Isobutyric anhydride and application thereof in preparation of reagent for inhibiting activity of pathogenic microorganisms

Abstract

The invention relates to the technical field of biology, in particular to isobutyric anhydride and application thereof in preparation of reagents for inhibiting activity of pathogenic microorganisms. The isobutyric anhydride provided by the invention is separated and identified from bacillus subtilis VR19, and it is found that the isobutyric anhydride can be used as a bacteriostatic active component and used for inhibiting pepper blast pathogen chlamydospore with poor pesticide effect of a conventional bactericide in a soil bag, and the pesticide can be accurately applied at a fixed point under the condition that soil is not turned over, so that the effect of efficiently preventing and treating pepper blast is achieved. Tests prove that under three test concentrations of 1.12 ppm, 0.56 ppm and 0.33 ppm and the like, the bacteriostasis rate of the isobutyric anhydride to the pepper blast pathogenic bacteria reaches 83.30%-100%. Isobutyric anhydride has the characteristics of high efficiency, low toxicity, safety and the like in application, meets the creation requirements of current new pesticides, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to isobutyric anhydride and its application in the preparation of a reagent for inhibiting the activity of pathogenic microorganisms. Background Art

[0002] Piper nigrum Linn. is a perennial tropical spice crop, known as the "king of spices", and the pepper production in China ranks fifth in the world. Pepper blight is a devastating soil-borne disease that once caused the pepper planting area in China to shrink by nearly 30% within several years. Its pathogen is Phytophthora capsici, which has a wide host range and can infect various crops such as pepper, chili, eggplant, and cucumber, causing symptoms such as plant wilting, root and stem rot, and fruit rot, resulting in serious yield reduction or even crop failure. The oospores of this pathogen can generally survive in the soil for 3 years and are difficult to kill by conventional spraying. With the prohibition of methyl bromide, chloropicrin, dazomet and other soil disinfectants with high lethality, there is an urgent need in production for soil pathogen killing means with strong pertinence and environmental friendliness. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide isobutyric anhydride and its application in the preparation of a reagent for inhibiting the activity of pathogenic microorganisms. The present invention provides the application of isobutyric anhydride in the prevention and treatment of pepper blight, providing a natural source bactericidal active substance for the prevention and treatment of pepper blight.

[0004] The present invention provides the application of isobutyric anhydride in the preparation of a reagent for inhibiting the activity of pathogenic microorganisms, and the chemical structural formula of the isobutyric anhydride is shown as formula (I):

[0005] Formula (I).

[0006] In some embodiments, the isobutyric anhydride is isolated from a strain with a preservation number of CGMCC NO.14450.

[0007] In some embodiments, the pathogenic microorganisms include bacteria and / or fungi.

[0008] In some embodiments, the pathogenic microorganisms include Phytophthora capsici of pepper blight.

[0009] In some embodiments, the inhibition of the activity of pathogenic microorganisms includes inhibiting the mycelial growth of pathogenic microorganisms.

[0010] In some embodiments, the inhibition of the activity of pathogenic microorganisms includes destroying the cell membrane structure of pathogenic microorganisms.

[0011] In some embodiments, the inhibition of the activity of pathogenic microorganisms includes inhibiting the spore germination of pathogenic microorganisms.

[0012] In some embodiments, the reagent comprises isobutyric anhydride and an agriculturally acceptable adjuvant.

[0013] In some embodiments, the adjuvant comprises one or more of a carrier, a surfactant, a stabilizer, a thickener, a dispersant, an adhesive, an antifreeze, a preservative, a colorant, a synergist, a buffer, and an antifoaming agent.

[0014] In some embodiments, the dosage form of the reagent is an injection, a tablet, a pill, a powder, a capsule, a suspension, or an emulsion.

[0015] The present invention provides a method for planting plants. The plants are planted in the soil, and isobutyric anhydride is buried in the basal soil pocket of the plants after being sealed with a hydrophobic and breathable membrane.

[0016] Compared with the prior art, the isobutyric anhydride provided by the present invention is isolated and identified from Bacillus subtilis VR19, and it is found that it can be used as an antibacterial active ingredient to inhibit the chlamydospores of Phytophthora capsici for which the efficacy of conventional fungicides in the soil pocket is poor. It can achieve the effect of highly efficient control of Phytophthora capsici by targeted and precise application of the drug without tilling the soil. Experiments have proved that at three tested concentrations of 1.12 ppm, 0.56 ppm, and 0.33 ppm, the antibacterial rate of isobutyric anhydride against Phytophthora capsici reaches 83.30% - 100%. Isobutyric anhydride has the characteristics of high efficiency, low toxicity, and safety in application, meets the requirements for the creation of current new pesticides, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Showing the chemical structure of isobutyric anhydride;

[0018] Figure 2 Showing the inhibition of the mycelial growth of Phytophthora capsici by the volatile products of Bacillus subtilis VR19;

[0019] Figure 3 Showing the effects of isobutyric anhydride, metalaxyl, and carvacrol at a tested concentration of 0.56 ppm on the mycelial growth inhibition of Phytophthora capsici;

[0020] Figure 4 Showing the control effects of isobutyric anhydride, metalaxyl, and carvacrol at a tested concentration of 10 ppm on Phytophthora capsici in a pot experiment; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The present invention provides isobutyric anhydride and its application in the preparation of reagents for inhibiting the activity of pathogenic microorganisms. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0022] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The present invention will be further described below in conjunction with embodiments.

[0023] Example 1

[0024] In view of the rapid spread and strong destructiveness of pepper blight caused by Phytophthora capsici, its oospores can survive in the soil for several years, and there is a lack of targeted soil disinfection means. Bacillus subtilis is a broad-spectrum and green plant protection product. In recent years, its share in the international market has increased rapidly and has become one of the indispensable means for pest control. Bacillus subtilis VR19 (the preservation number of the strain is CGMCC NO. 14450) was isolated from the rhizosphere soil of plants in the hot area. The inhibition rate of the spore germination of Phytophthora capsici was 96.25%, and the control effect on pepper blight was 76.86%. This strain can produce a large number of antibacterial substances such as surfactin, thujaplicin, bacillibactin, bacteriolysin, bacillaene, and bacitracin A, as well as volatile compounds such as 3-hydroxy-2-butanone (acetoin) and 2,3-butanediol. However, these widely reported biocontrol substances have no inhibitory activity against Phytophthora capsici. The present invention discovers that the volatile substance - isobutyric anhydride produced by Bacillus subtilis VR19 has a good effect on inhibiting Phytophthora capsici. Isobutyric anhydride, also known as 2-methylpropanoic anhydride, has the following chemical formula structure Figure 1 As shown, the chemical formula is C 8 H 14 O 3 , with a relative molecular weight of 158.195. It is mainly used as an intermediate for organic synthesis to prepare isobutyrate plasticizers and essences, flavors, etc. There has been no report and application of its antibacterial activity. The present invention discovers that this substance has both the characteristics of inhibiting Phytophthora capsici and volatility, and has good prospects for the development of a soil fumigant for pepper blight.

[0025] In this example, the functions of isobutyric anhydride and its structurally similar molecules in inhibiting crop pathogenic bacteria were explored. The specific experiments are as follows:

[0026] Pour 10 mL of PDA medium into one grid on one side of a two-compartment flat plate (with a volume of 90 mL). After solidification, inoculate a mycelial block of Phytophthora capsici as a control. On the basis of the control group, drop volatile compounds into the other grid. For each compound, set 3 treatments with dosages of 1.0 μL, 0.5 μL, and 0.3 μL. Seal the flat plate with parafilm sealing film. Set 3 replicates for each treatment, and 3 flat plates for each replicate. After culturing at 30 °C for 5 d, measure the colony diameter. Calculate the inhibition rate using the formula "(average diameter of the control group - average diameter of the treatment group) ÷ (average diameter of the control group - diameter of the inoculated mycelial block)". Use the least significant difference method (Fisher’s LSD) in SPSS software for one-way multiple comparisons to analyze the significance of differences. The specific substances and their antibacterial effects are shown in Table 1 below.

[0027] Table 1 Effects of volatile antibacterial substances on inhibiting Phytophthora capsici at three tested concentrations

[0028]

[0029] Note: Data with the same letter in the same column are not significantly different (LSD, P < 0.05).

[0030] As can be seen from Table 1, at the three tested concentrations, the inhibition rate of isobutyric anhydride against Phytophthora capsici reached 83.30% - 100%, and the inhibition rates of the other 4 structurally similar molecules were between 65.33% - 97.62%, indicating that this type of substance has good activity in inhibiting Phytophthora capsici. However, the fungicides metalaxyl and carvacrol had poor antibacterial effects due to their low volatility.

[0031] Using the volatile products of Bacillus subtilis VR19, isobutyric anhydride, metalaxyl, and carvacrol with a tested concentration of 0.56 ppm were used to inhibit the mycelial growth of Phytophthora capsici respectively, and the results are as Figure 2 and Figure 3 shown. The results showed that compared with the volatile products of Bacillus subtilis VR19, metalaxyl, and carvacrol, isobutyric anhydride had a more obvious effect in inhibiting the mycelial growth of Phytophthora capsici.

[0032] Example 2

[0033] This example explored the effect of isobutyric anhydride in controlling Phytophthora capsici in potted plants. The specific experiment is as follows:

[0034] The Phytophthora capsici was inoculated onto a cucumber agar (CA) medium plate and cultured at 26°C for about 2 weeks. The dark brown mycelia (containing a large number of chlamydospores) were collected, crushed by a blender, and mixed with nutrient soil at a ratio of 1‰, and then filled into a disposable plastic pot (with a volume of 300 mL). After removing the lid of a 200 μL EP centrifuge tube, isobutyric anhydride was added, and two treatments of 6 μL and 3 μL were set according to the dosage. After sealing with a hydrophobic and breathable PTFE membrane (φ = 0.45 μm), it was buried in the soil at the base of the plant. Without adding volatile compounds was used as the control. At the same time, the fungicides metalaxyl and carvacrol were set as references, and two treatments of 6 μL and 3 μL were also set. The use method was to uniformly mix the fungicides into the nutrient soil. One pepper seedling at the 3-5 true leaf stage was transplanted into each pot of soil, and the cup mouth was sealed with parafilm. There were 3 replicates for each treatment, and 5 pots for each replicate. It was placed in a light incubator and cultured for 7 days under the conditions of 26°C, 12 h of light / 12 h of darkness, and then the disease incidence was observed. The results are as Figure 4 shown.

[0035] Disease grading standard (refer to the agricultural industry standard "NY / T 3816-2020 Technical Regulations for Monitoring of Tropical Crop Diseases and Pests - Phytophthora blight of pepper"):

[0036] Grade 0: Normal growth;

[0037] Grade 1: The top leaves wilt and there are no lesions at the base of the stem;

[0038] Grade 3: The upper leaves wilt or the lesion at the base of the stem ≤ 0.5 cm;

[0039] Grade 5: All leaves wilt or 0.5 < lesion at the base of the stem ≤ 2.0 cm;

[0040] Grade 7: The stem lodges or the lesion at the base of the stem ≥ 2 cm;

[0041] Grade 9: The whole plant withers.

[0042] Disease index = ∑(number of diseased plants at each level × relative level value) / (total number of plants surveyed) × (highest disease level value) × 100

[0043] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0044] One-way ANOVA with the least significant difference method (Fisher's LSD) was used through SPSS software to analyze the significance of differences.

[0045] Table 2 Control effect on Phytophthora blight in the pot experiment

[0046]

[0047] Through Table 2 andFigure 4 It can be seen that at the two tested concentrations, the control effects of isobutyric anhydride against Phytophthora capsici were 80.00% and 75.56%, which were significantly better than those of the reference agents metalaxyl (42.21% and 28.89%) and carvacrol (15.35% and 8.87%). Although the literature reports that metalaxyl and carvacrol have good inhibitory effects on the mycelial growth of Phytophthora capsici, the control effects against Phytophthora capsici caused by chlamydospores in the soil were relatively low in this experiment. This indicates that conventional oomyceteicides are mainly applied in the form of surface spraying and are difficult to be applied to the prevention and control of soil-borne pathogens. Although mixing metalaxyl evenly into the soil had a certain control effect against Phytophthora capsici in this experiment, there was a possibility of improving the control effect by increasing the tested concentration. However, pepper is a perennial plant and it is difficult to carry out soil tillage and mixing treatment with pesticides, and mixing a large amount of pesticides into the soil does not meet the requirements of national pesticide safety management and environmental protection. Isobutyric anhydride administered precisely to the soil at the base of pepper not only has good effects but also uses less amount, without residue and pollution problems.

[0048] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of isobutyric anhydride in the preparation of an agent for inhibiting the activity of pathogenic microorganisms, wherein the chemical structural formula of the isobutyric anhydride is shown in formula (I): Formula (I).

2. The use according to claim 1, characterized in that: The isobutyric anhydride is separated from the strain with the deposit number of CGMCCNO.14450.

3. The use according to claim 1 or 2, characterized in that: The pathogenic microorganisms include bacteria and / or fungi.

4. The use according to claim 3, characterized in that: The pathogenic microorganisms include pepper blast pathogen.

5. The use according to any one of claims 1 to 4, characterized in that: The inhibiting activity of pathogenic microorganisms includes inhibiting the growth of pathogenic microorganism hyphae.

6. The use according to any one of claims 1 to 4, characterized in that: The inhibiting of the activity of pathogenic microorganisms includes destroying the cell membrane structure of pathogenic microorganisms.

7. The use according to any one of claims 1 to 4, characterized in that: The activity of inhibiting pathogenic microorganisms includes inhibiting the germination of spores of pathogenic microorganisms.

8. The use according to any one of claims 1 to 7, characterized in that: The reagent comprises isobutyric anhydride and an agriculturally acceptable auxiliary material.

9. The use according to claim 8, characterized in that: The auxiliary materials include one or more of carriers, surfactants, stabilizers, thickeners, dispersants, adhesives, antifreeze agents, preservatives, colorants, synergists, buffers and defoamers.

10. A method for growing plants, characterized in that: Plants are planted in soil, and isobutyric anhydride is buried in a soil bag at the base of the plant after being sealed with a hydrophobic breathable membrane.

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