CN112471153A

CN112471153A - Application of kojic acid in inhibition of sclerotinia sclerotiorum growth

Info

Publication number: CN112471153A
Application number: CN202011407969.2A
Authority: CN
Inventors: 拉博德·佩德罗; 施欣驰; 祝桂洋; 王苏妍
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2021-03-12

Abstract

The invention discloses an application of kojic acid in inhibition of sclerotinia sclerotiorum growth, belonging to the technical field of biological agriculture. According to the invention, the kojic acid can inhibit the growth of sclerotinia sclerotiorum through research, so that the kojic acid is further applied to a preparation for treating and/or preventing the sclerotinia rot of green soy beans. The invention does not need complex molecular operation, the main active substance kojic acid in the preparation is a natural source, the preparation can be produced by adopting a biological method, and the kojic acid can be degraded on green soybean pods along with the time, thus being environment-friendly.

Description

Application of kojic acid in inhibition of sclerotinia sclerotiorum growth

Technical Field

The invention belongs to the technical field of biological agriculture, and particularly relates to application of kojic acid in inhibiting the growth of sclerotinia sclerotiorum and treating and/or preventing green soybean sclerotiniose.

Background

Green soy beans (Glycine max), also called vegetable soybeans, are vegetable soybeans that are used exclusively for fresh-eating tender pods in soybean crops. The green soy bean is originated in China, has about 5000 years of cultivation history in China, and is rich in nutrient substances such as vegetable protein, vitamin B group, dietary fiber and the like. However, green beans are significantly lost during growth and harvest due to climate and disease.

Sclerotinia sclerotiorum (sclerotiorum) is a fungus, can infect more than 400 kinds of plants, endanger stems, leaves and fruit pods of the plants, generate cotton-shaped hypha on the surface of a diseased part, and seriously cause the whole plant to die and rot. So far, the market often sprays 500 times of 40% dimetachlone wettable powder or 800 times of 50% carbendazim wettable powder in the early stage of disease. However, the methods have common effects, and the problems of pesticide residues and environmental pollution caused by the use of chemical agents are increasingly prominent. Therefore, the development of an effective, safe and environment-friendly fungicide has important significance for preventing and treating the sclerotinia rot of green soy beans and promoting the stable development of the green soy bean industry.

Kojic acid is an organic acid, mainly produced by filamentous fungi and by aspergillus, penicillium and acetobacter bacteria, and is one of the oldest natural antibiotics. It has wide application in the fields of cosmetics and food processing, but the research of using kojic acid to prevent and treat plant pathogenic fungi is still blank.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of kojic acid, which has a significant effect of inhibiting the growth of sclerotinia sclerotiorum and can be used for the preparation of related drugs.

Kojic Acid (KA), 5-hydroxy-2-hydroxymethyl-1, 4-pyrone, molecular formula C₆H₆O₄The structural formula is as follows:

the invention discovers that kojic acid can inhibit the growth of plant pathogenic fungi sclerotinia sclerotiorum through research, so the invention provides the application of the kojic acid in inhibiting the growth of the sclerotinia sclerotiorum.

According to the application, the invention provides a preparation for inhibiting the growth of sclerotinia sclerotiorum, and the active ingredient of the preparation is kojic acid.

According to the literature (DH Jiang, YP Fu, GQ Li, et al. viruses of the plant pathogenic fungi microorganisms. Advances in viruses research.2013,86:215-248.), Sclerotinia sclerotiorum can cause Sclerotinia sclerotiorum, so the invention further uses kojic acid as a control agent for Sclerotinia sclerotiorum and researches.

According to the invention, water is used as a control, green soy bean pods (prevention group) inoculated with a sclerotinia sclerotiorum cake after being sprayed with 50mM kojic acid and green soy bean pods (treatment group) inoculated with a sclerotinia sclerotiorum cake and sprayed with a 50mM kojic acid solution are used as test groups, and the growth condition of sclerotinia sclerotiorum is observed after 24h, so that the growth of germs at the inoculated part of the control green soy bean pods in the prevention group is found to be serious and the invasion degree is large, after the sclerotinia sclerotiorum is remarkably inhibited after the control green soy bean pods are subjected to the prevention treatment of the kojic acid solution, and the infection degree of sclerotinia; in the treatment group, the part of the sclerotinia sclerotiorum inoculated by the control green soybean pod is infected in a large area, and the growth of the sclerotinia sclerotiorum is completely inhibited after the treatment by the kojic acid solution, which indicates that the kojic acid can effectively treat the sclerotiniose.

Compared with the prior art, the invention applies kojic acid to inhibit the growth of sclerotinia sclerotiorum and provides a preparation for treating and/or preventing green soybean sclerotiniose. The invention does not need complex molecular operation, the main active substance kojic acid in the preparation is a natural source, the preparation can be produced by adopting a biological method, and the kojic acid can be degraded on green soybean pods along with the time, thus being environment-friendly. In particular, the preparation for treating and/or preventing the sclerotinia rot of green soybean is 50mM kojic acid solution, the solvent is water, the treatment and/or prevention effect is optimal compared with other concentration preparations, and the preparation method is simple.

Drawings

FIG. 1 is a histogram showing the colony diameters of Sclerotinia sclerotiorum cultured in the PDA medium prepared in example 1 using kojic acid at concentrations of 0mM, 5mM, 10mM and 15 mM.

FIG. 2 is a graph showing the growth of sclerotinia in example 1 on PDA solid medium containing kojic acid at 0mM, 5mM, 10mM, 15 mM.

FIG. 3 is a histogram of conidia aggregate counts in YEPD medium at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 in example 2, with and without 10mM kojic acid.

FIG. 4 shows the presence of different complex metal ions (Fe) in example 3²⁺、Ni²⁺、Zn²⁺、Co²⁺、Cu²⁺) The YEPD medium of (1) shows a histogram of conidium aggregate counts with and without kojic acid.

FIG. 5 is a graph showing the growth of Sclerotinia leguminosa obtained in the control experiment and the kojic acid treatment experiment of the preventive group in example 4.

FIG. 6 is a graph showing the growth of Sclerotinia leguminosa obtained in the control experiment and the kojic acid treatment experiment of the treatment group in example 4.

FIG. 7 is a time-varying line graph of kojic acid content in green soybean pods from example 5.

Detailed Description

The invention discloses the application of kojic acid, and the technical personnel can appropriately improve the technological parameters for realization by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples.

Example 1

Antifungal Activity detection of kojic acid

Preparing a fungus cake: inoculating the sclerotinia sclerotiorum strain stored on the inclined plane into a PDA solid culture medium, culturing for 3 days in a constant temperature box at 28 ℃ in a dark place, cutting the edge of a bacterial colony by using a hole puncher with the diameter of 7mm, and preparing into a bacterial cake for later use.

The PDA culture medium is prepared according to the formula shown in Table 1, boiled for about 30min, and sterilized by wet heat sterilization at 121 deg.C for 15min after each component is dissolved.

TABLE 1 PDA culture medium formulation (1L)

Reagent	Quality of
		Potato	200g
Glucose	20g
		Agar powder	15g
Kojic acid aqueous solutions of different concentrations	1L

PDA medium containing 0mM, 5mM, 10mM, 15mM kojic acid was prepared separately, and the cake was inoculated at the center position of PDA medium, three sets of replicates for each concentration were set. The plates were incubated at 28 ℃ in the dark and the colony diameter in each plate was determined three days later.

The measurement results are shown in FIG. 1, which is a histogram of the colony diameters of Sclerotinia sclerotiorum obtained by culturing PDA medium containing kojic acid at various concentrations in FIG. 1. From FIG. 1, it was found that the colony diameter of Sclerotinia sclerotiorum obtained by the PDA culture medium prepared with kojic acid aqueous solution having a concentration of 15mM was the smallest, indicating that the higher the concentration of kojic acid aqueous solution was, the more significant the inhibitory effect on Sclerotinia sclerotiorum was.

The growth of Sclerotinia sclerotiorum was observed on one of the groups of aqueous kojic acid solutions with concentrations of 0mM, 5mM, 10mM, and 15mM for PDA preparation. As shown in FIG. 2, FIG. 2 is a graph showing the growth of Sclerotinia sclerotiorum on PDA solid medium containing kojic acid at 0mM, 5mM, 10mM, and 15 mM. According to the graph, the smaller the diameter of the sclerotinia sclerotiorum colony is, the better the bacteriostasis effect is, can be found along with the increase of the concentration of the kojic acid aqueous solution.

Example 2

Sclerotinia resistance of 10mM kojic acid solution under different pH environments

The sclerotinia sclerotiorum cake was prepared in the same manner as in example 1.

The formula of the barley-honey-peptone culture medium is shown in table 2, and after the preparation of the raw materials, the raw materials are sterilized by a damp-heat method at 121 ℃ for 1 hour.

TABLE 2 barley-Honey-peptone Medium formulation (40mL)

Reagent	Quality of
		Barley	100mg
Honey	600mg
		Peptone	200mg
Distilled water	40mL

Conidium production induction and extraction: fresh sclerotinia sclerotiorum cake is inoculated into 40mL barley-honey-peptone culture medium, and after shaking culture at 28 ℃ and 200rpm for 12h, the bacterial liquid is divided into 20 parts. The cells were centrifuged at 5000rpm for 5min at 4 ℃ and the supernatant was discarded to retain the cells. After 1mL of sterilized water is added into each tube to resuspend the fungi, the supernatant is centrifuged again and discarded, and the step is repeated twice to obtain fresh conidia.

Conidia were resuspended in 1mL of YEPD solutions at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, two flasks were set at each pH gradient, 71.05mg of kojic acid was added to one flask to prepare YEPD liquid medium with a final concentration of 10mM kojic acid, and the other flask was used as a Control (Control). Culturing at 28 deg.C and 200rpm for 12 hr, sucking 0.5 μ L bacterial solution with pipette gun, and counting the aggregates formed by aggregation of spores under microscope. YEPD culture medium formula is shown in Table 3, relying on HCl and Na₂CO₃The pH of the YEPD medium was adjusted.

TABLE 3 YEPD culture solution formula (50mL)

Reagent	Quality of
		Yeast extract	150mg
Tryptone	500mg
		Glucose	1g
Distilled water	50mL

The counting results are shown in FIG. 3, and FIG. 3 is a histogram of the count of conidium aggregates in 0.5. mu.L of the bacterial suspension of Sclerotinia sclerotiorum cultured in the control and experimental groups at pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0. From FIG. 3, it can be found that the number of conidium aggregates decreases with an increase in pH, and that the number of conidium aggregates is large when pH is 3 or 4, while Sclerotinia sclerotiorum hardly grows under alkaline conditions.

In addition, the inhibition rate of kojic acid on the growth of sclerotinia (inhibition rate ═ total number of aggregates in the control group/total number of aggregates in the control group (total number of aggregates in the control group-10 mM kojic acid group) was calculated under acidic and neutral conditions in which sclerotinia can grow, i.e., pH 3.0, 4.0, 5.0, 6.0, 7.0. The inhibition rates of the compounds at pH values of 3.0, 4.0, 5.0, 6.0 and 7.0 are 42%, 43%, 57%, 47% and 38%, respectively, and the growth inhibition ability of kojic acid against Sclerotinia sclerotiorum is not greatly different between different pH values. It is shown that the ability of kojic acid to inhibit the growth of sclerotinia is less affected by pH in a pH environment where sclerotinia can grow.

Example 3

Effect of different complexing Metal ions on the anti-Sclerotinia resistance of kojic acid

Sclerotinia sclerotiorum cake was prepared in the same manner as in example 1, and conidium production was induced and extracted in the same manner as in example 2.

Separately resuspending conidia with 50mL YEPD solution with pH of 4.0, preparing 12 bottles, and using two bottles as control group without adding metal ion, Fe²⁺、Zn²⁺、Ni²⁺、Co²⁺、Cu²⁺Each metal ion is provided with 2 bottles, and FeCl is added₂、NiCl₂、ZnCl₂、CoCl₂、CuCl₂The final concentration of each metal ion in YEPD medium was controlled to 1 mM. 2 flasks contained YEPD medium containing the same metal ion, one flask was supplemented with 71.05mg of kojic acid to prepare YEPD medium at a final concentration of 10mM kojic acid, and the other flask served as a control group. After culturing at 28 ℃ and 200rpm for 12 hours, 0.5. mu.L of the inoculum was aspirated by a pipette and the aggregates formed by the agglutination of the spores were counted under a microscope.

The results are shown in FIG. 4, where FIG. 4 shows the presence of different complex metal ions (Fe)²⁺、Ni²⁺、Zn²⁺、Co²⁺、Cu²⁺) The YEPD medium of (1) shows a histogram of conidium aggregate counts with and without kojic acid. It can be found that Fe is in contrast to the group without metal ions and kojic acid²⁺、Co²⁺No significant difference in aggregate number when present alone, Cu²⁺、Zn²⁺And Ni²⁺The number of aggregates decreases when present alone. In contrast to the kojic acid group without metal ions, Fe²⁺The aggregate number has no obvious difference after being mixed with kojic acid, and Co² ⁺The aggregate number increases after mixing with kojic acid. Explanation, Fe²⁺、Co²⁺No inhibition of sclerotinia growth when existing alone, Cu² ⁺、Zn²⁺And Ni²⁺When the compound exists alone, the compound has an inhibiting effect on the growth of sclerotinia sclerotiorum; fe²⁺Co does not alter the antibacterial activity of kojic acid after mixing with kojic acid²⁺Can reduce the anti-sclerotinia activity of kojic acid after being mixed with kojic acid.

Example 4

The medicine with kojic acid as main component can be used for preventing and treating soybean pod sclerotinia rot.

The sclerotinia sclerotiorum cake was prepared in the same manner as in example 1. 50mL of 10mM, 20mM, and 50mM aqueous solutions of kojic acid were prepared with purified water, respectively. Picked green soybean pods with similar growth conditions were taken and used for the experiments.

Prevention group: the aqueous solution was uniformly sprayed onto three groups of green soy bean pods (15 per group) respectively, with water spray set as a negative control. Pricking small holes with diameter of about 2mm on the surface of the pod by using toothpicks, inoculating a fungus cake on each hole by using sterilized tweezers, then putting the fungus cake into a tray, winding and fixing the fungus cake by using a preservative film, keeping the fungus cake moist, and observing the infection condition of the wound after three days.

Treatment groups: pricking a small hole with the diameter of 7mm on the surface of the pod by using a toothpick, inoculating a sclerotinia sclerotiorum cake on the hole by using a sterilized forceps, then putting the sclerotinia sclerotiorum cake into a tray, winding and fixing the sclerotinia sclerotiorum cake by using a preservative film, keeping the sclerotinia sclerotiorum cake moist, and removing the sclerotinia cake after 48 hours. 50mM kojic acid solution was uniformly sprayed onto the green soybean pods, and the same volume of water spray was set as a negative control. Wound infection was observed after 24 h.

The results are shown in fig. 5 and 6, and fig. 5 is a graph showing the growth of sclerotinia leguminosa obtained in the control experiment and the kojic acid treatment experiment of the prevention group; FIG. 6 is a graph showing the growth of Sclerotinia leguminosa in the control experiment and the kojic acid treatment experiment of the treated group.

From fig. 5, it can be found that, in the prevention group, compared with the green soybean pods obtained in the control experiment (water spraying), the wound color of the part of the green soybean pods inoculated with sclerotinia sclerotiorum in the kojic acid treatment experiment group is lighter, and the lighter the wound color is with the increase of the concentration of kojic acid, which indicates that the kojic acid can effectively prevent sclerotinia sclerotiorum, and the higher the concentration of the kojic acid is, the better the prevention effect is.

From fig. 6, it can be found that, in the treatment group, after the sclerotinia sclerotiorum is inoculated to the soybean pods of the control experiment group (water spraying), the colony infection range spreads to the outside of the inoculated part, and after the sclerotinia sclerotiorum is treated by 50mM kojic acid, the sclerotinia sclerotiorum almost does not diffuse outwards, which indicates that the kojic acid can effectively control the disease diffusion.

Example 5

Stability detection of kojic acid in green soy bean pods

25 green soy bean pods were soaked in 100mL of 10mM kojic acid solution for 30min, removed, placed in a tray, and allowed to stand at room temperature. On

days

0, 1, 3, 5, and 10, 0.5g of each of 5 green soybean pods was cut out with a knife and placed in 5 centrifuge tubes of 1.5mL, respectively, and stored at 4 ℃. After all green soybean pod samples are prepared, 200 mu L of methanol is added into each tube, vortex and shake are carried out for 2min, and the kojic acid concentration is detected by a High Performance Liquid Chromatography (HPLC).

HPLC method, the apparatus is Agilent 1200(California, USA), using C18 chromatographic column (250mm × 4.6mm, Phenomenex), ultraviolet detecting wavelength is 283nm, and H is₂O is mobile phase, the flow rate is 1mL/min, the elution time is 20min, and the column temperature box is 60 ℃. The residence time of kojic acid was 5.9 min. The kojic acid was quantified using an external standard method.

The results are shown in FIG. 7, which is a time-varying line graph of kojic acid content in green soybean pods. From fig. 7 it can be seen that the concentration of kojic acid decreased gradually over time and that the presence of kojic acid could not be detected at day ten, indicating that kojic acid was degraded over time. The degradable property can avoid the adverse effect of the sustained existence of kojic acid on plants and environment.

In the examples of the present invention, the unit symbol mM is mmol/L, and M is mol/L.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. Application of kojic acid in inhibiting sclerotinia sclerotiorum growth is provided.

2. A formulation for inhibiting the growth of sclerotinia in a plant, characterized by: the active ingredient of the preparation is kojic acid.

3. A preparation for treating and/or preventing sclerotinia rot of green soybean, characterized by: the main active ingredient of the preparation is kojic acid.

4. The formulation of claim 3, wherein: the preparation is kojic acid solution.

5. The formulation of claim 4, wherein: the concentration of the kojic acid solution is 50 mM.