Application of allurea in prevention and treatment of fungal diseases of food crops

Abstract

The invention relates to application of allurea in prevention and treatment of fungal diseases of food crops. The invention finds that the allurea can inhibit the growth of the pathogenic bacteria of the food crops, especially inhibit the pathogenic bacteria of the food crops from infecting host cells or inhibiting the pathogenic bacteria of the food crops from expanding in the host cells, so that the allurea can be used as a pesticide to prevent and inhibit the pathogenic bacteria of the food crops, especially pyricularia grisea, bipolaris maydis and ustilaginoidea virens.

Description

Technical Field

[0001] The present invention relates to the field of biocides, in particular to fungicides. Background Art

[0002] Fungal diseases pose a significant threat to agricultural cash crops, severely impacting both yield and quality. Rice blast and false smut are common fungal diseases of rice, causing significant annual yield losses and even total crop failure during blast outbreaks. U.S. smut can produce toxins that threaten human and animal health. The long-term use of a single fungicide has led to the growing problem of pathogen resistance. Summary of the Invention

[0003] The present invention provides the use of alloxan in inhibiting pathogenic bacteria of food crops.

[0004] In a specific embodiment, the food crop pathogen is at least one of: rice blast fungus, corn leaf blight fungus and rice smut fungus.

[0005] In a specific embodiment, alloxan is used to inhibit the food crop pathogens from infecting host cells or inhibiting the food crop pathogens from expanding in host cells.

[0006] In a specific embodiment, the food crop is at least one of rice, wheat and corn.

[0007] In a specific embodiment, the alloxirane is used for preventing and controlling fungal diseases of food crops.

[0008] In one embodiment, the fungal disease is caused by at least one of: rice blast fungus, southern blight fungus, and urticaria.

[0009] Beneficial effects of the present invention: The present invention found that alloxan can inhibit the growth of food crop pathogens, especially inhibit food crop pathogens from infecting host cells or inhibiting the expansion of food crop pathogens in host cells. This shows that alloxan can be used as a pesticide to prevent and control food crop pathogens, especially rice blast fungus, corn leaf blight fungus and rice smut fungus. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Photographs showing the inhibition of mycelial growth of Magnaporthe grisea by alloxan.

[0011] Figure 2 The figure shows the inhibitory effect of alloxan on mycelial growth of Magnaporthe grisea at different concentrations. The bar graphs are mean ± SEM, with n = 3 data (n represents biological replicates). Student's t test, *P < 0.05, ****P < 0.0001.

[0012] Figure 3 This figure shows the incidence of rice blast fungus on rice leaves after different treatments. A is a photograph of rice leaves with diseased lesions, and B is a bar graph showing the percentage of blast lesion area to the total leaf area. The bar graphs are mean ± SEM, n = 3 (n represents biological replicates). Student's t test, ***P < 0.001, ****P < 0.0001.

[0013] Figure 4 Shows the infection of rice sheath cells by rice blast fungus after different treatments. Scale bar = 10 μm.

[0014] Figure 5 Figure 2 shows the incidence of rice blast fungus on barley leaves after different treatments. A is a photograph of barley leaves with diseased leaves, and B is a bar graph showing the length of rice blast lesions on barley. The bar graphs are mean ± SEM, n = 3 (n represents biological replicates). Student's t test, **P < 0.01, ****P < 0.0001.

[0015] Figure 6 Shows the infection of barley epidermal cells by rice blast fungus after different treatments. Scale bar = 10 μm.

[0016] Figure 7 Shown are photographs of mycelial growth of Botrytis cinerea after different treatments and a histogram of colony diameters. A shows the photograph, and B shows the histogram of colony diameters. The histograms are mean ± SEM, with n = 3 (n represents biological replicates). ns indicates no significant difference.

[0017] Figure 8 Shown are photographs of mycelial growth of Fusarium oxysporum f. bananai after different treatments and a histogram of colony diameters. A shows the photograph, and B shows the histogram of colony diameters. The histograms are mean ± SEM, with n = 3 (n represents biological replicates); ns indicates no significant difference.

[0018] Figure 9 Photographs of mycelial growth of T. maydis after different treatments and a histogram of colony diameters are shown. (A) Photographs, (B) Colony diameter histogram. Histograms are mean ± SEM, n = 3 (n represents biological replicates). Student's t test, **P < 0.01.

[0019] Figure 10Shown are photographs of mycelial growth of Aspergillus oryzae (Rice) after different treatments and a bar graph showing colony diameters. (A) Photographs, (B) colony diameter bar graphs. Bar graphs are mean ± SEM, n = 3 (n represents biological replicates). Student's t test, **P < 0.01. DETAILED DESCRIPTION

[0020] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation of the present invention.

[0021] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0022] Complete Medium (CM): Add 25 mL of 40× Nitrate Salts, 1 mL of 1000× Trace Elements, 1 mL of 1000× Vitamin Solution, 10 g of D-Glucose, 2 g of Peptone, 1 g of Yeast Extract, and 1 g of Casamino Acids. Add ddH2O to make up to 1 L. To prepare solid culture medium, add 1.5% agar powder after aliquoting. Autoclave at 121°C for 20 min.

[0023] 40× Nitrate Salts: NaNO3 240 g, KCl 20.8 g, MgSO4·7H2O 20.8 g, KH2PO4 60.8 g, add ddH2O to 1 L, autoclave at 121°C for 20 min, and store at 4°C.

[0024] 1000× Trace Elements: zinc sulfate heptahydrate (ZnSO4·7H2O) 2.2 g, boric acid (H3BO3) 1.1 g, manganese chloride tetrahydrate (MnCl2·4H2O) 0.5 g, ferric sulfate heptahydrate (FeSO4·7H2O) 0.5 g, cobalt chloride hexahydrate (CoCl2·6H2O) 0.17 g, copper sulfate pentahydrate (CuSO4·5H2O) 0.16 g, sodium molybdate pentahydrate (Na2MoO4·5H2O) 0.15 g, tetrasodium ethylenediaminetetraacetic acid (Na4EDTA) 5 g. Add ddH2O to 100 mL and store at 4°C.

[0025] 1000× Vitamin solution: 0.01 g each of biotin, thiamine, pyridoxin, nicotinic acid, and para-aminobenzoic acid. Add ddH2O to a volume of 100 mL. Store at 4°C in the dark.

[0026] Alloxan solution: 0.5 g of alloxan monohydrate was weighed and dissolved in 15.6 mL of dimethyl sulfoxide (DMSO) using an analytical balance to prepare a 0.2 M alloxan solution.

[0027] Rice blast fungus ( Magnaporthe oryzae ) For wild-type strain P131, see Chen XL, Shi T, Yang J, Shi W, Gao X, Chen D, et al. N-glycosylation of effector proteins by analpha-1,3-mannosyltransferase is required for the rice blast fungus to evadehost innate immunity. Plant Cell 2014, 26(3): 1360-1376. Tomato gray mold ( Botrytis cinerea ): Liu,S.et al.Resistance to boscalid inBotrytis cinerea from greenhouse-grown tomato.Plant Disease 2021, 105(3):628-635. Banana Fusarium ( Fusarium oxysporum f .sp .cubense, Foc ) For details, see Chen D, Ju M, Xie J, Chen XL, Peng J. Current progress on pathogenicity-related genes in Fusarium oxysporum f. sp. cubense tropical race 4. Phytopathology Research2024, 6(1). Corn leaf spot fungus ( Cochliobolus heterostrophus ) For details, see Hu H, Liu T, Xie X, Li F, Liu C, Jiang J , et al.GPI anchoring controls cell wall integrity, immuneevasion and surface localization of ChFEM1 for infection of Cochlibolusheterostrophus1. Journal of Integrative Agriculture 2024. Rice koji fungus ( Ustilaginoidea virens ) For details, see Chen X, Li X, Li P, Chen X, Liu H, Huang J , et al. Comprehensive identification of lysine 2-hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals theinvolvement of lysine 2-hydroxyisobutyrylation in fungal virulence. J IntegrPlant Biol 2021, 63(2): 409—425. Example 1

[0028] Before preparing CM culture medium plates (6 cm culture dishes), add alloxan solution to the CM culture medium so that the final concentration of alloxan in the culture medium is 1 mM, 2 mM, and 4 mM, respectively. Then, perform the inoculation as follows: light an alcohol lamp to burn a 0.5 cm diameter puncher red. After cooling, punch holes in the rice blast fungus plate grown on the CM culture medium for 5 days. Use a sterilized toothpick to pick out the punched rice blast fungus together with the culture medium and turn it upside down on the culture medium. CM culture medium without adding alloxan or DMSO was used as the blank control group, and CM culture medium with the same volume of DMSO solvent as the 4 mM alloxan solution was used as the solvent control group. Set up 3 replicates, with 1 plate as 1 replicate. Place the plate upside down in an incubator at 28 degrees Celsius for 24 hours of light (light intensity 5000Lx), culture for 5 days, and take photos of the colony morphology, see. Figure 1 ; Determine the colony growth diameter, calculate the mycelial growth inhibition rate, and perform statistical analysis on the data. The results are shown in Figure 2 The calculation formula of mycelial growth inhibition rate is shown in (1).

[0029] Inhibition rate = (colony diameter of blank control group - colony diameter of treated group) / colony diameter of blank control group × 100% (1).

[0030] Figure 2The results showed that alloxan had an inhibitory effect on the growth of rice blast fungus, and the inhibitory effect increased with the increase of the alloxan dosage. Example 2

[0031] Conidia of rice blast fungus were cultured in CM, and the conidia were washed with 0.025% (v / v) Tween 20 aqueous solution. The final concentration of the conidia was adjusted to 1×10^5 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as the blank control group; an alloxan solution was added to the spore suspension to make the final concentration of the conidia 1×10^5 cells / mL, and the final concentrations of alloxan were 0.5 mM, 1 mM and 2 mM to obtain spore suspensions containing different concentrations of alloxan. The treatment groups using these spore suspensions were used as the alloxan experimental treatment groups; the same volume of DMSO as that in the spore suspension with a final alloxan concentration of 2 mM was added to the spore suspension to make the final concentration of the conidia 1×10^5 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0032] Ten milliliters of a spore suspension containing varying concentrations of alloxan was sprayed onto all leaves of ten one-month-old Lijiang Xintuan Black Valley rice plants. A blank control group and a solvent control group were set up. The rice seedlings were then incubated at 28°C in the dark (90% humidity) for 24 hours. Subsequently, they were incubated at 28°C with a 12-hour light cycle and 12-hour dark (90% humidity) for four days. The leaves were then examined for disease. Diseased leaves were trimmed, attached to A4 paper with double-sided tape, and scanned. Image J software was used to calculate the percentage of lesion area on the entire leaf for statistical analysis.

[0033] The results are as follows Figure 3 As shown in the figure, with the increase of alloxan concentration, the effect of inhibiting the occurrence of rice blast became more obvious, and when the alloxan concentration was 2 mM, the rice blast fungus almost lost its infection ability, indicating that alloxan can significantly inhibit the pathogenicity of rice blast fungus. Example 3

[0034] The infection cycle of rice blast fungus consists of conidia germination, appressorium formation, penetration of the host epidermal cells by the appressorium to form primary infectious hyphae, and expansion of the infectious hyphae within the host cells. Generally, appressorium penetrates the host epidermis and enters the host within 24 hours, and a large number of infectious hyphae will expand within 48 hours. A higher proportion of appressorium indicates a lower or slower infection rate, which is reflected in a decreased virulence.

[0035] Conidia of rice blast fungus were cultured in CM, and the conidia were washed with 0.025% (v / v) Tween 20 aqueous solution to adjust the final concentration of the conidia to 1×10^6 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as the blank control group; an alloxan solution was added to the spore suspension to make the final concentration of the conidia 1×10^6 cells / mL and the final concentration of alloxan was 2 mM to obtain a spore suspension containing alloxan. The treatment group using this spore suspension was used as the alloxan treatment group; the same volume of DMSO as that in the spore suspension with a final alloxan concentration of 2 mM was added to the spore suspension to make the final concentration of the conidia 1×10^6 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0036] A spore suspension containing alloxan was injected into the leaf sheaths of one-month-old Lijiang Xintuan Black Valley rice plants using a 1 mL syringe. The entire leaf sheath was filled, placed downward, and incubated at 28°C in a dark, moisturized environment (90% humidity). The leaf sheaths were excised 24 and 48 hours after inoculation and observed under an optical microscope to determine the infection status of the rice blast fungus. The number of conidia forming appressoria and the number of conidia forming infectious hyphae were recorded. The percentage of appressoria was calculated based on formula (1), and the percentage of infectious hyphae was calculated based on formula (2). Three replicates were set up, with 100 conidia counted per leaf sheath as one replicate. A blank control group and a solvent control group were also set up.

[0037] Percentage of appressoria = number of molecular spores forming appressoria / (number of molecular spores forming appressoria + number of conidia forming infective hyphae) (1).

[0038] Percentage of infectious hyphae = number of molecular spores forming infectious hyphae / (number of molecular spores forming appressorium + number of conidia forming infectious hyphae) (2).

[0039] See the results Figure 4 According to the results, there was no significant difference in the percentage of appressorium and the percentage of infected hyphae between the blank control group and the solvent control group at 24h and 48h; the percentage of appressorium in the alloxan-treated group was significantly higher than that in the blank control group and the solvent control group, and the percentage of infected hyphae was significantly lower than that in the blank control group and the solvent control group. In other words, at 24h of infection, most of the blank control group and the solvent control group were infected, with only a few being in the uninfected appressorium state; most of the treatment groups with 2mM alloxan were in the uninfected appressorium state, with only a few being in the infected hyphae state; at 48h of infection, most of the blank control group and the solvent control group formed secondary infected hyphae, while the treatment group with 2mM alloxan was still mostly in the appressorium state, and most of the infected hyphae that had formed were primary infected hyphae.

[0040] The above results indicate that alloxan significantly inhibits the ability of appressoria to infect rice cells and expand within rice cells. Example 4

[0041] Conidia of rice blast fungus were cultured in CM, and the conidia were washed with 0.025% (v / v) Tween 20 aqueous solution. The final concentration of the conidia was adjusted to 3×10^4 conidia / mL to obtain a spore suspension. The treatment group using this spore suspension was used as the blank control group. An alloxan solution was added to the spore suspension to make the final concentration of the conidia 3×10^4 conidia / mL. The final concentrations of alloxan were 0.05 mM, 0.1 mM and 0.5 mM to obtain spore suspensions containing different concentrations of alloxan. The treatment groups using these spore suspensions were used as the alloxan treatment group. The same volume of DMSO as that in the spore suspension with a final alloxan concentration of 0.5 mM was added to the spore suspension to make the final concentration of the conidia 3×10^4 conidia / mL. The treatment group using this spore suspension was used as the solvent control group.

[0042] A spore suspension containing varying concentrations of alloxan was spot-coated on the front of barley leaves, with five drops (5 μl) applied staggered onto each leaf. Each barley leaf constituted a replicate, with three replicates per treatment. A blank control group and a solvent control group were also established. The spot-coated barley leaves were incubated at 28°C in the dark (90% humidity) for 24 hours, followed by incubation at 28°C with 12 hours of light and 12 hours of darkness (90% humidity) for three days. To investigate leaf disease, diseased leaves were trimmed, attached to A4 paper with double-sided tape, and scanned. Image J software was used to calculate the area of ​​lesions on the leaves, and differential analysis was performed.

[0043] The results are as follows Figure 5 As shown in the figure, with the increase of alloxan concentration, the effect of inhibiting the occurrence of rice blast became more obvious, which further demonstrated that alloxan can significantly inhibit the pathogenicity of rice blast fungus. Example 5

[0044] Conidia of rice blast fungus were cultured in CM, and the conidia were washed with 0.025% (v / v) Tween 20 aqueous solution to adjust the final concentration of the conidia to 1×10^6 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as the blank control group. An alloxan solution was added to the spore suspension to make the final concentration of the conidia 1×10^6 cells / mL and the final concentration of alloxan was 0.5 mM to obtain a spore suspension containing alloxan. The treatment group using this spore suspension was used as the alloxan treatment group. The same volume of DMSO as that in the spore suspension with a final alloxan concentration of 0.5 mM was added to the spore suspension to make the final concentration of the conidia 1×10^6 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0045] A spore suspension containing alloxan was dripped onto the back of barley leaves, with 8 drops of 2 μl per drop staggered onto each leaf. The leaves were then placed in a dark, moisturizing environment (90% humidity) at 28 degrees Celsius for 24 and 48 hours. The cuticles on the back of the barley leaves were removed after 24 and 48 hours of incubation, and the leaves were observed and counted under an optical microscope for the infection status of the rice blast fungus. The number of conidia that formed appressoria and the number of conidia that formed infectious hyphae were recorded. The percentage of appressoria was calculated based on formula (1), and the percentage of infectious hyphae was calculated based on formula (2). Three replicates were set, with 100 conidia counted on one leaf as one replicate. A blank control group and a solvent control group were also set.

[0046] See the results Figure 6 . According to the results, there was no significant difference in the percentage of appressorium and the percentage of infected hyphae between the blank control group and the solvent control group at 24h and 48h; the percentage of appressorium in the alloxan-treated group was significantly higher than that in the blank control group and the solvent control group, and the percentage of infected hyphae was significantly lower than that in the blank control group and the solvent control group. In other words, at 24h of infection, most of the blank control group and the solvent control group were infected, with only a few being in the state of uninfected appressorium. Most of the treatment groups with 0.5mM alloxan were in the state of uninfected appressorium, with only a few being in the state of infected hyphae. At 48h of infection, most of the blank control group and the solvent control group formed secondary infected hyphae. Most of the treatment groups with 0.5mM alloxan were in the state of appressorium, and those that had formed infected hyphae were primary infected hyphae.

[0047] The above results indicate that alloxan significantly inhibited the ability of appressoria to infect barley epidermal cells and expand within barley epidermal cells. Example 6

[0048] Tomato gray mold: After the tomato gray mold cultured on PDA medium for 2 days was punched with a 0.5 cm diameter puncher, it was inoculated on a PDA medium plate containing 2 mM alloxan and placed in a 25°C incubator for 3 days. The colony diameter was measured and the inhibition rate was calculated. A PDA medium plate containing neither alloxan nor DMSO was used as a blank control, and a PDA medium plate with an equal volume of DMSO added to 2 mM alloxan was used as a solvent control. One plate was a biological replicate, and three replicates were set. The inhibition photos and colony diameter bar graph results are shown in Figure 7 Compared with the blank control group, the inhibition rate of the alloxanide-treated group was (-0.79±0.01)%, which had no significant difference from the blank control group.

[0049] Banana Fusarium: After the Fusarium graminearum cultured on PDA medium for 2 days was punched with a 0.5 cm diameter puncher, it was inoculated on a PDA medium plate containing 2 mM alloxan and placed in a 25°C incubator for 3 days. The colony diameter was measured and the inhibition rate was calculated. A PDA medium plate containing neither alloxan nor DMSO was used as a blank control, and a PDA medium plate with an equal volume of DMSO added to 2 mM alloxan was used as a solvent control. One plate was used as one biological replicate, and three replicates were set. The inhibition photos and colony diameter bar graph results are shown in Figure 8 Compared with the blank control group, the inhibition rate of the alloxanide-treated group was (-1±0.02)%, which had no significant difference from the blank control group.

[0050] Corn leaf spot fungus: After being cultured on PDA medium for 2 days, the corn leaf spot fungus was punched with a 0.5 cm diameter puncher and inoculated on a PDA medium plate containing 2 mM alloxan. The plate was placed in a 25°C incubator for 3 days, the colony diameter was measured, and the inhibition rate was calculated. A PDA medium plate containing neither alloxan nor DMSO was used as a blank control, and a PDA medium plate with an equal volume of DMSO added to 2 mM alloxan was used as a solvent control. One plate was used as one biological replicate, and three replicates were set. The inhibition photos and colony diameter bar graph results are shown in Figure 9 Compared with the blank control group, the inhibition rate of the alloxanide-treated group was (17.64±0.05)%, which was significantly different from the blank control group (P<0.05).

[0051] U.S. oryzae: After the U.S. oryzae cultured on PDA medium for 15 days was punched with a 0.5 cm diameter puncher, it was inoculated on a PDA medium plate containing 2 mM alloxan and cultured in a 28-degree Celsius incubator for 5 days. The colony diameter was measured and the inhibition rate was calculated. A PDA medium plate containing neither alloxan nor DMSO was used as a blank control, and a PDA medium plate with an equal volume of DMSO added to 2 mM alloxan was used as a solvent control. One plate was used as one biological replicate, and three replicates were set. The results of the inhibition photos and colony diameter bar graph are shown in Figure 10 Compared with the blank control group, the inhibition rate of the alloxanide-treated group was (12.36±0.02)%, which was significantly different from the blank control group (P<0.05).

[0052] The results showed that alloxan had an inhibitory effect on the pathogenic fungi U. oryzae and Micromyces zea on crops, but had no inhibitory effect on the tomato gray mold on vegetables and the banana Fusarium solani on fruit trees. The results indicate that alloxan can effectively inhibit fungal diseases on corn and rice.

Claims

1. Application of alloxan in inhibiting pathogenic fungi of food crops.

2. The use according to claim 1, characterized in that The food crop pathogenic fungus is at least one of rice blast fungus, corn leaf blight fungus and rice smut fungus.

3. The use according to claim 1 or 2, characterized in that The alloxan is used for inhibiting the food crop pathogenic fungi from infecting host cells or inhibiting the food crop pathogenic fungi from expanding in host cells.

4. The use according to claim 1 or 2, characterized in that The food crop is at least one of rice, wheat and corn.

5. The use according to claim 1 or 2, characterized in that: The application of the alloxan in preventing and controlling fungal diseases of grain crops.

6. The use according to claim 1 or 2, characterized in that The fungal disease is caused by at least one of rice blast fungus, corn leaf blight fungus and rice smut fungus.

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