Ralstonia solanacearum phage missible oil preparation and preparation method thereof

Abstract

The invention relates to the technical field of bactericides, and provides a ralstonia solanacearum bacteriophage missible oil preparation and a preparation method thereof. The ralstonia solanacearum bacteriophage missible oil preparation is prepared from the following components: a ralstonia solanacearum bacteriophage RSP46 suspension, turpentine oil and an emulsifying agent BY-130, and is characterized in that the ralstonia solanacearum bacteriophage RSP46 suspension is added into the emulsifying agent BY-130; the preservation number of the ralstonia solanacearum bacteriophage RSP46 is CCTCC (China Center for Type Culture Collection) NO: M20231467. According to the missible oil preparation, the ralstonia solanacearum phage RSP46 is used as a biological control component and is matched with the turpentine and the emulsifier BY-130 for use, so that the missible oil preparation is good in dispersion effect and high in phage titer, and the stability of the missible oil preparation is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of fungicides, and in particular to a solanacearum phage emulsifiable concentrate preparation and a preparation method thereof. Background Art

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

[0003] As a biological control method, bacteriophages have gradually attracted attention due to their advantages such as high specificity, no residue, and low resistance, such as the solanacearum phages disclosed in the invention patents with publication numbers CN118345051A and CN108251560A. However, if bacteriophages are directly applied to emulsifiable concentrates, the organic solvents or emulsifiers occupied by the emulsifiable concentrates will directly affect the protein structure of the bacteriophages, thereby reducing their activity or losing their activity. Therefore, it is necessary to develop a solanacearum phage emulsifiable concentrate preparation with good stability. Summary of the invention

[0004] In view of this, the present invention provides a solanacearum phage emulsifiable concentrate preparation with good stability and a preparation method thereof.

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

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

[0007] Specifically, as a natural solvent, turpentine can protect bacteriophages from environmental factors (such as oxygen, light, and temperature) to a certain extent, preventing them from being inactivated. Turpentine can be used as a medium for dissolving and dispersing bacteriophages, so that they can exist in an emulsifiable concentrate in a stable manner to prevent precipitation or separation. Turpentine also has good penetration properties, which helps to more effectively deliver bacteriophages to the location of plant pathogens and improve attack efficiency.

[0008] Emulsifier BY-130 can reduce the interfacial tension between the components in the preparation, form a stable emulsion, prevent stratification and separation, and thus improve storage stability. Emulsifier BY-130 helps to evenly disperse the phage particles, ensure their consistency throughout the preparation, and improve the uniformity of drug efficacy. Emulsifier BY-130 can also form a protective coating on the surface of the phage, which to a certain extent resists the influence of adverse external conditions on the activity of the phage.

[0009] Through the above mechanism, turpentine and emulsifier BY-130 work together to achieve the goal of improving the stability and biological efficacy of the bacterial phage emulsifiable concentrate preparation.

[0010] On the basis of the above technical scheme, preferably, calculated based on the volume percentage of 100%, the preparation includes 8%-15% of Ralstonia solanacearum bacteriophage RSP46 suspension, 10%-15% of emulsifier BY-130, and the balance is turpentine.

[0011] On the basis of the above technical solution, preferably, persimmon leaf extract and onion extract are also included.

[0012] Specifically, persimmon leaf extract contains a variety of active ingredients, such as polyphenols and flavonoids, which have natural antibacterial properties and can directly inhibit the growth of pathogens. The active ingredients in persimmon leaf extract can produce a synergistic effect with bacteriophages, enhancing the ability of bacteriophages to kill Ralstonia solanacearum. Persimmon leaf extract has antioxidant properties and can also protect bacteriophages from oxidative damage, thereby maintaining their activity. In addition, the polyphenol compounds contained in persimmon leaf extract have natural adhesion properties. These compounds can increase the adhesion of the preparation on the plant surface and further increase the bactericidal power of the emulsifiable concentrate preparation.

[0013] Onion extracts are rich in sulfur compounds and quercetin, which have significant antibacterial and antiviral activity and can directly destroy the cell walls of pathogens or inhibit their metabolism. Onion extracts may indirectly enhance the bactericidal effect of phages by enhancing the immune response of plants. Certain components in onion extracts may increase the permeability of the preparation, making it easier for phages to reach the site where the pathogens are located.

[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 bactericidal effect of the entire emulsion preparation.

[0015] On the basis of the above technical solution, preferably, the volume concentration of the persimmon leaf extract is 10%-15%; the volume concentration of the onion extract is 5%-10%.

[0016] On the basis of the above technical scheme, preferably, the preparation method of the persimmon leaf extract and the onion extract is: collect persimmon leaves and onions, wash them separately, dry them, crush them and sieve them; take crushed samples of each plant, add ethyl acetate and shake them for 3-4 times, each time for 24 hours, combine the filtrates for 3-4 times, and then concentrate them under reduced pressure to a paste, cool them and store them for later use.

[0017] On the basis of the above technical solution, preferably, the method for preparing the phage suspension is: mixing the Ralstonia solanacearum phage RSP46 with a buffer solution to form a phage suspension.

[0018] On the basis of the above technical solution, preferably, the concentration of RSP46 of Ralstonia solanacearum phage in the phage suspension is 2×10 7 PFU / mL.

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

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

[0021] The suspension of R. solanacearum bacteriophage RSP46, persimmon leaf extract, onion extract, emulsifier BY-130 and turpentine oil were uniformly mixed, the pH value was adjusted to 6.5-6.8 with Tris-HCl, and the mixture was continuously stirred to form a uniform emulsifiable concentrate preparation.

[0022] Compared with the prior art, the solanacearum phage emulsifiable concentrate preparation and the preparation method thereof of the present invention have the following advantages:

[0023] Beneficial effects:

[0024] (1) The present invention uses bacterial wilt phage as a biological control component, which has the advantages of high specificity, no residue, and not easy to produce resistance. The use of turpentine and emulsifier BY-130 makes the emulsifiable concentrate preparation have a good dispersion effect and a high phage titer, thereby improving the stability of the emulsifiable concentrate preparation.

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

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

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The Ralstonia solanacearumphage RSP46 used in the present invention was sent to the China Center for Type Culture Collection for preservation on August 14, 2023. The preservation address is: Wuhan University, Wuhan, China; the preservation number is CCTCCNO: M20231467.

[0029] Emulsifier BY-130 was purchased from Hai'an Petrochemical Plant, Jiangsu Province, with the product number BY-130.

[0030] Example 1

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

[0032] The preparation method of the phage suspension is as follows: the R. solanacearum phage RSP46 is mixed with SM buffer to form a phage suspension, wherein the components of the SM buffer are: 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4, and 0.01 wt% gelatin. The concentration of the R. solanacearum phage RSP46 in the phage suspension is 2×10 7 PFU / mL.

[0033] The preparation method of the solanacearum phage plant emulsifiable concentrate preparation is as follows: uniformly mix the solanacearum phage RSP46 suspension, the emulsifier BY-130 and the turpentine oil, adjust the pH value to 6.5 with Tris-HCl, and continue to stir to form a uniform emulsifiable concentrate preparation.

[0034] Example 2

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

[0036] The solanacearum phage emulsifiable concentrate preparation of this embodiment includes 10 mL of 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.

[0037] The preparation method of the phage suspension is as follows: the R. solanacearum phage RSP46 is mixed with SM buffer to form a phage suspension, wherein the components of the SM buffer are: 50 mM Tris-HCl, 100 mM NaCl, 10 mM MgSO4, and 0.01 wt% gelatin. The concentration of the R. solanacearum phage RSP46 in the phage suspension is 2×10 7 PFU / mL.

[0038] The preparation method of the plant extract is as follows: collect persimmon leaves and onions, clean them separately, dry them, crush them and sieve them; take 100 g of each plant crushed sample, add 500 mL of ethyl acetate and shake and extract for 3 times, each time for 24 hours, combine the filtrates for 3 times and concentrate them under reduced pressure to a paste, and cool and store them for later use.

[0039] The preparation method of the solanacearum phage plant emulsifiable concentrate preparation is as follows: uniformly mix the solanacearum phage RSP46 suspension, persimmon leaf extract, onion extract, emulsifier BY-130 and turpentine oil, adjust the pH value to 6.5 with Tris-HCl, and continue stirring to form a uniform emulsifiable concentrate preparation.

[0040] Example 3

[0041] The solanacearum phage emulsifiable concentrate preparation of this embodiment includes 8 mL of 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.

[0042] The preparation method of the phage suspension is as follows: the R. solanacearum phage RSP46 is mixed with SM buffer to form a phage suspension, wherein the components of the SM buffer are: 40 mM Tris-HCl, 110 mM NaCl, 5 mM MgSO4, and 0.02 wt% gelatin. The concentration of the R. solanacearum phage RSP46 in the phage suspension is 2×10 7 PFU / mL.

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

[0044] The preparation method of the solanacearum phage plant emulsifiable concentrate preparation is as follows: uniformly mix the solanacearum phage RSP46 suspension, plant extract, emulsifier BY-130 and turpentine oil, adjust the pH value to 6.5 with Tris-HCl, and continue stirring to form a uniform emulsifiable concentrate preparation.

[0045] Example 4

[0046] The solanacearum phage emulsifiable concentrate preparation of this embodiment includes, by weight, 15 mL of 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.

[0047] The preparation method of the phage suspension is as follows: the R. solanacearum phage RSP46 is mixed with SM buffer to form a phage suspension, wherein the components of the SM buffer are: 60 mM Tris-HCl, 90 mM NaCl, 15 mM MgSO4, and 0.01 wt% gelatin. The concentration of the R. solanacearum phage RSP46 in the phage suspension is 2×10 7 PFU / mL.

[0048] The preparation method of the plant extract is as follows: collect persimmon leaves and onions, clean them separately, dry them, crush them and sieve them; take 100 mg of each plant crushed sample, add 500 mL of ethyl acetate and shake and extract them 3 times, each time for 24 hours, combine the filtrates for 3 times and concentrate them under reduced pressure to a paste, and cool and store them for later use.

[0049] The preparation method of the solanacearum phage plant emulsifiable concentrate preparation is as follows: uniformly mix the solanacearum phage RSP46 suspension, plant extract, emulsifier BY-130 and turpentine oil, adjust the pH value to 6.5 with Tris-HCl, and continue stirring to form a uniform emulsifiable concentrate preparation.

[0050] Comparative Example 1

[0051] Compared with Example 2, Comparative Example 1 lacks onion extract and is replaced by turpentine, and the rest of the contents are the same.

[0052] 1. Solvent selection

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

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

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

[0056] As shown in Table 1, the dissolution and diffusion rates of the two plant extracts of persimmon leaves and onions in the four solvents are: ethyl acetate = turpentine > acetone > methanol. The phage storage period in the four solvents is turpentine > ethyl acetate > acetone > methanol, so turpentine is selected as the best solvent.

[0057] 2. Selection 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) extract and the solanacearum phage suspension were evenly mixed and then emulsified with turpentine 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, namely Tween-80, Tween-20, diethanolamine and BY-130, BY-13 has the best effect, and the best effect is achieved when the concentration is 10%-15%.

[0062] 3. Long-term storage stability test

[0063] The emulsifiable concentrates prepared in the examples and comparative examples were subjected to quality tests, including the activity of bacteriophages (initial titer 1.5×10 7 ), pH value (initial pH value 6.5), antibacterial properties and stability. The results are shown in Tables 3-5. The storage condition is 25℃ room temperature.

[0064] The antibacterial property of the emulsifiable concentrate preparation was determined by the activity of bacteriophage and the mycelial growth rate. The mycelial growth rate determination method was as follows: the PDA medium was heated to melt, and when it was cooled to about 45°C, 1 mL of the emulsifiable concentrate preparation prepared in the embodiment and the comparative example was added to 9 mL of the PDA medium to prepare a medium containing the emulsifiable concentrate preparation, and after being fully shaken, it was quickly poured into a sterile culture dish (9 cm in diameter), and a PDA plate without emulsifiable concentrate preparation to which an equal volume of sterile water was added was used as a control. After the culture medium solidified, a 7 mm diameter bacterial butterfly block was cut from the edge of the cultivated Ralstonia solanacearum colony with a hole puncher, and the mycelium was placed face down in the center of the PDA plate containing the emulsifiable concentrate preparation, one piece per dish, and three repetitions per treatment. After being placed in a 25°C constant temperature incubator and cultured for 5 days, the colony diameter was determined by the cross method, and the inhibition rate of the preparation treatment on mycelial growth was calculated.

[0065] The calculation formula of mycelium growth inhibition rate is: mycelium growth inhibition rate = {(average colony diameter - average colony diameter of the treatment group) / average colony diameter of the 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 decline in phage titer, thereby improving the stability of phage during storage.

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

[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 also slowed down the rate of decline in the phage titer, thereby improving the antibacterial activity of the phage.

[0072] Table 5 Stability of emulsifiers

[0073]

[0074]

[0075] As shown in Table 5, after the emulsifiable concentrate of the present invention was stored for one year, the stability and dispersibility were maintained at a relatively high level, indicating that the emulsifiable concentrate 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 in the protection scope of the present invention.

Claims

1. A solanacearum phage emulsifiable concentrate preparation, characterized in that: The invention comprises the following components: Ralstonia solanacearumphage RSP46 suspension, turpentine oil and emulsifier BY-130; The deposit number of the Ralstonia solanacearum phage RSP46 is CCTCC NO: M20231467.

2. A solanacearum phage emulsifiable concentrate preparation as claimed in claim 1, characterized in that: Calculated based on the volume percentage of 100%, the preparation includes 8%-15% of Ralstonia solanacearum bacteriophage RSP46 suspension, 10%-15% of emulsifier BY-130, and the balance is turpentine oil.

3. A solanacearum phage emulsifiable concentrate preparation as claimed in claim 2, characterized in that: Also includes persimmon leaf extract and onion extract.

4. A solanacearum phage emulsifiable concentrate preparation as claimed in claim 3, characterized in that: The volume concentration of the persimmon leaf extract is 10%-15%, and the volume concentration of the onion extract is 5%-10%.

5. The solanacearum phage emulsifiable concentrate preparation according to claim 3, characterized in that: The preparation method of the persimmon leaf extract and the onion extract is as follows: Collect persimmon leaves and onions, clean them separately, dry them, crush them and sieve them; take crushed samples of each plant, add ethyl acetate and shake to extract 3-4 times, each time for 24 hours, combine 3-4 filtrates and concentrate them under reduced pressure to a paste, cool and store them for later use.

6. The solanacearum phage emulsifiable concentrate preparation according to claim 1, characterized in that: The preparation method of the phage suspension is as follows: mixing the solanacearum phage RSP46 with a buffer solution to form a phage suspension.

7. A solanacearum phage emulsifiable concentrate preparation as claimed in claim 6, characterized in that: The concentration of RSP46 of Ralstonia solanacearum phage in the phage suspension was 2×10 7 PFU / mL.

8. The solanacearum phage emulsifiable concentrate preparation according to claim 6, characterized in that: The components of the buffer solution are: 40-60 mM Tris-HCl, 90-110 mM NaCl, 5-15 mM MgSO4, and 0.01%-0.02% mass concentration of gelatin.

9. A method for preparing a solanacearum phage emulsifiable concentrate preparation according to any one of claims 3 to 8, characterized in that: The following steps are involved: The suspension of R. solanacearum bacteriophage RSP46, persimmon leaf extract, onion extract, emulsifier BY-130 and turpentine oil were uniformly mixed, the pH value was adjusted to 6.5-6.8 with Tris-HCl, and the mixture was continuously stirred to form a uniform emulsifiable concentrate preparation.

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