPP-0-GZL7 has the abilities of degrading organic phosphorus and inorganic phosphorus, but does not have the ability of degrading potassium.

[0077] (2) Determination of straw degrading ability

[0078] a. Qualitative determination of cellulose degrading ability of the strain

[0079] Take the activated HPP-0-GZL7 strain and adjust the OD 600 value to 1, inoculate it into cellulose degrading medium, and incubate at 37°C for 2 days. Observe whether cellulose degradation rings appear around the colonies, and take photos to record the test results.

[0080] As shown in Figure 6 , strain HPP-0-GZL7 has the ability to degrade cellulose.

[0081] b. Determination of cellulase activity of the strain

[0082] Preparation of crude enzyme solution: inoculate HPP-0-GZL7 bacterial suspension into liquid fermentation enzyme production medium, and shake culture at 190 rpm and 30°C for 24 h, 48 h and 72 h, respectively. After centrifugation at 4000 rpm for 10 min, the obtained supernatant is the crude enzyme solution.

[0083] Fermentation enzyme production medium: sodium carboxymethyl cellulose 10 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 5 g / L, KH2PO4 1 g / L and MgSO4 0.5 g / L.

[0084] Preparation of glucose standard curve: glucose solutions with different concentration gradients (0 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, 1.2 mg / mL, 1.4 mg / mL) were prepared, 2 mL of each concentration of glucose solution was taken, then an equal volume of DNS reagent was added, mixed thoroughly, to ensure that each sample was subjected to the same reaction conditions, the mixed sample was reacted in a boiling water bath, which accelerated the process of glucose reducing DNS reagent, after 5 min of reaction, it was cooled to room temperature, and the OD 540 values of different concentrations were recorded, and a glucose standard curve was constructed using software analysis, and a linear regression equation was obtained. 540 values of different concentrations were recorded, and a glucose standard curve was constructed using software analysis, and a linear regression equation was obtained.

[0085] The carboxymethyl cellulase (CMCase), β-glucosidase and filter paper enzyme (FPA) activities of HPP-0-GZL7 were determined and calculated.

[0086] The results (Table 2) showed that the strain HPP-0-GZL7 had the ability to produce carboxymethyl cellulase (CMCase), β-glucosidase and filter paper enzyme (FPA), and the strain produced carboxymethyl cellulase (CMCase) and β-glucosidase best at 48 h, and the filter paper enzyme activity was highest at 24 h, reaching 5.83 U / mL.

[0087] Table 2 Determination of various enzyme activities of strain HPP-0-GZL7 (U / mL)

[0088]

[0089] Note: — means not detected.

[0090] c. Determination of straw weight loss rate

[0091] The corn straw was washed and cut to an appropriate size, and dried to constant weight in an oven at 102°C. In a flask, 4 g of straw was weighed and 4 mL of activated HPP-0-GZL7 bacterial solution and 12 mL of Mandels nutrient solution were added respectively, and corn straw inoculated with sterile water was used as a control. After standing at room temperature for 14 d, the treated corn straw was washed and dried, and the weight loss rate of the corn straw was calculated.

[0092] Corn straw weight loss rate = (dry weight before fermentation - dry weight after fermentation) / dry weight before fermentation x 100%

[0093] The results (Table 3) showed that the corn straw weight loss rate of strain HPP-0-GZL7 was 8.33%, which had a certain ability to degrade straw.

[0094] Table 3 Strain HPP-0-GZL7 straw weight loss rate determination results

[0095]

[0096] (3) Strain HPP-0-GZL7 siderophore production ability determination

[0097] Qualitative determination: inoculate strain HPP-0-GZL7 on the surface of CAS solid medium, and place in a 37°C dark incubator for 48h, observe whether there is an orange halo around the strain, and determine whether the strain has the ability to secrete siderophores.

[0098] Quantitative determination: inoculate strain HPP-0-GZL7 in MKB liquid medium in the siderophore detection kit, and after 24h culture at 30°C, 180rpm on a shaker, centrifuge at 5000rpm for 10min, add each culture supernatant and CAS detection solution in the test tube in equal proportion, avoid light and stand for 1h, measure the absorbance value (As) at wavelength OD 630 630 The absorbance value (Ar) at OD 630 was determined by adjusting the zero with distilled water. The siderophore activity was calculated, and the calculation formula was: siderophore activity (%) = (Ar-As) / Ar x 100%.

[0099] The qualitative determination results showed that strain HPP-0-GZL7 had the ability to secrete siderophores, and the quantitative determination results (Table 4) showed that the siderophore production ability of strain HPP-0-GZL7 reached 15.03%, which could better provide corn nutrition and induce corn resistance.

[0100] Table 4 Strain HPP-0-GZL7 siderophore production quantitative determination results

[0101]

[0102] Example 3 Strain compatibility test

[0103] The HPP-0-GZL7 bacterial solution with activated OD 600 value reaching 1 was used as the dominant strain, and was added to the dissolved and cooled LB solid medium at a volume ratio of 1%, and then poured into a flat plate after being mixed evenly. Inoculate HPP-0-TS7 bacterial solution with OD 600 value of 1 on the flat plate, and place in a 37°C constant temperature incubator for 2d, and observe whether there is a transparent circle around each strain colony. If a transparent circle appears at the junction around the colony, it indicates that there is antagonism between the two strains, and they are not compatible with each other; if no transparent circle appears, it indicates that the growth of the two strains does not affect each other, and they can be compatible.

[0104] The compatibility test was carried out between the dominant strain HPP-0-TS7 and the strain HPP-0-GZL7, and the results showed that no inhibition zone appeared between the strains HPP-0-TS7 and HPP-0-GZL7, and they were not antagonistic to each other, which indicated that the two strains could be compatible and could be compounded. Figure 3

[0105] Example 4 Antagonistic effect of different compounding ratios of strains

[0106] The OD values of the activated strains HPP-0-TS7 and HPP-0-GZL7 were adjusted to 1, and they were mixed in different ratios of HPP-0-TS7:HPP-0-GZL7 1:0, 1:1, 2:1, 1:2, 3:2 and 2:3, and the plate confrontation method was used to determine the bacteriostatic ability of the strains, and the best compounding ratio was screened according to the bacteriostatic ability. 600

[0107] In order to determine the best compounding ratio of the strains HPP-0-TS7 and HPP-0-GZL7, the plate confrontation method was used to determine the bacteriostatic ability of the strains against F. graminearum, F. pseudocorticium, F. oxysporum, F. verticillioides and C. sasakii in different compounding ratios. The results (Table 5) showed that the two strains still had certain bacteriostatic effect on the five pathogenic fungi after being mixed in different ratios. By comparing the bacteriostatic ability, it was found that the bacteriostatic effect was the highest when the compounding ratio of the strains HPP-0-TS7 and HPP-0-GZL7 was 3:2, and the bacteriostatic ability against F. graminearum, F. verticillioides, F. pseudocorticium, F. oxysporum and C. sasakii was stronger than that of the strain HPP-0-TS7 alone, which was increased by 1.88%, 20.14%, 7.29%, 0.92% and 1.61%, respectively.

[0108] Table 5 Inhibition rate of different compounding ratios of strains on multiple pathogenic fungi

[0109]

[0110]

[0111] Example 4 Grain inoculation antagonistic test of compounded strains

[0112] The spore suspension of F. pseudocorticium was adjusted to a concentration of 1×10 6 cfu / mL, and the concentration of the antagonistic strain was adjusted to 1×10 8 ​​cfu / mL (the concentration of the mixed bacterial solution remained unchanged, and the strains HPP-0-TS7 and HPP-0-GZL7 were compounded in a quantitative ratio of 3:2). A total of four treatments were set up, namely the sterile water control group, the HPP-0-TS7 bacterial solution group, the HPP-0-GZL7 bacterial solution group, and the HPP-0-TS7+GZL7 bacterial solution group. Among them, the corn kernels in the HPP-0-TS7 bacterial solution group were soaked in the HPP-0-TS7 bacterial suspension and the spore suspension for 30 minutes, respectively; the corn kernels in the HPP-0-GZL7 bacterial solution group were soaked in the HPP-0-GZL7 bacterial suspension and the spore suspension for 30 minutes, respectively; the HPP-0-TS7+GZL7 bacterial solution group was soaked in the mixed bacterial solution and the spore suspension for 30 minutes, respectively; and the sterile water control group was soaked in sterile water and the spore suspension for 30 minutes, respectively. After soaking, each group was placed in a sterile dish, two sterile filter papers were placed in the dish, and 3 mL of sterile water was added to keep it moist. The dishes were cultured in an incubator for 7 days and the results were recorded by taking photos.

[0113] result( Figure 4 ) showed that the mycelial growth of corn kernels treated with the composite bacteria was significantly reduced compared with the control, and the growth of pathogens on corn kernels was less than that treated with strains HPP-0-TS7 and HPP-0-GZL7 alone, further indicating that the antagonistic effect of the combination of strains HPP-0-TS7 and HPP-0-GZL7 on pathogens was better.

[0114] Example 5 Effect of composite strains on controlling stem rot in greenhouse

[0115] The spores of Fusarium graminearum were inoculated into PDA medium for activation. 7 to 10 5 mm bacterial cakes were inoculated into CMC-Na medium. After incubation at 25 °C and 180 rpm in a shaker for 5 to 7 days, the culture was centrifuged and the precipitate was resuspended in sterile water to a spore concentration of 1 × 10 6 The maize inbred line B73 was used as the planting variety. The seeds were disinfected with 10% sodium hypochlorite for 3 minutes, rinsed with sterile water several times, and then germinated. After germination, the seeds were transplanted into flower pots. During this period, they were watered moderately to meet the water needs of the plants. The first treatment was carried out when the maize grew to thick stems. 20 mL of HPP-0-TS7 bacterial suspension (concentration of 1×10 8 cfu / mL), HPP-0-GZL7 bacterial suspension (concentration of 1×10 8 cfu / mL) or HPP-0-TS7+HPP-0-GZL7 bacterial suspension (total concentration of 1×10 8The corn roots were irrigated with the compound strains HPP-0-TS7 and HPP-0-GZL7 (3:2) at a concentration of 1 x 10

[0116] Table 6. Effect of the compound strains on corn stalk rot

[0117]

[0118] As shown in Table 6 and Figure 5 The results showed that the compound strains HPP-0-TS7 and HPP-0-GZL7 reduced the lesion area at the corn stem base by 51.55%, and to some extent, inhibited the occurrence of corn stalk rot.

[0119] Example 6. In-vitro growth promotion test of the compound strains in different formulations

[0120] The seed coating agent was prepared by mixing the bacterial suspension of the strains HPP-0-TS7 and HPP-0-GZL7 (total concentration of 1 x 10 8 cfu / mL) with the film-forming agent (purchased from Beinong Haili Company) at a volume ratio of 1:5, so that the final total concentration of the strains was 1 x 10 8 After the seed coating operation, the seeds were placed in a ventilated and cool environment for natural air drying.

[0121] The liquid bacterial agent was prepared by inoculating the strains HPP-0-TS7 and HPP-0-GZL7 in LB medium, and then incubating at 220 r / min and 37°C for 12 h in a shaker. The seed liquid was then inoculated into LB liquid medium at a ratio of 1%, and further incubated at 220 r / min and 37°C for 48 h. The bacterial liquid was then centrifuged and resuspended, and the concentration was adjusted to 1 x 10 8 cfu / mL. The bacterial suspensions of HPP-0-TS7 and HPP-0-GZL7 were mixed at a volume ratio of 3:2.

[0122] The granules were prepared by mixing the bacterial liquid of the strains HPP-0-TS7 and HPP-0-GZL7 after centrifugation and resuspension at a ratio of 3:2, and then uniformly spraying onto the wheat bran granules, so that the final concentration of the bacteria in the wheat bran granules was 1 x 10 8 cfu / g for indoor potting, and dried in a ventilated and cool place.

[0123] Zhengdan 958 as planting variety, three treatments were set, seed coating agent, liquid inoculant, granular inoculant, the final concentration of different treatments was 1 x 10 8 cfu / mL, the untreated seeds were used as blank control. Three parallel repeats were set in each group.

[0124] Application method:

[0125] Seed coating agent treatment, the coated seeds were planted in flowerpots, and appropriate watering was performed to keep them moist.

[0126] Liquid inoculant treatment, the seeds were planted in flowerpots, and appropriate watering was performed to keep them moist. When the corn grew to the three-leaf-one-heart stage, the liquid inoculant was applied at the root of each corn seedling, and 20 mL of liquid inoculant was applied to each corn seedling.

[0127] Granular inoculant treatment, the seeds were placed in the prepared holes, 3 g of granular inoculant was mixed with nutrient soil, and the mixed granular inoculant and nutrient soil were uniformly applied to the holes containing the seeds. Then, the holes were covered with nutrient soil to make the surface flat and gently compacted, and appropriate watering was performed to keep the soil moist.

[0128] After 7 days of root irrigation treatment with the liquid inoculant, the agronomic traits of the corn in each group were measured and counted, and the untreated corn was used as control. The effects of different formulations of the compound strains on the growth of corn were evaluated.

[0129] Table 7 Promoting effect of compound strains in different formulations on corn in greenhouse

[0130]

[0131]

[0132] As shown in Table 7 and Figure 6 The results showed that the corn treated with different formulations of HPP-0-TS7+HPP-0-GZL7 strains had different degrees of promotion, and the liquid inoculant treatment had the best promoting effect. After the corn was treated with the liquid inoculant, the root system was more developed, and the plant height, leaf length, leaf width, and stem diameter of the aboveground part were increased by 19.36%, 20.11%, 29.27%, and 21.40%, respectively, compared with the control. The granular inoculant treatment had the second best promoting effect, and the plant height, leaf length, leaf width, and stem diameter were increased by 18.00%, 11.61%, 21.46%, and 10.93%, respectively, compared with the control.

[0133] Example 7 Effect of Bacillus velezensis HPP-0-TS7 on the growth of corn under salt stress in greenhouse

[0134] A salt solution with a salt content of 6 g / kg (NaCl:Na2CO3=5:1) was configured, and its pH value was 10.57, so that the salt content in 1 kg of nutrient soil was 6 g. Zhengdan 958 was planted in a flowerpot and transplanted into the prepared saline-alkali soil after germination. Bacillus velezensis HPP-0-TS7 bacterial liquid (1×10 8 cfu / mL) was used for the first time to irrigate the roots of corn at the three-leaf stage (20 mL per seedling), and the second time was 7 days later. The normal soil treated with sterile water was used as a positive control, and the saline-alkali soil treated with sterile water was used as a negative control. After 15 days, the agronomic traits of corn were measured to evaluate the effect of Bacillus velezensis HPP-0-TS7 on plant growth in a saline-alkali environment.

[0135] Table 8 Indoor growth-promoting index determination of strain HPP-0-TS7 under saline-alkali conditions

[0136]

[0137] Table 8 and Figure 7 Among them, the plant height, leaf length, leaf width, and chlorophyll content of the group treated with the bacterial liquid of strain HPP-0-TS7 were significantly improved compared with the negative control group. It shows that strain HPP-0-TS7 can enhance the salt-alkali tolerance of corn and has a promoting effect on corn growth.

[0138] Example 8 Field growth-promoting effect evaluation of composite strains

[0139] The field tests of the two strains were both carried out using Zhengdan 958 as the test material, and the seeds were planted manually using a dibbler with a plant spacing of 30 cm and a row spacing of 60 cm. The area of each plot was 32 m2, and each treatment was repeated three times.

[0140] Single-strain granule preparation method: After centrifugation and resuspension of the fermentation broth of strain HPP-0-GZL7 or HPP-0-TS7, the fermentation broth was uniformly sprayed onto the wheat bran granules, so that the final concentration of bacteria in the wheat bran granules was 1×10 8 cfu / g for field test, and dried in a ventilated and cool place.

[0141] Composite granule preparation method: After centrifugation and resuspension of the fermentation broth of strain HPP-0-TS7 and HPP-0-GZL7, the fermentation broth was uniformly mixed according to the quantity ratio of 3:2, and then uniformly sprayed onto the wheat bran granules, so that the final concentration of bacteria in the wheat bran granules was 1×10 8 cfu / g for field test, and dried in a ventilated and cool place.

[0142] Granule application method: When corn is sown, 5 g of granules are applied per hole, and the soil is covered with water to ensure soil moisture.

[0143] Seed coating agent application method: using commercial seed coating agent to prepare coated seeds.

[0144] When the corns grow to the trumpet stage, the plant height, leaf length, leaf width, and stem diameter of the corns in each treatment group were measured and statistically analyzed using the five-point sampling method.

[0145] Table 9: Effect of compound strain granules on the growth of corns in the field

[0146]

[0147] The corns were sowed, and the agronomic traits were measured to determine the effect of the strains on the growth of corns in the field. As shown in Table 9, the corns treated with the compound strain granules had a better growth, and the plant height, leaf length, leaf width, and stem diameter were increased by 7.54%, 5.80%, 8.17%, and 19.03%, respectively, compared with the control. This indicates that the compound strains have a good promoting effect on the growth of corns.

[0148] Example 9: Field control effect of compound strains

[0149] On the basis of Example 8, a granule + liquid inoculant treatment group was added. The liquid inoculant was applied by irrigating the roots of each corn with 50 mL of a bacterial solution (HPP-0-TS7 and HPP-0-GZL7 in a quantity ratio of 3:2) having a concentration of 1 x 10 9 cfu / mL at the large trumpet stage.

[0150] When the corns reached the ripening stage, 30 corns were randomly selected from each treatment group for investigation, and the occurrence of ear rot, leaf spot, and stem rot of the corns in each treatment group was counted and calculated to determine the disease index and control effect of the compound inoculant (Example 6).

[0151]

[0152] Table 10: Control effect of different inoculants on ear rot, stem rot, and leaf spot (curvularia leaf spot and small spot) of corns

[0153]

[0154] The results in Table 10 show that the compound strain treatment has a certain degree of control effect on ear rot and leaf spot of corns, and the combination of granules + liquid inoculants has the best control effect on diseases, with a control effect of 57.05%, 45.03%, and 59.20% on ear rot, stem rot, and leaf spot of corns, respectively.

[0155] After the corn matures, 30 ears are randomly taken from each plot, dried, and then the relevant indicators such as the number of corn ear rows, the number of grains per row, ear length, ear thickness, 100-grain weight, and water content are measured to calculate the corn yield under the treatment of different formulations of the compound microbial agent.

[0156] Yield (kg / hm 2 ) = plant number per mu x ear row number x grain number per row x 1000-grain weight x 8.5 x 10 -1 / (1000 x 1000).

[0157] Table 11 Effect of different microbial agents on the yield of corn

[0158]

[0159] The effect of different microbial agents on the yield of corn was determined for two consecutive years from 2023 to 2024. The results in Table 11 show that the treatment of the compound microbial agent in the form of granules + liquid microbial agent has the best effect on the yield of corn, and the yield is increased by 10.00% compared with the control commercial seed coating agent, indicating that the compound microbial agent HPP-0-TS7 + HPP-0-GZL7 granules + liquid microbial agent can be better applied and effectively improve the yield of corn.

[0160] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A biocontrol bacteria combination for use in corn planting, characterized in that: The biocontrol bacteria combination includes Bacillus velezensis HPP-0-TS7 and Bacillus subtilis HPP-0-GZL7; The Bacillus velezensis HPP-0-TS7 was deposited on August 8, 2024 at the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31638; The Bacillus subtilis HPP-0-GZL7 was deposited in the General Microbiology Center of the China Culture Collection Administration on August 8, 2024, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 31639.

2. Use of the biocontrol bacteria combination according to claim 1 in the preparation of a biological agent with the function of preventing and controlling corn diseases and / or promoting growth.

3. The use according to claim 2, characterized in that The corn diseases are corn leaf blight, corn leaf spot, corn stalk rot, corn sheath rot, corn ear rot and / or corn root rot.

4. The use according to claim 2, characterized in that The biological agent is a liquid agent, a coating agent or a granule.

5. A biological agent for preventing and controlling corn diseases and / or promoting growth, characterized in that: The active ingredient comprises the biocontrol bacteria combination according to claim 1.

6. The biological agent according to claim 5, characterized in that The corn diseases are corn leaf blight, corn leaf spot, corn stalk rot, corn sheath rot, corn ear rot and / or corn root rot.

7. The biological agent according to claim 5, characterized in that The biological microbial agent also includes auxiliary materials.

8. The biological agent according to claim 7, characterized in that The dosage form of the biological agent is liquid agent, coating agent or granule.

9. The biological agent according to claim 7, characterized in that When the dosage form of the biological agent is a coating agent, the excipient is a film-forming agent; When the dosage form of the biological agent is granules, the auxiliary material is wheat bran.

10. Use of the biocontrol bacteria combination according to claim 1 or the biological microbial agent according to any one of claims 5 to 9 in preventing and controlling corn diseases and / or increasing corn yield.

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