CN114009440A

CN114009440A - Application of abafungin in preventing and treating agricultural pathogenic bacteria

Info

Publication number: CN114009440A
Application number: CN202111387805.2A
Authority: CN
Inventors: 刘映前; 马越; 贺颖慧; 张保琪; 安俊霞; 罗雄飞; 周勇; 张智军; 赵文斌; 胡勇梅
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-02-08

Abstract

The invention belongs to the field of medicinal chemistry, and discloses abafungin for preventing and treating agricultural diseases caused by agricultural pathogenic bacteria of rice bacterial leaf blight pathogenic bacteria Xanthomonas Oryzae ACCC 11602, citrus canker pathogenic bacteria Xanthomonas anopodis pv. citri, potato black shank pathogenic bacteria Pebacterium atroseptica ACCC 19901, agricultural pathogenic fungi of Sclerotinia sclerotiorum, Rhizoctonia Solani Solani, Fusarium Graminearum, Botrytis cinerea, rice blast pathogenic bacteria Magnaporthe Oryza and Phytophtora Capsici. The abafungin serving as a novel agricultural bactericide has the characteristics of novel structure, broad spectrum and high efficiency, and has the value of further researching and developing into the novel agricultural bactericide. The compound has the following structural formula:

Description

Application of abafungin in preventing and treating agricultural pathogenic bacteria

Technical Field

The invention belongs to the field of medicinal chemistry, and discloses a new application of abafungin in preventing and treating plant diseases caused by agricultural pathogenic bacteria, namely, rice bacterial leaf blight pathogenic bacteria Xanthomonas Oryzae, citrus canker pathogenic bacteria Xanthomonas axonodis pv. citri, potato black shank pathogenic bacteria Pectobacterium atroseptica, agricultural pathogenic fungi, namely, Sclerotinia sclerotiorum, Rhizoctonia Rhizoctonia Solani, wheat scab Fusarium Graminearum, Botrytis cinerea, rice blast fungus Magnaporthe Oryzae Oryza and Phytophthora Capsici.

Background

Agricultural civilization has been developed for nearly ten thousand years, China is also a big agricultural country, and crops are infected by various plant diseases in the process of agricultural production, such as bacterial diseases of rice bacterial leaf blight pathogenic bacteria, citrus canker pathogenic bacteria, potato phytophthora parasitica pathogenic bacteria and the like, and fungal diseases of sclerotinia sclerotiorum, rhizoctonia solani, wheat scab pathogenic bacteria, tomato botrytis cinerea, rice blast pathogenic bacteria, pepper phytophthora blight and the like. In order to control these diseases, the use of chemical agricultural fungicides is particularly critical, in addition to the necessary protective measures used in agricultural production.

At present, the types of effective chemical agricultural bactericides are few, the curative effect is weak, the actual prevention effect of the existing agricultural chemical bactericides used in the field is not ideal, and the long-term use of the agricultural chemical bactericides can cause the drug resistance of agricultural pathogenic bacteria to be enhanced and the environmental pesticide residue to exceed the standard. The incidence of the infected crops tends to increase, and the income increase and benefit improvement of farmers are seriously influenced. The agricultural pathogenic bacteria are various in types and wide in distribution, plants do not have an immune system which is completely evolved like animals, once the plants are attacked, the plants are extremely difficult to treat, so that the discovery of chemical agricultural bactericides with brand-new structures or the search for new applications in the existing medicines is urgently needed to deal with the crisis.

In the early stage of the subject group, the inventor discovers that Abafungin (Abafungin) shows excellent inhibitory action on various agricultural pathogenic bacteria in a large number of commercial medical drug screens by using a new strategy of old drugs. However, at present, abafungin has no reported activity against agricultural pathogenic bacteria, and can be used for innovation of novel agricultural bactericides.

Disclosure of Invention

The invention aims to provide a new application of abafungin in resisting agricultural pathogenic bacteria, which is used for preventing and controlling agricultural pathogenic bacteria of rice bacterial leaf blight pathogenic bacteria Xanthomonas Oryzae, citrus canker pathogenic bacteria Xanthomonas anopodis pv. citri, potato black shank pathogenic bacteria Pectobacterium atroseptica, agricultural pathogenic fungi Sclerotinia sclerotiorum, Rhizoctonia Solani Solani, Fusarium Graminearum, Botrytis cinerea, rice blast pathogenic bacteria Magnaporthe Oryza and Phytophtora Capsici Phytophtora Caprici.

In order to achieve the purpose, the invention provides the following technical method:

the new application of abafungin in resisting agricultural pathogenic bacteria comprises that the dosage concentration of abafungin to agricultural pathogenic bacteria of rice bacterial leaf blight pathogenic bacteria Xanthomonas oryzae, citrus canker pathogenic bacteria Xanthomonas anopodis pv. citri and potato black shank pathogenic bacteria Pebacterium atroseptica is 100, 50, 25, 12.5, 6.25, 3.12 and 1.56 mug/mL; the concentrations of the agricultural pathogenic fungi Sclerotinia sclerotiorum, Rhizoctonia Solani, Fusarium Graminearum, Botrytis cinerea, Magnaporthe Oryzae Oryzae and Phytophthora Capsici administered were 100, 50, 20, 10, 5, 2, 1, 0.5, 0.25. mu.g/mL.

The bactericide provided by the invention has the following advantages:

1) the invention discovers that the abafungin has excellent inhibiting effect on agricultural pathogenic bacteria for the first time, and can be further developed into a lead molecule with higher activity by taking the abafungin as a lead model.

2) The abafungin has simple structure, easy synthesis, low toxicity and wide antibacterial spectrum, and has the potential of being further developed into novel agricultural bactericides.

Detailed Description

The foregoing invention will be described in further detail by way of the following specific examples for a better understanding of the invention. This is not to be construed as limiting the invention. The experimental procedures described in the following examples are conventional unless otherwise specified.

Example 1: the structural formula of abafungin is as follows:

example 2: determination of Abafungin Activity against Agrogenic bacteria

The strain used in the experiment is a strain frozen and stored with 30% glycerol at-80 ℃ in a laboratory. The frozen strains were taken out, streaked on NB solid medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, agar: 15g, distilled water: 1L, pH 7.0; sterilized at 121 ℃ for 20min) of agricultural bacteria, respectively, and cultured at a constant temperature of 28 ℃ until single colonies grew out. Respectively picking single colony on the solid culture medium to an agricultural bacteria NB liquid culture medium (beef extract: 3g, peptone: 5g, yeast powder: 1g, sucrose: 10g, distilled water: 1L; sterilizing at 121 ℃ for 20min), and carrying out shake culture at 28 ℃ and 180rpm on a constant temperature shaking table until logarithmic phase. The strain in logarithmic growth phase was diluted to about 10 with the corresponding liquid medium⁶CFU/mL is ready for use. Respectively dissolving the compounds in DMSO, adding into liquid culture medium, mixing well, and preparing into liquid culture medium containing medicine with concentration of 200 μ g/mL. Taking 50 μ L of medicated culture medium and the same volume of the medicated culture medium containing 10⁶CFU/mL bacterial culture was added to the wells of a 96-well plate at a final dosing concentration of 100. mu.g/mL. 100 μ L of the same concentration of the bacterial suspension containing the same amount of DMSO was used as a control. Culturing 96-well plate in 28 deg.C incubator for 24-48 hr until reaching control group bacteriaThe solution was grown out, and the OD (OD) of the bacterial solution in the well was measured on a microplate reader₆₀₀). In addition, OD values of 100. mu.L of the liquid medium and the drug at a concentration of 100. mu.g/mL were measured, and the OD values of the medium and the drug themselves were corrected. The calculation formula for correcting the OD value and the inhibition rate is as follows:

correcting OD value-bacteria-containing culture medium OD value-sterile culture OD value;

inhibition rate (OD value of control culture medium liquid after correction-OD value of drug-containing culture medium after correction)/OD value of control culture medium liquid after correction × 100%

The drug-containing liquid medium of abafungin was diluted in a 96-well plate by a double dilution method to obtain 50. mu.L of drug-containing medium of serial concentrations, and then the inhibition rate according to the serial concentrations was measured according to the same test method as described above.

All experiments were performed in triplicate and the inhibition rates of the resulting compounds were determined as shown in table 1.

TABLE 1 Activity of Abafungin against Agriopathogenic bacteria

Note: "-" indicates that the antibacterial activity at this concentration was not determined

The minimum concentration at which the inhibition rate was greater than 90% was defined as the MIC, and the activity data obtained by the assay are shown in Table 2.

TABLE 2 MIC values of Abafungin against Agrogenic bacteria

As can be seen from the results of the bioassay in tables 1 and 2, abafungin according to the present invention shows excellent inhibitory activity against all strains tested, and has stronger activity against pathogenic bacteria of bacterial blight of rice and pathogenic bacteria of citrus canker than commercial tiotropium at 100. mu.g/mL. Wherein the action on pathogenic bacteria of citrus canker is strongest, and the minimum MIC value can reach 6.25 mu g/mL

Example 3: determination of activity of abafungin against agricultural pathogenic fungi

The agricultural pathogenic bacteria used in the experiment are strains preserved at 4 ℃ in a laboratory, and the adopted culture medium is a potato agar glucose culture medium (PDA for short). The PDA culture medium formula comprises: potato (peeled) 200g, glucose 20g, agar 15g, distilled water 1000mL, natural pH.

The PDA culture medium configuration method comprises the following steps: cleaning potato, peeling, weighing 200g, cutting into small pieces, boiling with distilled water for about 20min (the potato pieces are soft but not rotten), filtering with eight layers of gauze, adding distilled water to 1000mL of filtrate, adding 15g of agar and 20g of glucose, stirring to fully dissolve, subpackaging in triangular flasks, sterilizing at 121 ℃ for 20min, and cooling for later use. The indoor activity is measured by a hypha growth rate method.

Activating strains: culturing the agricultural pathogenic bacteria on a PDA flat plate at 25 ℃ for 3-6 days.

Preparing a medicine board: heating and melting PDA culture medium, cooling to 45-50 deg.C, and adding the mixture with different concentrations to make into flat plate with medicine.

Inoculating and culturing: in a super clean bench, a punch is used for beating a fungus cake (the diameter is 5mm) at the edge of hypha cultured for 3-6 days (the growth condition is as consistent as possible), then an inoculating needle is used for picking the fungus cake to the center of a medicine plate, and then the fungus cake is inversely cultured in an incubator (25 ℃).

And (4) determining the result: after the blank control group is full of hyphae, the growth diameter of the hyphae of the administration group is measured by a cross method, and the inhibition rate is calculated.

The inhibition rate (%) was (control hypha diameter-treated hypha diameter)/(control hypha diameter-cake diameter) × 100, and 3 parallel experiments were performed for each concentration, and the inhibition rate of the obtained compound was measured and shown in table 3.

TABLE 3 Activity of Abafungin against Agriopathogenic fungi

Half Effect Concentration (EC) was determined using SPSS software₅₀). The activity data obtained are shown in Table 4.

TABLE 4 Abafungin's EC against Agriopathogenic fungi₅₀Value of

Note: "-" indicates that the antibacterial activity of the compound was not determined

As is clear from the results of the bioassay in tables 3 and 4, abafungin according to the present invention shows excellent inhibitory activity against all the strains tested and half-Effect Concentration (EC) against agricultural pathogenic fungi₅₀) Are all stronger than the commercialized azoxystrobin. Wherein the abafungin has the best inhibitory activity on sclerotium of colza, EC₅₀The value was 0.39. mu.g/mL.

In conclusion, the abafungin of the invention shows broad-spectrum and high-activity characteristics to agricultural pathogenic bacteria and fungi, and has further research and development values.

Claims

1. The invention relates to a new application of abafungin in resisting agricultural pathogenic bacteria.

2. Use according to claim 1, wherein the molecular structural characteristics of abafungin are as follows:

。

3. use according to claim 1, wherein abafungin is used for controlling phytopathogenic bacteria and fungi.

4. Use according to claims 1 to 3, wherein abafungin is used for controlling bacterial blight of rice.

5. Use according to claims 1 to 3, wherein abafungin is used for the control of citrus canker.

6. Use according to claims 1 to 3, wherein abafungin is used for controlling potato blackleg.

7. Use according to claims 1 to 3, wherein abafungin is used for the control of Rhizoctonia solani.

8. Use according to claims 1 to 3, wherein abafungin is used for the control of Sclerotinia sclerotiorum.

9. Use according to claims 1 to 3, wherein abafungin is used for controlling wheat scab.

10. Use according to claims 1 to 3, wherein abafungin is used for the control of Botrytis cinerea.

11. Use according to claims 1 to 3, wherein abafungin is used for controlling Pyricularia oryzae.

12. Use according to claims 1 to 3, wherein abafungin is used for controlling Phytophthora capsici.