A ralstonia solanacearum phage emulsion formulation and a preparation method thereof

Abstract

The present application relates to the technical field of fungicides, and proposes a Ralstonia solanacearum phage emulsion preparation and a preparation method thereof.The Ralstonia solanacearum phage emulsion preparation comprises the following components: Ralstonia solanacearum phage RSP46 suspension, turpentine oil and emulsifier BY-130;the preservation number of the Ralstonia solanacearum phage RSP46 is CCTCC NO: M20231467.The present application uses Ralstonia solanacearum phage RSP46 as a biological control component, cooperates with turpentine oil and emulsifier BY-130, so that the dispersion effect of the emulsion preparation is good, the phage titer is high, and the stability of the emulsion preparation is improved.

Description

Technical Field

[0001] This invention relates to the field of bactericide technology, and in particular to a bacteriophage emulsifiable concentrate formulation of Ralstonia solanacearum and its preparation method. Background Technology

[0002] Bacterial wilt is a serious plant disease caused by Ralstonia solanacearum, which causes great damage to a variety of crops. Traditional control methods mostly rely on chemical pesticides, but these methods have problems such as high residues and easy development of pesticide resistance.

[0003] Bacteriophages, as a biological control method, have gradually attracted attention due to their advantages such as high specificity, no residue, and low likelihood of resistance development, as exemplified by the Ralstonia solanacearum phage disclosed in invention patents CN118345051A and CN108251560A. However, if bacteriophages are directly applied to emulsifiable concentrate formulations, the organic solvents or emulsifiers used in the emulsifiable concentrates can directly affect the protein structure of the bacteriophages, thereby reducing or eliminating their activity. Therefore, it is necessary to develop a stable Ralstonia solanacearum phage emulsifiable concentrate formulation. Summary of the Invention

[0004] In view of this, the present invention proposes a stable Ralstonia solanacearum emulsifiable concentrate formulation and its preparation method.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a Ralstonia solanacearumphage emulsifiable concentrate formulation, comprising the following components: Ralstonia solanacearumphage RSP46 suspension, turpentine oil and emulsifier BY-130;

[0006] The preservation number of the Ralstonia solanacearum phage RSP46 is CCTCC NO: M20231467.

[0007] Specifically, turpentine, as a natural solvent, can protect bacteriophages to some extent from environmental factors (such as oxygen, light, and temperature), preventing their inactivation. Turpentine can be used as a medium for dissolving and dispersing bacteriophages, ensuring their stable existence in emulsifiable concentrates and preventing precipitation or separation. Turpentine also has good permeability, helping to deliver bacteriophages more effectively to the location of plant pathogens, thus improving attack efficiency.

[0008] Emulsifier BY-130 reduces the interfacial tension between components in a formulation, forming a stable emulsion, preventing stratification and separation, and thus improving storage stability. Emulsifier BY-130 assists in the uniform dispersion of phage particles, ensuring their consistency throughout the formulation and improving the uniformity of efficacy. Emulsifier BY-130 can also form a protective coating on the phage surface, mitigating the impact of adverse external conditions on phage activity to some extent.

[0009] Through the above mechanism, turpentine oil and emulsifier BY-130 work together to achieve the goal of improving the stability and bioavailability of Ralstonia solanacearum emulsifiable concentrate.

[0010] Based on the above technical solutions, preferably, the formulation comprises 8%-15% Ralstonia solanacearum phage suspension, 10%-15% emulsifier BY-130, and the remainder is turpentine oil, calculated as 100% by volume.

[0011] Based on the above technical solutions, preferred alternatives include persimmon leaf extract and onion extract.

[0012] Specifically, persimmon leaf extract contains various active ingredients, such as polyphenols and flavonoids, which possess natural antibacterial properties and can directly inhibit the growth of pathogens. The active ingredients in persimmon leaf extract can synergistically interact with bacteriophages, enhancing their ability to kill Ralstonia solanacearum. Persimmon leaf extract also has antioxidant properties, protecting bacteriophages from oxidative damage and maintaining their activity. Furthermore, the polyphenolic compounds in persimmon leaf extract have natural adhesive properties, increasing the adhesion of formulations to plant surfaces and further enhancing the bactericidal efficacy of emulsifiable concentrates.

[0013] Onion extract is rich in sulfur compounds and quercetin, which possess significant antibacterial and antiviral activities, directly disrupting the cell walls of pathogens or inhibiting their metabolism. Onion extract may indirectly enhance the bactericidal effect of bacteriophages by strengthening the plant's immune response. Certain components in onion extract may improve the permeability of formulations, making it easier for bacteriophages to reach the pathogen's location.

[0014] By combining persimmon leaf and onion extracts, these natural ingredients not only directly inhibit pathogens but also enhance the efficacy of bacteriophages through multiple mechanisms, thereby improving the overall bactericidal effect of the emulsifiable concentrate.

[0015] Based on the above technical solutions, preferably, the volume concentration of the persimmon leaf extract is 10%-15% and the volume concentration of the onion extract is 5%-10%.

[0016] Based on the above technical solutions, the preferred method for preparing the persimmon leaf extract and onion extract is as follows: collect persimmon leaves and onions, wash them clean, dry them, crush them, and sieve them; take the crushed samples of each plant, add ethyl acetate and shake to extract them 3-4 times, 24 hours each time, combine the filtrates from 3-4 times, concentrate them under reduced pressure to a paste, and cool and store them for later use.

[0017] Based on the above technical solutions, preferably, the method for preparing the phage suspension is as follows: mixing Ralstonia solanacearum phage RSP46 with a buffer solution to form a phage suspension.

[0018] Based on the above technical solutions, the preferred concentration of Ralstonia solanacearum phage RSP46 in the phage suspension is 2 × 10⁻⁶. 7 PFU / mL.

[0019] Based on the above technical solutions, preferably, the components of the buffer solution are: 40-60mM Tris-HCl, 90-110mM NaCl, 5-15mM MgSO4, and 0.01%-0.02% gelatin (mass concentration).

[0020] On the other hand, the present invention also provides a method for preparing a plant emulsifiable concentrate of Ralstonia solanacearum, comprising the following steps:

[0021] Mix Ralstonia solanacearum phage suspension RSP46, persimmon leaf extract, onion extract, emulsifier BY-130 and turpentine oil evenly, adjust the pH to 6.5-6.8 with Tris-HCl, and continue stirring to form a uniform emulsifiable concentrate.

[0022] The present invention provides a *Ralstonia solanacearum* phage emulsifiable concentrate formulation and its preparation method, which have the following advantages over the prior art:

[0023] Beneficial effects:

[0024] (1) This invention utilizes Ralstonia solanacearum phage as a biocontrol component, which has the advantages of high specificity, no residue, and low resistance. The combined use of turpentine oil and emulsifier BY-130 results in good dispersion of the emulsifiable concentrate, high phage titer, and thus improves the stability of the emulsifiable concentrate.

[0025] (2) By adding plant (persimmon leaf and onion) extracts, the present invention enhances the antibacterial effect and stability of the emulsifiable concentrate, and also improves its adhesion to plant surfaces.

[0026] (3) The bacterial wilt phage emulsifiable concentrate of the present invention has a significant control effect on bacterial wilt, and is safe and harmless to the environment and plants. Moreover, the production process is simple and easy to operate, and is suitable for large-scale production and application. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] The Ralstonia solanacearum phage RSP46 used in this invention was deposited at the China Center for Type Culture Collection on August 14, 2023, at Wuhan University, Wuhan, China; accession number CCTCCNO: M20231467.

[0029] The emulsifier BY-130 was purchased from Haian Petrochemical Plant in Jiangsu Province, product number BY-130.

[0030] Example 1

[0031] The Ralstonia solanacearum emulsifiable concentrate formulation in this embodiment includes 10 mL of Ralstonia solanacearum phage RSP46 suspension, 15 mL of emulsifier BY-130, and 75 mL of turpentine oil.

[0032] The phage suspension was prepared by mixing Ralstonia solanacearum phage RSP46 with SM buffer. The SM buffer consisted of 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4, and 0.01 wt% gelatin. The concentration of Ralstonia solanacearum phage RSP46 in the phage suspension was 2 × 10⁻⁶. 7 PFU / mL.

[0033] The preparation method of the plant emulsifiable concentrate of Ralstonia solanacearum phage is as follows: Ralstonia solanacearum phage RSP46 suspension, emulsifier BY-130 and turpentine are mixed evenly, the pH is adjusted to 6.5 with Tris-HCl, and the mixture is stirred to form a uniform emulsifiable concentrate.

[0034] Example 2

[0035] Compared with Example 1, this embodiment adds persimmon leaf extract and onion extract. The specific scheme is as follows:

[0036] This embodiment of the Ralstonia solanacearum phage emulsifiable concentrate comprises 10 mL of Ralstonia solanacearum phage RSP46 suspension, 10 mL of persimmon leaf extract, 10 mL of onion extract, 15 mL of emulsifier BY-130, and 55 mL of turpentine oil.

[0037] The phage suspension was prepared by mixing Ralstonia solanacearum phage RSP46 with SM buffer. The SM buffer consisted of 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4, and 0.01 wt% gelatin. The concentration of Ralstonia solanacearum phage RSP46 in the phage suspension was 2 × 10⁻⁶. 7 PFU / mL.

[0038] The preparation method of plant extracts is as follows: collect persimmon leaves and onions, wash them clean, dry them, crush them and sieve them; take 100g of each crushed plant sample, add 500mL of ethyl acetate and shake to extract three times, each time for 24h, combine the three filtrates and concentrate them under reduced pressure to a paste, cool and store for later use.

[0039] The preparation method of the plant emulsifiable concentrate of Ralstonia solanacearum phage is as follows: Ralstonia solanacearum phage RSP46 suspension, persimmon leaf extract, onion extract, emulsifier BY-130 and turpentine are mixed evenly, the pH value is adjusted to 6.5 with Tris-HCl, and the mixture is stirred to form a uniform emulsifiable concentrate.

[0040] Example 3

[0041] The Ralstonia solanacearum phage emulsifiable concentrate formulation of this embodiment includes 8 mL of Ralstonia solanacearum phage RSP46 suspension, 15 mL of persimmon leaf extract, 8 mL of onion extract, 18 mL of emulsifier BY-130, and 51 mL of turpentine oil.

[0042] The phage suspension was prepared by mixing Ralstonia solanacearum phage RSP46 with SM buffer. The SM buffer consisted of 40 mM Tris-HCl, 110 mM NaCl, 5 mM MgSO4, and 0.02 wt% gelatin. The concentration of Ralstonia solanacearum phage RSP46 in the phage suspension was 2 × 10⁻⁶. 7 PFU / mL.

[0043] The preparation method of plant extracts is as follows: collect persimmon leaves and onions, wash them clean, dry them, crush them and sieve them; take 100g of each crushed plant sample, add 500mL of ethyl acetate and shake to extract 4 times, 24h each time, combine the 4 filtrates and concentrate them under reduced pressure to a paste, cool and store for later use.

[0044] The preparation method of the plant emulsifiable concentrate of Ralstonia solanacearum phage is as follows: Ralstonia solanacearum phage RSP46 suspension, plant extract, emulsifier BY-130 and turpentine are mixed evenly, the pH is adjusted to 6.5 with Tris-HCl, and the mixture is stirred to form a uniform emulsifiable concentrate.

[0045] Example 4

[0046] The Ralstonia solanacearum emulsifiable concentrate formulation of this embodiment, calculated by weight, includes 15 mL of Ralstonia solanacearum phage RSP46 suspension, 12 mL of persimmon leaf extract, 5 mL of onion extract, 12 mL of emulsifier BY-130, and 56 parts of turpentine oil.

[0047] The phage suspension was prepared by mixing Ralstonia solanacearum phage RSP46 with SM buffer. The SM buffer consisted of 60 mM Tris-HCl, 90 mM NaCl, 15 mM MgSO4, and 0.01 wt% gelatin. The concentration of Ralstonia solanacearum phage RSP46 in the phage suspension was 2 × 10⁻⁶. 7 PFU / mL.

[0048] The preparation method of the plant extract is as follows: collect persimmon leaves and onions, wash them clean, dry them, crush them and sieve them; take 100mg of each crushed plant sample, add 500mL of ethyl acetate and shake to extract three times, each time for 24h, combine the three filtrates and concentrate them under reduced pressure to a paste, cool and store for later use.

[0049] The preparation method of the plant emulsifiable concentrate of Ralstonia solanacearum phage is as follows: Ralstonia solanacearum phage RSP46 suspension, plant extract, emulsifier BY-130 and turpentine are mixed evenly, the pH is adjusted to 6.5 with Tris-HCl, and the mixture is stirred to form a uniform emulsifiable concentrate.

[0050] Comparative Example 1

[0051] Compared with Example 2, Comparative Example 1 lacked onion extract and was replaced with turpentine oil; all other contents were the same.

[0052] I. Solvent Screening

[0053] Taking Example 1 as an example, other solvents were selected: ethyl acetate, methanol and acetone, in the same amount, and the effect of different solvents on the emulsifiable concentrate was analyzed. The results are shown in Table 1.

[0054] Table 1. Effects of different solvents on phage titer and extract solubility in emulsifiable concentrates.

[0055] solvent Ethyl acetate turpentine methanol acetone Solubility (mg / uL) 0.25 0.25 0.052 0.16 Bacteriophage titer (pfu / mL) <![CDATA[1.2x10 7 ]]> <![CDATA[2.5x10 7 ]]> <![CDATA[3.28x10 5 ]]> <![CDATA[1.14x10 6 ]]>

[0056] Table 1 shows that the dissolution and diffusion rates of persimmon leaf and onion extracts in four solvents are as follows: ethyl acetate = turpentine > acetone > methanol. The bacteriophage preservation period in the four solvents is as follows: turpentine > ethyl acetate > acetone > methanol. Therefore, turpentine is selected as the best solvent.

[0057] II. Screening of Emulsifiers

[0058] Taking Example 1 as an example, four emulsifiers were selected: Tween-80, Tween-20, diethanolamine and BY-130, with concentrations of 5%, 10%, 15%, 20% and 25%. The plant (persimmon leaf and onion) extracts and the wilt phage suspension were mixed evenly and then emulsified with turpentine oil and the four emulsifiers respectively. The results are shown in Table 2.

[0059] Table 2. Effects of different emulsifiers on the stability and dispersibility of emulsifiable concentrates.

[0060]

[0061] As shown in Table 2, among the four emulsifiers Tween-80, Tween-20, diethanolamine, and BY-130, BY-13 has the best effect, with the optimal effect at a concentration of 10%-15%.

[0062] III. Long-term storage stability test

[0063] The emulsifiable concentrate formulations prepared in the examples and comparative examples were subjected to quality testing, including phage activity (initial titer 1.5 × 10⁻⁶). 7 The results of the assays (pH value (initial pH 6.5), antibacterial activity, and stability) are shown in Table 3-5. Storage conditions: 25℃ (room temperature).

[0064] The antibacterial activity of emulsifiable concentrates was determined by measuring phage activity and mycelial growth rate. The mycelial growth rate was measured as follows: PDA medium was heated to melt, and after cooling to approximately 45°C, 1 mL of the emulsifiable concentrate prepared in the examples and comparative examples was added to 9 mL of PDA medium to prepare a medium containing the emulsifiable concentrate. The medium was thoroughly mixed and quickly poured into sterile petri dishes (9 cm in diameter). A PDA plate without the emulsifiable concentrate and an equal volume of sterile water was used as a control. After the medium solidified, 7 mm diameter mycelial discs were cut from the edge of the cultured *Ralstonia solanacearum* colonies using a punch. The discs were placed hyphae-side down in the center of the PDA plate containing the emulsifiable concentrate, one disc per plate, with each treatment repeated three times. The plates were incubated at 25°C for 5 days. The colony diameter was then measured using the cross-hatching method, and the inhibition rate of mycelial growth by the concentrate treatment was calculated.

[0065] Formula for calculating mycelial growth inhibition rate: Mycelial growth inhibition rate = {(average colony diameter - average colony diameter of treatment group) / average colony diameter of control group} * 100%.

[0066] Table 3. Phage activity and pH stability

[0067]

[0068] As shown in Table 3, the addition of persimmon leaf extract and onion extract slowed down the rate of decrease in phage titer, thereby improving the stability of phage during storage.

[0069] Table 4. Inhibitory effect of plant extract emulsifiable concentrate on bacterial wilt pathogen.

[0070]

[0071] As shown in Table 4, the addition of active ingredients from persimmon leaf extract and onion extract enhanced the antibacterial activity of the phage and slowed down the rate of decrease in phage titer, thereby improving the antibacterial activity of the phage.

[0072] Table 5. Stability of Emulsifiers

[0073]

[0074]

[0075] As shown in Table 5, the emulsifiable concentrate formulation of the present invention maintained a high level of stability and dispersibility after one year of storage, indicating that the emulsifiable concentrate formulation of the present invention can be stored for a long time.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A virucidal phage emulsifiable concentrate formulation, characterized in that: Includes the following components: Ralstonia solanacearum phage ( Ralstonia solanacearum phage RSP46 suspension, turpentine oil, and emulsifier BY-130; The preservation number of the Ralstonia solanacearum phage RSP46 is CCTCC NO: M20231467; Based on a volume percentage of 100%, the Ralstonia solanacearum phage RSP46 suspension contains 8%-15%, emulsifier BY-130 contains 10%-15%, and the remainder is turpentine.

2. The efflorescence phage emulsifiable concentrate formulation as described in claim 1, characterized in that: The method for preparing the phage suspension is as follows: Ralstonia solanacearum phage RSP46 is mixed with buffer solution to form a phage suspension.

3. The efflorescence phage emulsifiable concentrate formulation as described in claim 2, characterized in that: The concentration of Ralstonia solanacearum phage RSP46 in the phage suspension was 2 × 10⁻⁶. 7 PFU / mL.

4. The efflorescence phage emulsifiable concentrate formulation as described in claim 2, characterized in that: The buffer solution consists of: 40-60 mM Tris−HCl, 90-110 mM NaCl, 5-15 mM MgSO4, and 0.01%-0.02% gelatin (by mass).

Images