A biological bactericide for controlling maize ear rot, its preparation method and application

Abstract

The present invention belongs to the technical field of maize disease control methods, and particularly relates to a biological bactericide for controlling maize ear rot, its preparation method and application. The biological bactericide comprises a water dispersible granule carrier mixture and a biocontrol bacteria culture solution, and the mass ratio of the water dispersible granule carrier mixture to the biocontrol bacteria culture solution is 2-3:1; the water dispersible granule carrier mixture comprises diatomaceous earth, chitosan and sucrose, and the mass ratio of diatomaceous earth, chitosan and sucrose is 1-2:1-2:1; the biocontrol bacteria culture solution is obtained by mixing fermentation broths of Bacillus subtilis, Streptomyces microflavus, Paenibacillus polymyxa and Bacillus velezensis. The biocontrol bactericide provided by the present invention has a very significant effect on controlling maize ear rot caused by Fusarium verticillioides, has the potential to be used as a new biological pesticide for controlling ear rot caused by Fusarium verticillioides in maize production, and has the value of further research.

Description

Technical Field

[0001] The present invention belongs to the technical field of maize disease control methods, and specifically relates to a biological bactericide for controlling maize ear rot, its preparation method and application. Background Art

[0002] Maize is an annual herbaceous plant belonging to the genus Zea in the family Poaceae. Its stalk is erect, usually unbranched, 1 - 4 meters high, and the leaves are flat and broad. According to management classification, the growth period of maize is divided into sowing period, emergence period, jointing period, tasseling period, flowering period, silking period, and maturity period.

[0003] There are many pathogenic species of maize ear rot, among which Fusarium verticillioides is the dominant pathogenic bacterium. Fusarium verticillioides infects the maize ear, and this disease occurs widely in maize planting areas and the damage is increasing year by year. The average incidence rate reaches 10% - 20%, and can reach 30% - 40% in severe years. In addition, the mycotoxins produced by the metabolism of Fusarium verticillioides have strong toxic side effects. The grains of infected ears, when used as food, feed or processed products, can threaten the health of humans and livestock, causing serious food safety problems.

[0004] At present, the existing control methods for Fusarium ear rot of maize mainly use chemical agents. For example, chemical agents are used for seed coating treatment; chemical agents such as iprodione and difenoconazole are sprayed during the silking period of maize growth. The control methods using chemical agents for Fusarium ear rot of maize have problems of unsatisfactory control effect and pesticide residues. Therefore, it is very necessary to develop new control methods that can control Fusarium ear rot of maize and are environmentally friendly. Summary of the Invention

[0005] In order to solve the problems of unsatisfactory control effect and pesticide residues in the existing control methods using chemical agents for Fusarium ear rot of maize, and to achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a biological bactericide for controlling maize ear rot. The biological bactericide includes a water - dispersible granule carrier mixture and a biocontrol bacterium culture solution, and the mass ratio of the water - dispersible granule carrier mixture to the biocontrol bacterium culture solution is 2 - 3:1;

[0007] The water - dispersible granule carrier mixture includes diatomaceous earth, chitosan and sucrose, and the mass ratio of diatomaceous earth, chitosan and sucrose is 1 - 2:1 - 2:1;

[0008] The biocontrol bacterium culture solution is obtained by mixing the fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis.

[0009] Preferably, the volume ratio of the Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth is 1:2-3:1-2:1-2.

[0010] The present invention also provides a preparation method of the biological bactericide for preventing and controlling maize ear rot, comprising the following steps:

[0011] Preparation of Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth: Bacillus subtilis, Streptomyces microflavus, Paenibacillus polymyxa, and Bacillus velezensis are respectively inoculated into different culture media for culture. After centrifuging the culture, the cells are collected, resuspended in sterile normal saline to obtain a cell suspension, and the cell suspension is further cultured to obtain Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth respectively;

[0012] Mix the Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth to obtain a biocontrol bacteria culture solution;

[0013] Mix diatomaceous earth, chitosan, and sucrose to obtain a water-dispersible granule carrier mixture;

[0014] Mix the water-dispersible granule carrier mixture and the biocontrol bacteria culture solution evenly to obtain a biocontrol bactericide.

[0015] Preferably, the viable bacteria count in the Bacillus subtilis fermentation broth is greater than 1×10 7 CFU / mL.

[0016] Preferably, the viable bacteria count in the Streptomyces microflavus fermentation broth is greater than 1×10 8 CFU / mL.

[0017] Preferably, the viable bacteria count in the Paenibacillus polymyxa fermentation broth is greater than 1×10 7 CFU / mL.

[0018] Preferably, the viable bacteria count in the Bacillus velezensis fermentation broth is greater than 1×10 7 CFU / mL.

[0019] The present invention also provides the application of the biological bactericide for preventing and controlling maize ear rot in the biological control of maize ear rot, and the maize ear rot is caused by Fusarium verticillioides.

[0020] Preferably, the biocontrol bactericide is sprayed once at the silking stage of the maize ear, and the biocontrol bactericide is sprayed once 20-30 days before the maturity stage.

[0021] Preferably, the silking stage of the maize female ear is the stage when the silk of the maize female ear protrudes 1-3 cm from the bract leaf.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides a biological bactericide for controlling maize ear rot. Using this biological bactericide to control maize ear rot can solve the problem that the control effect of chemical agents on Fusarium ear rot of maize is not ideal in the prior art. The biocontrol bactericide provided by the present invention has a significant inhibitory effect on Fusarium verticillioides. The inhibitory effects of the original solution, 10-fold dilution and 25-fold dilution of the biocontrol bactericide on Fusarium verticillioides are all relatively high, indicating that the biocontrol bactericide provided by the present invention can effectively inhibit Fusarium verticillioides, and the original solution of the biocontrol bactericide has the best antibacterial effect. The preparation method of this biocontrol bactericide is simple. It only needs to mix the water-dispersible granule carrier mixture and the biocontrol bacteria culture solution in a certain proportion and can be applied after dilution with water; and all components in the biocontrol bactericide are environmentally friendly and will not cause environmental and food safety problems.

[0024] 2. The control effect of the biocontrol bactericide provided by the present invention on ear rot caused by Fusarium verticillioides is above 95%, indicating that the biocontrol bactericide prepared by the present invention can control maize ear rot caused by Fusarium verticillioides and has a good control effect.

[0025] 3. The control effect of the biocontrol bactericide provided by the present invention on ear rot caused by Fusarium verticillioides in maize is more than 60% higher than that of carbendazim, and more than 70% higher than that of difenoconazole on ear rot caused by Fusarium verticillioides in maize. The biocontrol bactericide prepared by the present invention has a very significant effect on controlling maize ear rot caused by Fusarium verticillioides, has the potential to be a new biological pesticide for controlling maize ear rot caused by Fusarium verticillioides in maize production, and has the value of further research. Detailed Embodiments

[0026] The present invention will be described in detail below with reference to specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0027] The experimental materials used in the following examples are as follows:

[0028] 1. Strains

[0029] Bacillus subtilis E72, purchased from the China General Microbiological Culture Collection Center.

[0030] Streptomyces microflavus var. liaoningensis, preserved in the China General Microbiological Culture Collection Center, preservation date: 2018-02-12, preservation number: CGMCC No. 15355.

[0031] Paenibacillus polymyxa Jsa-9, purchased from the China General Microbiological Culture Collection Center.

[0032] Bacillus velezensis 10, purchased from the China General Microbiological Culture Collection Center.

[0033] The pathogen is Fusarium verticillioides, which causes maize ear rot.

[0034] 2. Culture medium

[0035] The formula of LB liquid medium is: Tryptone 10g, Yeast Extract 5g, NaCl 10g, deionized water 1000 mL.

[0036] The formula of LB solid medium is: LB liquid medium plus 1.5% - 2% agar.

[0037] Among them, the formula of LB liquid medium is Tryptone 10g, Yeast Extract 5g, NaCl 10g, deionized water 1000 mL.

[0038] The formula of Gao's No. 1 liquid medium is: soluble starch 20g, NaCl 0.5g, KNO3 1g, K2HPO4·3H2O 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, deionized water 1000 mL. Adjust the pH to 7.4 - 7.6.

[0039] The formula of Gao's No. 1 solid medium is: Gao's No. 1 liquid medium plus 1.5% - 2% agar.

[0040] Among them, the formula of Gao's No. 1 liquid medium is soluble starch 20g, NaCl 0.5g, KNO3 1g, K2HPO4·3H2O 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, deionized water 1000 mL. Adjust the pH to 7.4 - 7.6.

[0041] The formula of CM solid medium is: 50 mL of 20×Nitrate, 1 mL of 1000×Trace Element, 1 mL of 1000×Vitamin, 10 g of D-Glucose, 2 g of Peptone, 1 g of Yeast Extract, 1 g of CasaminoAcids, 15 g of agar, and add deionized water to 1000 mL.

[0042] The formula of 20×Nitrate is: 120 g of NaNO3, 10.4 g of KCl, 10.4 g of MgSO4·7H2O, 30.4 g of KH2PO4, and add deionized water to 1000 mL.

[0043] The formula of 1000×Trace Element is: 1.1 g of ZnSO4·7H2O, 0.5 g of MnCl2·4H2O, 0.5 g of FeSO4·7H2O, 0.17 g of CoCl2·6H2O, 0.16 g of CuSO4·5H2O, 0.15 g of Na2Mo4·2H2O, 5 g of Na2EDTA, 1.1 g of H3BO3, add deionized water to 100 mL, heat to 60 °C to dissolve, and adjust the pH to 6.5 with KOH.

[0044] The formula of 1000×Vitamin is: 0.01 g of Biotin, 0.01 g of Pyridoxin, 0.01 g of Thiamine, 0.01 g of Riboflavin, 0.01 g of p-aminobenzoic acid, 0.01 g of Nicotinic acid, and add deionized water to 100 mL.

[0045] The above media are sterilized at 121 °C for 15 min.

[0046] Example 1

[0047] A preparation method of a biological bactericide for controlling maize ear rot includes the following steps:

[0048] 1. Preparation of Bacillus subtilis fermentation broth

[0049] Inoculate Bacillus subtilis E72 on the plate of LB solid medium, and then purify it 4 times to obtain single colonies. Pick the single colonies with an inoculation needle into 250 mL of LB liquid medium, and culture it at 37 °C with shaking at 180 rpm for 48 h. Take 10 mL of the aforementioned culture, centrifuge at 2500 rpm for 10 min at room temperature to collect the bacteria. Resuspend the bacteria with sterile normal saline (Solarbio, IN9000) and adjust the OD600 to 7.0, then suck the bacterial suspension according to an inoculation amount of 2% and add it to the LB liquid medium, and culture it at 37 °C with shaking at 180 rpm for 24 h. The obtained product is the Bacillus subtilis fermentation broth.

[0050] The viable count in the above Bacillus subtilis fermentation broth is greater than 1×10 7 CFU / mL.

[0051] 2. Preparation of Streptomyces microflavus Fermentation Broth

[0052] Inoculate Streptomyces microflavus var. liaoningensis on a plate of Gao's No. 1 solid medium, and then purify it 4 times to obtain single colonies. Pick a single colony with an inoculation needle into 250 mL of Gao's No. 1 liquid medium, and culture it with shaking at 37 °C and 180 rpm for 48 h. Take 10 mL of the aforementioned culture, centrifuge at 2500 rpm for 10 min at room temperature to collect the thalli. Resuspend the thalli with sterile normal saline (Solarbio, IN9000) and adjust the OD600 to 7.0, then pipette 2% of the bacterial suspension according to the inoculation amount and add it to Gao's No. 1 liquid medium, and culture it with shaking at 37 °C and 180 rpm for 24 h. What is obtained is the Streptomyces microflavus fermentation broth.

[0053] The viable count in the above Streptomyces microflavus fermentation broth is greater than 1×10 8 CFU / mL.

[0054] 3. Preparation of Paenibacillus polymyxa Fermentation Broth

[0055] Inoculate Paenibacillus polymyxa Jsa-9 on a plate of LB solid medium, and then purify it 4 times to obtain single colonies. Pick a single colony with an inoculation needle into 250 mL of liquid LB medium, and culture it with shaking at 37 °C and 180 rpm for 48 h. Take 10 mL of the aforementioned culture, centrifuge at 2500 rpm for 10 min at room temperature to collect the thalli. Resuspend the thalli with sterile normal saline (Solarbio, IN9000) and adjust the OD600 to 7.0, then pipette 2% of the bacterial suspension according to the inoculation amount and add it to LB liquid medium, and culture it with shaking at 37 °C and 180 rpm for 24 h. What is obtained is the Paenibacillus polymyxa fermentation broth.

[0056] The viable count in the above Paenibacillus polymyxa fermentation broth is greater than 1×10 7 CFU / mL.

[0057] 4. Preparation of Bacillus velezensis Fermentation Broth

[0058] Bacillus velezensis 10 was inoculated on the plate of LB solid medium, and then purified 4 times to obtain single colonies. A single colony was picked with an inoculation needle and transferred into 250 mL of LB liquid medium, and cultured with shaking at 37 °C and 180 rpm for 48 h. 10 mL of the aforementioned culture was taken, and the cells were collected by centrifugation at 2500 rpm for 10 min at room temperature. The cells were resuspended with sterile normal saline (Solarbio, IN9000) and the OD600 was adjusted to 7.0. Then, the bacterial suspension was aspirated according to 2% of the inoculation amount and added into the LB liquid medium, and the obtained product after culturing with shaking at 37 °C and 180 rpm for 24 h was the Bacillus velezensis fermentation broth.

[0059] The viable count in the above-mentioned Bacillus velezensis fermentation broth is greater than 1×10 7 CFU / mL.

[0060] 5. Preparation of biocontrol bacteria culture solution

[0061] The Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth prepared above were mixed according to a volume ratio of 2:3:1:2 to obtain a biocontrol bacteria culture solution.

[0062] 6. Preparation of biocontrol agent

[0063] Diatomite, chitosan, and sucrose were mixed according to a mass ratio of 1:2:1 to prepare a carrier mixture for water-dispersible granules. According to the dosage ratio of the carrier mixture for water-dispersible granules to the above-mentioned biocontrol bacteria culture solution of 10 g:100 mL, the carrier mixture for water-dispersible granules was added to the biocontrol bacteria culture solution and mixed evenly to obtain a biocontrol agent.

[0064] The implementation method of the biocontrol agent for preventing and controlling maize ear rot includes the following steps:

[0065] The biocontrol agent prepared in the above steps was diluted with 10 L of water, and the diluted biocontrol agent was sprayed once during the silking stage of maize ear, and the biocontrol agent with the same preparation method and concentration was sprayed a second time 20 days before the maturity stage.

[0066] Example 2

[0067] A preparation method of a biological bactericide for preventing and controlling maize ear rot includes the following steps:

[0068] In this example, the preparation methods of Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth are the same as those in Example 1. The difference lies in that the steps of preparing the biocontrol bacteria culture solution and the biocontrol agent are different. The specific differences are as follows:

[0069] (1) In this embodiment, the prepared biocontrol agent Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth are mixed in a volume ratio of 2:2:1:2 to obtain a biocontrol bacteria culture solution.

[0070] (2) In the biocontrol agent prepared in this embodiment, the total mass of diatomaceous earth, chitosan, and sucrose accounts for 6% of the mass of the biocontrol agent, and the mass ratio of diatomaceous earth, chitosan, and sucrose is 2:1:1.

[0071] The implementation method of this biocontrol agent for controlling maize ear rot includes the following steps:

[0072] The implementation method of the biological control agent prepared in this embodiment is basically the same as that in Example 1, except that:

[0073] Spray the biocontrol agent once during the silking stage of the maize female ear, and spray the biocontrol agent a second time 25 days before maturity.

[0074] Example 3

[0075] A preparation method of a biological control agent for controlling maize ear rot includes the following steps:

[0076] In this embodiment, the preparation methods of Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth are the same as those in Example 1. The difference is that the steps of preparing the biocontrol bacteria culture solution and the biocontrol agent are different. The specific differences are as follows:

[0077] (1) In this embodiment, the prepared biocontrol agent Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth, and Bacillus velezensis fermentation broth are mixed in a volume ratio of 2:3:1:1 to obtain a biocontrol bacteria culture solution.

[0078] (2) In the biocontrol agent prepared in this embodiment, the total mass of diatomaceous earth, chitosan, and sucrose accounts for 4% of the mass of the biocontrol agent, and the mass ratio of diatomaceous earth, chitosan, and sucrose is 1:1:1.

[0079] The implementation method of this biocontrol agent for controlling maize ear rot includes the following steps:

[0080] The implementation method of the biological control agent prepared in this embodiment is basically the same as that in Example 1, except that:

[0081] Spray the biocontrol agent once during the silking stage of the maize female ear, and spray the biocontrol agent a second time 30 days before maturity.

[0082] Example 4

[0083] A preparation method of a biological bactericide for preventing and controlling maize ear rot, comprising the following steps:

[0084] In this example, the preparation methods of Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth and Bacillus velezensis fermentation broth are the same as those in Example 1. The difference lies in that the steps of preparing the biocontrol bacteria culture solution and the biocontrol bactericide are different. The specific differences are as follows:

[0085] (1) In this example, the prepared biocontrol bactericides, namely Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth and Bacillus velezensis fermentation broth, are mixed in a volume ratio of 2:2:1:2 to obtain the biocontrol bacteria culture solution.

[0086] (2) In the biocontrol bactericide prepared in this example, the total mass of diatomaceous earth, chitosan and sucrose accounts for 6% of the mass of the biocontrol bactericide, and the mass ratio of diatomaceous earth, chitosan and sucrose is 2:1:1.

[0087] The implementation method of this biocontrol bactericide for preventing and controlling maize ear rot comprises the following steps:

[0088] In this example, the implementation method of the prepared biological bactericide is the same as that in Example 1.

[0089] Example 5

[0090] A preparation of a biological bactericide for preventing and controlling maize ear rot, comprising the following steps:

[0091] In this example, the preparation methods of Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth and Bacillus velezensis fermentation broth are the same as those in Example 1. The difference lies in that the steps of preparing the biocontrol bacteria culture solution and the biocontrol bactericide are different. The specific differences are as follows:

[0092] (1) In this example, the prepared biocontrol bactericides, namely Bacillus subtilis fermentation broth, Streptomyces microflavus fermentation broth, Paenibacillus polymyxa fermentation broth and Bacillus velezensis fermentation broth, are mixed in a volume ratio of 2:3:1:1 to obtain the biocontrol bacteria culture solution.

[0093] (2) In the biocontrol bactericide prepared in this example, the total mass of diatomaceous earth, chitosan and sucrose accounts for 4% of the mass of the biocontrol bactericide, and the mass ratio of diatomaceous earth, chitosan and sucrose is 1:1:1.

[0094] The implementation method of this biocontrol bactericide for preventing and controlling maize ear rot comprises the following steps:

[0095] In this example, the implementation method of the prepared biological bactericide is the same as that in Example 1.

[0096] Example 6

[0097] A preparation method of a biological bactericide for controlling maize ear rot comprises the following steps:

[0098] In this example, the preparation method of the biological bactericide is the same as that in Example 1.

[0099] The implementation method of this biocontrol bactericide for controlling maize ear rot comprises the following steps:

[0100] In this example, the implementation method of the prepared biological bactericide is basically the same as that in Example 1, except that:

[0101] Spray the biocontrol bactericide once during the silking stage of the maize ear, and spray the biocontrol bactericide a second time 25 days before maturity.

[0102] Example 7

[0103] The preparation of a biological bactericide for controlling maize ear rot comprises the following steps:

[0104] In this example, the preparation method of the biological bactericide is the same as that in Example 1.

[0105] 2. The implementation method of the biocontrol bactericide for controlling maize ear rot

[0106] In this example, the implementation method of the prepared biological bactericide is basically the same as that in Example 1, except that:

[0107] Spray the biocontrol bactericide once during the silking stage of the maize ear, and spray the biocontrol bactericide a second time 30 days before maturity.

[0108] The present invention provides a biological bactericide for controlling maize ear rot, and biological control is carried out by applying the biological bactericide prepared by the present invention. In order to illustrate the biological control effect of the biological bactericide provided by the present invention, the following research was carried out:

[0109] 1. Bacteriostatic test (inhibitory effect of different dilution solutions of the biocontrol bactericide on Fusarium verticillioides)

[0110] (1) Prepare CM solid medium, pour 15 mL of the medium into each petri dish, and wait for it to cool and solidify for later use.

[0111] (2) The plate confrontation culture method was adopted. A purified Verticillium dahliae Kleb. cake (0.5 cm) was picked and inoculated into the center of the above-mentioned CM solid medium plate. Oxford cups were placed at four points, 2.5 cm above, below, left, and right from the center of the plate. 200 μL of the biocontrol agent prepared in Example 1 above was added into the Oxford cups. The plate was cultured at 28 °C for 7 d. At the same time, Oxford cups with 200 μL of LB liquid medium added at the four points were set as blank controls. The cross method was used to measure the diameter of the Verticillium dahliae Kleb. colony, and the growth inhibition rate of the biocontrol agent against Verticillium dahliae Kleb. was calculated. Three biological replicates were set for the experiment. The calculation formula for the growth inhibition rate is as follows:

[0112] Growth inhibition rate = (control colony diameter - treated colony diameter) / control colony diameter × 100%;

[0113] Here, the control bacteria refer to Verticillium dahliae Kleb. without any treatment, and the treated bacteria refer to Verticillium dahliae Kleb. growing under the treatment conditions of the biocontrol agent.

[0114] The biocontrol agent was diluted 10-fold, 25-fold, 50-fold, 75-fold, and 100-fold respectively, and a total of six groups of experiments were carried out, which were respectively labeled as Group I (biocontrol agent stock solution), Group II (10-fold dilution of the biocontrol agent), Group III (25-fold dilution of the biocontrol agent), Group IV (50-fold dilution of the biocontrol agent), Group V (75-fold dilution of the biocontrol agent), and Group VI (100-fold dilution of the biocontrol agent).

[0115] Through the above plate confrontation test, the colony diameter was measured and the average value was calculated. The results are shown in Table 1.

[0116] Table 1 Antibacterial effects of different treatment groups on Verticillium dahliae Kleb.

[0117] Group Colony diameter (cm) Group I 0.67 Group II 1.17 Group III 1.77 Group IV 3.33 Group V 4.80 Group VI 6.70 Blank control group 8.13

[0118] As can be seen from Table 2, the biocontrol agent prepared by the present invention has a significant inhibitory effect on Verticillium dahliae Kleb. The biocontrol agent stock solution, 10-fold dilution of the biocontrol agent, and 25-fold dilution of the biocontrol agent all have a high inhibitory effect on Verticillium dahliae Kleb., indicating that the biocontrol agent prepared by the present invention can effectively inhibit Verticillium dahliae Kleb., and the antibacterial effect of the biocontrol agent stock solution is the best.

[0119] 2. Field experiment (control effect of the biocontrol agent on Fusarium verticillioides ear rot of maize)

[0120] The selected corn variety is B73. The corn is planted in the experimental field of Shenyang Agricultural University, with double-row planting, a row spacing of 60 cm, and a plant spacing of 30 cm. Field management is carried out normally. Every 20 corn plants are set as a group, and a total of eight groups are set. Each group is sprayed once with the biocontrol agent prepared in Example 1 above during the silking stage of the corn ear, and the biocontrol agent prepared in Example 1 above is sprayed a second time 20 days before maturity. The appropriate amount for each spraying is 10 mL / ear. The Fusarium verticillioides spore suspension is inoculated 72 h after the first spraying.

[0121] Method for inoculating Fusarium verticillioides: Collect Fusarium verticillioides spores from the edge of the colony of Fusarium verticillioides cultured on CM solid medium, and then transfer them to CM liquid medium. Shake-culture at 28 °C and 120 rpm for 48 h, filter to remove the mycelium, and collect the spore suspension to obtain the Fusarium verticillioides spore suspension. Aspirate the Fusarium verticillioides spore suspension and dilute it to a spore concentration of 10 6 mL -1 to obtain the Fusarium verticillioides spore solution. Aspirate the spore solution with a syringe for standby. Peel off the husk of the corn ear at the milk-ripe stage, insert the syringe 5 cm from the top of the ear, and inoculate 2 mL of the Fusarium verticillioides spore solution into each ear.

[0122] The biocontrol agent is diluted 10 times, 25 times, 50 times, 75 times, and 100 times respectively. At the same time, an LB liquid medium treatment is set as a negative control; for the chemical agents (chemical fungicides) currently used to control Fusarium ear rot of corn: carbendazim (Andochem Huifeng (Jiangsu) Co., Ltd.) with a mass concentration of 4 μg / mL and difenoconazole (Jiangsu Huifeng Bio-Agriculture Co., Ltd.) with a mass concentration of 20 mg / mL are used to treat Fusarium verticillioides as positive controls. The specific grouping and implementation are as follows:

[0123] The experimental group is sprayed once with the biocontrol agent prepared in Example 1 of the present invention during the silking stage of the corn ear and 20 days before maturity, with 10 mL sprayed per ear each time; blank control group: other conditions are the same as those in the experimental group, except that an equal amount of LB liquid medium is sprayed; positive control group I: other conditions are the same as those in the experimental group, except that an equal amount of 4 μg / mL carbendazim is sprayed; positive control group II: other conditions are the same as those in the experimental group, except that an equal amount of 20 mg / mL difenoconazole is sprayed.

[0124] According to the following table, the disease grading of ear rot caused by Fusarium verticillioides in corn is counted, and the disease index of corn is calculated according to the formula (Table 2), as well as the control effects of the biological agent provided in Example 1, carbendazim provided in Comparative Example 1, and difenoconazole provided in Comparative Example 2 (Table 3).

[0125] Table 2 Disease grading criteria for ear rot caused by Fusarium verticillioides in corn

[0126] Disease grading Description 1 Disease area accounts for 0% - 1% of the female ear area 3 Disease area accounts for 2% - 10% of the female ear area 5 Disease area accounts for 11% - 25% of the female ear area 7 Disease area accounts for 26% - 50% of the female ear area 9 Disease area accounts for 51% - 100% of the female ear area

[0127] Disease index calculation formula:

[0128]

[0129] Control effect calculation formula:

[0130]

[0131] Table 3 Disease index and control effect of ear rot caused by Fusarium verticillioides on corn

[0132] Group Disease index Control effect Blank control group 79.67 — Experimental group 3.36 95.78％ Positive control group I 52.60 33.98％ Positive control group II 59.93 24.78％

[0133] Note: — indicates that the blank control group has no control effect, and no data statistics on the control effect were made.

[0134] As can be seen from Table 3, the biocontrol agent prepared in Example 1 of the present invention has a control effect of more than 95% on ear rot caused by Fusarium verticillioides on corn, indicating that the biocontrol agent prepared by the present invention can control ear rot caused by Fusarium verticillioides on corn, and the control effect is better.

[0135] Compared with positive control I, the biocontrol agent prepared in Example 1 of the present invention has a control effect on ear rot caused by Fusarium verticillioides on corn that is more than 60% higher than that of carbendazim; compared with positive control II, the biocontrol agent prepared in Example 1 of the present invention has a control effect on ear rot caused by Fusarium verticillioides on corn that is more than 70% higher than that of difenoconazole. The biocontrol agent prepared by the present invention has a very significant effect on controlling ear rot caused by Fusarium verticillioides on corn, has the potential to be a new biological pesticide for controlling ear rot caused by Fusarium verticillioides in corn production, and has the value of further research.

[0136] The above results show that the control effect of the biological bacterial agent for controlling corn ear rot provided by the present invention far exceeds that of the existing chemical agents on Fusarium ear rot of corn. And the existing chemical agents will cause serious pesticide residue problems when used, while the raw materials used in the biological bacterial agent prepared by the present invention are all substances or organisms widely existing in nature or organisms, so it will not cause environmental pollution and there will be no problem of pesticide residue when used.

[0137] The present invention provides a biological bacterial agent for controlling corn ear rot. Using this biological bacterial agent to control corn ear rot can solve the problem that the control effect of using chemical agents on Fusarium ear rot of corn is not ideal in the prior art. The biocontrol agent provided by the present invention has a control effect of more than 95% on ear rot caused by Fusarium verticillioides, indicating that the biocontrol agent prepared by the present invention can control corn ear rot caused by Fusarium verticillioides, and the control effect is better.

[0138] The biocontrol agent prepared by the present invention has a very significant effect on controlling Fusarium verticillioides-induced ear rot of maize, has the potential to be a new biological pesticide for controlling ear rot caused by Fusarium verticillioides in maize production, and is worthy of further research.

[0139] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, preferred embodiments of the present invention are described.

[0140] Although the preferred embodiments of the present invention have been described, once those skilled in the art learn the basic creative concepts, additional changes and modifications can be made to these embodiments. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications that fall within the scope of the present invention.

[0141] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A biological bactericide for controlling maize ear rot, characterized in that, The maize ear rot is caused by *Verticillium* Fusarium verticillioides sp. The biological bactericide includes a water dispersible granule carrier mixture and a biocontrol bacteria culture solution, and the mass ratio of the water dispersible granule carrier mixture to the biocontrol bacteria culture solution is 2-3:

1. The carrier mixture of the water dispersible granules comprises diatomite, chitosan and sucrose, and the mass ratio of diatomite, chitosan and sucrose is 1-2:1-2:1; The biocontrol bacteria culture solution is obtained by mixing the fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis; The Bacillus subtilis fermentation broth is the fermentation broth of Bacillus subtilis ( Bacillus subtilis ); the Streptomyces microflavus fermentation broth is the fermentation broth of Streptomyces microflavus var. liaoningensis ( Streptomyces microflavus ); the Paenibacillus polymyxa fermentation broth is the fermentation broth of Paenibacillus polymyxa ( Paenibacillus polymyxa ); the Bacillus velezensis fermentation broth is the fermentation broth of Bacillus velezensis ( Bacillus velezensis ). Among them, the Liaoning variant of Streptomyces microflavus is currently preserved in the General Microbiology Center of the China Microbial Culture Collection Center, and its preservation number is CGMCC No. 15355.

2. The biological bactericide for preventing and controlling maize ear rot according to claim 1, characterized in that, The volume ratio of the fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis is 1:2-3:1-2:1-2.

3. The preparation method of the biological bactericide for preventing and controlling maize ear rot according to claim 1, characterized in that, Comprising the following steps: Preparation of the fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis: Bacillus subtilis E72, the Liaoning variant of Streptomyces microflavus, Paenibacillus polymyxa Jsa-9 and Bacillus velezensis 10 are respectively inoculated into different culture media for culturing. After centrifuging the cultures, the thalli are collected, and the thalli are resuspended with sterile normal saline to obtain a bacterial suspension. The fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis are respectively obtained by further culturing the bacterial suspension; Mix the fermentation broth of Bacillus subtilis, the fermentation broth of Streptomyces microflavus, the fermentation broth of Paenibacillus polymyxa and the fermentation broth of Bacillus velezensis to obtain the biocontrol bacteria culture solution; Mix diatomite, chitosan and sucrose to obtain the carrier mixture of the water dispersible granules; Mix the carrier mixture of the water dispersible granules and the biocontrol bacteria culture solution evenly to obtain the biocontrol agent.

4. The preparation method according to claim 3, characterized in that, The viable count in the Bacillus subtilis fermentation broth is greater than 1×10 7 CFU / mL.

5. The preparation method according to claim 3, characterized in that, The viable count in the fermentation broth of Streptomyces microflavus is greater than 1×10 8 CFU / mL.

6. The preparation method according to claim 3, characterized in that, The viable count in the Paenibacillus polymyxa fermentation broth is greater than 1×10 7 CFU / mL.

7. The preparation method according to claim 3, characterized in that, The viable count in the Bacillus velezensis fermentation broth is greater than 1×10 7 CFU / mL.

8. Use of the biological bacterial agent for preventing and controlling maize ear rot in the biological control of maize ear rot, characterized in that, Spray the biocontrol agent once during the silking stage of the maize ear, and spray the biocontrol agent once 20-30 days before maturity; the maize ear rot is caused by Fusarium verticillioides.

9. The application according to claim 8, wherein The silking stage of the maize ear is the stage when the silk of the maize ear exposes 1-3 cm of the bract leaf.

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