Use of 2-cyano-3-aminoacrylate compound in control of fungal diseases caused by pestalotiopsis-like fungi

Abstract

Disclosed in the present invention is a use of a 2-cyano-3-aminoacrylate compound in control of fungal diseases caused by Pestalotiopsis-like fungi. The compound exhibits generally ultrahigh ex-vivo antifungal activity against Pestalotiopsis-like fungi, including Pestalotiopsis, Neopestalotiopsis, and Pseudopestalotiopsis. Phenamacril exhibits a median effective concentration (EC50) ranging from 0.1455 to 0.6025 μg / mL, a mean value of 0.2800 μg / mL and a standard deviation of 0.095 μg / mL against 56 strains of Pestalotiopsis-like pathogenic fungi isolated from Camellia sinensis, Prunus persica, and Vitis vinifera. The Phenamacril suspension formulation spray treatment scheme for Camellia sinensis provided by the present invention can significantly increase tea yield, and provide a basis for controlling diseases caused by Pestalotiopsis, Neopestalotiopsis, and Pseudopestalotiopsis pathogens in tea plantations as well as for monitoring fungicide resistance.

Description

Application of 2-cyano-3-aminoacrylate compounds in the control of fungal diseases caused by *Pterocarya spp.* Technical Field

[0001] This invention relates to the field of agricultural pest control technology, specifically to the application of 2-cyano-3-aminoacrylate compounds in the control of fungal diseases caused by *Plasmodium spp.* Background Technology

[0002] Pestalotiopsis-like fungi, including the genera *Pestalotiopsis*, *Neopestalotiopsis*, and *Pseudopestalotiopsis*, are asexual fungi belonging to the family Amphisphaeriaceae. Their conidia possess appendages. This fungal group is widely distributed globally, exhibiting particularly rich biodiversity in tropical and subtropical ecosystems. They exhibit low host specificity, causing a variety of diseases in various plants, making them important plant pathogens. Neopestalotiopsis is an important pathogen affecting the growth of crops such as tea and fruit trees. For example, Neopestalotiopsis, which causes ring spot disease in tea, can also cause red leaf root rot in strawberries, dry flower disease in macadamia nuts, leaf spot in areca nut, ring spot disease in guava fruits, shoot blight in grapes, branch blight in blueberries, Neopestalotiopsis leaf spot in rubber trees, and leaf spot in beans. Neopestalotiopsis can survive for many years in infected crop debris and soil. Environmental conditions, such as temperatures around 35°C and relative humidity >85%, favor disease development.

[0003] Grapes, pears, peaches, apples, tea, and other fruit and tea cultivation typically result in perennial orchards, undergoing numerous agronomic operations such as pruning throughout their life cycle. *Pseudomonas aeruginosa* fungi primarily invade through natural wounds created by agricultural operations like pruning; some pathogens can also enter through new shoots and / or tender leaves. Currently, control of these diseases relies mainly on spraying chemical pesticides, supplemented by biological pesticides. Commonly used fungicides are primarily demethylation inhibitors (such as tebuconazole, difenoconazole, and cyazofamid) and benzimidazole fungicides (including benomyl, thiophanate-methyl, and carbendazim). However, years of using single pesticides have led to pathogen resistance, resulting in a gradual decline in pesticide efficacy, and even complete ineffectiveness. Therefore, it is necessary to discover low-toxicity, low-residue pesticides with different modes of action, and to rotate or combine them with currently used fungicides to reduce the development of resistance.

[0004] 2-Cyano-3-amino-3-phenylacrylate compounds are novel fungicides with a novel structure and unique mode of action. Cyanobacterium oxychloride has been successfully industrialized. Studies have shown that cyanobacterium oxychloride is a slightly toxic fungicide, with low to moderate toxicity levels in four environmental organisms: bees, quails, silkworms, and fish, indicating good environmental compatibility. Cyanobacterium oxychloride is a highly specific and selective fungicide, mainly exhibiting inhibitory activity against Fusarium, particularly showing a strong inhibitory effect on the mycelial growth of Fusarium graminearum and Fusarium moniliforme. (EC) 50 The values ​​were 0.141 μg / mL and 0.459 μg / mL, respectively, with relative toxicity 4.06 and 1.43 times that of carbendazim, and no cross-resistance with existing fungicides was observed. Currently, there are no clear reports on whether cyazofamid has an inhibitory effect on *Pseudomonas aeruginosa*, the causal agent of fruit and tea crops. Summary of the Invention

[0005] Purpose of the invention: Addressing the problem of increasing resistance to *Polytrichum candida* strains of existing fungicides, this invention provides a novel fungicide for controlling *Polytrichum candida* fungi. Specifically, this invention provides the application of 2-cyano-3-aminoacrylate compounds in controlling fungal diseases caused by *Polytrichum candida*, and for the first time, it has been found that 2-cyano-3-aminoacrylate compounds are significantly effective in controlling *Polytrichum candida* fungal diseases.

[0006] The present invention also provides the aforementioned fungicidal composition for controlling diseases caused by Polychaete spp.

[0007] Technical Solution: To achieve the above objectives, this invention relates to the application of the 2-cyano-3-aminoacrylate compound of Formula I in the prevention and control of fungal diseases caused by *Polytrichum gloeosporioides*, wherein *Polytrichum gloeosporioides* includes fungi of the genera *Polytrichum*, *Polytrichum* pseudo*, and *Polytrichum* neo*, and the structure of the 2-cyano-3-aminoacrylate compound is shown in Formula I:

[0008] Wherein: R1 is hydrogen, amino, substituted amino, cyano, nitro, phenyl, halophenyl, halogen, C1-C6 alkyl, aromatic heterocyclic, hydroxyl or ester group;

[0009] R2 is a C1-C6 alkyl group or a C3-C6 cycloalkyl group.

[0010] The substituted amino groups are optionally substituted by 0 to 2 substituents independently selected from -OH, halogen, -CN or NO2, or are primary, secondary or tertiary substituted amino groups; the aromatic heterocyclic group is a 5 to 10-membered heteroaryl containing 1 to 4 heteroatoms independently selected from N, O or S; the halogen is fluorine, chlorine, bromine or iodine.

[0011] The 2-cyano-3-aminoacrylate compound is selected from any one of the following compounds:

[0012] The application of the 2-cyano-3-aminoacrylate compound of Formula I described in this invention in the preparation of drugs or reagents for preventing and treating diseases caused by fungal pathogens of the genera *Polytrichum*, *Polytrichum pseudosporidis*, and *Polytrichum neosporidis*.

[0013] Among them, the 2-cyano-3-aminoacrylate compounds can prevent and control diseases caused by fungi such as *Pterocarya spp.*, including gray spot and ring spot, as well as diseases caused by wounds created by horticultural practices such as pruning or trimming in fruit trees or tea trees.

[0014] The fungicidal composition for controlling diseases caused by Polytrichum candidae fungi of the present invention comprises 2-cyano-3-aminoacrylate compounds as active ingredients, with the addition of pesticide adjuvants or excipients.

[0015] The fungicidal composition for controlling diseases caused by Polytrichum candidae fungi of the present invention includes the aforementioned 2-cyano-3-aminoacrylate compound as one of the active ingredients, which is combined with other fungicidal active ingredients and pesticide adjuvants or excipients to form a compound formulation.

[0016] The composition is an emulsifiable concentrate, a suspension concentrate, a wettable powder, a water-dispersible granule, an emulsion, or a microemulsion.

[0017] The 2-cyano-3-aminoacrylate compounds of this invention can be used as single-agent formulations for the control of diseases caused by *Polytrichum spp.* These compounds, with 2-cyano-3-aminoacrylate compounds as the active ingredient, can be formulated into emulsifiable concentrates, suspensions, wettable powders, water-dispersible granules, emulsions, microemulsions, seed treatments, and other formulations by adding pesticide adjuvants. This invention can also be used as a fungicidal composition for the control of diseases caused by *Polytrichum spp.* This composition uses 2-cyano-3-aminoacrylate compounds as one of the fungicidal active ingredients, combined with other fungicidal active ingredients and pesticide adjuvants to form compound formulations, thereby enhancing efficacy or expanding the scope of application.

[0018] The application of the fungicidal composition for controlling diseases caused by Polychaete spp. described in this invention in the control of diseases caused by Polychaete spp.

[0019] The method for controlling the fungal pathogen *Polytrichum candida* described in this invention involves spraying a solution containing the 2-cyano-3-aminoacrylate compound as an active ingredient, or spraying the fungicidal composition for controlling *Polytrichum candida* diseases, after pruning trees in the field.

[0020] Among them, the field trees refer to fruit trees (such as peach, pear, grape, apple, etc.) and tea trees that require artificial pruning or gardening measures that cause wounds.

[0021] This invention provides a method for determining the toxicity of a fungicide for *Plasmodium spp.* fungi and its application, comprising the following steps:

[0022] (1) Culture the tested pathogens;

[0023] The tested pathogens were single-spore strains of *Pseudomonas spp.* isolated and purified from diseased branches of tea, peach, and grape trees.

[0024] The sample isolation and purification process was as follows: The sample was cut into 5 mm pieces at the junction of diseased and healthy tissue. In a sterile operating table, the pieces were disinfected with 2% sodium hypochlorite for 5 seconds, rinsed three times with sterile water, blotted dry with absorbent paper, and then transferred to potato dextrose agar (PDA) containing 100 μg / mL streptomycin sulfate. After incubation at 25°C for 3 days, the mycelial blocks of each strain were transferred to new PDA medium and incubated in the dark at 25°C for 14 days. The colonies were then rinsed with 5 mL of distilled water to collect conidia, which were then prepared into 1x10⁻⁶ cells / mL. 4 A spore suspension of 1 conidia / mL was prepared by adding 1% Tween 20. 10 μl of the spore suspension was then evenly spread onto a PDA plate containing 100 μg / mL streptomycin sulfate. After incubating at 25°C for 1 day, single spores were picked and transferred to a fresh PDA plate. This yielded single-spore colonies of the fungus *Plasmodium spp.*, which were then transferred to cryovials and stored at 4°C for later use.

[0025] The potato culture medium (PDA) consists of 200g potatoes, 15g agar powder, and 20g glucose, diluted to 1L with pure water, sterilized under high temperature and high pressure, and then stored at room temperature.

[0026] (2) Preparation of pesticide fungicide

[0027] The fungicide is cyazofamid, and the technical grade of 2-cyano-3-aminoacrylate compound is prepared with methanol to a concentration of 1×10⁻⁶. 4 μg / mL stock solution, for later use.

[0028] (3) Determination of the effective intermediate concentration (EC) of 2-cyano-3-aminoacrylate compounds against *Pterocarya spp.* in tea, peach, and grape plants. 50 )

[0029] Perforated discs were created at the edges of the test strain colonies using a 5 mm punch. The stock solution of the reagent was uniformly mixed with the culture medium to obtain final concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μg / mL. The mixture was poured into 90 mm diameter petri dishes to prepare plates. The perforated discs were placed in the center of PDA plates containing the series of concentrations of 2-cyano-3-aminoacrylate compounds, one disc per plate. The plates were incubated at 25°C in the dark until the control colonies covered 60% of the plate. The colony diameter was measured using the cross-crossing method. Each treatment was repeated three times. The growth inhibition rate was calculated from the average colony diameter (mm).

[0030] The mycelial growth inhibition rate (%) was calculated as follows: (Control colony diameter - Colony diameter of each treatment) / (Control colony diameter - 5) × 100%

[0031] (4) Statistically analyze the effective medium concentration data of 2-cyano-3-aminoacrylate compounds against the tested strains and plot the sensitivity curves.

[0032] In the step of statistically analyzing the effective intermediate concentration data of 2-cyano-3-aminoacrylate compounds against the tested strains and plotting sensitivity curves: the toxicity regression equation Y = a + bX was calculated using DPS software, representing the effective inhibition of the intermediate concentration (EC50) of the active ingredient. 50 The correlation coefficient (r) and 95% confidence interval were calculated for the tested strain EC. 50 Mean and standard deviation, according to EC 50 Distribution frequency, plot sensitivity baseline.

[0033] (5) In newly pruned tea gardens, randomly select plots and prepare a 25% suspension using the aforementioned agent, applying 200 ml / mu at a rate of 250 L / hm. 2 Dilute with water and spray with a sprayer when there is no wind. Use clean water as a control. Each treatment has 3 replicates. Spray a second time after 10 days. After 6 weeks, use the five-point sampling method to record the weight of 100 buds and the number of buds (33×33cm) in each plot.

[0034] The 2-cyano-3-aminoacrylate compounds of this invention exhibit broad and ultra-high antibacterial activity against *Polytrichum spp.* fungi in vitro, including *Polytrichum spp.*, *Polytrichum neoplasm*, and *Polytrichum pseudoplasm*. Specifically, cyazofamid showed an effective median concentration (EC50) against 56 *Polytrichum spp.* pathogens collected from tea, peach, and grape plants. 50The effective concentration ranged from 0.1455 to 0.6025 μg / mL, with an average of 0.2800 μg / mL and a standard deviation of 0.095 μg / mL. The azoxystrobin suspension spray treatment method for tea plants provided by this invention can significantly increase tea yield and provides a basis for controlling diseases caused by *Pseudomonas aeruginosa*, *Pseudomonas neotrichum*, and *Pseudomonas pseudotrichum* pathogens in tea gardens, as well as for monitoring drug resistance.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0036] (1) The fungicide 2-cyano-3-aminoacrylate compound of the present invention has a novel mechanism of action and has excellent inhibitory activity against Polychaete spp. It is the first time that it can be used to control Polychaete spp. fungi and improve the control effect of diseases including ring spot and gray spot.

[0037] (2) The sensitivity baseline of the fungus *Pseudomonas aeruginosa* to cyazofamid provided by this invention, and the EC50 of *Pseudomonas aeruginosa* to cyazofamid. 50 The average value was 0.2800 μg / mL, and the standard deviation was 0.095 μg / mL. This provides a scientific basis for the field control of *Pseudomonas aeruginosa* pathogens by using cyazofamid. Attached Figure Description

[0038] Figure 1 shows the frequency distribution of susceptibility to cyazofamid among 56 strains of Pseudomonas aeruginosa fungi.

[0039] Figure 2 shows a comparison of the inhibitory effects of cyazofamid on the hyphal growth of *Pseudomonas aeruginosa*.

[0040] Figure 3 shows a field comparison of the control effects of cyazofamid on the fungus *Polytrichum gloeosporioides*. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0042] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] The various pathogenic fungi used in this invention, such as MJ-1, MJ-5, MJ-7, MJ-127, and MJ-13, are all wild-type fungal strains of *Plasmodium spp.*, obtained by isolating and purifying pathogens from diseased branches collected from tea, grape, and peach trees. The pathogenic fungi used in this invention can also be obtained by purchasing. All pathogenic fungi used in this invention were provided by Jiangsu Provincial Pesticide Research Institute Co., Ltd.

[0044] MJ-1 was identified as *Pestalotiopsis*; MJ-5, MJ-7, and MJ-127 were identified as *Neopestalotiopsis*; and MJ-13 was identified as *Pseudopestalotiopsis*. Specifically, MJ-1, MJ-5, MJ-7, MJ-127, and MJ-13 are *Pestalotiopsis camelliae*, *N. clavispora*, *N. ellipsospora*, *N. asiatica*, and *Pseudopestalotiopsis cocos*, respectively.

[0045] The compounds cyazofamid (CAS: 3336-69-4), ZJS158 (CAS: 2810129-50-9), ZJS159 (CAS: 2810129-52-1), and ZJS178 (CAS: 2810129-51-0) used in this invention are all known compounds.

[0046] The synthesis of ZJS232 and ZJS288 can be carried out with reference to CN101417962A, as well as the known synthesis methods of ZJS158, ZJS159, and ZJS178.

[0047] Example 1

[0048] Activity screening of cyanoacrylate bactericides

[0049] Experimental subjects: *Polytrichum pseudosporidis* strains MJ-1, MJ-5, MJ-7, MJ-127, and MJ-13, obtained by isolating and purifying pathogens from diseased branches collected from tea gardens.

[0050] The tested pathogens were single-spore strains isolated and purified from diseased branches of tea trees, grapes, and peaches collected from different tea gardens in Nanjing, Jiangsu Province in 2022.

[0051] Sample isolation and purification were performed as follows: Diseased branches were cut into 5 mm pieces at the junction of diseased and healthy tissue. In a sterile environment, the pieces were disinfected with 2% sodium hypochlorite for 5 seconds, rinsed three times with sterile water, blotted dry with absorbent paper, and transferred to potato dextrose agar (PDA) containing 100 μg / mL streptomycin sulfate. After incubation at 25°C for 3 days, mycelial blocks of each strain were transferred to new PDA medium and incubated in the dark at 25°C for 14 days. The colonies were then rinsed with 5 mL of distilled water to collect conidia, which were then prepared into 1x10⁻¹⁰ samples. 4A spore suspension of 1 conidia / mL was prepared by adding 1% Tween 20. 10 μL of the spore suspension was then evenly spread onto a PDA plate containing 100 μg / mL streptomycin sulfate. After incubating at 25°C for 1 day, single spores were picked and transferred to fresh PDA plates. Single spore colonies of various types of *Plasmodium spp.* were obtained and named MJ-1, MJ-5, MJ-7, MJ-127, and MJ-13, respectively. These colonies were then transferred to cryovials and stored at 4°C for later use.

[0052] At the edge of colonies of the test strains cultured to the logarithmic growth phase, 5 mm perforators were used to create bacterial discs. The stock solution of the reagent was thoroughly mixed with the culture medium. The reagent concentrations were set at 0, 0.0625, 0.025, 0.05, 0.1, 0.2, 0.25, 0.4, 0.8, 1, and 1.6 μg / mL. The mixture was poured into 90 mm diameter petri dishes to prepare plates. The bacterial discs were placed in the center of each PDA plate containing the series of test reagent concentrations, with one disc inoculated per plate. The plates were incubated at 25°C in the dark until the control colonies covered 60% of the petri dish. The colony diameter was measured using the cross-sectional method. Each treatment was repeated three times. The growth inhibition rate was calculated from the average colony diameter (mm).

[0053] The mycelial growth inhibition rate (%) was calculated as follows: (control colony diameter - diameter of each treatment colony) / (control colony diameter - 5) × 100%. The test results are shown in Table 1 below.

[0054] Table 1 Inhibitory activity of different compounds

[0055] The experimental results show that the various 2-cyano-3-aminoacrylate compounds in Table 1 exhibit certain antibacterial activity against *Polytrichum spp.* at relatively low concentrations. In particular, compound ZJS178 showed an inhibition rate of over 90% against *Polytrichum spp.*. The experimental compounds of this invention all exhibited extremely high activity against *Polytrichum spp.* strains.

[0056] Example 2

[0057] Virulence determination of cyazofamid against 56 strains of *Pseudomonas aeruginosa* pathogenic fungi.

[0058] At the edge of colonies of the tested strains cultured to the logarithmic growth phase, 5 mm perforators were used to create bacterial discs. The stock solution of the fungicide was uniformly mixed with the culture medium at concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 μg / mL. The mixture was poured into 90 mm diameter petri dishes to prepare plates. The bacterial discs were placed in the center of PDA plates containing the series of concentrations of cyazofamid, with one disc inoculated per plate. The plates were incubated at 25°C in the dark until the control colonies covered 60% of the petri dish. The colony diameter was measured using the cross-crossing method. Each treatment was repeated three times. The growth inhibition rate was calculated from the average colony diameter (mm), as shown in Figure 2.

[0059] The mycelial growth inhibition rate (%) is calculated as follows: (control colony diameter - diameter of each treatment colony) / (control colony diameter - 5) × 100%.

[0060] The effective concentration data of cyazofamid technical for the tested strains were statistically analyzed, and sensitivity curves were plotted.

[0061] In the step of statistically analyzing the effective intermediate concentration data of cyazofamid technical against the tested strains and plotting the sensitivity curve: the toxicity regression equation Y = a + bx was calculated using DPS software, representing the effective inhibition of the intermediate concentration (EC50) of cyazofamid. 50 Correlation coefficient (r). Calculate the EC of the tested strain. 50 Mean and standard deviation, according to EC 50 Distribution frequency, plot sensitivity baseline.

[0062] Table 2. Toxicity of cyazofamid against 56 strains of *Pseudomonas aeruginosa*

[0063] The susceptibility of 56 single-spore strains of *Pestalotiopsis* sp. and *Neopestalotiopsis* sp. isolated from Jiangsu Province in 2022 to cyazofamid was determined using the mycelial growth rate assay. EC50 50 The values ​​ranged from 0.1455 to 0.6025 μg / mL, with an average EC50. 50 The concentration was 0.2800 ± 0.0952 μg / mL, with a standard deviation of 0.095 μg / mL. The frequencies of strains with different sensitivities showed a continuous unimodal distribution, as shown in Table 2 and Figure 1, respectively. This further demonstrates that the compounds of the present invention have excellent antifungal effects against the fungus *Plasmodium spp.*.

[0064] Example 3

[0065] Field efficacy testing

[0066] The fungus *Pseudomonas aeruginosa* causes tea buds to be significantly smaller and fewer in number. This embodiment further demonstrates the effectiveness of the compound of the present invention in controlling *Pseudomonas aeruginosa* through field efficacy testing.

[0067] (1) Experimental subjects: Tea trees in tea gardens in Jiangning District, Nanjing City, Jiangsu Province.

[0068] (2) Chemical treatment

[0069] In the newly pruned tea garden, randomly selected plots were treated with (1) 25% cyazofamid SC suspension at 200 ml / mu, at a rate of 250 L / hm. 2 Dilute with water, (2) 80% carbendazim WP at 100 g / mu, at 250 L / hm 2 (3) Water control. Spraying was carried out with a sprayer when there was no wind. Each treatment was replicated 3 times. The first application was carried out on June 11, 2023; the second application was carried out on June 20, 2023. Six weeks after the second application, the five-point sampling method was used to record the weight of 100 buds and the number of buds (33×33cm) and the control effect in each plot.

[0070] The weight of 100 shoots refers to the total weight of 100 new shoots harvested, used to measure the quality and robustness of the shoots. The quality of a new shoot can be judged by its weight of 100 shoots. The method for calculating the weight of 100 shoots is to weigh 100 randomly selected new shoots or mixed new shoots with the same number of leaves unfolded, and record the average value at least three times. The results are shown in Table 3.

[0071] Table 3. Effects of cyazofamid treatment on tea yield

[0072] The weight of 100 buds and the number of buds (33×33cm) of tea trees in Jiangning District, Nanjing City, Jiangsu Province were determined using a five-point sampling method. The average weight of 100 buds of tea trees treated with 200 ml / mu of 25% cyazofamid SC suspension was 10.05 g, which was higher than the average weight of 8.05 g of the water control. At the same time, the average number of buds of tea trees treated with 25% cyazofamid SC suspension was 29.8±4.20, which was significantly higher than the average number of buds of the water control (13±1.58) (Table 3, Figure 3), and significantly higher than the average weight of 8.81 g and the number of buds of 18.2 of the 80% carbendazim treatment.

Claims

1. The application of the 2-cyano-3-aminoacrylate compound of Formula I in the control of fungal diseases caused by *Polytrichum gloeosporioides*, wherein *Polytrichum gloeosporioides* includes fungi of the genera *Polytrichum*, *Polytrichum pseudosporioides*, and *Polytrichum neosporioides*, and the structure of the 2-cyano-3-aminoacrylate compound is shown in Formula I: in: R1 is hydrogen, amino, substituted amino, cyano, nitro, phenyl, halophenyl, halogen, C1-C6 alkyl, aromatic heterocyclic, hydroxyl or ester group; R2 is a C1-C6 alkyl group or a C3-C6 cycloalkyl group.

2. The application according to claim 1, characterized in that, The substituted amino groups are optionally substituted by 0 to 2 substituents independently selected from -OH, halogen, -CN or NO2, or are primary, secondary or tertiary substituted amino groups; the aromatic heterocyclic group is a 5 to 10-membered heteroaryl containing 1 to 4 heteroatoms independently selected from N, O or S; the halogen is fluorine, chlorine, bromine or iodine.

3. The application according to claim 1, characterized in that, The 2-cyano-3-aminoacrylate compound is preferably selected from any one of the following compounds:

4. The use of 2-cyano-3-aminoacrylate compounds as described in Formula I in the preparation of drugs or reagents for the prevention and treatment of diseases caused by fungal pathogens of the genera *Polytrichum*, *Polytrichum pseudosporidis*, and *Polytrichum neosporidis*.

5. The application according to any one of claims 1-4, characterized in that, The 2-cyano-3-aminoacrylate compounds, in addition to preventing gray spot and ring spot caused by Polytrichum speciosus fungi, can also prevent diseases caused by wounds created by horticultural practices such as pruning or trimming of fruit trees or tea trees.

6. A fungicidal composition for treating diseases caused by *Plasmodium spp.* fungi, characterized in that, It includes the 2-cyano-3-aminoacrylate compound as described in claim 1 as the active ingredient, with the addition of pesticide adjuvants or excipients.

7. A fungicidal composition for controlling diseases caused by *Plasmodium spp.*, characterized in that, It includes the 2-cyano-3-aminoacrylate compound of claim 1 as one of the active ingredients, combined with other bactericidal active ingredients, and added with pesticide adjuvants or excipients to form a compound formulation.

8. The bactericidal composition according to claim 6 or 7, characterized in that, The composition is an emulsifiable concentrate, suspension concentrate, wettable powder, water-dispersible granule, water emulsion, or microemulsion.

9. A method for controlling and preventing the pathogenic fungus *Plasmodium spp.*, characterized in that, After pruning trees in the field, spray a solution containing the 2-cyano-3-aminoacrylate compound of claim 1 as the active ingredient, or spray the bactericidal composition of claim 5 or 6.

10. The sterilization method according to claim 9, characterized in that, The term "field trees" refers to fruit trees or tea trees that require artificial pruning or trimming, which may result in wounds.

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