Use of 4,4'-difluorobenzophenone oxime in improving plant resistance to biotic stress

Abstract

The application discloses application of 4,4'-difluorobenzophenone oxime in improving plant resistance to biological stress, and further discloses a plant immune inducer with 4,4'-difluorobenzophenone oxime as an active ingredient. The 4,4'-difluorobenzophenone oxime can be used for preventing and controlling main disease types in agricultural production, including oomycete diseases (such as soybean Phytophthora root rot), fungal diseases (such as soybean purple spot and oilseed rape sclerotinia stem rot) and bacterial diseases (such as rice bacterial leaf blight), and shows high broad-spectrum immune induction activity. The 4,4'-difluorobenzophenone oxime is convenient to use, has the functions of prevention and protection, can effectively reduce the harm of various biological stresses to plants, reduce the amount of chemical pesticides, reduce production cost, and meets the development requirements 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 4,4'-difluorobenzophenone oxime in improving plant resistance to biotic stress. Background Technology

[0002] Plant diseases are a significant factor restricting the quality of agricultural products. For a long time, chemical fungicides have dominated disease control, but their extensive use easily leads to a surge in pathogen resistance, excessive residues in agricultural products, and damage to soil and the ecological environment, severely hindering the development of green agriculture and sustainable plant protection. Against this backdrop, plant immune inducers, with their unique advantage of activating the plant's own innate immune system and inducing systemic resistance / specific resistance (ISR / SAR) rather than directly killing pathogens, have become a research hotspot in new green control technologies and are hailed as plant "immune vaccines."

[0003] Plant immune inducers recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), initiating core defense pathways such as calcium ion signaling, MAPK cascade reactions, salicylic acid (SA), and jasmonic acid (JA). This upregulates the synthesis of pathogenesis-associated proteins (PRs), defense enzyme systems (PAL, POD, SOD, CAT), and antimicrobial substances such as phytoalexins and lignin, achieving broad-spectrum resistance against various pathogens including fungi, bacteria, and viruses. They possess significant advantages such as being environmentally friendly, less prone to inducing resistance, and having a mild and long-lasting effect. In recent years, small molecule compounds have gradually become an important direction in the research and development of plant immune inducers due to their simple structure, high safety, and easy absorption and metabolism by plants.

[0004] 4,4'-Difluorobenzophenone oxime, with the molecular formula C 13 H9F2NO, with a molecular weight of 233.22, belongs to the ketoxime class of small molecule compounds. Current research on 4,4'-difluorobenzophenone oxime mainly focuses on optimizing its synthetic processes as a pharmaceutical or pesticide intermediate, developing new derivatives, and its applications in catalysis and other fields. There are no reports on its use as a plant immune inducer. Therefore, developing novel plant immune inducers with 4,4'-difluorobenzophenone 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 4,4'-difluorobenzophenone oxime in improving plant resistance to biotic stress, thereby overcoming the shortcomings of the prior art.

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

[0007] Application of 4,4'-Difluorobenzophenone oxime in improving plant resistance to biotic stress, wherein the biotic stress is selected from any one or more of oomycete diseases, fungal diseases, and bacterial diseases; wherein the oomycete diseases include Phytophthora root rot; wherein the fungal diseases include purple blotch and sclerotinia rot; and wherein the bacterial diseases include bacterial leaf blight.

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

[0009] Furthermore, the food crops include rice ( Oryza sativa L., the oilseed crop includes soybean (L.), Glycine max (L.) Merr.), rapeseed ( Brassica napus L.).

[0010] Furthermore, the Phytophthora root rot includes soybean Phytophthora root rot (… Phytophthora sojae Kaufmann & Gerdemann); the purple blotch disease includes soybean purple blotch (Kaufmann & Gerdemann); Cercospora kikuchii (Matsumoto & Tomoyasu) Chupp); the sclerotinia disease includes rapeseed sclerotinia disease ( Sclerotinia sclerotiorum (Lib.) deBary); the bacterial leaf blight includes rice bacterial leaf blight ( Xanthomonas oryzae pv. oryzae (Ishiyama) Swings).

[0011] Furthermore, the 4,4'-difluorobenzophenone oxime is applied in formulation form, and the application method includes spraying or irrigating the target plants.

[0012] A plant immune inducer, wherein the plant immune inducer uses 4,4'-difluorobenzophenone oxime as its active ingredient.

[0013] The above-mentioned plant immune inducers are used to improve the resistance of plants to biological stress, wherein the biological stress is selected from any one or more of oomycete diseases, fungal diseases, and bacterial diseases; the oomycete diseases include Phytophthora root rot; the fungal diseases include purple blotch and sclerotinia rot; and the bacterial diseases include bacterial leaf blight.

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

[0015] Furthermore, the grain crops include rice, and the oil crops include soybeans and rapeseed.

[0016] Furthermore, the Phytophthora root rot includes soybean Phytophthora root rot; the purple blotch disease includes soybean purple blotch; the sclerotinia rot includes rapeseed sclerotinia rot; and the bacterial leaf blight includes rice bacterial leaf blight.

[0017] The beneficial effects of this invention are:

[0018] This invention confirms that 4,4'-difluorobenzophenone oxime can induce immune activity in plants against a variety of major diseases in agricultural production, possessing the potential to be developed into a plant immune inducer and effectively enhancing the plant's resistance to biotic stress. 4,4'-difluorobenzophenone 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 soybean purple blotch and rapeseed sclerotinia rot), and bacterial diseases (such as rice bacterial blight), exhibiting high broad-spectrum immune-inducing activity.

[0019] Specifically, 4,4'-difluorobenzophenone oxime significantly promotes the release of reactive oxygen species in plants, activating immune defense responses. At a concentration of 1.95 µg / mL, 4,4'-difluorobenzophenone oxime induces an immune response in soybeans. At a concentration of 50 µg / mL, 4,4'-difluorobenzophenone oxime significantly reduces the incidence of soybean purple blotch in the field; it significantly inhibits the infection of rice bacterial blight pathogen PXO99 on rice leaves, shortens the length of lesions on rice leaves, and significantly improves rice resistance to rice bacterial blight; it effectively inhibits the infection of Sclerotinia sclerotiorum on rapeseed, significantly reduces the diseased area of ​​rapeseed leaves, and significantly improves rapeseed resistance to rapeseed sclerotinia stem rot. At a concentration of 125 µg / mL, 4,4'-difluorobenzophenone oxime significantly improves soybean resistance to soybean Phytophthora root rot.

[0020] 4,4'-Difluorobenzophenone 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. 256 in Example 1.

[0022] Figure 2 The image shows the antibacterial activity of oxime compound No. 256 in Example 2 against Phytophthora soybean P6497 and Fusarium graminearum.

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

[0024] Figure 4This is a morphological image of soybean etiolated seedlings treated with 31.25 µg / mL oxime compound No. 256 in Example 3.

[0025] Figure 5 This is a morphological image of soybean etiolated seedlings treated with 7.81 µg / mL oxime compound No. 256 in Example 3.

[0026] Figure 6 This is a morphological image of soybean etiolated seedlings treated with 1.95 µg / mL oxime compound No. 256 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. 256 in Example 4.

[0030] Figure 10 This is a statistical chart showing the survival rate of potted soybean seedlings under different treatments in Example 4.

[0031] Figure 11 The image shows the control effect of different treatments on soybean purple blotch in the field in Example 5.

[0032] Figure 12 This is a statistical chart showing the incidence rate of soybean purple blotch in the field under different treatments in Example 5.

[0033] Figure 13 This is a diagram showing the incidence of sclerotinia stem rot on rapeseed leaves under different treatments in Example 6.

[0034] Figure 14 This is a statistical chart showing the area of ​​rapeseed leaves affected by sclerotinia stem rot under different treatments in Example 6.

[0035] Figure 15 This is a diagram showing the incidence of bacterial blight on rice leaves under different treatments in Example 7.

[0036] Figure 16 This is a statistical chart showing the length of bacterial blight lesions on rice leaves under different treatments in Example 7.

[0037] 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

[0038] 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.

[0039] 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.

[0040] The culture medium formulations involved in the following examples are as follows:

[0041] V8 liquid culture 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, and sterilize at 121℃ for 20min.

[0042] 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.

[0043] 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.

[0044] PDB 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 and sterilize at 121℃ for 20 minutes.

[0045] 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.

[0046] 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.

[0047] Agar powder solution: Dissolve 0.05g of agar powder in deionized water and bring the volume to 50mL. Sterilize at 121℃ for 20min.

[0048] Unless otherwise specified, the plates (petition dishes) used in the following examples all have a diameter of 90 mm.

[0049] Example 1: Reactive oxygen bursts in oxime-type small molecule compound No. 256

[0050] Our research group screened a series of oxime small molecule compounds using reactive oxygen species (ROS) emission as an indicator, and identified oxime small molecule compound number 256, 4,4'-difluorobenzophenone oxime, with the following structural formula:

[0051] .

[0052] The reactive oxygen species bursting test procedure for oxime small molecule compound No. 256 is as follows:

[0053] 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 compound No. 256 (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 release 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 oxime compound 256, 4,4'-difluorobenzophenone oxime, significantly promoted ROS release and activated the plant's immune response. Figure 1 ).

[0054] Example 2: Antibacterial test of oxime small molecule compound No. 256

[0055] 2.1 The liquid culture method was used to determine the presence of *Phytophthora sojae* (a type of mold) on V8 agar plates that had been incubated upside down for 6 days at 25°C in the dark. Phytophthora sojae P6497 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 a 5mm mycelial cake around the edge of the colony and set it aside.

[0056] 2.2 Stock solutions of oxime compound No. 256, prepared with DMSO at concentrations of 2 mg / mL, 4 mg / mL, 8 mg / mL, 16 mg / mL, and 32 mg / mL, were added to V8 liquid medium to achieve final concentrations of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL, respectively. One mL of each concentration of the drug-containing medium was added to each well of a 24-well plate, and a 5 mm mycelial cake of *Phytophthora sojae* P6497 was inoculated into each well as the experimental group. A control group (CK) was also set up, consisting of V8 liquid medium containing the same volume of DMSO as the experimental group but without oxime compound No. 256. Each treatment was replicated in triplicate, with one well per replicate. The 24-well plates were incubated in the dark at 25°C for 3 days. Observation and recording were performed after the mycelial growth in the control group was complete.

[0057] 2.3 Stock solutions of oxime compound 256 at concentrations of 2 mg / mL, 4 mg / mL, 8 mg / mL, 16 mg / mL, and 32 mg / mL were prepared using DMSO and added to PDB medium, resulting in final concentrations of oxime compound 256 of 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL, respectively. 1 mL of each concentration of the drug-containing medium was added to 24-well plates, and a 5 mm *Fusarium graminearum* cake was inoculated into each well as the experimental group. A control group (CK) was also set up, consisting of PDB medium containing the same volume of DMSO as the experimental group but without oxime compound 256. Each treatment was replicated in triplicate, with one well per replicate. The 24-well plates were incubated in the dark at 25°C for 3 days. Observation and recording were performed after the mycelium in the control group had fully grown.

[0058] The results are as follows Figure 2 As shown, oxime compound No. 256 has no direct inhibitory effect on Phytophthora soybean P6497 and Fusarium graminearum.

[0059] Example 3: Effects of oxime compound No. 256 on the growth of etiolated soybean seedlings

[0060] 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 oxime small molecule compound solution No. 256 were poured in: the 31.25 mg / mL, 7.81 mg / mL and 1.95 mg / mL of oxime small molecule compound No. 256 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. 256 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.

[0061] The results are as follows Figures 3-7 As shown, compared with the control group, oxime compound No. 256 has a certain promoting effect on the growth of soybean etiolated seedlings.

[0062] Example 4: Oxime compound No. 256 enhances soybean resistance to Phytophthora root rot.

[0063] 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.

[0064] 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.

[0065] 4.3 After 10 days, soybean seedlings were treated in two groups: an experimental group (treated with a solution of oxime compound No. 256: a 125 mg / mL stock solution of oxime compound No. 256 prepared in DMSO was diluted with sterile water to a concentration of 125 µg / mL) and a control group (CK: treated with DMSO-sterile water, the volume of DMSO added was the same as that added in the experimental group). Each treatment was replicated in four batches, with one pot per replicate. The experimental group received 50 mL of the 125 µg / mL solution of oxime compound No. 256 per pot daily for 5 days. The control group received 50 mL of DMSO-sterile water per pot daily for 5 days. No watering was required during the treatment period.

[0066] 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.

[0067] The results are as follows Figures 8-10 As shown, the control group of soybean seedlings exhibited yellowing leaves, lodging, and an inverted "V" shape, which are symptoms of soybean root rot caused by Phytophthora infestans. The survival rate of soybean seedlings treated with root irrigation of a solution of oxime No. 256 was significantly higher than that of the control group, indicating that oxime No. 256 can significantly improve the resistance of soybeans to soybean root rot.

[0068] Example 5: Effects of oxime compound No. 256 on soybean purple blotch in the field

[0069] 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. 256) was first sprayed with a solution of oxime compound No. 256 (prepared with sterile water, containing 50 µg / mL of oxime compound No. 256 (stock liquor concentration of 50 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. 2Seven days later (September 3rd), a second spray of oxime compound solution No. 256 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. 2 Fifty 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%.

[0070] The results are as follows Figures 11-12 As shown, a small molecule compound of oxime No. 256 at a concentration of 50 µg / mL can significantly reduce the incidence of soybean purple blotch in the field.

[0071] Example 6: Oxime compound No. 256 enhances the resistance of rapeseed to sclerotinia stem rot.

[0072] 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.

[0073] 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. 256, 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. 256 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.

[0074] 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.

[0075] The area of ​​diseased leaves was calculated using ImageJ software, and the results are as follows: Figure 13-14 As shown, the diseased area of ​​rapeseed leaves sprayed with oxime compound No. 256 was significantly lower than that of the control group, indicating that oxime compound No. 256 can significantly improve the resistance of rapeseed to sclerotinia stem rot.

[0076] Example 7: Oxime compound No. 256 enhances rice resistance to bacterial blight.

[0077] 7.1 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.

[0078] 7.2 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. 256 (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.

[0079] 7.3 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 7.1 (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 of 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.

[0080] The results are as follows Figure 15-16 As shown, compared with the control group, the length of rice leaf lesions was significantly shortened and the severity of rice bacterial blight was significantly reduced when the solution of oxime No. 256 was sprayed, indicating that oxime No. 256 can significantly improve the resistance of rice to rice bacterial blight.

[0081] 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

The application of 1,4'-difluorobenzophenone oxime in improving plant resistance to biotic stress is characterized by, The biological stress is one or more of the following: soybean root rot caused by Phytophthora infestans, soybean purple blotch, rapeseed sclerotinia stem rot, and rice bacterial blight.

2. The application according to claim 1, characterized in that, The 4,4'-difluorobenzophenone oxime is applied in formulation form, and the application method includes spraying or irrigating the target plants.

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