Use of allylurea for controlling fungal diseases of food crops

Abstract

The present application relates to the application of allantoin in the prevention and treatment of fungal diseases of grain crops. The present application finds that allantoin can inhibit the growth of pathogenic fungi of grain crops, especially inhibit the infection of pathogenic fungi of grain crops to host cells or inhibit the expansion of pathogenic fungi of grain crops in host cells, thus indicating that allantoin can be used as a pesticide to prevent and treat pathogenic fungi of grain crops, especially Magnaporthe oryzae, Cochliobolus miyabeanus and Ustilaginoidea virens.

Description

Technical Field

[0001] This invention relates to the field of biocides, and particularly to fungicides. Background Technology

[0002] Fungal diseases pose a significant threat to agricultural and economic crops, severely impacting crop yield and quality. Rice blast and rice false smut are common fungal diseases of rice, causing large-scale yield reductions every year. During outbreaks of rice blast, they can even lead to total crop failure. The fungus *Aspergillus oryzae* can produce toxins that threaten human and animal health. Long-term use of single fungicides has led to increasingly serious problems of pathogen resistance. Summary of the Invention

[0003] This invention provides the application of allourea in the inhibition of pathogens in food crops.

[0004] In one specific embodiment, the pathogenic fungus for grain crops is at least one of rice blast fungus, corn leaf blight fungus, and rice aspergillus.

[0005] In one specific embodiment, allourea is used to inhibit the infection of host cells by the crop pathogen or to inhibit the spread of the crop pathogen within host cells.

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

[0007] In one specific embodiment, the allourea is used for the prevention and control of fungal diseases in food crops.

[0008] In one specific embodiment, the fungal disease is a disease caused by at least one of rice blast fungus, corn leaf blight fungus, and rice aspergillus.

[0009] The beneficial effects of this invention are as follows: This invention has found that allourea can inhibit the growth of pathogens in grain crops, especially inhibiting the infection of host cells by grain crop pathogens or inhibiting the spread of grain crop pathogens within host cells. This indicates that allourea can be used as a pesticide to control and inhibit pathogens in grain crops, especially rice blast fungus, corn leaf blight fungus and rice aspergillus. Attached Figure Description

[0010] Figure 1 The image shows a photograph illustrating how allourea inhibits the growth of rice blast fungus mycelia.

[0011] Figure 2 The inhibition rates of different concentrations of alloxan on the mycelial growth of *Magnapordica oryzae* are shown. The bar charts represent the mean ± SEM values, with a statistical count of n = 3 (n represents biological replicates); Student's t-test, *P<0.05, ****P<0.0001.

[0012] Figure 3 The study shows the disease incidence of rice blast fungus on rice leaves after different treatments. A is a photograph of the diseased rice leaves, and B is a bar chart showing the percentage of rice blast lesions on the entire leaf area. The bar charts represent mean ± SEM values, with a statistical count of n=3 (n represents biological replications); Student's t-test was used, with ***P<0.001 and ****P<0.0001.

[0013] Figure 4 The image shows the infection of rice leaf sheath cells by *Bacillus oryzae* after different treatments. Scale bar = 10 micrometers.

[0014] Figure 5 The study shows the disease incidence of rice blast fungus on barley leaves after different treatments. A is a photograph of diseased barley leaves, and B is a bar chart showing the length of rice blast lesions on barley. The bar charts represent mean ± SEM, with a statistical count of n=3 (n represents biological replications); Student's t-test was used, with **P<0.01 and ****P<0.0001.

[0015] Figure 6 The image shows the infection of barley epidermal cells by rice blast fungus after different treatments. Scale bar = 10 micrometers.

[0016] Figure 7 The images show photographs of *Botrytis cinerea* mycelial growth after different treatments and bar charts of *Botrytis cinerea* colony diameters. A represents a photograph, and B represents the bar chart of colony diameters. The bar charts represent mean ± SEM, with a statistical count of n=3 (n indicates biological replication); ns indicates no significant difference.

[0017] Figure 8 The images show photographs of Fusarium moniliforme mycelial growth after different treatments and bar charts of colony diameters. A represents a photograph, and B represents the colony diameter bar chart. The bar charts represent mean ± SEM, with a statistical count of n=3 (n indicates biological replication); ns indicates no significant difference.

[0018] Figure 9 The images show photographs and bar charts of colony diameters of *S. cornensis* after different treatments. In the image, A is a photograph, and B is a bar chart of colony diameters. The bar charts represent the mean ± SEM, with a statistical count of n=3 (n represents biological replicates); Student's t-test, **P<0.01.

[0019] Figure 10The images show photographs of *Aspergillus oryzae* mycelial growth after different treatments and bar charts of *Aspergillus oryzae* colony diameters. In the images, A is a photograph, and B is a bar chart of colony diameters. The bar charts represent mean ± SEM, with a statistical count of n = 3 (n represents biological replicates); Student's t-test, **P < 0.01. Detailed Implementation

[0020] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0021] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0022] Complete Medium (CM): Add 25 mL 40× nitrate salts, 1 mL 1000× trace elements, 1 mL 1000× vitamin solution, 10 g D-glucose, 2 g peptone, 1 g yeast extract, and 1 g casamino acids. Add ddH2O to bring the volume to 1 L. For solid medium, aliquot and add 1.5% agar powder. Autoclave at 121°C for 20 min.

[0023] 40× Nitrate Salts: 240g NaNO3, 20.8g KCl, 20.8g MgSO4·7H2O, 60.8g KH2PO4, add ddH2O to bring the volume 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 ethylenediaminetetraacetate (Na4EDTA) 5 g. Add ddH2O to a final volume of 100 mL and store at 4°C.

[0025] 1000× Vitamin Solution: 0.01g each of Biotin, Thiamine, Pyridoxin, Nicotinic Acid, and Para-Aminobenzoic Acid, add ddH2O to bring the volume to 100 mL, and store at 4°C protected from light.

[0026] Albumin solution: Weigh 0.5 g of alliin monohydrate using an analytical balance and dissolve it in 15.6 mL of dimethyl sulfoxide (DMSO) to prepare an alliin solution with a concentration of 0.2 M.

[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.

[0028] tomato gray mold ( Botrytis cinerea ): Liu, S. et al. Resistance to boscalid in Botrytis cinerea from greenhouse-grown tomato. Plant Disease 2021, 105(3):628-635.

[0029] 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).

[0030] Corn spores ( Cochliobolus heterostrophus(See Hu H, Liu T, Xie X, Li F, Liu C, Jiang J for details) , et al. GPI anchoring controls cell wall integrity, immuneevasion and surface localization of ChFEM1 for infection of Cochlibolusheterostrophus1. Journal of Integrative Agriculture 2024.

[0031] Rice Aspergillus ( 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

[0032] Before preparing CM agar plates (6cm petri dishes), allourea solution was added to the CM agar to achieve final concentrations of 1mM, 2mM, and 4mM. Inoculation was then performed as follows: A 0.5cm diameter punch was heated to red-hot temperature with an alcohol lamp. After cooling, holes were punched in plates containing *Blastoma oryzae* that had grown on CM agar for 5 days. The *Blastoma oryzae* bacteria, along with the culture medium, were removed from the holes using a sterile toothpick and inverted onto the agar plate. CM agar containing neither allourea nor DMSO served as a blank control, while CM agar containing an equal volume of DMSO (4mM allourea solution) served as a solvent control. Three replicates were set up, with one plate constituting one replicate. The plates were inverted and incubated at 28°C for 24 hours under 5000 Lx light for 5 days. Colony morphology was photographed and recorded. Figure 1 The colony diameter was measured, the hyphal growth inhibition rate was calculated, and the data were statistically analyzed. The results are shown in [the table below]. Figure 2 The formula for calculating the mycelial growth inhibition rate is shown in (1).

[0033] Inhibition rate = (colon diameter of blank control group - colony diameter of treatment group) / colony diameter of blank control group × 100% (1).

[0034] Figure 2 The results showed that allourea had an inhibitory effect on the growth of rice blast fungus, and the inhibitory effect increased with the increase of allourea dosage. Example 2

[0035] Conidia of *Magnaporum oryzae* were cultured in CM. The conidia were washed with a 0.025% (v / v) Tween 20 aqueous solution, and the final concentration of conidia was adjusted to 1×10^5 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as a blank control group. Aloxine solution was added to the spore suspension to achieve a final conidia concentration of 1×10^5 cells / mL. The final concentrations of allourea were 0.5 mM, 1 mM, and 2 mM, resulting in spore suspensions containing different concentrations of allourea. The treatment group using these spore suspensions was used as the allourea experimental treatment group. The same volume of DMSO as the 2 mM allourea spore suspension was added to the spore suspension to achieve a final conidia concentration of 1×10^5 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0036] Ten mL of spore suspensions containing different concentrations of allourea were sprayed onto all leaves of ten 1-month-old Lijiang Xintuan Black Rice seedlings, with a blank control group and a solvent control group included. The rice seedlings were then cultured in darkness at 28°C with 90% humidity for 24 hours, followed by 12 hours of light followed by 12 hours of darkness at 28°C with 90% humidity for 4 days. Leaf disease incidence was investigated; diseased leaves were scanned after being pruned and attached to A4 white paper with double-sided tape. ImageJ software was used to calculate the percentage of lesion area on the entire leaf, and statistical analysis was performed.

[0037] The results are as follows Figure 3 As shown, the effect of allourea in inhibiting rice blast disease becomes more obvious with increasing concentration. Furthermore, when the concentration of allourea is 2 mM, rice blast fungus almost loses its infectivity, indicating that allourea can significantly inhibit the pathogenicity of rice blast fungus. Example 3

[0038] The infection cycle of *Magnapordica oryzae* consists of conidial germination, appressorium formation, appressorium penetration of host epidermal cells to form primary infectious hyphae, and the spread of infectious hyphae within host cells. Generally, appressoriums penetrate the host epidermis and enter the host within 24 hours, and a large number of infectious hyphae spread within 48 hours. A higher proportion of appressoriums indicates a lower infection rate or slower infection, which is reflected in a decreased pathogenicity phenotype.

[0039] Conidia of *Magnapordica oryzae* were cultured in CM. The conidia were washed off with a 0.025% (v / v) Tween 20 aqueous solution, and the final concentration of conidia was adjusted to 1×10^6 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as a blank control group. Aloxine solution was added to the spore suspension to achieve a final conidia concentration of 1×10^6 cells / mL and a final alloureine concentration of 2 mM, resulting in an alloureine-containing spore suspension. The treatment group using this spore suspension was used as the alloureine treatment group. The same volume of DMSO was added to the spore suspension to achieve a final conidia concentration of 1×10^6 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0040] A 1 mL syringe was used to inject a spore suspension containing allopurinol into the leaf sheaths of 1-month-old Lijiang Xintuan Black Rice plants, filling the entire leaf sheath. The leaf sheaths were placed downwards and cultured in the dark at 28 degrees Celsius with 90% humidity. The leaf sheaths were cut off at 24 and 48 hours after inoculation and observed under an optical microscope to count the infection status of rice blast fungus. The number of molecular spores 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 from one leaf sheath constituting one replicate. A blank control group and a solvent control group were also included.

[0041] Appressorium percentage = number of molecular spores forming appressorium / (number of molecular spores forming appressorium + number of conidia forming infective hyphae) (1).

[0042] Percentage of infected hyphae = number of molecular spores forming infected hyphae / (number of molecular spores forming appressorium + number of conidia forming infected hyphae) (2).

[0043] See results Figure 4 The results showed that at 24h and 48h, there were no significant differences in the percentage of appressoria and infected hyphae between the blank control group and the solvent control group; however, the percentage of appressoria in the allourea treatment group was significantly higher than that in the blank control group and the solvent control group, while 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 appressoria in the blank control group and the solvent control group were infected, with only a few remaining as uninfected appressoria; the majority of the appressoria in the 2mM allourea treatment group remained uninfected, with only a few remaining as infected hyphae; at 48h of infection, most of the appressoria in the blank control group and the solvent control group developed secondary infected hyphae, while the majority of the appressoria in the 2mM allourea treatment group remained in the appressoria state, and the infected hyphae were mostly primary infected hyphae.

[0044] The above results indicate that allourea significantly inhibits the ability of appressorium to infect rice cells and spread within rice cells. Example 4

[0045] Conidia of *Magnapordica oryzae* were cultured in CM. The conidia were washed off with a 0.025% (v / v) Tween 20 aqueous solution, and the final concentration of conidia was adjusted to 3 × 10^4 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as a blank control group. Aloxine solution was added to the spore suspension to achieve a final conidia concentration of 3 × 10^4 cells / mL. The final concentrations of aloxine were 0.05 mM, 0.1 mM, and 0.5 mM, resulting in spore suspensions containing different concentrations of aloxine. The treatment group using these spore suspensions was used as the aloxine treatment group. The same volume of DMSO as the 0.5 mM aloxine-concentrated spore suspension was added to the spore suspension to achieve a final conidia concentration of 3 × 10^4 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0046] Five drops of spore suspensions containing different concentrations of allourea were spot-inoculated onto the upper surface of barley leaves, staggered across each leaf, with each drop containing 5 μL. One barley leaf constituted one replicate, with three replicates for each treatment. A blank control group and a solvent control group were also included. The spot-inoculated barley leaves were incubated at 28°C in the dark with 90% humidity for 24 hours, followed by 12 hours of light followed by 12 hours of darkness with 90% humidity at 28°C for 3 days. Leaf disease incidence was assessed. Diseased leaves were cleaned, mounted on A4 white paper with double-sided tape, and scanned. ImageJ software was used to calculate the area of ​​lesions on the leaves, and differential analysis was performed.

[0047] The results are as follows Figure 5 As shown, the effect of alloxan on inhibiting rice blast disease becomes more obvious with increasing concentration, further demonstrating that alloxan can significantly inhibit the pathogenicity of rice blast fungus. Example 5

[0048] Conidia of *Magnaporum oryzae* were cultured in CM. The conidia were washed off with a 0.025% (v / v) Tween 20 aqueous solution, and the final concentration of conidia was adjusted to 1×10^6 cells / mL to obtain a spore suspension. The treatment group using this spore suspension was used as a blank control group. Aloxine solution was added to the spore suspension to achieve a final conidia concentration of 1×10^6 cells / mL and a final alloureine concentration of 0.5 mM, resulting in an alloureine-containing spore suspension. The treatment group using this spore suspension was used as the alloureine treatment group. The same volume of DMSO as the 0.5 mM alloureine-containing spore suspension was added to the spore suspension to achieve a final conidia concentration of 1×10^6 cells / mL. The treatment group using this spore suspension was used as the solvent control group.

[0049] Eight drops of a spore suspension containing allopurinol were dropped onto the underside of barley leaves, staggered across each leaf, with each drop containing 2 μL. The leaves were then incubated at 28°C in the dark with 90% humidity for 24 and 48 hours. After 24 and 48 hours of incubation, the cuticle of the underside of the barley leaves was peeled off, and the infection status of *Strombus haematobium* was observed and counted under an optical microscope. The number of molecular spores 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 from one leaf constituting one replicate. A blank control group and a solvent control group were also set up.

[0050] See results Figure 6 The results showed that at 24h and 48h, there were no significant differences in the percentage of appressoria and infected hyphae between the blank control group and the solvent control group; however, the percentage of appressoria in the allourea treatment group was significantly higher than that in the blank control group and the solvent control group, while 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 appressoria in the blank control group and the solvent control group were infected, with only a few remaining as uninfected appressoria; the majority of the appressoria in the 0.5mM allourea treatment group were uninfected, with only a few remaining as infected hyphae; at 48h of infection, most of the appressoria in the blank control group and the solvent control group developed secondary infected hyphae; the majority of the appressoria in the 0.5mM allourea treatment group were in the appressoria state, and those that had developed infected hyphae were mostly primary infected hyphae.

[0051] The above results indicate that allourea significantly inhibits the ability of appressorium to infect barley epidermal cells and spread within them. Example 6

[0052] Tomato gray mold: Tomato gray mold cultured on PDA medium for 2 days was perforated using a 0.5 cm diameter punch and inoculated onto PDA medium plates containing 2 mM allopurinol. The plates were incubated at 25°C for 3 days, and colony diameters were measured to calculate the inhibition rate. PDA medium plates containing neither allopurinol nor DMSO served as a blank control, while PDA medium plates containing an equal volume of DMSO (2 mM allopurinol) served as a solvent control. One plate constituted one biological replicate, with three replicates. The inhibition rate photos and colony diameter bar chart results are shown below. Figure 7 Compared with the blank control group, the inhibition rate of the allourea treatment group was (-0.79±0.01)%, which was not significantly different from that of the blank control group.

[0053] Fusarium graminearum: Fusarium graminearum cultured on PDA medium for 2 days was perforated using a 0.5 cm diameter punch and inoculated onto PDA medium plates containing 2 mM allopurinol. The plates were incubated at 25°C for 3 days, and colony diameters were measured to calculate the inhibition rate. PDA medium plates containing neither allopurinol nor DMSO served as a blank control, while PDA medium plates containing an equal volume of DMSO (2 mM allopurinol) served as a solvent control. One plate constituted one biological replicate, with three replicates. The inhibition rate photos and colony diameter bar charts are shown below. Figure 8 Compared with the blank control group, the inhibition rate of the allourea treatment group was (-1±0.02)%, which was not significantly different from that of the blank control group.

[0054] Corn leaf spot pathogens: Corn leaf spot pathogens cultured on PDA medium for 2 days were perforated using a 0.5 cm diameter punch and inoculated onto PDA medium plates containing 2 mM allopurinol. The plates were incubated at 25°C for 3 days, and colony diameters were measured to calculate the inhibition rate. PDA medium plates containing neither allopurinol nor DMSO served as a blank control, while PDA medium plates containing an equal volume of DMSO (2 mM allopurinol) served as a solvent control. One plate constituted one biological replicate, with three replicates. The inhibition rate photos and colony diameter bar charts are shown below. Figure 9 Compared with the blank control group, the inhibition rate of the allourea treatment group was (17.64±0.05)%, which was significantly different from that of the blank control group (P<0.05).

[0055] Aspergillus oryzae: Aspergillus oryzae cultured on PDA medium for 15 days were perforated using a 0.5 cm diameter punch and inoculated onto PDA medium plates containing 2 mM allopurinol. The plates were incubated at 28°C for 5 days, and colony diameters were measured to calculate the inhibition rate. PDA medium plates containing neither allopurinol nor DMSO served as a blank control, while PDA medium plates containing an equal volume of DMSO (2 mM allopurinol) served as a solvent control. One plate constituted one biological replicate, with three replicates. The inhibition rate photos and colony diameter bar charts are shown below. Figure 10 Compared with the blank control group, the inhibition rate of the allourea treatment group was (12.36±0.02)%, which was significantly different from that of the blank control group (P<0.05).

[0056] The results showed that allourea had an inhibitory effect on the pathogenic fungi Aspergillus oryzae and Microsporum simonii on crops, but no inhibitory effect on the pathogenic fungi Botrytis cinerea on vegetables and Fusarium oxysporum on fruit trees. The results indicate that allourea can effectively inhibit fungal diseases on corn and rice.

Claims

1. The application of allourea in the inhibition of pathogenic fungi of grain crops, wherein the pathogenic fungi of grain crops are at least one of rice blast fungus, maize leaf spot fungus and rice aspergillus.

2. The application according to claim 1, characterized in that, The use of allourea in inhibiting the infection of host cells by the pathogenic fungi of the food crop or inhibiting the spread of the pathogenic fungi of the food crop within the host cells.

3. The application according to claim 1, characterized in that, The food crop is at least one of rice, wheat, and corn.

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