Microbial complex microbial inoculant for preventing and treating corn grey speck disease and preparation method of microbial complex microbial inoculant

Abstract

The invention discloses a microbial complex microbial inoculant for preventing and treating corn grey speck disease and a preparation method thereof, and relates to the technical field of biology. The active components of the microbial composite inoculant comprise a microbial combination; the present invention relates to a microbial composition comprising Burkholderia latii (Burkholderia latii) and Paecilomyces variotii (Paecilomyces variotii), and the microbial composition comprising Burkholderia latii (Burkholderia latii) and Paecilomyces variotii (Paecilomyces variotii). The research finds that the pathogenic fungi of the corn grey leaf spot can be effectively inhibited by combined application of the burkholderia breviscapus and the paecilomyces variotii. The invention provides a technical support for developing a microbial composite bacterial agent for preventing and controlling the corn gray leaf spot, which plays an important positive role in realizing safe and efficient prevention and control of the corn gray leaf spot.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a microbial composite agent for preventing and treating corn gray spot disease and a preparation method thereof. Background Art

[0002] Corn gray spot is also known as Cercospora leaf spot. The pathogenic fungus is Cercospora zeae-maydis Tehon & E.Y.Daniels or Cercospora zeina Crous & U.Braun. Corn gray spot is one of the diseases that has risen rapidly and caused serious damage in recent years. The disease mainly infects corn leaves, and also infects leaf sheaths and bracts. In the early stage of the disease, the lesions are light brown, gradually expanding into long strips or rectangular lesions of gray-brown, gray to yellow-brown, mostly limited to parallel leaf veins, with a size of 0.5-40×0.5-6 (mm). When the humidity is high, gray mold-like substances grow on both sides of the lesions, namely the conidiophores and conidia of the pathogen. When the climatic conditions are suitable, the lesions can spread to the entire plant leaves, causing the leaves to die, and eventually leading to the death and premature aging of the diseased plants.

[0003] Chemical control is currently the main method for preventing and controlling corn gray spot disease, but long-term use of chemical agents can easily cause drug resistance in pathogens, thereby reducing drug efficacy, and the harm of agent residues to the environment and humans is becoming increasingly significant. Biological control has attracted widespread attention due to its safety and environmental friendliness, especially the use of biocontrol bacteria for disease prevention and control is a current research hotspot. Compared with chemical pesticides, biocontrol bacteria have attracted much attention due to their diverse mechanisms of action and relatively low risk of pathogenic microorganisms developing drug resistance. The present invention intends to develop a microbial composite agent for preventing and controlling corn gray spot disease, thereby safely and efficiently achieving prevention and control of corn gray spot disease. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial composite agent for preventing and treating corn gray spot disease and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art. The microbial composite agent can effectively prevent and treat corn gray spot disease, and the prevention effect can reach more than 70%.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a microbial combination for preventing and controlling corn gray spot disease, which comprises Burkholderia lata and Paecilomyces variotii.

[0007] Furthermore, the ratio of the number of live bacteria of the Burkholderia broadleaf and the Paecilomyces variotii is 7:(3-28).

[0008] Preferably, the ratio of the number of live bacteria of the Burkholderia broadleaf and the Paecilomyces variotii is 3:(2-3).

[0009] More preferably, the ratio of the number of live bacteria of the Burkholderia broadleaf and the Paecilomyces variotii is 3:2.

[0010] The present invention also provides application of the above-mentioned microbial combination in preparing a microbial composite agent for preventing and controlling corn gray spot disease.

[0011] The present invention also provides application of the above-mentioned microbial combination in preventing and controlling corn gray spot disease.

[0012] The present invention also provides a composite microbial agent for preventing and treating corn gray spot disease, wherein the active ingredients include the above-mentioned microbial combination.

[0013] The present invention also provides a method for preparing the above-mentioned microbial composite bacterial agent, comprising the steps of uniformly mixing a Burkholderia broadleaf suspension and a Paecilomyces variotii suspension to obtain the microbial composite bacterial agent;

[0014] The Burkholderia broada bacterial suspension is obtained by fermenting and culturing Burkholderia broada, isolating bacterial bodies, and then resuspending them in sterile physiological saline;

[0015] The Paecilomyces variotii suspension is obtained by fermenting and culturing Paecilomyces variotii, separating spores, and then resuspending them in sterile physiological saline.

[0016] The present invention also provides application of the above-mentioned microbial composite agent in preventing and controlling corn gray spot disease.

[0017] The present invention also provides a method for preventing and controlling corn gray spot disease, comprising the step of spraying the above-mentioned microbial composite agent on the leaves of plants.

[0018] The present invention discloses the following technical effects:

[0019] The present invention has found that the combined use of Burkholderia broad and Paecilomyces variotii can effectively inhibit the pathogenic fungus of corn gray spot disease, Cercosporazeae-maydis Tehon & Daniels. When the ratio of the number of live bacteria of Burkholderia broad and Paecilomyces variotii is in the range of 7: (3-28), different degrees of synergistic inhibition are shown for Cercosporazeae, among which the synergistic effect of the 6:4 ratio is the best, with a synergistic ratio of 1.35 and an actual inhibition rate of 68.27%, followed by a 5:5 ratio, with a synergistic ratio of 1.23 and an actual inhibition rate of 62.39%. The present invention provides technical support for the development of a microbial composite agent for the prevention and control of corn gray spot disease, which plays an important and positive role in achieving safe and efficient prevention and control of corn gray spot disease. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] Biocontrol bacteria refer to beneficial microorganisms that can prevent and control plant diseases through a variety of mechanisms. Biocontrol bacteria mainly include bacteria, fungi, actinomycetes and viruses. Bacterial biocontrol bacteria such as Bacillus and Pseudomonas have the characteristics of many species, high reproductive capacity and complex metabolic activities. They can inhibit the growth of pathogens through antagonism and competition.

[0026] The mechanisms of action of biocontrol bacteria are diverse, mainly including:

[0027] Competition: Biocontrol bacteria compete with pathogens for infection sites on the plant surface, for water and nutrients, thereby inhibiting the growth of pathogens.

[0028] Antagonism: Biocontrol bacteria inhibit the survival and activity of pathogens by producing metabolites.

[0029] Parasitic effect: Some biocontrol bacteria parasitize on pathogens and use the nutrients of pathogens to inhibit their growth.

[0030] Induced resistance: Induce plants to develop disease resistance through the metabolites of microorganisms, thereby enhancing the plants' self-protection capabilities.

[0031] Biocontrol bacteria are widely used in agricultural production, and can effectively prevent and control plant diseases, promote plant growth, and improve the soil environment. For example, Bacillus can activate soil, dissolve phosphorus and potassium, promote crop rooting, and enhance crop immunity and disease resistance. In addition, research hotspots of biocontrol bacteria also include strains with strong stress resistance such as Trichoderma harzianum, which perform well in the ecological environment and have high application value.

[0032] The strains and culture medium used in the following examples are as follows:

[0033] The pathogenic fungus Cercospora zeae-maydis Tehon & Daniels was provided by the Institute of Plant Protection, Liaoning Academy of Agricultural Sciences.

[0034] Burkholderia lata was purchased from China Industrial Culture Collection Center (CICC) with the strain number CICC 24958.

[0035] Paecilomyces variotii was purchased from China Industrial Microbiological Culture Collection Center with the strain number CICC 4025.

[0036] Tryptone soy liquid medium: tryptone 15.0 g / L, soy peptone 5.0 g / L and NaCl 5.0 g / L, pH 7.3.

[0037] Tryptone soy agar solid culture medium: tryptone 15.0 g / L, soy peptone 5.0 g / L, NaCl 5.0 g / L and agar 15.0 g / L, pH 7.3.

[0038] Czapek liquid medium: sucrose 30.0 g, NaNO3 3.0 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4·4H2O 0.01 g / L and K2HPO4 1.0 g / L, pH 6.0-6.5.

[0039] Czapek agar solid culture medium: sucrose 30.0 g, NaNO3 3.0 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4·4H2O 0.01 g / L, K2HPO4 1.0 g / L and agar 15.0 g / L, pH 6.0-6.5.

[0040] The fermentation culture method of Burkholderia broadleaf is as follows:

[0041] (1) inoculating Burkholderia latum into a tryptone soy liquid culture medium, and culturing with shaking at 30° C. and 160 rpm for 24 h to obtain a Burkholderia latum bacterial liquid;

[0042] (2) streaking the Burkholderia broadleaf liquid obtained in step (1) onto a tryptone soy agar solid medium plate, and culturing in an incubator at 30° C. for 48 h;

[0043] (3) Pick a single colony from the plate in step (2) and inoculate it into a shake flask loaded with tryptone soy liquid culture medium, and culture it at 30° C. and 180 r / min for 48 h to obtain a Burkholderia broaden fermentation broth.

[0044] The fermentation culture method of Paecilomyces variotii is as follows:

[0045] (1) picking up a block of Paecilomyces variotii and inoculating it into a Czapek liquid culture medium, and shaking and culturing it at 28°C and 160 rpm for 72 h to obtain a culture medium;

[0046] (2) diluting the culture solution obtained in step (1) and applying it onto a solid culture medium of Czapek agar, and culturing it in an incubator at 28° C. for 5 days;

[0047] (3) Pick a single colony from the plate in step (2) and inoculate it into a shake flask filled with Czapek liquid culture medium, and culture it at 28° C. and 200 r / min for 72 h to obtain a fermentation broth of Paecilomyces variotii.

[0048] Example 1 Detection of biocontrol bacteria affinity

[0049] Use a 5mm diameter cork puncher to punch out mycelial blocks from the edge of a 2-day-old Paecilomyces variotii colony and transfer them to the center of a PDA plate. Inoculate activated Burkholderia latum at a distance of 20mm from the center. Use Paecilomyces variotii without inoculation of Burkholderia latum as a control, and set up 3 replicates for each strain. Co-culture at 28°C for 5 days to observe whether inhibition zones are produced between the strains.

[0050] The results showed that there was no obvious inhibition zone between Burkholderia broadleaf and Paecilomyces variotii, indicating that the two had good affinity.

[0051] Example 2

[0052] The mycelial growth rate method was used to determine the half effective concentration (EC) of Burkholderia broadleaf and Paecilomyces variotii to inhibit Cercosporangiate of mays. 50 ), as follows:

[0053] After the strain to be tested was fermented and cultured, the bacterial cells (Paecilomyces variotii refers to spores) were isolated and resuspended in sterile saline to a concentration of 10 and 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU / mL of bacterial suspension, and then add 1 mL of bacterial suspension to each 20 mL of PDA solid medium to prepare PDA plates containing different concentrations of bacterial suspension. The control treatment is to add 1 mL of sterile saline to the PDA plate, and each treatment is set up for 3 replicates.

[0054] Take the corn cercospora pathogen that has been cultured for 40 days, and use a 5mm diameter puncher to punch the bacterial cake at the edge of the colony. Inoculate the bacterial cake in the center of the PDA plate containing different concentrations of bacterial suspension, place it in a 28℃ constant temperature incubator and culture it upside down for 40 days. Use the cross method to measure the growth diameter of each treatment colony. According to the measurement results, calculate the inhibition rate of each treatment on the pathogen, and calculate the EC 50 value.

[0055] Inhibition rate = [(colony diameter of control group - diameter of bacterial cake) - (colony diameter of treatment group - diameter of bacterial cake)] / (colony diameter of control group - diameter of bacterial cake) × 100%.

[0056] The results showed that Burkholderia latum and Paecilomyces variotii had a certain inhibitory effect on Cercospore of Zea mays. 50 4.8×10 7 CFU / mL, EC of Paecilomyces variotii 50 2.5×10 6 CFU / mL.

[0057] Example 3

[0058] EC selection for Burkholderia latum and Paecilomyces variotii 50Concentration, prepare PDA plates with different bacterial suspension ratios. Mix according to volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10, and prepare a total of 10 composite culture media. The control treatment is to add 1 mL of sterile saline to the PDA plate, and each group of treatments is repeated 3 times. The inoculation method of Cercospore maydis and the subsequent plate culture method are the same as in Example 2.

[0059] After inverted culture in a 28°C constant temperature incubator for 40 days, the growth diameter of the colonies in each treatment was measured using the cross method. Based on the measurement results, the actual and theoretical inhibition rates of each treatment were calculated.

[0060] Calculation method of joint toxicity enhancement:

[0061] The Horsfall method was used to calculate the combined virulence enhancement, and the synergistic effect of the mixed bacteria in different proportions was determined according to the enhancement ratio (IR): synergistic effect: IR value > 1; additive effect: IR value = 1; antagonistic effect: IR value < 1.

[0062] IR = actual inhibition rate / theoretical inhibition rate;

[0063] Theoretical inhibition rate = strain AEC 50 The actual antibacterial rate when the concentration is used alone × the percentage of strain A in the mixture + the EC of strain B 50 The actual inhibition rate when the concentration is used alone × the percentage of strain B in the mixture.

[0064] The results of the combined toxicity test of each treatment group against Cercospore of Zea mays are shown in Table 1. The results showed that when the volume ratio of Burkholderia broadleaf and Paecilomyces variotii suspension (i.e., the ratio of the number of viable bacteria) was in the range of 7: (3-28), different degrees of synergistic enhancement were exhibited, among which the synergistic enhancement effect of the treatment group with a ratio of 6:4 was the best, with a synergistic ratio of 1.35 and an actual inhibition rate of 68.27%, followed by the treatment group with a ratio of 5:5, with a synergistic ratio of 1.23 and an actual inhibition rate of 62.39%. The two treatment groups with better synergistic effects were selected for potted plant control experiments in the later stage.

[0065] Table 1 The results of the combined toxicity test of each treatment group against Cercosporium maydis

[0066]

[0067]

[0068] Example 4

[0069] Healthy potted corn plants (plant height 20-25 cm) were selected and randomly divided into 6 groups. The leaves were sprayed as shown in Table 2 and kept moisturized for 12 hours. After 30 days, the disease situation of each group was observed. The disease index of each group of potted corn was counted and the prevention effect was calculated. The formula is:

[0070] Preventive effect (%) = (disease index of negative control group - disease index of treatment group) / disease index of negative control group × 100%.

[0071] Table 2 Experimental groups

[0072]

[0073] The statistical results of the control effect of each treatment group are shown in Table 3. The results show that the combination of Burkholderia broadleaf and Paecilomyces variotii can effectively control corn gray spot disease, and the control effect can reach more than 70%.

[0074] Table 3 Statistical results of the control effect of each treatment group

[0075]

[0076]

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A microbial combination for preventing and controlling corn gray spot disease, characterized in that: These include Burkholderia lata and Paecilomyces variotii.

2. The microbial combination according to claim 1, characterized in that The ratio of the number of live bacteria of the broad Burkholderia to that of the Paecilomyces variotii is 7:(3-28).

3. The microbial combination according to claim 2, characterized in that: The ratio of the number of live bacteria of the broad Burkholderia to that of the Paecilomyces variotii is 3:(2-3).

4. The microbial combination according to claim 3, characterized in that The ratio of the number of live bacteria of the Burkholderia broadleaf and the Paecilomyces variotii is 3:

2.

5. Use of the microbial combination as described in any one of claims 1 to 4 in the preparation of a microbial composite agent for preventing and controlling corn gray spot disease.

6. Use of the microbial combination according to any one of claims 1 to 4 in preventing and controlling corn gray spot disease.

7. A microbial composite agent for preventing and treating corn gray spot disease, characterized in that: The active ingredient comprises the microbial combination according to any one of claims 1 to 4.

8. A method for preparing the microbial composite agent according to claim 7, characterized in that: The method comprises the steps of uniformly mixing a suspension of Burkholderia broadleaf and a suspension of Paecilomyces variotii to obtain the microbial composite agent; The Burkholderia broada bacterial suspension is obtained by fermenting and culturing Burkholderia broada, isolating bacterial bodies, and then resuspending them in sterile physiological saline; The Paecilomyces variotii suspension is obtained by fermenting and culturing Paecilomyces variotii, separating spores, and then resuspending them in sterile physiological saline.

9. Use of the microbial composite agent as claimed in claim 7 in preventing and controlling corn gray spot disease.

10. A method for preventing and controlling corn gray spot disease, characterized in that: The method comprises the step of spraying the microbial composite agent described in claim 7 on the leaves of plants.

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