Application of benzaldehyde oxime in improving plant resistance to biotic stress

Abstract

This invention discloses the application of benzaldehyde oxime in improving plant resistance to biotic stress. It also discloses a plant immune inducer with benzaldehyde oxime as the active ingredient. Benzaldehyde oxime can be used to control major types of diseases in agricultural production, including oomycete diseases (such as soybean Phytophthora root rot), fungal diseases (such as wheat scab, soybean purple blotch, and rapeseed sclerotinia rot), bacterial diseases (such as rice bacterial blight), and viral diseases (such as turnip mosaic virus), exhibiting high broad-spectrum immune-inducing activity. Benzaldehyde oxime is convenient to use, has both preventative and protective effects, effectively reduces the harm of various biotic stresses to plants, reduces the amount of chemical pesticides used, lowers production costs, and meets the requirements for the development of green and efficient biological pesticides.

Description

Technical Field

[0001] This invention relates to the field of plant immune inducer technology, specifically to the application of benzaldehyde oxime in improving plant resistance to biological stress. Background Technology

[0002] Crops are continuously attacked by various diseases and pests throughout their growth cycle. Once some diseases break out, they can easily cause large-scale and severe yield reductions, or even total crop failure. Currently, agricultural production still heavily relies on chemical fungicides for the control of plant diseases and pests. However, long-term use of pesticides leads to a series of ecological and environmental problems, such as pesticide residues, increased pesticide resistance in pests, reduced biodiversity, and potential risks to the quality and safety of agricultural products, seriously threatening food security and sustainable agricultural development. Therefore, developing plant immune inducers with novel mechanisms of action and environmental friendliness, which can activate and enhance the plant's own immune defense capabilities before or in the early stages of disease, effectively reducing or inhibiting disease occurrence, and thus achieving the reduction or even replacement of chemical fungicides, has significant theoretical and practical value for promoting green agricultural development and ensuring the quality and safety of agricultural products.

[0003] Plant immune inducers enhance crop disease resistance by activating the host's own defense system (such as the phenylpropane metabolic pathway and the mitogen-activated protein kinase signaling pathway). Due to their advantages such as low dosage, good environmental compatibility, and broad spectrum of induced resistance, they have become a research hotspot in the field of green control of plant diseases.

[0004] Benzaldehyde oxime, with the molecular formula C7H7NO and a molecular weight of 121.14, belongs to the class of small molecule aldehyde oximes. International patent application WO2025026555A1 discloses that aldehyde oxime can release nitric oxide in vivo and can be used to treat nitric oxide-sensitive diseases. This patent application also discloses that, based on the nitric oxide-releasing characteristic, aldehyde oxime can be used as a plant rooting agent, plant architecture improver, biotic / abiotic stress tolerance inducer, and germination inducer. The abiotic stresses described and experimentally verified in this patent application are water stress and pure ammonium nitrogen nutrient stress, but biotic stresses have not been further studied. Existing research has not yet reported any use of benzaldehyde oxime as a plant immune inducer. Therefore, the development of novel plant immune inducers with benzaldehyde oxime as the active molecule has significant research and application value. Summary of the Invention

[0005] The purpose of this invention is to provide the application of benzaldehyde oxime in improving the resistance of plants to biological stress, thereby overcoming the shortcomings of the prior art.

[0006] To address the above shortcomings, the present invention adopts the following technical solution:

[0007] The application of benzaldehyde oxime in improving plant resistance to biotic stress, wherein the biotic stress is selected from any one or more of oomycete diseases, fungal diseases, bacterial diseases, and viral diseases; the oomycete diseases include Phytophthora root rot; the fungal diseases include Fusarium head blight, purple blotch, and sclerotinia rot; the bacterial diseases include bacterial leaf blight; and the viral diseases include mosaic virus.

[0008] Furthermore, the plant is selected from any one or more of food crops, oil crops, and cash crops.

[0009] Furthermore, the food crops include wheat and rice, the oil crops include soybeans and rapeseed, and the cash crops include tobacco.

[0010] Furthermore, the Phytophthora root rot includes soybean Phytophthora root rot; the Fusarium head blight includes wheat Fusarium head blight; the purple blotch includes soybean purple blotch; the sclerotinia rot includes rapeseed sclerotinia rot; the bacterial leaf blight includes rice bacterial leaf blight; and the mosaic virus includes turnip mosaic virus.

[0011] Furthermore, the benzaldehyde oxime is applied in formulation form, and the application method includes spraying or irrigating the target plants.

[0012] A plant immune inducer, wherein benzaldehyde oxime is the active ingredient.

[0013] The application of the above-mentioned plant immune inducers in improving plant resistance to biological stress, wherein the biological stress is selected from any one or more of oomycete diseases, fungal diseases, bacterial diseases, and viral diseases; the oomycete diseases include Phytophthora root rot; the fungal diseases include Fusarium head blight, purple blotch, and sclerotinia rot; the bacterial diseases include bacterial leaf blight; and the viral diseases include mosaic virus.

[0014] Furthermore, the plant is selected from any one or more of food crops, oil crops, and cash crops.

[0015] Furthermore, the food crops include wheat and rice, the oil crops include soybeans and rapeseed, and the cash crops include tobacco.

[0016] Furthermore, the Phytophthora root rot includes soybean Phytophthora root rot; the Fusarium head blight includes wheat Fusarium head blight; the purple blotch includes soybean purple blotch; the sclerotinia rot includes rapeseed sclerotinia rot; the bacterial leaf blight includes rice bacterial leaf blight; and the mosaic virus includes turnip mosaic virus.

[0017] The beneficial effects of this invention are:

[0018] This invention confirms that benzaldehyde oxime can induce immune activity in plants against a variety of major diseases in agricultural production, and has the potential to be developed into a plant immune inducer, effectively enhancing the plant's resistance to biotic stress. Benzaldehyde oxime can be used to control major types of diseases in agricultural production, including oomycete diseases (such as soybean Phytophthora root rot), fungal diseases (such as wheat scab, soybean purple blotch, and rapeseed sclerotinia rot), bacterial diseases (such as rice bacterial blight), and viral diseases (such as turnip mosaic virus), showing high broad-spectrum immune-inducing activity.

[0019] Specifically, benzaldehyde oxime can significantly promote the release of reactive oxygen species in plants and activate immune defense responses. At a concentration of 7.81 µg / mL, benzaldehyde oxime can induce an immune response in soybeans, significantly improving soybean resistance to soybean root rot. At a concentration of 50 µg / mL, benzaldehyde oxime can significantly reduce the incidence of soybean purple blotch in the field; significantly improve wheat resistance to wheat scab; significantly inhibit the infection of rice bacterial blight pathogen PXO99 on rice leaves, shorten the length of rice leaf lesions, and significantly improve rice resistance to rice bacterial blight; effectively inhibit the infection of Sclerotinia sclerotiorum on rapeseed, significantly reduce the diseased area of ​​rapeseed leaves, and significantly improve rapeseed resistance to rapeseed sclerotinia rot; and significantly inhibit the infection of turnip mosaic virus (TuMV) on tobacco, and significantly improve tobacco resistance to turnip mosaic virus disease.

[0020] Benzaldehyde oxime is easy to use and has both preventive and protective effects. It can effectively reduce the harm of various biological stresses to plants, reduce the amount of chemical pesticides used, and lower production costs, which meets the requirements for the development of green and efficient biological pesticides. Attached Figure Description

[0021] Figure 1 The graph shows the reactive oxygen species (ROS) bursts induced in soybean roots by the solution of oxime compound No. 237 in Example 1.

[0022] Figure 2 The image shows the inhibition of Phytophthora soybeani P6497, Fusarium graminearum, and Phytophthora capsici in Example 2 by oxime compound No. 237.

[0023] Figure 3 This is a morphological diagram of soybean etiolated seedlings under CK treatment in Example 3.

[0024] Figure 4 This is a morphological image of soybean etiolated seedlings treated with 31.25 µg / mL oxime compound No. 237 in Example 3.

[0025] Figure 5 This is a morphological image of soybean etiolated seedlings treated with 7.81 µg / mL oxime compound No. 237 in Example 3.

[0026] Figure 6 This is a morphological image of soybean etiolated seedlings treated with 1.95 µg / mL oxime compound No. 237 in Example 3.

[0027] Figure 7 This is a statistical chart showing the length of soybean etiolated seedlings under different treatments in Example 3.

[0028] Figure 8 This is a diagram showing the survival rate of potted soybean seedlings under the CK treatment in Example 4.

[0029] Figure 9 The image shows the survival rate of potted soybean seedlings treated with 125 µg / mL oxime compound No. 237 in Example 4.

[0030] Figure 10 The image shows the survival rate of potted soybean seedlings treated with 31.25 µg / mL oxime compound No. 237 in Example 4.

[0031] Figure 11 The image shows the survival rate of potted soybean seedlings treated with 7.81 µg / mL oxime compound No. 237 in Example 4.

[0032] Figure 12 The image shows the survival rate of potted soybean seedlings treated with 1.95 µg / mL oxime compound No. 237 in Example 4.

[0033] Figure 13 This is a statistical chart showing the survival rate of potted soybean seedlings under different treatments in Example 4.

[0034] Figure 14 The image shows the control effect of different treatments on soybean purple blotch in the field in Example 5.

[0035] Figure 15 This is a statistical chart showing the incidence rate of soybean purple blotch in the field under different treatments in Example 5.

[0036] Figure 16 This is a diagram showing the incidence of sclerotinia stem rot on rapeseed leaves under different treatments in Example 6.

[0037] Figure 17 This is a statistical chart showing the area affected by sclerotinia stem rot in rapeseed leaves under different treatments in Example 6.

[0038] Figure 18 This is a diagram showing the incidence of wheat scab under different treatments in Example 7.

[0039] Figure 19 This is a statistical chart showing the incidence of wheat scab in different treatments in Example 7.

[0040] Figure 20This is a statistical chart of wheat scab disease severity index under different treatments in Example 7.

[0041] Figure 21 This is a diagram showing the incidence of bacterial blight on rice leaves under different treatments in Example 8.

[0042] Figure 22 This is a statistical chart showing the length of bacterial blight lesions on rice leaves under different treatments in Example 8.

[0043] Figure 23 This is a graph showing the effects of different treatments in Example 9 on turnip mosaic virus disease (TuMV) induced by turnip mosaic virus on tobacco.

[0044] The above bar chart * indicates ( P <0.05), ** indicates ( P <0.01), *** indicates ( P <0.001), **** indicates a significant difference ( P <0.0001). Different letters indicate different significance levels. p ≤0.05). Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are intended to facilitate a better understanding of the present invention, but do not limit the present invention.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0047] The culture medium formulations involved in the following examples are as follows:

[0048] V8 medium: Add 1g CaCO3 to 100mL V8 juice, mix well on a magnetic stirrer for 10min, then centrifuge at 3000rpm for 5min, take the supernatant, make up to 1L with deionized water, then add 15g agar powder and stir well, sterilize at 121℃ for 20min.

[0049] PDA medium: Cut 200g of peeled potatoes into small pieces. Add 800mL of deionized water to a pot. After the water boils, add the potatoes and cook for 20 minutes until the water becomes viscous. Add 20g of glucose to a beaker. Filter the viscous potato liquid through three layers of gauze and pour it into the beaker. Then, make up the volume to 1L with deionized water. Add 20g of agar powder and sterilize at 121℃ for 20 minutes.

[0050] NA medium: Dissolve 3g beef extract, 5g peptone, 1g yeast extract, and 10g sucrose in deionized water and bring the volume to 1L. Adjust the pH to 7.2, add 16g agar powder, and sterilize at 121℃ for 20min.

[0051] LB medium: Dissolve 10g sodium chloride, 5g yeast extract and 10g peptone in deionized water and bring the volume to 1L. Sterilize at 121℃ for 20min.

[0052] Mung bean culture medium: Take 60g of mung beans and add 800mL of deionized water to boil. After boiling thoroughly, filter through three layers of gauze to collect the filtrate, then add deionized water to make up to 1L and sterilize at 121℃ for 20min.

[0053] Agar powder solution: Dissolve 0.05g of agar powder in deionized water and bring the volume to 50mL. Sterilize at 121℃ for 20min.

[0054] Unless otherwise specified, the plates (petition dishes) used in the following examples all have a diameter of 90 mm.

[0055] Example 1: Reactive oxygen bursts in oxime-type small molecule compounds No. 237

[0056] Our research group screened a series of oxime small molecule compounds using reactive oxygen species (ROS) emission as an indicator. We identified oxime compound number 237, benzaldehyde oxime, with the following structural formula:

[0057] .

[0058] The reactive oxygen species bursting test procedure for oxime small molecule compound No. 237 is as follows:

[0059] According to literature, the root tip meristem region of plants does not stimulate reactive oxygen species (ROS) to produce an immune response, while the elongation zone is the main region that can stimulate ROS to produce an immune response. Therefore, soybean etiolated seedlings grown in a greenhouse at 25℃ and 70% relative humidity for 4-5 days in darkness were harvested. Based on the regional division of the soybean root tip, the root tip was cut off approximately 1 cm from the top, and an 8 cm root segment was measured as experimental material for the ROS burst test. The cut soybean root was then cut into 5 mm segments, and two segments were randomly selected and placed in one well of a 96-well plate (each well containing 200 µL of sterile water). The plates were then placed in a 25℃ constant temperature incubator in darkness for 30 minutes. Afterward, all the sterile water in each well was aspirated, and 200 µL of ROS reaction solution was added, forming the experimental group. The reactive oxygen species (ROS) reaction solution (200 µL system) included horseradish peroxidase (P6782-10 mg, SIGMA, final concentration 10 µg / mL; stock solution was 1000 times diluted and prepared with sterile water), enhanced luminol (8511-5 G, SIGMA, final concentration 17.7 µg / mL; stock solution was 1000 times diluted and prepared with sterile water), oxime compounds No. 237 (final concentrations of 1.95 µg / mL, 7.81 µg / mL, 31.25 µg / mL, and 125 µg / mL; corresponding stock solution concentrations of 1.95 mg / mL, 7.81 mg / mL, 31.25 mg / mL, and 125 mg / mL, prepared with dimethyl sulfoxide (DMSO), respectively), and sterile water. A control group (CK) was also included, with 200 µL of DMSO-sterile water added, the same volume of DMSO added as the experimental group. Each treatment was performed in triplicate, with one well per replicate. Immediately after adding the reactive oxygen species reaction solution, the 96-well plate was placed in a GLOMAX container. TM The emission of reactive oxygen species (ROS) in soybean roots was detected using a 96-well microplate luminescence analyzer (Promega, USA). Light was avoided during the experiment. Results showed that benzaldehyde oxime (oxime 237), a small molecule oxime, effectively promoted ROS emission and activated the plant's immune response. Figure 1 ).

[0060] Example 2: Antibacterial test of oxime small molecule compound No. 237

[0061] 2.1 The mycelial growth method was used to determine the mycelial growth of *Phytophthora sojae* (a type of fungus) cultured upside down on V8 agar plates at 25°C in the dark for 6 days. Phytophthora sojae P6497, Phytophthora capsici ( Phytophthora capsici ) and Fusarium graminearum that had been incubated upside down on PDA agar plates at 25°C in the dark for 5 days. Fusarium graminearum Create 5mm mycelial cakes around the edge of the colony for later use.

[0062] 2.2 Stock solutions of oxime compound 237, prepared with DMSO at concentrations of 1.95 mg / mL, 7.81 mg / mL, 31.25 mg / mL, and 125 mg / mL, were added to sterilized V8 medium cooled to 50-55°C, respectively, to achieve final concentrations of oxime compound 237 of 1.95 μg / mL, 7.81 μg / mL, 31.25 μg / mL, and 125 μg / mL. 2 mL V8 agar plates (35 mm in diameter) containing different concentrations of oxime compound 237 were then prepared. A 5 mm mycelial cake of *Phytophthora sojae* P6497 was inoculated in the center of each plate as the experimental group. A control group (CK) was also established, consisting of V8 medium containing the same volume of DMSO as the experimental groups but without oxime compound 237. Each treatment was performed in triplicate, with one plate per replicate. The plates were placed upside down in a 25℃ constant temperature incubator and cultured in the dark for 3 days. The observation and recording were made when the mycelial growth in the control group exceeded half of the petri dish.

[0063] 2.3 Stock solutions of oxime compound 237, prepared with DMSO at concentrations of 1.95 mg / mL, 7.81 mg / mL, 31.25 mg / mL, and 125 mg / mL, were added to sterilized V8 medium cooled to 50-55°C, respectively, to achieve final concentrations of oxime compound 237 of 1.95 μg / mL, 7.81 μg / mL, 31.25 μg / mL, and 125 μg / mL. 2 mL V8 agar plates (35 mm in diameter) containing different concentrations of oxime compound 237 were then prepared. A 5 mm *Phytophthora capsici* clod was inoculated in the center of each plate as the experimental group. A control group (CK) was also established, consisting of V8 medium containing the same volume of DMSO as the experimental groups but without oxime compound 237. Each treatment was performed in triplicate, with one plate per replicate. The plates were placed upside down in a 25℃ constant temperature incubator and cultured in the dark for 3 days. The observation and recording were made when the mycelial growth in the control group exceeded half of the petri dish.

[0064] 2.4 Stock solutions of oxime compound 237, prepared with DMSO at concentrations of 1.95 mg / mL, 7.81 mg / mL, 31.25 mg / mL, and 125 mg / mL, were added to sterilized PDA medium cooled to 50-55°C, respectively, to achieve final concentrations of oxime compound 237 of 1.95 μg / mL, 7.81 μg / mL, 31.25 μg / mL, and 125 μg / mL. 2 mL PDA agar plates (35 mm in diameter) containing different concentrations of oxime compound 237 were then prepared. A 5 mm Fusarium graminearum mycelium cake was inoculated in the center of each plate as the experimental group. A control group (CK) was also included, consisting of PDA medium containing the same volume of DMSO as the experimental groups but without oxime compound 237. Each treatment was performed in triplicate, with one plate per replicate. The plates were placed upside down in a 25℃ constant temperature incubator and cultured in the dark for 3 days. The observation and recording were made when the mycelial growth in the control group exceeded half of the petri dish.

[0065] The results are as follows Figure 2 As shown, oxime compound No. 237 has no direct inhibitory effect on Phytophthora soybean P6497, Fusarium graminearum, and Phytophthora capsici.

[0066] Example 3: Effects of oxime compound No. 237 on the growth of soybean etiolated seedlings

[0067] Soybeans (Hefeng 47) were planted in cylindrical pots filled with sterile vermiculite (the inner diameter of the pot is about 13 cm and the inner height is about 13 cm). Ten soybeans were planted in each pot (the height of vermiculite above the soybeans is about 1 cm). An experimental group (different concentrations of 237 oxime small molecule compound solution were poured in: the 31.25 mg / mL, 7.81 mg / mL and 1.95 mg / mL of 237 oxime small molecule compound stock solution prepared with DMSO were diluted with sterile water to 31.25 µg / mL, 7.81 µg / mL and 1.95 µg / mL respectively) and a control group (CK, poured in DMSO-sterile water, the volume of DMSO added is the same as that added to the experimental group). After soybeans were planted, 50 mL of a solution of oxime compound No. 237 at different concentrations (31.25 µg / mL, 7.81 µg / mL, and 1.95 µg / mL, respectively) was applied to the roots of one potted plant in the experimental group (root irrigation). 50 mL of DMSO-sterile water was applied to the roots of one potted plant in the control group (root irrigation). The plants were then grown in darkness for 4 days at 25℃ and 70% relative humidity, without watering. After 4 days, the length of the yellowed soybean seedlings (i.e., the height of each soybean plant emerging from the soil) was recorded.

[0068] The results are as follows Figures 3-7As shown, compared with the control group, oxime compound No. 237 has a certain promoting effect on the growth of soybean etiolated seedlings.

[0069] Example 4: Oxime compound No. 237 enhances soybean resistance to Phytophthora root rot.

[0070] 4.1 Inoculate *Phytophthora sojae* P6497 into a petri dish containing 15 mL of V8 medium and incubate upside down at 25°C in the dark for 6 days until the culture medium completely covers the dish. *Phytophthora sojae* P6497 plates are used for preparing *Phytophthora sojae* P6497 mycelial cakes. Use a 5 mm diameter punch to create 5 mm mycelial cakes around the edge of the colony for later use.

[0071] 4.2 Soybeans (Hefeng 47) were planted in cylindrical pots filled with sterile vermiculite (the inner diameter of the pot was about 15cm and the inner height was about 13cm). 15 soybeans were sown in each pot (the height of the vermiculite above the soybeans was about 1cm). The plants were grown in a greenhouse at 25℃, 16h light / 8h darkness, and 70% relative humidity for 10 days, and watered once every 3 days.

[0072] 4.3 After 10 days, soybean seedlings were treated, and experimental groups (different concentrations of 237 oxime small molecule compound solution were poured in: 125 mg / mL, 31.25 mg / mL, 7.81 mg / mL, and 1.95 mg / mL of 237 oxime small molecule compound stock solution prepared by DMSO were diluted with sterile water to 125 µg / mL, 31.25 µg / mL, 7.81 µg / mL, and 1.95 µg / mL, respectively) and control group (CK, poured in DMSO-sterile water, the volume of DMSO added was the same as that added to the experimental group) were set up. Each treatment was set up with 4 replicates, and each replicate was in a pot. The experimental group received 50 mL of a solution of oxime compound No. 237 at different concentrations (125 µg / mL, 31.25 µg / mL, 7.81 µg / mL, and 1.95 µg / mL) daily as a root drenching for 5 days. The control group received 50 mL of sterile DMSO water daily as a root drenching for 5 days. No watering was required during the treatment period.

[0073] 4.4 Twenty-four hours after the final treatment, soybean seedlings were inoculated using a wound inoculation method. Using a sterile blade, a longitudinal wound approximately 1 cm long was made downwards at the base of the soybean seedling stem (2-3 cm from the vermiculite surface). A 5 mm diameter mycelial cake of *Phytophthora soybeanis* P6497 was then placed at the wound site (wound inoculation), with the mycelial side in contact with the wound. After wound inoculation, the soybean potted plants were placed in a glass dome to maintain humidity and cultured for 48 hours in a greenhouse at 25°C, 16 hours light / 8 hours darkness, and 70% relative humidity (no watering required during this period). The soybean potted plants were then removed from the glass dome and cultured under the same greenhouse conditions for another 7 days, watering every 3 days. Finally, the incidence of *Phytophthora soybeanis* root rot in each treatment was statistically analyzed, and the survival rate was calculated using the formula: Survival rate = Number of healthy surviving plants / Total number of potted plants.

[0074] The results are as follows Figures 8-13 As shown, the control group exhibited yellowing leaves, lodging, and an inverted "V" shape in soybean seedlings, symptoms characteristic of Phytophthora root rot caused by Phytophthora soybeanis. After root drenching with a solution of oxime compound No. 237, the survival rates of soybean seedlings in the experimental groups with concentrations of 125 µg / mL, 31.25 µg / mL, and 7.81 µg / mL were significantly higher than those in the control group. The highest survival rate was observed at a concentration of 31.25 µg / mL. This indicates that oxime compound No. 237 can significantly improve the resistance of soybeans to Phytophthora root rot.

[0075] Example 5: Effects of oxime compound No. 237 on soybean purple blotch in the field

[0076] This experiment was conducted in a field in Suzhou City, Anhui Province, using the soybean variety Nannong 77. Field management was carried out according to standard procedures, without the application of pesticides. The experimental group (containing oxime compound No. 237) was first sprayed with a solution of oxime compound No. 237 (prepared with sterile water, containing 50 µg / mL of oxime compound No. 237 (stock solution concentration of 125 mg / ml, prepared with DMSO) and 0.1 wt% surface dispersant Tween 20; sprayed on soybean leaves; the same applies below), at a rate of 1 L / 4.5 m³. 2 Seven days later (September 3rd), a second spray of the oxime-type small molecule compound solution No. 237 was applied, with the same spray volume as the first spray. The control group (CK) was sprayed with an equal volume of 0.1 wt% surface dispersant Tween 20 (prepared with sterile water and containing the same volume of DMSO as the experimental group). Each treatment was replicated in triplicate, with each replicate constituting one experimental plot, and the plot area was 4.5 m². 2Fifty days after the second spraying, soybean plants from the field were collected. Ten plants were randomly selected from each experimental plot for each treatment for threshing. The incidence of purple blotch disease in the field was statistically analyzed, and the incidence rate was calculated. The formula for calculating the incidence rate of purple blotch disease is: Incidence rate of purple blotch disease = (Number of infected grains / Total number of grains surveyed) × 100%.

[0077] The results are as follows Figures 14-15 As shown, a small molecule compound of oxime No. 237 at a concentration of 50 µg / mL can significantly reduce the incidence of soybean purple blotch in the field.

[0078] Example 6: Oxime compound No. 237 enhances the resistance of rapeseed to sclerotinia stem rot.

[0079] 6.1 Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Disinfect the sclerotia once with a 15w / v% sodium hypochlorite solution (containing 0.1wt% Tween 20) (the contact time with the disinfectant should not exceed 1 minute). After disinfection, dry them and bury them in PDA medium (make a slit in the PDA medium with a pipette tip and insert the sclerotia into it), place them in an incubator at 20℃ and 60% humidity, and incubate them upside down in the dark for 3 days. When fresh and tender mycelia grow, use a pipette tip (about 3mm in diameter) to break the mycelia along the edge into mycelial cakes about 3mm in diameter for later use.

[0080] 6.2 Rapeseed (Xiangyou 15, two-leaf-one-heart stage) was planted in cylindrical pots (approximately 13cm in diameter and 13cm in height) filled with substrate (Xingxing Xiangnong brand special substrate produced by Jiangsu Xingnong Substrate Technology Co., Ltd., product number: 161102G0097N). One plant was planted in each pot and cultivated in a greenhouse at 22°C, 16h light / 8h darkness, and 60% humidity. Watering was done every 2 days. When the plants reached the initial stage of four true leaves unfolding, spraying treatment was started. Two groups were set up: the experimental group (sprayed with a solution of oxime compound No. 237, with the stock solution of DMSO prepared at a concentration of 50mg / mL diluted with sterile water to 50µg / mL) and the control group (CK, sprayed with sterile water (containing the same volume of DMSO as the experimental group)). Each treatment was replicated 4 times, with 1 pot per replicate. 3 mL of a 50 µg / mL solution of oxime compound No. 237 was evenly sprayed onto the leaves of each rapeseed plant in the experimental group. The control group (CK) was sprayed with an equal volume of sterile water (containing the same volume of DMSO as the experimental group). The spraying was performed twice, with a 3-day interval between the two applications. The culture conditions remained unchanged during the spraying period.

[0081] 6.3 Twenty-four hours after the second spraying, select the largest leaf with the best growth. Place a *Sclerotinia sclerotiorum* mycelium (with 80 µL of agar powder solution added to prevent mycelial death during inoculation) upside down onto the leaf (mycelial side in contact with the leaf). Secure the leaf by wrapping a layer of sealing film around the center (using a veinless area) to prevent the mycelium from falling off during subsequent cultivation. Place the entire plant in a sealed glass container at 22°C, 16 hours of light / 8 hours of darkness, and 60% humidity. No watering is required during this period. After 3 days, select inoculated leaves for phenotypic observation.

[0082] The area of ​​diseased leaves was calculated using ImageJ software, and the results are as follows: Figures 16-17 As shown, the diseased area of ​​rapeseed leaves sprayed with oxime compound No. 237 was significantly lower than that of the control group, indicating that oxime compound No. 237 can significantly improve the resistance of rapeseed to sclerotinia stem rot.

[0083] Example 7: Efficacy test of oxime small molecule compound No. 237 against wheat scab in greenhouse

[0084] Test site and materials: Wheat field in greenhouse of Huai'an Academy of Agricultural Sciences, and the variety of material was Huaimai 33.

[0085] Experimental Methods: Sowing was carried out on October 30, 2024, using machine row sowing at a seeding rate of 35 kg / mu. Field management was conducted according to conventional methods, without the application of pesticides. The experimental group (using oxime small molecule compound No. 237, sprayed a total of 3 times) received the first application of oxime small molecule compound No. 237 solution (prepared with sterile water, containing 50 µg / mL of oxime small molecule compound No. 237 (stock liquor concentration of 50 mg / mL, prepared with DMSO) and 0.1 wt% surface dispersant Tween 20; sprayed on wheat ears and leaves; the same applies below) at the wheat ear and leaf area, using a dosage of 750 mL / 13 m 2 The control group (CK) was sprayed with an equal volume of 0.1 wt% Tween 20 surface dispersant (prepared with sterile water and containing the same volume of DMSO as the experimental group). Each treatment was repeated in triplicate, with one plot per replicate, and the plot area was 13 m². 2 Two days after spraying, inoculate against Fusarium head blight using the artificial single-flower drip method. The spore solution used for inoculation is a mixed strain of wheat Fusarium head blight strains F0301, F0609, F0980, and F1312 (cultured in mung bean medium). Before use, dilute the spore concentration to 10% with sterile water. 5 spores / mL. 20µL of spore solution was dripped into one spikelet in the middle of the wheat ear. Each treatment was inoculated with 600 spikelets, i.e., 200 spikelets per plot. A second spray was applied 3 days after inoculation (same as the first spray), and a third spray was applied 7 days after inoculation (same as the first spray).

[0086] On May 28, 2025, a survey on the severity of wheat scab was conducted to calculate the disease incidence rate and disease index. The disease incidence rate was calculated as follows: Disease incidence rate (%) = (Number of diseased ears / Total number of ears surveyed (i.e., 200 ears)) × 100%; The disease index was calculated as follows: Disease index = ∑(Number of ears with disease at each level × Disease level) / (Total number of ears surveyed (i.e., 200 ears) × Highest disease level (i.e., level 4)) × 100.

[0087] Disease classification:

[0088] Level 0: No disease;

[0089] Grade 1: The number of diseased spikelets accounts for less than 25% of all spikelets (i.e., the diseased spikelet rate is <25%).

[0090] Level 2: Diseased spikelets account for 25%-50% of all spikelets (i.e., 25% ≤ diseased spikelet rate < 50%).

[0091] Level 3: Diseased spikelets account for 50%-75% of all spikelets (i.e., 50% ≤ diseased spikelet rate < 75%).

[0092] Level 4: Diseased spikelets account for more than 75% of all spikelets (i.e., diseased spikelet rate ≥ 75%).

[0093] The results are as follows Figure 18-20 As shown, after spraying the solution of oxime-like small molecule compound No. 237, the incidence of wheat scab and the disease index were significantly reduced, indicating that oxime-like small molecule compound No. 237 can significantly improve wheat resistance to wheat scab.

[0094] Example 8: Oxime compound No. 237 enhances rice resistance to bacterial blight.

[0095] 8.1 Remove the PXO99 strain (the pathogen of rice bacterial blight) stored at -80℃. Under aseptic conditions, take 50 μL of the bacterial suspension and spread it evenly on the surface of NA medium containing 50 μg / mL kanamycin. Incubate in the dark at 28℃ for 48 h. After a uniform yellow colony formation on the plate, add a small amount of sterile water to the petri dish. Use a sterile spatula to scrape the bacteria and transfer them to a 50 mL centrifuge tube. Gently invert the tube to mix the bacteria thoroughly. Centrifuge at 4500g for 10 min at room temperature and discard the supernatant. Resuspend the bacteria in sterile water and mix well. Measure the OD600 using a NanoDrop ultraviolet-visible spectrophotometer (Thermo Fisher Scientific, USA). Adjust the bacterial suspension concentration to OD600 = 0.5 with sterile water.

[0096] 8.2 Germinated rice seeds (CO39) were planted in cylindrical pots (approximately 13 cm in diameter and 13 cm in height) filled with black soil (from Baishan City, Jilin Province), with 30 seedlings per pot. They were cultivated in soil at 28℃, 16h light / 8h darkness, and 70% relative humidity, watered every 3 days. After 20 days of soil cultivation, each pot of rice seedlings was evenly sprayed (on the leaves) with 5 mL of a 50 μg / mL solution of oxime compound No. 237 (a 50 mg / mL stock solution of DMSO diluted with sterile water) as the experimental group. The control group (CK) was sprayed with the same volume of sterile water (containing the same volume of DMSO as the experimental group). After spraying, the pots were placed in a transparent humidity chamber at 28℃, 16h light / 8h darkness, and 70% relative humidity for 24 hours without watering.

[0097] 8.3 After 24 hours of cultivation, select the second-to-last functional leaf of each plant for inoculation with the PXO99 strain. During the procedure, dip the blade of the scissors into the bacterial suspension prepared in 8.1 (ensuring the blade is evenly coated with droplets), and cut the leaf tip approximately 1 cm from the tip, ensuring the wound is fully in contact with the bacterial suspension. After inoculation, place the plants directly in an artificial climate incubator with the following conditions: 28℃, 70% relative humidity, 16 hours light / 8 hours darkness, and watering every 3 days. Based on the disease response, 10-14 days after inoculation (measurements were taken at the same time for each treatment), select 8 plants with consistent leaf size and growth status from each treatment. Measure the length of leaf lesions at the inoculation site using a ruler and record the data.

[0098] The results are as follows Figure 21-22 As shown, compared with the control group, the length of lesions on rice leaves sprayed with the solution of oxime No. 237 was significantly shortened and the severity of rice bacterial blight was significantly reduced, indicating that oxime No. 237 can significantly improve the resistance of rice to rice bacterial blight.

[0099] Example 9: Oxime compound No. 237 enhances tobacco resistance to turnip mosaic virus disease.

[0100] 9.1 The germinated tobacco (Tobacco Benedict) Nicotiana benthamiana The seedlings were planted in cylindrical plastic cups filled with vermiculite (approximately 7 cm in inner diameter and 7.5 cm in inner height), one seedling per cup, and cultivated in a greenhouse at 25°C, 16 h light / 8 h dark, and 70% relative humidity, watered every 3 days. Once the seedlings reached the 4-leaf stage and were of uniform size, they were used for plant virus inoculation experiments.

[0101] 9.2 Products containing turnip mosaic virus (CPV) Turnip mosaic virusAgrobacterium bifidum culture carrying the green fluorescent protein (GFP) gene (i.e., the GFP gene fragment is integrated into the TuMV genome) expression vector was added to a 50 mL centrifuge tube containing LB medium. Kanamycin and rifampin were then added at a ratio of 1:1:1000 (50 μg / mL kanamycin stock solution: 50 μg / mL rifampin stock solution: LB medium). The mixture was incubated at 28°C and 200 rpm for 16 h. After centrifugation at 6000 g at room temperature for 10 min, the supernatant was discarded, and a resuspension solution (sterile water containing 150 µmol / L acetosyringone (AS), 10 mmol / L 2-morpholinoethanesulfonic acid (MES), 10 mmol / L MgCl2, pH 5.6) was added to the precipitate to prepare a bacterial suspension (OD600=1). The suspension was then incubated at room temperature in the dark for 2 h before use.

[0102] 9.3 At 21 days of growth (i.e., the 4-leaf stage) of *Nicotiana benthamiana*, 3 mL of a 50 μg / mL solution of oxime compound No. 237 (prepared by diluting a 50 mg / mL stock solution of DMSO with sterile water 1000 times) was sprayed onto each cup of *Nicotiana benthamiana* leaves using a spray bottle as the experimental group. The control (CK) was sprayed with an equal volume of sterile water (containing the same volume of DMSO as the experimental group). Each treatment was repeated in triplicate, with one cup per replicate. Inoculation was performed 3 days later. The bacterial suspension containing TuMV prepared in 9.2 was drawn into a syringe and inoculated onto 3 true leaves (the 3 true leaves located below the fully unfolded young leaves). The injection volume was approximately 0.5 mL per leaf (2 injection points, approximately 0.25 mL at each injection point). After inoculation, the tobacco plants were placed in a greenhouse at 25°C, with 16 hours of light / 8 hours of darkness and 70% relative humidity for 10 days. Watering was carried out every 3 days. The disease incidence of the tobacco plants was then observed under 470nm long-wave ultraviolet light at 4, 6 and 10 days after inoculation.

[0103] The results are as follows Figure 23 As shown, inoculated leaves (leaves directly inoculated with turnip mosaic virus) and systemic leaves (new leaves above the inoculated leaves) were selected for observation. Compared with the control group, the leaves of Tobacco Benedictine plants sprayed with solution of oxime No. 237 showed a significant decrease in GFP fluorescence signal (reflecting turnip mosaic virus infection) under long-wave ultraviolet light, and the symptoms were significantly alleviated, indicating that oxime No. 237 can significantly improve the resistance of tobacco to turnip mosaic virus disease.

[0104] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The application of benzaldehyde oxime in improving plant resistance to biotic stress, characterized in that, The biological stresses are selected from one or more of the following: soybean Phytophthora root rot, wheat Fusarium head blight, soybean purple blotch, rapeseed sclerotinia stem rot, rice bacterial blight, and turnip mosaic virus.

2. The application according to claim 1, characterized in that, The benzaldehyde oxime is applied in formulation form, and the application method includes spraying or irrigating the target plants.

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