Application of usnic acid or usnic acid sodium in prevention and treatment of wheat scab

Abstract

The invention provides application of usnic acid or usnic acid sodium in prevention and treatment of wheat scab, and belongs to the technical field of pesticides. The natural product usnic acid and the sodium salt thereof disclosed by the invention have a bacteriostatic effect on phytopathogens including fusarium graminearum, pseudofusarium graminearum, magnaporthe oryzae and rhizoctonia cerealis, the content of DON toxin of the fusarium graminearum can be remarkably reduced, and the usnic acid and the sodium salt thereof have good inhibitory activity on fusarium graminearum indoors and in a field; and an important basis is provided for discovery and creation of natural product new pesticides.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, and in particular to application of usnic acid or sodium usnic acid in preventing and controlling wheat scab. Background Art

[0002] Wheat fusarium scab caused by Fusarium graminearum infection of wheat is an epidemic disease worldwide and is known as the "cancer" of wheat. In recent years, with global warming and changes in farming systems, the area of ​​wheat fusarium scab in my country has gradually expanded and is showing a worsening trend. In epidemic years, the yield loss is as high as 20%-50%. In addition to causing serious yield and economic losses, fungal toxins such as deoxynivalenol (DON) produced by fusarium graminearum on diseased grains can remain in the food chain for a long time, causing anorexia, vomiting, diarrhea, etc., and can also suppress immune function, posing a serious threat to human and animal health. How to reduce the occurrence of fusarium scab and toxin pollution is a practical production problem that needs to be solved urgently.

[0003] At present, the prevention and control of wheat fusarium head blight mainly relies on chemical control, and there are few effective chemical fungicides, mainly triazoles, 2-cyanoacrylates and SDHIs. Long-term use will lead to frequent problems of pathogen resistance and drug resistance. At the same time, the continuous and large-scale use of agents has brought about increasingly serious problems such as excessive environmental pesticide residues, water ecological pollution, and food safety. Natural products have the characteristics of low toxicity, environmental friendliness, and structural diversity, providing many excellent molecules for the development of new drugs and fungicides. Therefore, finding safe and effective natural products is of great significance for the prevention and control of wheat fusarium head blight and the development of new lead compounds or fungicides.

[0004] Usnic acid is one of the most common secondary metabolites in lichens. It was first isolated by German scientist Knop in 1844. Usnic acid has good antioxidant, anti-inflammatory, antibacterial and anti-tumor activities. However, poor solubility and low utilization in the human body limit the application and development of usnic acid in medicine. The problem to be solved by the present invention is to use usnic acid and its sodium salt to prevent and treat wheat scab, so as to overcome the negative effects of chemical agents and provide an important basis for the discovery and creation of new natural product pesticides. Summary of the invention

[0005] The natural product usnic acid and its sodium salt provided by the present invention show good inhibitory activity against wheat fusarium indoors and in the field, and can also effectively inhibit the growth of other plant pathogenic fungi such as pseudo-Fusarium graminearum, rice blast and sheath blight.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention discloses a use of usnic acid or sodium usnic acid as an antibacterial agent for plant pathogenic fungi Fusarium graminearum, pseudo-Fusarium graminearum, rice blast fungus and Rhizoctonia graminearum.

[0008] The present invention also provides the use of usnic acid or sodium usnic acid in preventing and treating wheat fusarium scab, wherein the pathogen of wheat fusarium scab is the Fusarium graminearum described in claim 1.

[0009] Preferably, the usnic acid is sprayed in the form of a usnic acid suspension when preventing and controlling wheat fusarium head blight.

[0010] Preferably, the concentration of the usnic acid suspending agent is 28-32 wt %.

[0011] Preferably, the usnic acid suspension is sprayed during the flowering period of wheat.

[0012] Application of usnic acid or sodium usnic acid in reducing the content of deoxynivalenol in wheat infected with fusarium head blight.

[0013] The natural product usnic acid and its sodium salt described in the present invention show good inhibitory activity against wheat fusarium both indoors and in the field, and can also effectively inhibit the growth of other plant pathogenic fungi such as Fusarium graminearum, rice blast, and sheath blight. Conidia serve as the primary infection source of many plant fungal diseases such as wheat fusarium, wheat stem rot, wheat sheath blight and rice blast. Usnic acid and sodium usnic acid both show good spore germination inhibitory activity and can be used as spore germination inhibitors, providing an important basis for the discovery and creation of new natural product pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are pictures of Fusarium graminearum, Pseudomonas graminearum, Magnaporthe oryzae, and Rhizoctonia graminearum growing for different days under different concentrations of usnic acid in Example 1;

[0015] Figure 2 These are pictures of Fusarium graminearum, Pseudomonas graminearum, Magnaporthe oryzae, and Rhizoctonia graminearum growing for different days under different concentrations of sodium usnicotinate in Example 1;

[0016] Figure 3 The growth inhibition rates of different concentrations of usnic acid on Fusarium graminearum, Pseudofusarium graminearum, Magnaporthe oryzae, and Rhizoctonia graminearum in Example 1;

[0017] Figure 4 The growth inhibition rates of different concentrations of sodium usnicotinate on Fusarium graminearum, Pseudofusarium graminearum, Magnaporthe oryzae, and Rhizoctonia graminearum in Example 1;

[0018] Figure 5 This is the observation result of the edge of the Fusarium graminearum colony in Example 1;

[0019] Figure 6The inhibition rate of spore germination of Fusarium graminearum under the treatment of different concentrations of usnic acid and sodium usnic acid in Example 1 is statistically shown;

[0020] Figure 7 This is the transmission electron microscopy observation of Fusarium graminearum spores in Example 1, the scale bar is 1 μm;

[0021] Figure 8 The results of the determination of pathogenicity of wheat bud sheath in Example 2;

[0022] Fig. 9 The results of experimental example 1 on the toxicity of high concentration usnic acid to wheat seedlings;

[0023] Fig.10 In Experimental Example 2, 20 μg / ml was included or not included -1 The DON toxin content of Fusarium graminearum in the liquid toxin-producing induction culture medium of usnic acid and usnic acid sodium. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1

[0026] Usnic acid (98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The chemical structure is shown below:

[0027]

[0028] Sodium usnic acid (≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. The chemical structure is shown below:

[0029]

[0030] Preparation of usnic acid solution: Prepare 5 mg / ml in acetone;

[0031] Preparation of sodium usnicoate solution: dissolve 20 mg sodium usnicoate powder in 1 ml ethanol, then add 2 ml water.

[0032] Preparation of 30% usnic acid suspension: (1) Add accurately measured usnic acid (75 g), surfactant: SC3266 (6%), antifreeze: ethylene glycol (3%), thickener: xanthan gum (0.2%), preservative: kason (0.2%), defoamer: 1522 (0.2%) and water (make up to 100%) into a stirrer, stir evenly with the stirrer to obtain a mixed solution; (2) Add the mixed solution in step (1) into a vertical sand mill (grinding speed 2000 rpm, material: grinding medium = 1:1.5) for grinding. After grinding for 1 hour, take a sample to measure the particle size. The particle size DV90 is not greater than 5 μm. Stop grinding, pour into a beaker and mix evenly, a total of 250 ml.

[0033] Strains: standard strain of Fusarium graminearum PH-1, pseudo-Fusarium graminearum strain CF14047 (isolated by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences), standard strain of Magnaporthe oryzae 70-15, and Rhizoctonia graminearum strain R0301 (isolated by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences).

[0034] (1) Plate activity test

[0035] The standard strain of Fusarium graminearum PH-1 (Fg), the pseudo-Fusarium graminearum strain CF14047 (Fp), the standard strain of Magnaporthe oryzae 70-15 (Mo), and the Rhizoctonia graminearum strain R0301 (Rc) were inoculated on a 1 / 2CM plate containing medicine (5 g glucose, 1 g peptone, 0.5 g yeast extract, 0.5 g casamino acid, 3 g sodium nitrate, 0.25 g potassium chloride, 0.5 g magnesium sulfate heptahydrate, 0.75 g potassium dihydrogen phosphate, 15 g agar, 1 L distilled water, sterilized at 121°C for 20 min) (the concentrations of usnic acid and its sodium salt were 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml, respectively), and cultured at 25°C for 3 days (Fg), 4 days (Fp), 6 days (Rc), and 10 days (Mo), respectively, and then the colony diameters were measured and photographed and recorded ( Figure 1-2 ).

[0036] Inhibition rate (%) = (control mycelium diameter - treated mycelium diameter) / (control mycelium diameter - cake diameter) × 100, and three parallel experiments were set for each concentration.

[0037] The inhibition rate results show that the inhibition rate of the compound is: under usnic acid treatment ( Figure 3), the inhibition rates of Fg were 46.63%, 67.04%, 78.51%, 79.70% and 84.13%, respectively; the inhibition rates of Fp were 29.11%, 43.19%, 53.99%, 37.58% and 43.75%, respectively; the inhibition rates of Mo were 40.08%, 55.08%, 64.76%, 91.88% and 62.67%, respectively; the inhibition rates of Rc were 16.66%, 20.86%, 26.80%, 20.86% and 19.97%; under the treatment of sodium usnic acid ( Figure 4 ), the inhibition rates of Fg were 51.16%, 73.6%, 77.03%, 78.14% and 81.48% respectively; the inhibition rates of Fp were 37.02%, 46.03%, 65.63%, 59.83% and 63.65% respectively; the inhibition rates of Mo were 37.98%, 56.25%, 98.56%, 100% and 100% respectively; the inhibition rates of Rc were 28.09%, 32.16%, 48.54%, 50.89% and 50.90% respectively.

[0038] In summary, at a concentration of 16 μg / ml, the inhibition rates of usnic acid against the four pathogens were 84.13%, 43.75%, 62.67% and 19.97%, respectively. At the same concentration, the inhibition rates of sodium usnic acid against the four pathogens were 81.48%, 63.65%, 100% and 50.90%, respectively. This shows that usnic acid has a good inhibitory effect on Fusarium graminearum, Pseudomonas graminearum and Magnaporthe oryzae, and its sodium salt has a good inhibitory effect on the four pathogens, and the inhibitory activity against each pathogen is higher than that of usnic acid.

[0039] Compared with other pathogens, usnic acid has the highest inhibitory activity against Fusarium graminearum, with an inhibition rate of nearly 50% (46.63%) at a concentration of 1 μg / ml; the best inhibitory activity against Magnaporthe grisea is 91.88% at a concentration of 8 μg / ml, and the inhibitory activity against Rhizoctonia graminearum is poor at different concentrations. Sodium usnic acid has the highest inhibitory activity against Magnaporthe grisea, with inhibition rates of 37.98%, 56.25%, 98.56%, 100%, and 100% at 1 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, and 16 μg / ml, respectively. The second is Fusarium graminearum, with inhibition rates of 51.16%, 73.60%, 77.03%, 78.14%, and 81.48% at the following concentrations. The inhibition rates of Pseudofusarium graminearum and Rhizoctonia graminearum were significantly improved, and the concentrations were 37.02%, 46.03%, 65.63%, 59.83%, 63.65% for Pseudofusarium graminearum and 28.09%, 32.16%, 48.54%, 50.89%, 50.90% for Rhizoctonia graminearum. In general, usnic acid and sodium usnic acid can significantly inhibit the growth of Fusarium graminearum, Pseudofusarium graminearum, Magnaporthe oryzae and Rhizoctonia graminearum, especially Fusarium graminearum and Magnaporthe oryzae.

[0040] (2) Colony edge observation

[0041] Dissolve 1 / 2CM solid culture medium and pour into a glass dish with a slide, inoculate the standard strain PH-1 on the slide, and culture overnight for 16h (plate without drug addition) and 24h (plate containing 18μg / ml usnic acid and 12.5μg / ml sodium usnic acid). Microscopic observation of hyphae morphology at the edge of the colony ( Figure 5 ). Comparing the observation results of the colony edge of the wild type (CK) and the mutant (drug-treated group), the wild type had neat colony edges and linear hyphae, while the hyphae at the colony edge after usnic acid and usnic acid treatment were dense and the hyphae branches increased significantly, especially after sodium usnic acid treatment, the hyphae were highly dense and highly branched. Therefore, usnic acid and its sodium salt treatment affected the polar growth of hyphae, which may be the main reason for the slow growth of the colony.

[0042] (3) Determination of the inhibition rate of usnic acid and its sodium salt on the germination of conidia of Fusarium graminearum

[0043] The standard strain PH-1 was inoculated on a PDA plate (200 g of potato was boiled and filtered to remove the residue, 20 g of glucose, 15 g of agar, and the volume was adjusted to 1 L with pure water, and sterilized at high temperature and high pressure), cultured at 25°C for 3 days, and the mycelium block was inoculated with a CMC spore-forming medium (15 g of sodium carboxymethyl cellulose, 1 g of NH4NO3, 1 g of KH2PO3, 0.5 g of MgSO4·7H2O, 1 g of yeast extract, and the volume was adjusted to 1 L with pure water, and sterilized at high temperature and high pressure), and cultured at 25°C for 5 days. The spores were collected and transferred to a YEPD liquid medium containing different concentrations of usnic acid or sodium usnic acid (3 g of yeast extract, 10 g of peptone, 20 g of sucrose, 1 L of distilled water, and sterilized at 121°C for 20 min), and the spores were cultured for 6 hours (the germination rate of wild-type spores was more than 95%). The control and different concentrations of usnic acid (0 μg / ml, 0.625μg / ml, 1.25μg / ml, 2.5μg / ml, 5μg / ml, 10μg / ml, 20μg / ml) and sodium usnicoate (0μg / ml, 0.39μg / ml, 0.78μg / ml, 1.56μg / ml, 3.125μg / ml, 6.25μg / ml, 12.5μg / ml), and the germination rate was calculated. The results are shown in Figure 6 As shown. Usnic acid can inhibit 63.53% of spore germination at 5μg / ml, and 20μg / ml of usnic acid completely inhibits spore germination (inhibition rate 100%). Sodium usnic acid can inhibit 71.61% of spore germination at 3.125μg / ml, and 12.5μg / ml of sodium usnic acid completely inhibits spore germination (inhibition rate 100%). Therefore, usnic acid and its sodium salt show strong activity in inhibiting spore germination of Fusarium graminearum.

[0044] (4) Observation of the ultrastructure of conidia after treatment with usnic acid and its sodium salt (transmission electron microscopy)

[0045] PH-1 was inoculated on a PDA plate and cultured at 25°C for 3 days. Mycelial blocks were inoculated into CMC spore-forming medium and cultured at 25°C for 5 days. Spores were collected and transferred to YEPD liquid medium containing usnic acid (40 μg / ml) and its sodium salt (25 μg / ml). The spores were cultured for 4 hours (spores to be germinated before the emergence of the germ tube). Spores were collected and fixed in 2.5% glutaraldehyde solution and washed three times with ultrapure water for 10 minutes each time. The spores were ... the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2.5% glutaraldehyde solution and the spores were fixed in 2 Fix for 2 hours, wash three times with ultrapure water, each time for 10 minutes; dehydrate with 50%, 70%, 80%, and 90% acetone, respectively, for 15 minutes each; dehydrate with pure acetone three times, each time for 30 minutes; replace pure acetone: epoxy resin at a ratio of 3:1 (1 hour), 1:1 (3 hours), and 1:3 (overnight); embed the sample with pure resin, put the embedding plate in an oven at 37°C, 45°C, and 60°C for 24 hours, 24 hours, and 48 hours, respectively, use a Leica EMUC7 ultrathin section machine to make ultrathin sections, and observe with a Hitachi H-7650 transmission electron microscope after double staining with phosphotungstic acid-uranyl acetate.

[0046] The results are as follows Figure 7 As shown in the figure, the spores to be germinated normally have regular shapes, and the fat particles are round or oval, neatly arranged on the inner side of the cell membrane. However, after treatment with usnic acid and its sodium salt, the cells are deformed, the outermost layer of the cell wall is thickened, and almost no fat particles can be seen in the cells after treatment with usnic acid. After treatment with sodium usnic acid, the fat particles in the cells are not regular round or oval. The fat particles provide nutrients and substances for the germination of spores and the elongation of germ tubes. Therefore, the effects of usnic acid and its sodium salt on cell morphology, cell wall construction and fat particles are likely to be the main reason for inhibiting spore germination.

[0047] Example 2

[0048] Determination of the efficacy of usnic acid and its sodium salt against wheat scab indoors and outdoors

[0049] Wheat bud sheath

[0050] Two days after the wheat seeds were germinated, they were neatly placed in a glass dish covered with filter paper to keep them moist. The top of the bud sheath was cut off and wrapped with a small cotton ball. 150 μl of usnic acid diluted solutions with concentrations of 100 μg / ml, 200 μg / ml, and 400 μg / ml were dripped onto the cotton ball. After 2 hours, 1×10 5 2 μl of spore suspension was dried and placed in a humidifying box in a 25°C greenhouse for 7 days. The results showed that 400 μg / ml of usnic acid had the same control effect as 100 μg / ml of tebuconazole ( Figure 8 ).

[0051] Wheat ear inoculation

[0052] During the flowering period of wheat, 30% usnic acid suspension (200 ml / mu) was sprayed, and the control agent was 48% cyanobacterium-tebuconazole suspension (50 ml / mu). A control group (no agent treatment), usnic acid treatment group and control agent treatment group were set up, with three replicates each. Ten ears were randomly selected from each replicate, and the standard strain PH-1 spore suspension (concentration of 2×10 5 Inoculate the spikelets in the middle of the ear with 15 μl of 10 ...

[0053] The results are shown in Table 1. The disease index of the control was 2.43, the disease index of 30% usnic acid suspension was 1.48, and the disease index of the control agent was 0.42. This proves that although it cannot achieve the effect of chemical agents, the spraying of usnic acid can significantly reduce the incidence index of wheat fusarium rust and can be used as a potential natural compound for the prevention and treatment of wheat fusarium rust.

[0054] Table 1 Effect of 30% usnic acid suspension on wheat scab

[0055]

[0056] Note: Different letters after the data in the same column in the table indicate significant differences at the P<0.05 level tested by the least significant difference method (LSD method).

[0057] Experimental Example 1

[0058] Cell Trial

[0059] Set up four treatments: control, 30% usnic acid sprayed twice at the beginning and peak of flowering, 30% usnic acid sprayed once at the beginning of flowering, and 48% cyanobacterium-tebuconazole sprayed once at flowering. Each treatment was repeated in three plots, and each plot had an area of ​​20m 2 . The disease severity was investigated 20 days after the last spraying. Five sampling points were taken in each plot, and each point was investigated with a distance of 2.5 m 2 The diseased ears with fusarium scaly in the iron ring were graded according to the percentage of the diseased ear area to the whole ear area, and the number of diseased ears and the total number of ears in each grade were recorded.

[0060] Grading Standard:

[0061] Level 0: The whole ear is disease-free;

[0062] Level 1: The diseased ear area accounts for less than 1 / 4 of the total ear area;

[0063] Level 3: The diseased ear area accounts for 1 / 4 to 1 / 2 of the whole ear area;

[0064] Level 5: The diseased ear area accounts for 1 / 2 to 3 / 4 of the whole ear area;

[0065] Level 7: The diseased ear area accounts for more than 3 / 4 of the total ear area.

[0066] Diseased ear classification:

[0067]

[0068] Table 2 Field efficacy of 30% usnic acid suspension on wheat fusarium head blight

[0069]

[0070] Note: Different letters after the data in the same column in the table indicate significant differences at the P<0.1 level tested by the least significant difference method (LSD method).

[0071] The results of the plot test are shown in Table 2. The control effect of 30% usnic acid on wheat scab was close to 60% (59.48%) when applied once during the flowering period. The results also showed that usnic acid suspension can effectively reduce the content of DON on diseased grains.

[0072] At the same time, in order to clarify whether usnic acid has toxic effects on wheat, we compared wheat without usnic acid (DDW) and with usnic acid (400μg / ml Usnic acid) from germination to seedling stage. The results showed that usnic acid had no effect on wheat seed germination and seedling growth ( Fig. 9 ). This proves that usnic acid has no toxic side effects on wheat.

[0073] Experimental Example 2

[0074] Method: 1×10 5 The spore suspension was inoculated into toxin-producing inducing medium (TBI), shaken at 25°C and 100 rpm in the dark for 7 days, filtered, the mycelium was collected, dried and weighed; the filtrate was collected in a 50 ml centrifuge tube, centrifuged at 8000 rpm for 5 min, 5 ml of the supernatant was aspirated and extracted with 2 ml of ethyl acetate, 1 ml of the upper extract was aspirated, dried with a nitrogen stream at 50°C, 1 ml of chromatographic methanol: water (20:80) was added, all 1 ml of the sample was passed through a 0.22 μm organic microporous filter membrane, the filtrate was collected, and it was diluted 100 times and then determined by high performance liquid chromatography.

[0075] The results are as follows Fig.10 As shown, in the liquid toxin-producing induction medium, compared with the control, 20 μg / ml -1 The DON content of Fusarium graminearum was significantly reduced after treatment with usnic acid.

[0076] Combining the results of Experimental Examples 1 and 2, it is shown that usnic acid can effectively reduce the toxin synthesis of wheat fusarium sphaeroides.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of usnic acid or sodium usnic acid as an antibacterial agent for plant pathogenic fungi Fusarium graminearum, Pseudomonas graminearum, Magnaporthe oryzae and Rhizoctonia graminearum.

2. The use of usnic acid or sodium usnic acid in preventing and controlling wheat scab, characterized in that: The pathogen of wheat fusarium rust is the Fusarium graminearum described in claim 1.

3. The use according to claim 2, characterized in that When the usnic acid is used to prevent and treat wheat fusarium head blight, it is sprayed in the form of a usnic acid suspension.

4. The use according to claim 3, characterized in that The concentration of the usnic acid suspending agent is 28-32 wt %.

5. The use according to claim 3, characterized in that The usnic acid suspension is sprayed during the flowering period of wheat.

6. Application of usnic acid or sodium usnic acid in reducing the content of deoxynivalenol in wheat infected with fusarium head blight.

Images