CN112877221A - Pythium oligandrum graphene material composite biocontrol preparation and preparation method thereof - Google Patents
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Abstract
The invention discloses a pythium oligandrum graphene material composite biocontrol preparation and a preparation method thereof. The biological control agent of the pythium oligandrum composite graphene material provided by the invention has a much higher inhibition rate on alternaria solani, fusarium oxysporum, cucumber specialty type and fusarium solani than the common pythium oligandrum inoculant. The preparation method of the pythium oligandrum graphene-like material composite biocontrol preparation provided by the invention has the advantages of simple preparation process, low requirement on production equipment, low cost and suitability for large-scale production, and the composite biocontrol preparation has a good biocontrol effect and good popularization and application prospects.
Description
Technical Field
The invention belongs to the field of biological control, and particularly relates to a pythium oligandrum graphene material composite biocontrol preparation and a preparation method thereof.
Background
Crops are often threatened by various pathogenic bacteria when planted in large areas, so that the yield is reduced, for example, harmful pathogenic bacteria such as pythium and phytophthora oomycetes. Generally, the propagation path of pathogenic bacteria mainly comprises wind, diseased seedlings, soil, irrigation or rainwater and the like, when the pathogenic bacteria contact crops along with water flow, the pathogenic bacteria can parasitize on the surface to form resting spores which further infect the crops and enter host bodies, and the sapling pythium root rot can be caused after the crops are infected by the pathogenic bacteria in the seedling stage. Under proper environmental conditions, the outbreak can cause the seedling to gradually wither in a short period, and the seedling can be dead in severe cases.
At present, the control method for the crop epidemic disease mainly comprises the following two aspects: (1) and (3) cultivation control measures: selecting disease-resistant varieties, and removing accumulated water in rainy season in time; (2) and (3) medicine prevention and treatment: the common pesticides include DuPonkelu and DuPont Xinwangsheng. However, the first control method has the disadvantages of insignificant effect and ecological potential safety hazard, and the disadvantages of pesticide residue, soil and water source pollution and the like in the drug control method can not meet the requirements of human beings on healthy diet and green environment. Biological control has recently received great attention as an effective method having the obvious advantages of environmental protection, no residue and the like to replace the traditional control.
Pythium oligandrum (B)Pythium oligandrum) Is a parasitic fungus which is harmless to human and livestock and has strong aggressivity in the nature, can be colonized around roots of various crops,not only does not produce pathogenic effect on crops, but also can inhibit or kill other pathogenic fungi and soil-borne pathogenic bacteria, induce plants to produce defense reaction and reduce the invasion of pathogenic bacteria; meanwhile, secretion and various enzymes generated by pythium oligandrum are good plant growth promoting active agents, and can promote root system development of crops and improve nutrient absorption. The research results show that the Pythium oligandrum antibacterial agent has strong parasitism on more than 50 fungi and oomycetes, can effectively protect plants from diseases, and has practical application of biological products with antifungal effect of Pythium oligandrum microorganisms, for example, the 'multi-benefit vitamin' is a microbial preparation which is developed by Image Trade International Inc. and Czech biological preparation Co, utilizes Pythium oligandrum active spores to carry out broad-spectrum sterilization, is known as 'New century agriculture light' in Europe, specifies recommended products for European Union, and is widely applied to fields, economic crops, fruit trees, vegetables, golf courses and garden flowers. How to further improve the biocontrol effect of pythium oligandrum is a technical problem to be solved urgently.
Disclosure of Invention
The invention provides a pythium oligandrum graphene material composite biocontrol preparation and a preparation method thereof, aiming at the defects of the prior art.
The invention aims to provide a preparation method of a pythium oligandrum graphene material composite biocontrol preparation, which specifically comprises the following steps:
1. adding graphene materials into the sterilized fungus liquid culture medium, wherein the addition amount is 5-100mg/L, and uniformly stirring;
2. inoculating a pythium oligandrum strain into the culture medium prepared in the step 1, culturing at 28 ℃ at 150r/min until a large amount of black grey cluster flocculent pythium oligandrum mycelia grow out, and stopping culturing when the culture medium is clear and bright;
3. taking out the mycelium, and washing away the culture medium on the mycelium by using deionized water to obtain the pythium oligandrum graphene material composite biocontrol preparation.
Preferably, the graphene-based material is graphene, graphene oxide and three-dimensional network activated carbon.
Preferably, the addition amount of the graphene-based material is 5 mg/L.
Preferably, the fungus is pythium oligandrum, and other biocontrol fungi such as trichoderma, metarhizium anisopliae and the like can be used for replacing the fungus.
The invention also aims to provide the pythium oligandrum graphene-like material composite biocontrol preparation prepared by the method.
The third purpose of the invention is to provide the application of the pythium oligandrum graphene material composite biocontrol preparation, and the application is strong inhibition on pathogenic fungi such as alternaria solani, fusarium oxysporum cucumber professional type and fusarium solani.
According to the invention, the graphene-type carbon material is introduced into the liquid culture medium to be cultured to prepare the pythium oligandrum graphene-type material composite biocontrol preparation, so that the biocontrol performance of pythium oligandrum can be effectively improved. Under the same application amount, the inhibition rate of the pythium oligandrum composite graphene material biocontrol agent on alternaria solani, fusarium oxysporum, cucumber specialty type and fusarium solani is greatly higher than that of a common pythium oligandrum agent. The preparation method of the pythium oligandrum graphene-like material composite biocontrol preparation provided by the invention has the advantages of simple preparation process, low requirement on production equipment, low cost and suitability for large-scale production, and the composite biocontrol preparation has a good biocontrol effect and good popularization and application prospects.
Drawings
FIG. 1 is a photograph of mycelia when the culture was completed in step 2 described in example 2.
FIGS. 2 and 3 are photographs showing the inhibition of the Pythium oligandrum graphene-based composite biocontrol agents against Alternaria solani, Fusarium oxysporum Cucuminum professional type and Fusarium solani in example 5.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the present application.
In the following examples, the Pythium oligandrum strain was deposited at the institute of biological research, national academy of sciences of Gansu province, and other reagents were commercially available.
The media formulations used in the following examples were as follows:
PDA liquid culture medium: weighing 200g of potato, cleaning, peeling, cutting into pieces, adding 1000ml of water, boiling for half an hour, filtering with gauze, adding 20g of glucose, and sterilizing with high-pressure steam at 121 deg.C for 20 min;
v8 liquid medium: 200 mL of V8 juice, CaCO3 3 g, adding distilled water to 1000mL, and autoclaving at 121 ℃ for 20 min.
Example 1 Effect of different concentrations of graphene-like materials on growth of Pythium oligandrum
PDA liquid medium was dispensed into 100mL Erlenmeyer flasks, 40mL each. Experimental groups: adding three carbon materials, namely graphene, graphene oxide and three-dimensional network activated carbon, into a liquid culture medium according to the ratio of 5mg/L, 50 mg/L and 100mg/L respectively, and shaking up; the control group was not additionally added, but only PDA liquid medium. Each experimental group was set to 3 replicates.
And (3) inoculating the same amount of pythium oligandrum inoculant into the conical flask, performing shake culture in a constant-temperature shaking table at 28 ℃ for 150r/min until a large amount of flocculent mycelia grow out of each flask after 5-7 days, and stopping culture when the culture medium of the experimental group becomes clear and transparent.
Taking out the mycelia in each conical flask, washing with deionized water, carrying out freeze-drying treatment, and weighing, wherein the results show that no significant difference exists in the growth amount of the mycelia among experimental groups, experimental groups and control groups, and the result proves that the graphene material has no inhibition or promotion effect on the growth of pythium oligandrum.
Example 2 preparation of Pythium oligandrum graphene composite biocontrol agent
1. PDA liquid medium was dispensed into 100mL Erlenmeyer flasks at 40mL per flask. Adding graphene into the conical flask according to the concentration of 5mg/L, 50 mg/L and 100mg/L, and uniformly stirring;
2. inoculating pythium oligandrum to the liquid culture medium prepared in the step 1, culturing at 28 ℃ at 150r/min until a large amount of black grey cluster flocculent pythium oligandrum mycelia grow out after 5-7 days, and stopping culturing when the culture medium is clear and bright, as shown in figure 1;
3. taking out the mycelium, and washing away the culture medium on the mycelium by using deionized water to obtain the pythium oligandrum graphene composite biocontrol agent.
Example 3 preparation of Pythium oligandrum graphene oxide composite biocontrol agent
1. PDA liquid medium was dispensed into 100mL Erlenmeyer flasks at 40mL per flask. Adding graphene oxide into the conical flask according to the concentration of 5mg/L, 50 mg/L and 100mg/L, and uniformly stirring;
2. inoculating pythium oligandrum to the liquid culture medium prepared in the step 1, culturing at 28 ℃ at 150r/min until a large amount of black grey cluster flocculent pythium oligandrum mycelia grow out after 5-7 days, and stopping culturing when the culture medium is clear and bright;
3. taking out the mycelium, and washing away the culture medium on the mycelium by using deionized water to obtain the pythium oligandrum graphene oxide composite biocontrol agent.
EXAMPLE 4 preparation of Pythium oligandrum three-dimensional network active carbon composite biocontrol agent
1. PDA liquid medium was dispensed into 100mL Erlenmeyer flasks at 40mL per flask. Adding three-dimensional network activated carbon into the conical flask according to the proportion of 5mg/L, 50 mg/L and 100mg/L, and uniformly stirring;
2. inoculating pythium oligandrum to the liquid culture medium prepared in the step 1, culturing at 28 ℃ at 150r/min until a large amount of black grey cluster flocculent pythium oligandrum mycelia grow out after 5-7 days, and stopping culturing when the culture medium is clear and bright;
3. taking out the mycelium, and washing away the culture medium on the mycelium by using deionized water to obtain the pythium oligandrum three-dimensional network active carbon composite biocontrol agent.
Example 5 test of the inhibition efficiency of Pythium oligandrum graphene-based composite biocontrol agents against Alternaria solani, Fusarium oxysporum Cucumis professional type and Fusarium solani
The method comprises the steps of respectively inoculating fungus cakes of a pythium oligandrum graphene-based material composite biocontrol preparation (prepared in examples 2, 3 and 4) with the diameter of 7mm and phytopathogen alternaria solani, fusarium oxysporum cucumber professional type and fusarium solani fungus cakes with the same diameter on a PDA (personal digital assistant) plate with the diameter of 9 cm, enabling the distances between the composite biocontrol preparation and the phytopathogen fungus cakes to be equal, inoculating the phytopathogen fungus cakes with the same diameter as a reference, repeating the treatment for 3 times each time, culturing in a constant temperature box at 28 ℃, measuring the diameters D2 and D3 of the growth of the pythium oligandrum and the colonies of the phytopathogen and the diameter D1 of the reference pathogen, calculating the bacteriostasis rate according to the following formula, and calculating the average bacteriostasis rate of each group. The results are shown in Table 1
Bacteriostatic rate (%) = (D1-D3)/D1 × 100%.
TABLE 1 inhibition rate of Pythium oligandrum graphene-based composite biocontrol agent against Alternaria solani, Fusarium oxysporum, cucumber specialty type and Fusarium solani
The results of the confrontation culture of the invention show that the growth radius of the pathogenic bacteria colony in the confrontation culture medium is obviously lower than that of the contrast, the pythium oligandrum has an inhibiting effect on 3 plant pathogenic bacteria alternaria solani, fusarium oxysporum cucumber professional type and fusarium solani, fig. 2 and fig. 3 show that after the pythium oligandrum is stably combined with the graphene material, the antagonistic inhibiting effect on the pathogenic bacteria alternaria solani, fusarium oxysporum cucumber professional type and fusarium solani can be enhanced, and the bacteriostasis rate of the pythium oligandrum graphene material composite biocontrol agent is obviously higher than that of the pythium oligandrum protomycete. However, with the increase of the content of the graphene materials, the bacteriostatic efficiency of the composite biocontrol preparation does not show positive correlation increase, and three concentration levels of the carbon material have no significant bacteriostatic difference, so that the action mechanism is yet to be clarified, and the lowest addition amount, namely 5mg/L is taken as the optimal addition amount by considering the cost factor.
Claims (6)
1. A preparation method of a pythium oligandrum graphene material composite biocontrol preparation is characterized by comprising the following steps:
(1) adding graphene materials into the sterilized fungus liquid culture medium, wherein the addition amount is 5-100mg/L, and uniformly stirring;
(2) inoculating a pythium oligandrum strain into the culture medium prepared in the step (1), culturing at 28 ℃ at 150r/min until a large amount of black grey colony flocculent pythium oligandrum mycelia grow out, and stopping culturing when the culture medium is clear and bright;
(3) taking out the mycelium, and washing away the culture medium on the mycelium by using deionized water to obtain the pythium oligandrum graphene material composite biocontrol preparation.
2. The preparation method of the pythium oligandrum graphene-like material composite biocontrol preparation according to claim 1, characterized in that the addition amount of the graphene-like material in the step (1) is 5 mg/L.
3. A pythium oligandrum graphene-like material composite biocontrol preparation, characterized in that the preparation is prepared by the method of claim 1 or 2.
4. An application of a pythium oligandrum graphene material composite biocontrol preparation is characterized in that the application is that the pythium oligandrum graphene material composite biocontrol preparation is used for inhibiting alternaria solani.
5. An application of a pythium oligandrum graphene material composite biocontrol preparation is characterized in that the application is that the pythium oligandrum graphene material composite biocontrol preparation is used for inhibiting Fusarium oxysporum cucumber professional type.
6. An application of a pythium oligandrum graphene material composite biocontrol preparation is characterized in that the application is that the pythium oligandrum graphene material composite biocontrol preparation is used for inhibiting fusarium solani.
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| CN117089462A (en) * | 2023-02-28 | 2023-11-21 | 陕西师范大学 | Pythium oligandrum strain EMF915 and application thereof |
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| CN112877221B (en) | 2023-02-28 |
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