Use of usnic acid or sodium usnate to inhibit fusarium pseudograminearum and pyricularia oryzae

Abstract

The application provides application of usnic acid or sodium usnate in inhibition of false smut fungus and pyricularia oryzae, and belongs to the technical field of pesticides. The natural product usnic acid and its sodium salt have inhibitory effects on plant pathogenic fungi, such as fusarium graminearum, false smut fungus, pyricularia oryzae and fusarium graminearum, and can significantly reduce the content of DON toxin of fusarium graminearum, and also has good inhibitory activity on wheat scab fungus in indoor and field, thereby providing an important foundation for discovery and creation of new natural product pesticides.

Description

[0001] This invention is a divisional application of the patent filed on February 12, 2025, with application number 202510155769.9, entitled "Application of Usnea Acid or Sodium Usnea Acid in the Prevention and Control of Wheat Fusarium Head Blight". Technical Field

[0002] This invention relates to the field of pesticide technology, and in particular to the application of usnic acid or sodium usnic acid in inhibiting Fusarium graminearum and rice blast fungus. Background Technology

[0003] Fusarium head blight, caused by Fusarium graminearum infection of wheat, is a worldwide epidemic disease, often referred to as the "cancer" of wheat. Besides causing severe yield and economic losses, the mycotoxins produced by Fusarium head blight on infected grains, such as deoxynivalenol (DON), can persist in the food chain for extended periods, leading to symptoms like anorexia, vomiting, and diarrhea, as well as suppressing immune function, posing a serious threat to human and animal health. Reducing the incidence of Fusarium head blight and toxin contamination is a pressing practical problem in wheat production that needs to be addressed.

[0004] Currently, the control of wheat scab mainly relies on chemical control. However, the types of effective chemical fungicides are limited, primarily triazoles, 2-cyanoacrylates, and SDHIs. Long-term use leads to frequent occurrences of pathogen resistance and tolerance. Simultaneously, the continuous and excessive use of pesticides results in increasingly serious problems such as excessive pesticide residues in the environment, water pollution, and food safety. Natural products, with their characteristics of low toxicity, environmental friendliness, and structural diversity, provide many excellent molecules for the development of novel drugs and fungicides. Therefore, finding safe and effective natural products is of great significance for the control of wheat scab and the development of novel lead compounds or fungicides.

[0005] Usnea acid is one of the most common secondary metabolites in lichens, first isolated by the German scientist Knop in 1844. Usnea acid possesses excellent antioxidant, anti-inflammatory, antibacterial, and antitumor activities. However, its poor solubility and low bioavailability in the human body limit its application and development in medicine. The problem this invention aims to solve is to use usnea acid and its sodium salt to control wheat scab, overcoming the negative effects of chemical agents and providing an important foundation for the discovery and creation of new natural pesticides. Summary of the Invention

[0006] This invention provides the application of usnic acid or sodium usnic acid in inhibiting Fusarium graminearum, rice blast fungus, or Rhizoctonia graminearum. The natural product usnic acid and its sodium salt showed good inhibitory activity against Fusarium graminearum in the laboratory and in the field, and can also effectively inhibit the growth of other plant pathogenic fungi such as Fusarium graminearum and rice blast fungus.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Use of isocyanate or sodium isocyanate as an inhibitor of plant pathogens such as Fusarium graminearum, Fusarium pseudograminearum and rice blast fungus.

[0008] The present invention also provides the application of usnic acid or sodium usnic acid in the prevention and control of wheat scab, wherein the pathogen of wheat scab is Fusarium graminearum.

[0009] Preferably, the usnic acid is sprayed in the form of a usnic acid suspension when controlling wheat scab.

[0010] Preferably, the concentration of the isosine suspension is 28-32 wt%.

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

[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 products usnic acid and its sodium salt described in this invention exhibited good inhibitory activity against Fusarium graminearum, the causal agent of wheat scab, both indoors and in the field. They also effectively inhibited the growth of other plant pathogenic fungi such as Fusarium graminearum and rice blast fungus. Conidia are the primary source of infection for many plant fungal diseases, including wheat scab, wheat stem rot, wheat sheath blight, and rice blast. Both usnic acid and sodium usnic acid showed excellent inhibitory activity in spore germination, providing an important foundation for the discovery and creation of new natural product pesticides. Attached Figure Description

[0014] Figure 1 Images of *Fusarium graminearum*, *Fusarium pseudograminearum*, *Oryza sativa*, and *Rhizoctonia graminearum* after different growth days under different concentrations of isoflavones in Example 1. Figure 2 Images of *Fusarium graminearum*, *Fusarium pseudograminearum*, *Oryza sativa*, and *Rhizoctonia graminearum* after different growth days under different concentrations of sodium usnic acid in Example 1; Figure 3 Example 1 shows the growth inhibition rate of different concentrations of isoflavones on Fusarium graminearum, Fusarium pseudograminearum, rice blast fungus, and Rhizoctonia graminearum. Figure 4 Example 1 shows the growth inhibition rates of different concentrations of sodium usnic acid on Fusarium graminearum, Fusarium pseudograminearum, Oryza sativa, and Rhizoctonia graminearum. Figure 5 The results of observing the edge of Fusarium graminearum colonies in Example 1; Figure 6 This is a statistical analysis of the inhibition rate of Fusarium graminearum spore germination under different concentrations of isoflavone and sodium isoflavate treatment in Example 1. Figure 7 Transmission electron microscopy observation of Fusarium graminearum spores in Example 1, scale bar 1µm; Figure 8 The results of the pathogenicity test of wheat coleoptiles in Example 2; Figure 9 The results of the toxicity observation of high concentrations of isosine on wheat seedlings in Experiment Example 1; Figure 10 For example 2, whether or not it contains 20µg / ml -1 The DON toxin content of Fusarium graminearum in liquid toxin-inducing culture medium containing usnic acid and sodium usnicate. Detailed Implementation

[0015] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] Example 1

[0017] Usnea acid (98%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., and its chemical structure is shown below:

[0018] Sodium usnic acid (≥98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and its chemical structure is shown below:

[0019] Preparation of Usnea acid solution: Prepare a solution with acetone to a concentration of 5 mg / ml; Preparation of sodium usnic acid solution: Dissolve 20 mg sodium usnic acid powder in 1 ml of ethanol, then add 2 ml of water.

[0020] Preparation of 30% Usnea acid suspension: (1) Add accurately measured amounts of usnea acid (75g), surfactant: SC3266 (6%), antifreeze: ethylene glycol (3%), thickener: xanthan gum (0.2%), preservative: Kathon (0.2%), defoamer: 1522 (0.2%), and water (to make up to 100%) to a stirrer and stir evenly to obtain a mixed solution; (2) Add the mixed solution from step (1) to 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. If the particle size DV90 is not greater than 5µm, stop grinding, pour into a beaker and mix evenly, for a total of 250ml.

[0021] Strains: Fusarium graminearum standard strain PH-1, Fusarium pseudograminearum strain CF14047 (isolated by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences), Oryza sativa standard strain 70-15, Rhizoctonia graminearum strain R0301 (isolated by the Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences).

[0022] (1) Plate activity detection

[0023] Standard strains of *Fusarium graminearum* PH-1 (Fg), *Fusarium pseudogranatum* CF14047 (Fp), *Magnaporthe oryzae* 70-15 (Mo), and *Rhizoctonia graminearum* R0301 (Rc) were inoculated onto 1 / 2 cm agar plates containing the following ingredients: glucose 5g, peptone 1g, yeast extract 0.5g, casein amino acids 0.5g, sodium nitrate 3g, potassium chloride 0.25g, magnesium sulfate heptahydrate 0.5g, potassium dihydrogen phosphate 0.75g, agar 15g, distilled water 1L, sterilized at 121℃ for 20min (usually 1µg / ml, 2µg / ml, 4µg / ml, 8µg / ml, and 16µg / ml). After incubation at 25℃ for 3 days (Fg), 4 days (Fp), 6 days (Rc), and 10 days (Mo), the colony diameter was measured and photographed. Figure 1-2 ).

[0024] Inhibition rate (%) = (control mycelium diameter - treated mycelium diameter) / (control mycelium diameter - mycelium cake diameter) × 100, with 3 parallel experiments set up for each concentration.

[0025] The inhibition rate results show that the inhibition rate of the compound obtained was: under the treatment of usnic acid ( 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; and the inhibition rates of Rc were 16.66%, 20.86%, 26.80%, 20.86%, and 19.97%, respectively. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 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; and the inhibition rates of Rc were 28.09%, 32.16%, 48.54%, 50.89%, and 50.90%, respectively.

[0026] 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 usnicate against the four pathogens were 81.48%, 63.65%, 100%, and 50.90%, respectively. This indicates that usnic acid has a good inhibitory effect on Fusarium graminearum, Fusarium pseudograminearum, and Blast fungus, and its sodium salt has a good inhibitory effect on all four pathogens, with higher inhibitory activity against each pathogen than that of usnic acid.

[0027] Compared with other pathogens, usnic acid showed 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 blast fungus was observed at a concentration of 8 µg / ml, reaching 91.88%. Inhibitory activity against Rhizoctonia graminearum was poor at different concentrations. Sodium usnic acid showed the highest inhibitory activity against blast fungus, 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. Inhibitory activity against Fusarium graminearum was followed by 51.16%, 73.60%, 77.03%, 78.14%, and 81.48% at the corresponding concentrations. The inhibition rates against *Fusarium graminearum* and *Rhizoctonia graminearum* were significantly improved at the following concentrations: (*Fusarium graminearum*) 37.02%, 46.03%, 65.63%, 59.83%, 63.65%; (*Rhizoctonia graminearum*) 28.09%, 32.16%, 48.54%, 50.89%, 50.90%. Overall, usnic acid and sodium usnicate significantly inhibited the growth of *Fusarium graminearum*, *Fusarium graminearum*, *Magnaporum oryzae*, and *Rhizoctonia graminearum*, especially showing good inhibitory activity against *Fusarium graminearum* and *Magnaporum oryzae*.

[0028] (2) Observation of colony edges

[0029] Dissolve 1 / 2 cm solid culture medium and pour it into a glass dish with a slide. Inoculate the standard strain PH-1 onto the slide and incubate overnight for 16 h (without treatment) and 24 h (with 18 μg / ml usnic acid and 12.5 μg / ml sodium usnic acid). Observe the mycelial morphology at the colony edge under a microscope. Figure 5 Comparing the colony edges of wild-type (CK) and mutant (drug-treated group), wild-type colonies showed neat edges and linear hyphae. After treatment with usnic acid and its sodium salt, the colony edges exhibited denser hyphae and significantly increased branching, especially after sodium usnic acid treatment, where the hyphae became highly dense and branched. Therefore, treatment with usnic acid and its sodium salt affects the polarity of hyphae, which may be the main reason for the slow colony growth.

[0030] (3) Determine the inhibition rate of usnic acid and its sodium salt on the germination of Fusarium graminearum conidia.

[0031] Standard strain PH-1 was inoculated onto PDA plates (200g of boiled and filtered potatoes, 20g of glucose, 15g of agar, and purified water to a final volume of 1L, then autoclaved). The plates were cultured at 25°C for 3 days. Mycelial blocks were then inoculated onto CMC sporulation medium (15g sodium carboxymethyl cellulose, 1g NH4NO3, 1g KH2PO3, MgSO4). 0.5g of 7H2O, 1g of yeast extract, and 1L of pure water were added and brought to a final volume (autoclaved). The mixture was incubated at 25℃ for 5 days. Spores were collected and transferred to YEPD liquid medium containing different concentrations of isinic acid or sodium isinate (3g yeast extract, 10g peptone, 20g sucrose, 1L distilled water, sterilized at 121℃ for 20min). Spores were cultured for 6 hours (wild-type spore germination rate was over 95%). The results were compared with those of different concentrations of isinic acid (0μg). The number of conidia germinating under treatment with sodium aluminate (0 μg / ml, 0.625 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml) and sodium aluminate (0 μg / ml, 0.39 μg / ml, 0.78 μg / ml, 1.56 μg / ml, 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml) was recorded, and the germination rate was calculated. The results are as follows: Figure 6 As shown, usnic acid at 5 μg / ml inhibited 63.53% of spore germination, and 20 μg / ml completely inhibited spore germination (inhibition rate 100%). Sodium usnic acid at 3.125 μg / ml inhibited 71.61% of spore germination, and 12.5 μg / ml completely inhibited spore germination (inhibition rate 100%). Therefore, usnic acid and its sodium salt exhibit strong activity in inhibiting the germination of Fusarium graminearum spores.

[0032] (4) Observe the ultrastructure of conidia after treatment with usnic acid and its sodium salt (transmission electron microscope)

[0033] PH-1 was inoculated onto PDA plates and cultured at 25°C for 3 days. Mycelial blocks were then inoculated into CMC sporulation 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). Germination culture was carried out for 4 hours (spores to be germinated before germ tube emergence). Spores were collected and fixed in 2.5% glutaraldehyde solution, washed three times with ultrapure water for 10 min each time; fixed with 1% osmium tetroxide for 2 h, washed three times with ultrapure water for 10 min each time; dehydrated in gradients of 50%, 70%, 80%, and 90% acetone for 15 min each time; dehydrated three times with pure acetone for 30 min each time; pure acetone:epoxy resin was used at a ratio of 3:1 (1 h) and 1:1 (3 h). 1:3 (overnight) replacement; samples were embedded in pure resin, and the embedded plates were placed in an oven and immersed at 37℃, 45℃, and 60℃ for 24h, 24h, and 48h respectively. Ultrathin sections were prepared using a Leica EMUC7 ultramicrotome, and observed using a Hitachi H-7650 transmission electron microscope after phosphotungstic acid-uranium acetate double staining.

[0034] The results are as follows Figure 7 As shown, normal spores awaiting germination have a regular morphology, with round or oval fat granules neatly arranged inside the cell membrane. However, after treatment with usnic acid and its sodium salt, cells become deformed, the outermost layer of the cell wall thickens, and fat granules are almost invisible inside the cells after usnic acid treatment, while those after sodium usnic acid treatment are not regularly round or oval. Fat granules provide nutrients and substances for spore germination and germ tube elongation; therefore, the effects of usnic acid and its sodium salt treatment on cell morphology, cell wall formation, and fat granules are likely the main reason for inhibiting spore germination.

[0035] Example 2

[0036] Determining the control efficacy of usnic acid and its sodium salt against wheat scab in indoor and outdoor environments.

[0037] wheat coleoptile

[0038] Two days after germination, wheat seeds were neatly arranged in a glass dish kept moist with filter paper. The tips of the coleoptiles were cut off, and small cotton balls were wrapped around them. 150 μl of diluted isoflavone solution at concentrations of 100 μg / ml, 200 μg / ml, and 400 μg / ml were respectively added to the cotton balls. Two hours later, 1×10⁻⁶ drops of [a specific solution] were added. 5 Two μl of spore suspension was dried and then placed in a humidified container in a 25°C greenhouse for 7 days. Results showed that 400 µg / ml of usnic acid was as effective as 100 µg / ml of tebuconazole in controlling spore growth. Figure 8 ).

[0039] Wheat ear inoculation

[0040] During the wheat flowering stage, spray with 30% usnic acid suspension (200 ml / mu), with the control agent being 48% cyazofamid·tebuconazole suspension (50 ml / mu). A control group (no treatment), a usnic acid treatment group, and a control treatment group were set up, with three replicates in each group. Ten ears were randomly selected from each replicate, and the standard strain PH-1 spore suspension (concentration 2×10⁻⁶) was sprayed on each group. 5 15 μl of the inoculated solution (number per ml) was applied to the spikelets in the middle of the ear. The inoculation was carried out in a bag to maintain moisture for 48 hours, and the disease incidence was recorded after 14 days.

[0041] 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 shows that although it does not achieve the effect of chemical agents, the application of usnic acid can significantly reduce the disease index of wheat scab and can be used as a potential natural compound for the prevention and control of wheat scab.

[0042] Table 1. Effects of 30% Usnic acid suspension on wheat scab.

[0043] Note: Different letters after the data in the same column in the table indicate that the difference is significant at the P < 0.05 level according to the least significant difference test (LSD test).

[0044] Experimental Example 1

[0045] Community Trial

[0046] Four treatments were set up: a control, two applications of 30% isoflavone at the early and peak flowering stages, one application of 30% isoflavone at the early flowering stage, and one application of 48% cyazofamid·tebuconazole at the flowering stage, for a total of four treatments. Each treatment was replicated in three plots, with each plot measuring 20 m². 2 The severity of disease was assessed 20 days after the last spraying. Five sampling points were taken from each community, with each point measuring 2.5 meters. 2 Fusarium head blight-infected ears within the iron ring. The ears are graded by the percentage of infected ear area relative to the total ear area, and the number of infected ears at each grade and the total number of ears are recorded.

[0047] Grading standards: Grade 0: Disease-free entire ear of grain; Level 1: The area of ​​infected ears accounts for less than 1 / 4 of the total ear area; Level 3: The area of ​​infected ears accounts for 1 / 4 to 1 / 2 of the total ear area; Level 5: The area of ​​infected ears accounts for 1 / 2 to 3 / 4 of the total ear area; Level 7: The area of ​​infected ears accounts for more than 3 / 4 of the total ear area.

[0048] Diseased ear classification:

[0049] Table 2. Field control efficacy of 30% Usnic acid suspension against wheat scab.

[0050] Note: Different letters after the data in the same column in the table indicate that the difference is significant at the P < 0.1 level according to the least significant difference test (LSD test).

[0051] The results of the small-scale trial are shown in Table 2. A single application of 30% usinidin at the flowering stage achieved a control efficacy of nearly 60% (59.48%) against wheat scab. The results also indicate that usinidin suspension can effectively reduce the DON content in diseased grains.

[0052] Meanwhile, to clarify whether isosic acid has a toxic effect on wheat, we compared wheat germination to seedling stage between treatments without isosic acid (DDW) and treatments with isosic acid (400 μg / ml). The results showed that isosic acid had no effect on wheat seed germination or seedling growth. Figure 9 This proves that ournic acid has no toxic side effects on wheat.

[0053] Experiment Example 2

[0054] Method: 1×10 5 The spore suspension was inoculated into toxin-inducing medium (TBI) and cultured in the dark at 25°C and 100 rpm for 7 days. After filtration, the mycelia were collected, dried, and weighed. The filtrate was collected in a 50 ml centrifuge tube and centrifuged at 8000 rpm for 5 min. 5 ml of the supernatant was taken and extracted with 2 ml of ethyl acetate. 1 ml of the upper extract was taken and dried under nitrogen at 50°C. 1 ml of chromatographic methanol:water (20:80) was added. All 1 ml of the sample was filtered through a 0.22 μm organic microporous membrane. The filtrate was collected, diluted 100 times, and analyzed by high performance liquid chromatography.

[0055] The results are as follows Figure 10 As shown, in the liquid toxin-inducing medium, compared with the control, 20 µg / ml -1 Usnea acid treatment significantly reduced the DON content of Fusarium graminearum.

[0056] The results of Experiment 1 and Experiment 2 demonstrate that usnic acid can effectively reduce the toxin synthesis of Fusarium graminearum, the pathogen of wheat blight.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of usnic acid or sodium usnic acid in inhibiting Fusarium graminearum, rice blast fungus or Rhizoctonia graminearum.

2. The application according to claim 1, characterized in that, The concentration of the isosic acid or sodium isosic acid is selected from one of 1 µg / ml, 2 µg / ml, 4 µg / ml, 8 µg / ml and 16 µg / ml.

3. The application of usnic acid or sodium usnic acid in inhibiting Fusarium graminearum, characterized in that, Usnea acid or sodium usneaate inhibits the germination of Fusarium graminearum hyphae and conidia.

4. The application of usnic acid or sodium usnicate in the control of wheat scab, characterized in that, The pathogen causing wheat scab is Fusarium graminearum. The concentration of the isosic acid or sodium isosic acid is selected from one of 1 µg / ml, 2 µg / ml, 4 µg / ml, 8 µg / ml and 16 µg / ml; When controlling wheat scab, the isosine is sprayed in the form of an isosine suspension. The concentration of the isoflavone suspension is 28-32 wt%.

5. The application according to claim 4, characterized in that, The concentration of the isoflavone suspension is 30 wt%.

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