A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum and preparation method thereof

Abstract

The present invention belongs to the field of biological control technology, and specifically relates to a pesticide composition containing bacterial wilt phage and Trichoderma harzianum and a preparation method thereof. Specifically, the pesticide composition containing bacterial wilt phage and Trichoderma harzianum comprises 5×10 8 ‑1×10 10 pfu / g of bacterial wilt phage and 5×10 8 ‑1×10 10 The present invention compounds the bacterial wilt pathogen phage with Trichoderma harzianum, and finds that the bacterial wilt pathogen can synergistically control the population of the bacterial wilt pathogen, thereby reducing the occurrence of bacterial wilt.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and specifically relates to a pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum and a preparation method thereof. Background Art

[0002] Bacterial wilt is a systemic disease caused by infection by Ralstonia sola anacearum (referred to as bacterial wilt pathogen). It is a typical vascular disease with the most notable symptom of wilt. In severe cases, it can cause the death of the entire plant, resulting in total crop failure and significant economic losses to tobacco production. The occurrence of tobacco bacterial wilt is the result of the combined effects of tobacco, pathogens and the environment. Ambient temperature, humidity, soil pH and the number of bacterial wilt pathogens in the soil are closely related to the occurrence of tobacco bacterial wilt.

[0003] The traditional methods for controlling the number of bacterial wilt pathogens mainly include: timely cleaning of tobacco stem and leaf residues and other host plants of bacterial wilt in tobacco fields, using chemical agents to control the number of bacterial wilt pathogens, adding antagonistic bacteria to bacterial wilt pathogens through the use of biological organic fertilizers, soil disinfection, and reasonable tillage and cultivation measures to control the number of bacterial wilt pathogens in tobacco fields.

[0004] With the widespread use of antibiotics and the emergence of drug resistance or resistance, green control and biological control have regained attention as alternatives to traditional drug therapies. In recent years, the use of bacteriophages to control plant bacterial diseases and the use of antagonistic microorganisms of bacterial wilt for biological control have attracted attention and discussion. Summary of the invention

[0005] The present invention aims to provide a pesticide composition capable of controlling the number of bacterial wilt pathogens and resisting bacterial wilt, and a preparation method and application thereof.

[0006] Specifically, in one aspect, the present invention provides a pesticide composition containing a bacteriophage of bacterial wilt and Trichoderma harzianum, which comprises 5×10 8 pfu / g-1×10 10 pfu / g of bacterial wilt phage and 5×10 8 cfu / g-1×10 10 cfu / g of Trichoderma harzianum.

[0007] Furthermore, the mass ratio of the bacterial wilt phage to the Trichoderma harzianum in the pesticide composition is 2:3-3:2.

[0008] As used herein, the species of Ralstonia solanacearum phage and Trichoderma harzianum are not limited, and any commercially available Ralstonia solanacearum phage and Trichoderma harzianum can be used.

[0009] Furthermore, the pesticide composition also contains mannitol and trehalose as adjuvants.

[0010] Furthermore, the mass ratio of mannitol to trehalose is 1:0.8-1.2.

[0011] Furthermore, the ratio of the total mass of the mannitol and trehalose to the total mass of the bacterial wilt phage and the Trichoderma harzianum is 0.05-0.1:1.

[0012] Furthermore, the pesticide composition also contains other pesticide ingredients.

[0013] Furthermore, the other pesticide ingredients include one or more of sodium sulfamethoxam, fludioxonil, trichloroacetonitrile uric acid, methyl thiophanate, metalaxyl-M, cypermethrin acetate aqueous solution, and meconium chloride.

[0014] Furthermore, the pesticide composition is in the form of a water-soluble powder.

[0015] In another aspect, the present invention provides a method for preparing a pesticide composition as described herein, comprising:

[0016] (1) The phages of Ralstonia solanacearum were activated, propagated and counted, and then freeze-dried to obtain 5×10 8 -1×10 10 pfu / g phage powder of bacterial wilt phage;

[0017] (2) preparing spore liquid from Trichoderma harzianum, adding solid fermentation medium for solid fermentation culture, and then freeze-drying to obtain Trichoderma harzianum powder, counting the powder and controlling the content of Trichoderma harzianum powder to 5×10 8 -1×10 10 cfu / g of Trichoderma harzianum;

[0018] (3) uniformly mixing the bacteriophage powder with Trichoderma harzianum powder and optionally adjuvants or other pesticide components to obtain a pesticide composition.

[0019] In yet another aspect, the present invention provides use of the pesticide composition described herein in combating bacterial wilt.

[0020] Beneficial effects of the present invention

[0021] The present invention compounds bacterial wilt phage with Trichoderma harzianum, and finds that it can synergistically control the population of bacterial wilt, thereby reducing the occurrence of bacterial wilt. Furthermore, the present invention also adds an adjuvant consisting of mannitol and trehalose to the pesticide composition containing bacterial wilt phage and Trichoderma harzianum, and finds that it can stabilize the bacterial wilt phage and Trichoderma harzianum in the pesticide composition, and improve the effect of killing bacterial wilt. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0023] The tobacco bacterial wilt pathogen used in this embodiment was purchased from China Center for Type Culture Collection with a number of CCTCC NO: M 2020559; the bacterial wilt pathogen phage was purchased from China Center for Type Culture Collection with a number of CCTCC NO: M2020560; and the Trichoderma harzianum was purchased from China Center for Type Culture Collection with a number of CCTCC NO: HF 2008541.

[0024] The culture medium formula used in this embodiment is as follows:

[0025] NA liquid culture medium: 10 g glucose, 5 g peptone, 3 g beef extract, 0.5 g yeast powder, 1000 mL deionized water, adjust pH to 7.2-7.4, and sterilize at 115°C for 30 min.

[0026] M-SMSA solid medium: Add 50 mg of TTC, 5 mg of crystal violet, 50 mg of polymyxin, 25 mg of bacitracin, 5 mg of chloramphenicol, 50 mg of cycloheximide, and 5 mg of penicillin to 1000 mL of NA solid medium.

[0027] PDA liquid medium: 1L potato boiling liquid, 20g glucose, KH 2 PO 4 3g, MgSO 4 7H 2 O1.5g, vitamin B18mg, agar 20.0g, pH adjusted to 6.0±0.2, sterilized at 121℃ for 15min; Potato cooking liquid: weigh 200g peeled potato pieces, boil in boiling water for 30min, collect the filtrate and make up to 1L.

[0028] Solid fermentation medium: 30g corn flour, 90g bran powder, add 55ml water, add 2% urea, 1.5% magnesium sulfate, 0.05% potassium dihydrogen phosphate, in a 250ml Erlenmeyer flask, 121℃, sterilize for 30min.

[0029] SMSA solid medium: 10 g bacterial peptone, 5 mL glycerol, 1.0 g hydrolyzed casamino acids / casamino acids, 15 g bacterial agar powder, 1000 mL distilled water, pH 7.0. After sterilization, when cooled to 50°C, add 2.5 mL 1.0% polymyxin, 125 μL 1.0% crystal violet, 1.25 mL 1.0% triphenyltetrazolium chloride, 625 μL 1.0% bacitracin, 125 μL 0.1% penicillin, and 125 μL 1.0% chloramphenicol.

[0030] Example 1

[0031] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum is prepared as follows:

[0032] (1) Preparation of bacterial wilt pathogen phage powder: The freeze-dried powder of tobacco bacterial wilt pathogen was resuspended in NA liquid medium, streaked on M-SMSA solid medium for activation, and cultured in a constant temperature incubator at 30°C for 48 h. A single colony on the plate was picked and transferred to NA liquid medium, and cultured in a shaker at 30°C and 170 rpm for 24 h to obtain a bacterial solution of tobacco bacterial wilt pathogen in the logarithmic growth phase;

[0033] The lyophilized powder of bacterial wilt phage was resuspended in NA liquid culture medium, added to the above-mentioned tobacco bacterial wilt phage solution in the logarithmic growth phase, cultured in a shaker at 30°C and 170 rpm for 24 h, centrifuged at 13000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a phage solution. The phage content of bacterial wilt in the phage solution was counted by the double-layer agar counting method, and finally lyophilized to obtain a 1×10 10 pfu / g phage powder of bacterial wilt pathogen, stored at 4℃.

[0034] (2) Preparation of Trichoderma harzianum powder: The Trichoderma harzianum strain was inoculated into PDA liquid culture medium and cultured in a constant temperature oscillator at 28° C. and 180 rpm for 78 h to obtain seed liquid;

[0035] The seed liquid was inoculated into a qu dish containing 2 kg of solid fermentation medium at a mass ratio of 5%, and solid fermentation culture was carried out at 28°C for 7 days to obtain a solid Trichoderma harzianum agent, which was then freeze-dried to obtain Trichoderma harzianum powder, which was counted and the content of Trichoderma harzianum powder was controlled to be 1×10 10 cfu / g of Trichoderma harzianum.

[0036] (3) The bacteriophage powder and the Trichoderma harzianum powder are uniformly mixed in a ratio of 3:2 to obtain a pesticide composition.

[0037] Example 2

[0038] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are uniformly mixed in a ratio of 2:2.

[0039] Example 3

[0040] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are uniformly mixed in a ratio of 2:3.

[0041] Example 4

[0042] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are uniformly mixed in a ratio of 2:5.

[0043] Example 5

[0044] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are mixed evenly with mannitol at a ratio of 1:0.1.

[0045] Example 6

[0046] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are mixed evenly with trehalose in a ratio of 1:0.1.

[0047] Example 7

[0048] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are mixed evenly with glucose in a ratio of 1:0.1.

[0049] Example 8

[0050] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are also mixed evenly with a mixture consisting of equal amounts of mannitol and trehalose in a ratio of 1:0.1.

[0051] Example 9

[0052] A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, the preparation method of which is the same as that of Example 1, except that in step (3), the bacteriophage powder and Trichoderma harzianum powder are also mixed evenly with a mixture consisting of equal amounts of glucose and trehalose in a ratio of 1:0.1.

[0053] Example 10

[0054] A pesticide composition containing bacterial wilt bacteriophage is prepared as follows:

[0055] The freeze-dried powder of tobacco bacterial wilt pathogen was resuspended in NA liquid medium, streaked on M-SMSA solid medium for activation, cultured in a constant temperature incubator at 30°C for 48 hours, and a single colony on the plate was transferred to NA liquid medium, and cultured in a shaker at 30°C and 170 rpm for 24 hours to obtain a bacterial solution of tobacco bacterial wilt pathogen in the logarithmic growth phase;

[0056] The lyophilized powder of bacterial wilt phage was resuspended in NA liquid culture medium, added to the above-mentioned tobacco bacterial wilt phage solution in the logarithmic growth phase, cultured in a shaker at 30°C and 170 rpm for 24 h, centrifuged at 13000 rpm for 3 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain a phage solution. The phage content of bacterial wilt in the phage solution was counted by the double-layer agar counting method, and finally lyophilized to obtain a 1×10 10 pfu / g phage powder of bacterial wilt pathogenic bacteria phage is a pesticide composition containing bacterial wilt pathogenic bacteria phage.

[0057] Embodiment 11

[0058] A pesticide composition containing Trichoderma harzianum is prepared as follows:

[0059] The Trichoderma harzianum strain was inoculated into a PDA liquid culture medium, and cultured in a constant temperature oscillator at 28°C and 180 rpm for 78 h to obtain a seed solution;

[0060] The seed liquid was inoculated into a qu dish containing 2 kg of solid fermentation medium at a mass ratio of 5%, and solid fermentation culture was carried out at 28°C for 7 days to obtain a solid Trichoderma harzianum agent, which was then freeze-dried to obtain Trichoderma harzianum powder, which was counted and the content of Trichoderma harzianum powder was controlled to be 1×10 10 cfu / g of Trichoderma harzianum, that is, a pesticide composition containing Trichoderma harzianum.

[0061] Experimental Example 1:

[0062] The compositions of Examples 1 and 5-9 were redissolved in water (1:1 material ratio) and placed at room temperature for 20 days. The amounts of bacterial wilt phage and Trichoderma harzianum in each composition were then measured. The results are shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] From the results in Table 1, it can be found that when stored at room temperature after reconstitution, the addition of adjuvants mannitol, trehalose or glucose can increase the titer of bacterial wilt phage and Trichoderma harzianum after storage, but the combined use of mannitol and trehalose can significantly increase the survival rate of bacterial wilt phage and Trichoderma harzianum, while the combined use of glucose and trehalose has no such effect. Such an effect can increase the shelf life of the pesticide composition containing bacterial wilt phage and Trichoderma harzianum and can also increase its effective period of action in actual application, and has good application prospects.

[0067] Experimental Example 2

[0068] The compositions of Examples 1-11 were respectively reconstituted with water (1:1 material ratio) to form treatment group solutions, and the blank group, control group and treatment group solutions were directly dropped into the NA liquid culture medium containing tobacco bacterial wilt pathogen.

[0069] For the blank group, 40 μl of distilled water was added to 200 ml of NA liquid medium;

[0070] In the control group, 200 ml of NA liquid medium was inoculated with 20 μl of 1×10 10 CFU / ml NA liquid medium of tobacco solanacearum, and 20 μl of distilled water;

[0071] Treatment group, 200ml NA liquid medium was inoculated with 20μl containing 1×10 10 CFU / ml NA liquid culture medium of tobacco solanacearum, and 20 μl aqueous solution containing 0.02 g of the composition prepared in Example 1-11 were added respectively;

[0072] Each group was cultured in triplicate at 28°C with shaking, and samples were taken at 12, 24, 48, and 72 hours of culture. The population of tobacco bacterial wilt pathogen RsZP was detected using semi-selective medium SMSA culture combined with plate counting method. The results are shown in Table 1.

[0073] Table 1

[0074]

[0075] From the results in Table 1, it can be seen that the pesticide composition containing bacterial wilt phage and Trichoderma harzianum of the present invention has significantly stronger bacterial wilt inhibition ability than the pesticide composition containing bacterial wilt phage and Trichoderma harzianum alone, and the effect is better when the mass ratio of bacterial wilt phage to Trichoderma harzianum is 2:3-3:2. After adding the adjuvants mannitol and trehalose, the inhibitory ability of the pesticide composition to bacterial wilt is further enhanced.

[0076] It should be noted that the preferred embodiments of the present invention are given in the specification of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the present invention specification; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A pesticide composition containing bacterial wilt bacteriophage and Trichoderma harzianum, characterized in that: Contains 5×10 8 -1×10 10 pfu / g of bacterial wilt phage and 5×10 8 -1×10 10 cfu / g of Trichoderma harzianum; The mass ratio of the bacterial wilt phage to Trichoderma harzianum in the pesticide composition is 2:3-3:2; Among them, the bacteriophage number of the bacterial wilt pathogen is CCTCC NO: M 2020560; the number of Trichoderma harzianum is CCTCC NO: HF2008541.

2. The pesticide composition according to claim 1, characterized in that Mannitol and trehalose are also included as adjuvants.

3. The pesticide composition according to claim 2, characterized in that The mass ratio of mannitol to trehalose is 1:0.8-1.

2.

4. The pesticide composition according to claim 3, characterized in that The ratio of the total mass of the mannitol and trehalose to the total mass of the bacterial wilt phage and the Trichoderma harzianum is 0.05-0.1:

1.

5. A method for preparing a pesticide composition according to any one of claims 1 to 4, characterized in that: include: (1) The phages of Ralstonia solanacearum were activated, propagated and counted, and then freeze-dried to obtain 5×10 8 -1×10 10 pfu / g phage powder of bacterial wilt phage; (2) A spore liquid is prepared from Trichoderma harzianum, and then solid fermentation medium is added for solid fermentation culture, and then freeze-dried to obtain Trichoderma harzianum powder, which is counted and the content of the Trichoderma harzianum powder is controlled to be 5×10 8 -1×10 10 cfu / g of Trichoderma harzianum; (3) The bacteriophage powder is uniformly mixed with the Trichoderma harzianum powder and optionally an adjuvant to obtain a pesticide composition.

6. Use of the pesticide composition according to any one of claims 1 to 4 in combating bacterial wilt.

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