Application of arachidonic acid in induction improvement of corn leukoderma resistance

Abstract

The invention discloses application of arachidonic acid in induction improvement of corn leukoderma resistance, the arachidonic acid is applied to prevention and treatment of corn leukoderma for the first time, and experiments show that the arachidonic acid can improve the photosynthetic effective leaf area, Fm content, Fv / Fm content, NPQ content, soluble sugar content and soluble protein content of plants; and / or increasing the activity of disease-resistant-related defensive substances; and / or catalyzing synthesis of lignin and phenolic substances; and / or the content of Rfd, MDA and reducing sugar of the plant is reduced to induce and improve the corn leukoderma resistance, so that a new direction and basis can be provided for preventing and treating the corn leukoderma by adopting natural compounds. It is found that the arachidonic acid does not strictly depend on the total spraying amount for inducing and improving the leukoderma resistance, but when the spraying amount is the same and the concentration of the arachidonic acid in the spraying liquid is 15 [mu] M, the arachidonic acid is optimal for inducing and improving the leukoderma resistance. And the application of arachidonic acid is beneficial to reducing the particle size of starch particles in corn kernels under the pressure of leukoderma.

Description

Technical Field

[0001] This invention relates to the field of maize technology, and more particularly to the application of arachidonic acid in inducing and enhancing resistance to maize white spot disease. Background Technology

[0002] Corn is an important dual-purpose crop for food, feed, and cash crops, holding a pivotal position in China's agricultural production. Diseases, especially white spot disease, are among the major factors affecting corn production. In the past five years, white spot disease has occurred extensively in corn-producing areas of southern China, causing yield losses of over 60% in some susceptible varieties, severely impacting local food security. Therefore, improving resistance to and controlling white spot disease is crucial for corn cultivation. Currently, field control of white spot disease primarily relies on chemical pesticides, placing significant pressure on the ecological environment.

[0003] Natural compounds are widely considered a rich source of eco-friendly plant growth regulators for safe crop applications. Arachidonic acid (ARA), an ω-6 polyunsaturated fatty acid, primarily exists in the cell membrane as a phospholipid. Studies have investigated its effects on human and animal diseases, as well as its application to combat plant viral diseases. For example, ARA treatment has been shown to inhibit the growth and development of Alternaria alternata-induced black spot disease on mustard leaves and reduce susceptibility to Botrytis cinerea in tomatoes. Other studies have demonstrated that exogenous application of lipid-based ARA formulations can significantly reduce the occurrence of late blight, common blight, and root-knot rot in potato tubers, and significantly increase yield. However, there are currently no technical solutions for using arachidonic acid to control white spot disease in maize. Summary of the Invention

[0004] Therefore, based on the above background, this invention is the first attempt to apply arachidonic acid to the prevention and control of white spot disease in maize, and found that it can induce and enhance resistance to white spot disease in maize, thus providing a new direction and basis for the prevention and control of white spot disease in maize using natural compounds.

[0005] The technical solution provided by this invention is as follows:

[0006] Applications of arachidonic acid, including any one of the following i) to iii):

[0007] i) Application in inducing and enhancing resistance to white spot disease in maize;

[0008] ii) Application in improving maize kernel quality under white spot disease stress;

[0009] iii) Application in the preparation of agents for the prevention and control of white spot disease in maize.

[0010] Furthermore, in ii), improving the quality of maize kernels under white spot disease stress involves reducing the particle size of starch granules in maize kernels.

[0011] Based on the same inventive concept, the present invention provides an agent for improving resistance to white spot disease of maize, comprising an effective amount of arachidonic acid, wherein the agent has at least one of the following functions a) to c), the functions including:

[0012] a) Increase the photosynthetically effective leaf area, Fm, Fv / Fm, NPQ, and the content of soluble sugar and soluble protein in the plant;

[0013] b) Increase the activity of disease-resistant defense substances;

[0014] c) Catalyzes the synthesis of lignin and phenolic substances;

[0015] d) Reduce the Rfd, MDA and reducing sugar content of plants to induce and improve maize white spot disease resistance.

[0016] Furthermore, the arachidonic acid is the only active ingredient.

[0017] Based on the same inventive concept, the present invention also provides a corn spray solution for improving corn white spot disease resistance, the corn spray solution comprising an effective amount of arachidonic acid and a solvent.

[0018] Furthermore, the solvent used is sterile water.

[0019] Furthermore, the concentration of arachidonic acid is 10 μM-20 μM.

[0020] Furthermore, the concentration of arachidonic acid is 15 μM.

[0021] Based on the same inventive concept, the present invention also provides a corn planting method for achieving at least one of the following objectives 1) to 3), wherein the corn is sprayed at least once with the corn spray solution described in any one of claims 5 to 8 when the corn is planted to the four-leaf stage and / or to the large trumpet stage.

[0022] The objectives include:

[0023] 1) Inducing and enhancing resistance to white spot disease in maize;

[0024] 2) Reduce the particle size of starch granules in corn kernels under white spot disease stress;

[0025] 3) Control white spot disease in corn.

[0026] Furthermore, the spraying method involves spraying the entire corn plant with a fine mist until a water film forms on the surface of the plant.

[0027] The beneficial effects achieved by this invention are as follows:

[0028] This invention is the first to apply arachidonic acid to the prevention and control of white spot disease in maize. Experiments have shown that it can induce and improve maize resistance to white spot disease by increasing the photosynthetically effective leaf area, Fm, Fv / Fm, NPQ, soluble sugar and soluble protein content; and / or increasing the activity of disease resistance-related defense substances; and / or catalyzing the synthesis of lignin and phenolic substances; and / or reducing the Rfd, MDA and reducing sugar content of plants. This provides a new direction and basis for the use of natural compounds to control maize white spot disease.

[0029] This invention found that the effect of arachidonic acid in inducing and improving resistance to white spot disease does not strictly depend on the total amount of spraying. Rather, when the amount of spraying is the same, the concentration of arachidonic acid in the spray solution is 15 μM, which is the optimal concentration for inducing and improving resistance to white spot disease.

[0030] Experiments have shown that the application of arachidonic acid can improve the quality of corn kernels under white spot disease stress, especially by reducing the particle size of starch granules in corn kernels. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Appendix Figure 1 The effect of exogenous spraying of different concentrations of ARA solution on vitiligo resistance of ZHL908 and QB2816 in this embodiment of the invention: Figure 1 (A) Chlorophyll fluorescence imaging, photosynthetically effective leaf area; Figure 1 (B) SPAD value, Fo, Fm, Fv / Fm, NPQ, Rfd, soluble sugar, reducing sugar, and soluble protein content of ZHL908; Figure 1 (C) SPAD value, Fo, Fm, Fv / Fm, NPQ, Rfd, soluble sugar, reducing sugar, soluble protein content of QB2816.

[0033] Appendix Figure 2 This is an in vitro antibacterial diagram showing the effects of different ARA concentrations applied by exogenous spraying according to an embodiment of the present invention.

[0034] Appendix Figure 3 This invention illustrates the changes in photosynthetic traits, carbohydrate content, and disease resistance-related defense substances in ZHL908 and QB2816 bacteria after exogenous ARA application in accordance with embodiments of the present invention. Figure 3(A) shows the changes in photosynthetic traits and carbohydrate indicators of ZHL908 and QB2816; Figure 3 (B) shows the changes in disease-related defense material indicators of ZHL908 and QB2816.

[0035] Appendix Figure 4 This is a Class B classification diagram of total metabolites in an embodiment of the present invention.

[0036] Appendix Figure 5 The DAMs comparing T and CK at multiple stress time points (0h, 72h, 168h) for exogenous ARA-induced maize white spot disease resistance in this embodiment of the invention are as follows: Figure 5 (A) is a classification diagram of all DAMs for ZHL908 and QB2816. Figure 5 (B) shows all DAMsvenn plots for ZHL908 and QB2816 (left), and a total DAMs clustering plot (right).

[0037] Appendix Figure 6 This invention provides a comparison of control (CK) versus control (CK) and time (T) versus DAMs at multiple stress time points (0h, 72h, 168h) for exogenous ARA-induced maize white spot disease resistance: Figure 6 (A) represents the number of DAMs that are upregulated and downregulated in ZHL908 CK compared to CK; Figure 6 (B) represents the number of DAMs that were upregulated and downregulated when comparing QB2816CK with CK; Figure 6 Figure (C) shows the cluster heatmap of all differentially expressed metabolites between ZHL908T and CK (the red box contains the common DAMs) and the venn plot; Figure (D) shows the cluster heatmap of all differentially expressed metabolites between QB2816T and CK (the red box contains the common DAMs) and the venn plot.

[0038] Appendix Figure 7 The following is a bubble diagram showing the pathway enrichment of DAMs at multiple stress time points (0h, 72h, 168h) for exogenous ARA-induced maize white spot disease resistance in an embodiment of the present invention: Figure 7 (A) is a bubble diagram of the pathway enrichment of DAMs for Z72T_vs_Z0T; Figure 7 (B) is a bubble diagram of the pathway enrichment of DAMs for Z168T_vs_Z0T; Figure 7 (C) is a bubble diagram of the pathway enrichment of DAMs for Q72T_vs_Q0T; Figure 7 (D) is a bubble diagram of pathway enrichment of DAMs for Q168T_vs_Q0T.

[0039] Appendix Figure 8These are DAMs enriched in the Biosynthesis of various plant secondary metabolites (blue), Phenylalanine metabolism (red), and Tropane, piperidine, and pyridine alkaloid biosynthesis (yellow) of this invention.

[0040] Appendix Figure 9 The effect of exogenous ARA on the physicochemical properties of corn kernel starch infected with white spot disease, as described in this embodiment of the invention: Figure 9 (A) is an electron microscope scan; Figure 9 (B) represents the starch particle size; Figure 9 (C) represents X-ray diffraction. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] In the description of this invention, "first feature" and "second feature" may include one or more of the indicated features. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first" and "second" may explicitly or implicitly include one or more of the indicated features.

[0043] The invention will now be further described with reference to the accompanying drawings.

[0044] Example:

[0045] (1) Activation culture of pathogens and preparation of spore suspension

[0046] The white spot pathogen (Epicoccum sorghinum) was provided by the Institute of Plant Protection, Guizhou Academy of Agricultural Sciences.

[0047] The pathogen of white spot disease was cultured on potato dextrose agar and incubated in the dark for 7 days at 28°C and 70% relative humidity. After colonies emerged, pure cultures were obtained by single-spore isolation. The purified isolate was cultured in potato dextrose liquid medium with shaking (28°C, 160 rpm) for 7 days, filtered through sterile gauze, and a 2000:1 solution of 20% Tween was added to the conidial suspension for subsequent inoculation.

[0048] (2) ARA concentration screening test

[0049] From March to April 2024, maize was cultivated in pots at the Guizhou University Farm in Guiyang City, Guizhou Province, China (106.27-106.52 E, 26.11-26.34 N, altitude 1112 m, average annual sunshine hours 1040 h, average annual temperature 15.7℃, rainfall 1220 mm).

[0050] This experiment used a two-factor randomized block design:

[0051] Factor A consists of two inbred lines (ZHL908, highly resistant to white spot disease, and QB2816, highly susceptible to white spot disease, provided by the Dryland Grain Research Institute of the Guizhou Academy of Agricultural Sciences).

[0052] Factor B is the application of exogenous ARA (prepared with sterile water):

[0053] ARA concentrations were 0 μM (pure sterile water), 5 μM, 10 μM, 15 μM, 20 μM, 35 μM, and 50 μM.

[0054] Planting pot specifications: inner diameter 62cm, outer diameter 70cm, bottom diameter 42cm, height 39.5cm.

[0055] Substrate: Peat moss (sterilized by high-temperature steam).

[0056] Fertilizer: Add 100g of NPK compound fertilizer (N:P2O5:K2O ratio 15:15:15) to each pot. Set up 3 biological replicates, for a total of 42 flower pots.

[0057] Corn plants were conventionally cultured to the four-leaf stage. Corn plants with uniform growth (height difference not exceeding 2cm) were selected and treated with exogenous ARA spray. 24 hours later, the first visible leaf below the heart leaf was cut off and the white spot disease pathogen was artificially inoculated onto the detached leaf.

[0058] The spraying method involved finely spraying the entire corn plant until a thin film of water formed on the plant surface, without creating flowing runoff. In this experiment, 300 ml of the corresponding concentration of ARA solution was used per corn plant, and no more was added afterward. During spraying, the 300 ml solution was intended to evenly and finely cover the above-ground surface of the plant, including its stem, petioles, and both sides of the leaves.

[0059] First, the cut leaves were disinfected (quickly and gently wiped with 75% alcohol wipes, then rinsed with sterile water, and the surface moisture was absorbed with paper towels). The leaves were then placed, back side up, in a white dish lined with a damp gauze. The leaves were sprayed with the pathogenic bacteria (Zhao, 2024), the dish was sealed with plastic wrap, and placed in a 24°C incubator with alternating day and night light cycles of 12 hours each, maintaining a humidity of approximately 75%. After 72 hours, chlorophyll fluorescence imaging (instrument model: Fluor Cam7.0, measuring indicators: Size, Fo, Fm, Fv / Fm, NPQ, Rfd), SPAD value (instrument model: SPAD-502PLUS), soluble sugar, reducing sugar (Vadera at al., 2021), and soluble protein (Liu at al., 2019) were measured.

[0060] (3) In vitro antibacterial test

[0061] Using a punch, the qualitative filter paper is made into small round pieces (D=5mm), which are then autoclaved and kept for later use.

[0062] Prepare circular lesion rings (D=6mm) using a punch and place them upside down in the center of PDA medium. Then, take filter paper discs and evenly place 4 discs on each plate with the lesion as the center. Add 10μl of ARA solution (concentrations of 0μM, 5μM, 10μM, 15μM, 20μM, 35μM, and 50μM) to each filter paper disc. After culturing for 7 days, measure the size of the lesions using the cross measurement method and observe and record the antibacterial effect of each treatment group.

[0063] (4) Determination of photosynthetic traits, carbohydrates and disease-related defense substances

[0064] From April to October 2024, maize was cultivated in pots at the Guizhou University farm. This experiment used a two-factor randomized block design:

[0065] Factor A consists of two inbred lines (ZHL908, highly resistant to vitiligo, and QB2816, highly susceptible to vitiligo);

[0066] Factor B was exogenous ARA spraying at concentrations of 0 μM (CK) and 15 μM (T). Three replicates were set up (one replicate per pot), with three plants per pot, for a total of 12 pots. The spraying procedure was the same as above.

[0067] Corn plants were conventionally cultivated to the large trumpet stage, and then selected with uniform growth were treated with exogenous ARA spray; 24 hours later, artificial spraying of white spot pathogens was carried out.

[0068] At 0h, 72h, and 168h post-inoculation, SPAD values, photosynthetic traits (instrument model: LI-6400XT, measured indicators: Pn, Gs, Ci, Tr), carbohydrates (Vadera at al., 2021), and disease-related defense substances: laccase (Moshtaghioun at al., 2011), lignin (FUKUSHIMAR at al., 2001), MDA, PAL (Jogaiah at al., 2013), PPO, POD (Ali at al., 2016), CHI, LAC, and soluble protein (Liu at al., 2019) were measured using the first unfolded leaf below the central leaf. Simultaneously, metabolomics sampling was performed. The sampling site was the first unfolded leaf below the heart leaf, excluding leaf spots, edges, and veins. 6g samples were collected from 6 plants, mixed thoroughly, and divided into 3 portions as 3 biological replicates. The samples were flash-frozen in liquid nitrogen and stored at -80℃ for subsequent metabolomics experiments.

[0069] In this experiment, the maize plants were cultured normally after inoculation, self-pollinated and bagged at the appropriate time, and harvested at the appropriate time. The kernels were used for subsequent determination of starch physicochemical properties.

[0070] (5) Metabolomics Sample Extraction

[0071] Transfer 0.5-1.0 mL of sample to a centrifuge tube, add 2 times the volume of extraction buffer (methanol / acetonitrile, 1:1, v / v), vortex for 60 s, extract by low-temperature ultrasonication for 30 min, centrifuge at 12000 rpm / min for 10 min at 4 °C, collect the supernatant, place at -20 °C for 1 h to precipitate the protein, centrifuge at 12000 rpm / min for 10 min at 4 °C, collect the supernatant and vacuum dry it, add 100 μL of 30% acetonitrile solution to reconstitute, vortex, centrifuge at 12000 rpm / min for 10 min at 4 °C, collect the supernatant for instrumental analysis (Ghirardo atal., 2020).

[0072] (6) Metabolomics analysis

[0073] Metabolomics analysis of the extract was performed using UHPLC-Q Exactive HFX, Thermo, USA. High-performance liquid chromatography (HPLC) analysis was performed using a Waters HSS T3 column (100 × 2.1 mm, 1.8 μm).

[0074] Mobile phase A is an ultrapure aqueous solution (containing 0.1% formic acid), and mobile phase B is an acetonitrile solution (containing 0.1% formic acid).

[0075] Flow rate: 0.3 mL / min; column temperature: 40 °C; injection volume: 2 μL;

[0076] Elution gradient: 0 min A phase / B phase (100:0, v / v), 1 min A phase / B phase (100:0, v / v), 12 min A phase / B phase (5:95, v / v), 13 min A phase / B phase (5:95, v / v), 13.1 min A phase / B phase (100:0, v / v), 17 min A phase / B phase (100:0, v / v).

[0077] Primary and secondary mass spectra were acquired using a Thermo Q Exactive HFX high-resolution mass spectrometry system (USA). The scan mode was Full-ms-ddMS2, and the scan pattern was positive / negative ion. The primary mass spectrometry scan range was 70-1050 Da, with a primary resolution of 70,000 and a secondary resolution of 17,500.

[0078] Differentially accumulated metabolites (DAMs) were screened based on thresholds of VIP>1.0, FC>1.2 or FC<0.8375, and P_value<0.05 for subsequent bioinformatics analysis (volcano plot thresholds of FC>1.2 or FC<0.8375, and P_value<0.05). Enrichment pathway analysis was performed using the KEGG PATHWAY Database.

[0079] (7) Determination of physicochemical properties of grain starch

[0080] Test samples: Grains harvested at the appropriate time in the "Photosynthetic traits, carbohydrates and disease resistance-related defense substances test" above (4). At the same time, grains of ZHL908 and QB2816 in normal production (without white spot infection) were added as negative controls, and were recorded as ZHL908 group and QB2816 group respectively. "ZCK" is the electron micrograph of the starch of ZHL908 grains after being sprayed with sterile water and then subjected to white spot stress, as a positive control, reflecting the change of starch in the grains of ZHL908 after natural maturation in the face of white spot stress without exogenous intervention;

[0081] ZHL908 is a verified maize inbred line highly resistant to white spot disease. It can still produce a certain yield even after exposure to white spot disease stress without human intervention (meaning that a small number of maize kernels are ultimately harvested). QB2816 maize is a verified inbred line highly susceptible to white spot disease. Without treatment, it dies in the mid-to-late stages of growth after exposure to white spot disease stress, resulting in no yield. Therefore, there is no electron micrograph of QCK kernels. In contrast, QB2816 maize, after exposure to white spot disease stress, underwent ARA-induced enhancement of its resistance, resulting in a harvest of seeds. ZT and QT are electron micrographs of starch from the harvested kernels of ZHL908 and QB2816 maize after being sprayed with ARA solution and subjected to white spot disease stress, respectively. The morphological changes of starch are clearly observed in the images.

[0082] Scanning electron microscopy (SEM) observation: The dried and sieved starch sample was evenly sprinkled onto a stage coated with conductive adhesive, and excess starch particles were blown away with a bulb syringe. The treated starch was placed in the sample chamber for observation, and representative particle morphology photographs were taken at 500, 1000, and 2000x magnification.

[0083] Particle size determination: Using water as the dispersed phase, the starch particle size was determined using a Mastersizer 3000 laser particle size analyzer. The particle size was measured as the volume average particle size.

[0084] X-ray diffraction pattern of crystal structure: Starch was placed in an X-ray diffractometer (Rigaku Miniflex 600, Japan) with the following parameters set: tube current 40mA, tube voltage 40KV, Cu target, scanning range 10°-90°, and scanning speed 2° / min (BHA Tatal., 2017).

[0085] Data Analysis

[0086] Microsoft Excel 2020 was used for data processing and tabulation, and GraphPad Prism 7.0 software was used for analysis of variance and plotting. Significance was defined as * (P < 0.05) and ** (P < 0.01).

[0087] Results and Analysis

[0088] (1) Effects of different concentrations of ARA on the incidence of white spot disease in maize

[0089] Maize plants were treated with multiple concentrations of exogenous ARA (0 μM was the control for sterile water spraying), followed by artificial inoculation with white spot disease pathogen. Results after 72 hours showed significant differences between plants sprayed with ARA and those sprayed with sterile water in terms of photosynthetically active leaf area, relative chlorophyll content, chlorophyll fluorescence, sugar and protein content, and different concentrations of ARA exhibited varying induction effects on white spot disease resistance in maize. In ZHL908 and QB2816, except for the 5 μM and 50 μM ARA treatments, the photosynthetically active area of ​​all other concentrations was significantly increased compared to 0 μM, with the largest increase observed at 15 μM (Figure 1A). The SPAD values ​​of ZHL908 and QB2816 were highest at a spraying concentration of 15 μM, significantly increasing by 78.28% and 45.65% respectively compared to 0 μM. Fm, Fv / Fm, and NPQ were significantly higher at ARA concentrations of 10 μM, 15 μM, and 20 μM than at 0 μM, with the most significant differences observed at 15 μM, increasing by 49.23% and 52.47%, 8.9% and 30.91%, and 50.39% and 67.86% respectively compared to 0 μM. Rfd at 1... The lowest values ​​were observed at 5 μM ARA treatment, significantly decreasing by 15.53% and 17.90% compared to 0 μM, respectively. Reducing sugar content was significantly lower at all ARA concentrations than at 0 μM, with the lowest values ​​observed at 15 μM, decreasing by 16.08% and 24.15% compared to 0 μM, respectively. Soluble sugar and soluble protein contents reached their maximum values ​​at 15 μM treatment, significantly increasing by 28.07% and 22.32%, and 16.77% and 6.53% respectively compared to 0 μM (Figure 1B). These results indicate that under the same stress conditions caused by white spot disease infection, compared to maize sprayed with sterile water, spraying with 15 μM ARA induced a larger photosynthetically active leaf area, significantly increased SPAD value, Fm, Fv / Fm, NPQ, soluble sugar and soluble protein content, and significantly decreased Rfd and reducing sugar content. Meanwhile, after exogenous application of 15 μM ARA, the variation range of Fm, Fv / Fm, NPQ, Rfd and reducing sugar content in the highly susceptible inbred line QB2816 was significantly greater than that in the highly resistant inbred line ZHL908.

[0090] To determine whether the antibacterial effect of ARA against maize white spot disease is fungicidal or resistance-inducing, experiments were conducted to determine the effect of different concentrations of ARA on the growth of the pathogen. The plate method was used to measure colony growth. The results showed that ( Figure 2(Table 1) Compared to 0 μM, there was no significant difference in the in vitro culture of *Aureobasidium sorghum* treated with different concentrations of ARA, with colony diameters all around 5.5 cm. These results suggest that the inhibitory effect of ARA on *Aureobasidium sorghum* may be achieved through resistance induction. In summary, these results indicate that, under the same spraying dosage, 15 μM ARA is the optimal experimental concentration, providing support for further research on the mechanism of ARA-induced resistance to maize white spot disease.

[0091] Table 1: In vitro antibacterial effect of different ARA concentrations applied by exogenous spraying on vitiligo pathogens

[0092]

[0093] (2) Effects of exogenous ARA treatment on maize photosynthetic traits, carbohydrates and disease resistance-related defense substances

[0094] Maize plants at the large trumpet stage were treated with 15 μM exogenous ARA (T) and sterile water (CK), followed by inoculation with white spot disease pathogen. Related indicators were measured at three stress time points (0 h, 72 h, and 168 h). The results showed that white spot disease infection led to a decrease in SPAD values ​​in the leaves of both inbred lines of maize. With increasing stress duration, the SPAD value of T was significantly higher than that of CK. Analysis of photosynthetic traits and related products revealed that white spot disease infection also caused a decrease in Pn, Gs, and soluble sugar content, and an increase in Ci and reducing sugar content in ZHL908 and QB2186. After exogenous ARA application, the contents of Pn, Gs, and soluble sugars in both inbred lines were significantly higher than those in the control (CK), while the contents of reducing sugars were significantly lower. However, unlike the control, Pn, Gs, and reducing sugars showed significant differences from the CK at both 72 h and 168 h after inoculation of ZHL908, while QB2816 only showed significant differences from the CK at 168 h after inoculation. Figure 3 A). In the detection of disease-related defense substances in maize, infection with white spot disease fungus led to an increase in lignin, MDA, PPO, POD, and soluble protein content, and a decrease in CHI and LAC activities in ZHL908 and QB2186. Exogenous spraying of ARA significantly decreased MDA content and significantly increased lignin content, POD, PPO, and LAC activity in both inbred lines at 72 h and 168 h after inoculation. CHI, PAL, and soluble protein content were significantly higher in ZHL908 than in the control (CK) at both 72 h and 168 h after inoculation, while QB2816 was only significantly higher than the CK at 168 h after inoculation. Figure 3 B).

[0095] (3) Metabolomics evaluation of exogenous ARA-induced resistance to maize white spot disease

[0096] Maize plants at the large trumpet stage were treated with 15 μM exogenous ARA (T) and sterile water (CK), followed by inoculation with white spot disease pathogen. Non-target metabolomics analysis was used to analyze metabolite differences between T and CK at three stress time points. A total of 3456 metabolites were detected, and 530 metabolites were used for further analysis. HCA and PCA analyses showed significant differences in metabolites between T and CK. The samples from the three bioreproducible studies were distributed in similar regions, demonstrating good repeatability and reproducibility, and the experimental results were stable and reliable. The 530 metabolites were classified into 43 categories: others (47%), carboxylic acids and derivatives (16%), organic oxygen compounds (10%), and Benzene and substituted derivatives (4%). Figure 4 ).

[0097] (4) Screening and analysis of Class B classification map of total metabolites in exogenous ARA-induced maize white spot disease resistance

[0098] Using maize inbred lines with different resistances as research subjects, the differential metabolites of T and CK were analyzed. The results showed that a total of 206 DAMs were screened in the highly resistant inbred line ZHL908; a total of 249 DAMs were screened in the highly susceptible inbred line QB2816. After further screening using the VIP>1 criterion, 41 and 45 DAMs were screened in ZHL908 and QB2816, respectively. Figure 5 A), using Venn diagram visualization, it was found that there are a total of 9 DAMs (A). Figure 5B) contains Apodanthoside, Zeanoside C, (2Z)-2-hydroxy-3-phenylprop-2-enoic acid, (2S,3S)-2-amino-3-methylpentanoic acid, D-glu sose-6-phosphate, 5-Hydroxy-6-methoxy-7-O-beta-D-glucosylcoumarin, Avenanthramide L, Paracetamol sulfate, and 2,3-dihydroxypropanoic acid. ZHL908 has 32 unique DAMs, while QB2816 has 36 unique DAMs. These metabolites were classified as Class B as candidate metabolites for exogenous ARA-induced resistance to maize white spot disease. The highly resistant inbred line ZHL908 contained Triazines, Phenols, and Furanoid lignans, while the highly susceptible inbred line QB2816 contained Hydroxy acids and derivatives, Piperidines, and Flavonoids. These may represent different categories of inbred lines responding to ARA-induced white spot disease resistance, which may be related to race-specific resistance in maize.

[0099] Using the duration of stress from white spot disease fungus as the research object, this study analyzed the metabolite accumulation patterns in maize responding to exogenous ARA spraying for white spot disease. The results showed that ( Figure 6 A, Figure 6 B) With increasing stress duration, both the total number of DAMs and the number of upregulated DAMs in maize increased. In the sterile water inoculation treatment (CK), with increasing stress duration, the number of upregulated DAMs gradually increased, while the number of downregulated DAMs gradually decreased, with the highest number of upregulated DAMs and the lowest number of downregulated DAMs observed in 168CK_vs_72CK. In the ARA treatment (T), the number of both upregulated and downregulated DAMs was highest in 168T_vs_0T. At both stress time points (72h and 168h), after ARA treatment, the number of DAMs in the highly susceptible inbred line QB2816 was higher than that in the highly resistant inbred line ZHL908.

[0100] DAMs of ZHL908 and QB2816 after ARA application at 0h, 72h, and 168h of stress were analyzed, and 53 and 37 DAMs were selected, respectively (Tables S10-S15). The results were visualized using a venn diagram. Figure 6 C Figure 6D) Screening of the three comparative groups of ZHL908 and QB2816 revealed 10 and 9 common DAMs, respectively. ZHL908 mainly contained organooxygen compounds, carboxylic acids and derivatives, while QB2816 mainly contained Benzene and substitute dderivatives, Piperidines, and Purine nucleosides. This indicates that ARA treatment elicits different responses to white spot disease in different inbred lines at the metabolic level. Furthermore, we found that four differentially metabolized metabolites—(2E)-2-(2-methoxy-2-oxoethyl)but-2-enedioate, 1,5-Anhydro-4-deoxy-D-glycero-hex-3-en-2-ulose, (2S)-2-amino-3-phenylpropanoic acid, and 3-furoic acid—were included in both the 10 and 9 DAMs found in ZHL908 and QB2816. These metabolites may influence the systemic acquired resistance of maize to white spot disease.

[0101] (5) KEGG analysis of differential metabolites and screening of highly correlated pathways in exogenous ARA-induced maize white spot disease resistance

[0102] Pathway enrichment analysis of DAMs in maize treated with ARA for 72 h and 168 h under stress revealed that DAMs from ZHL908 and QB2816 were enriched in 16 and 11 pathways, respectively, after 72 h of stress, and in 19 and 17 pathways, respectively, after 168 h of stress. Tropane, piperidine and pyridine alkaloid biosynthesis, phenylalanine metabolism, and biosynthesis of various plant secondary metabolites showed significant enrichment. Figure 7 ).

[0103] The study focused on the biosynthesis of various plant secondary metabolites, phenylalanine metabolism, and the biosynthesis of tropane, piperidine, and pyridine alkaloids. In the biosynthesis pathway of various plant secondary metabolites, (2R)-2-beta-D-Glucopyranosyloxy-4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one was upregulated 72 h after inoculation with ZHL908, S-Adenosylmethionine and 3,4,5-Trihydroxybenzoate were upregulated 168 h after inoculation with QB2816, 1-O-Galloyl-beta-D-glucose was upregulated in ZHL908, and 6,7-Dihydroxycoumarin was upregulated in QB2816. In the phenylalanine metabolism pathway, (2S)-2-amino-3-phenylpropanoic acid was upregulated 168 h after QB2816 inoculation, while (2S)-2-amino-3-(4-hydroxyphenyl)propanoic acid and (2Z)-2-hydroxy-3-phenylprop-2-enoic acid were upregulated 72 h after QB2816 inoculation. In the Tropane, piperidine, and pyridine alkaloid biosynthesis pathway, 1,4-Diaminobutane, Piperidine, and (2S,3S)-2-amino-3-methylpentanoic acid were upregulated 168 h after QB2816 inoculation. Figure 8 ).

[0104] (6) Effects of exogenous ARA on the physicochemical properties of maize kernel starch under the adverse conditions of white spot disease

[0105] To observe the effect of exogenous ARA on maize quality under white spot disease stress, the relevant physicochemical properties of maize kernel starch at maturity were tested.

[0106] Since QB2816 is a maize inbred line highly susceptible to white spot disease, at harvest time, the infected plants (QCK) that had not been sprayed with exogenous ARA had already died completely. This treatment ultimately did not yield any yield, and relevant starch data for QCK are missing.

[0107] Figure 9 As can be seen from A, the starch granules of untreated ZHL908 are of varying sizes and have uneven surfaces; the surface of the starch granules is relatively rough, with many being dented or broken.

[0108] The starch granules treated with ZCK became smoother and rounder, with more smooth granules than those treated with ZHL908 and fewer fractures. Under ZT treatment, both uneven and smooth starch granules appeared, with fewer fractures, but fewer smooth starch granules than those treated with ZCK and more uneven granules.

[0109] Untreated QB2816 starch granules have two types of surfaces: uneven and smooth, with fewer depressions and breaks. Under QT treatment, the starch granules become rough, with uneven surfaces and more breaks and depressions.

[0110] Figure 9 As shown in Figure B, the average particle size and distribution range of ZHL908 starch (negative control) were 2.08 μm and 0.06–5.58 μm, respectively. The average particle size and distribution range of ARA-treated ZCK starch (ZHL908 positive control) were 2.13 μm and 0.10–5.56 μm, respectively, while those of ZT-treated starch were 2.08 μm and 0.03–5.63 μm, respectively. The average particle size and distribution range of QB2816 starch were 2.12 μm and 0.09–6.34 μm, respectively, while those of QT-treated starch were 2.13 μm and 0.07–6.44 μm, respectively. The highest peak values ​​were observed in the ZT and QT treatments, respectively, among the two inbred lines.

[0111] Figure 9 As can be seen from C, the two self-crossed starch crystal types are the same after ARA treatment, with obvious diffraction peaks near 15°, 18°, and 23°. The diffraction peaks at 18° and 23° are relatively sharp, which suggests that their crystal type is type A. The three most intense peaks of ZHL908 are at 15.30°, 18.10°, and 23.16°, respectively; ZCK is at 15.38°, 18.12°, and 23.08°; and ZT is at 15.26°, 17.28°, and 22.94°. The three most intense peaks of QB2816 are at 15.34°, 18.18°, and 23.06°, and the two most intense peaks of QT are at 20.10° and 22.60°.

[0112] in conclusion:

[0113] In the above experiment, exogenous spraying of maize with multiple concentrations of ARA induced an increase in maize's resistance to white spot disease. In the experiment, the photosynthetically effective leaf area, SPAD value, Fm, Fv / Fm, NPQ, and the contents of soluble sugar, reducing sugar and soluble protein in the in vitro inoculated leaves showed significant changes under treatments of 10 μM, 15 μM and 20 μM, among which the 15 μM concentration of ARA treatment had the best effect on improving resistance.

[0114] Meanwhile, the changes in Fm, Fv / Fm, NPQ, Rfd and reducing sugar content of the highly susceptible inbred line QB2816 under 15 μM treatment were significantly greater than those of the highly resistant inbred line ZHL908.

[0115] Photosynthesis provides raw materials and energy for the growth and immune defense of higher plants. After a plant is infected with a disease, the efficiency of light energy absorption, utilization and conversion in the leaves and its own heat dissipation capacity decrease significantly, inhibiting nutrient absorption. In addition to supporting plant growth, carbohydrates such as soluble sugars and reducing sugars can also act as auxiliary messengers under stress conditions, playing a key role in improving plant resistance (Su et al., 2024; Chen et al., 2020; Aslam et al., 2024; Zhang et al., 2021).

[0116] In the above experiment, after exogenous spraying of ARA, the SPAD value gradually increased with the duration of stress, and was significantly higher than the CK in both inbred lines. 168 h after inoculation, the SPAD value of the highly susceptible inbred line QB2816 showed a more pronounced change than that of the highly resistant inbred line ZHL908. Among the various photosynthetic indices, Pn, Gs, and Tr of ZHL908 showed a significant increase, while Pn and Gs of QB2816 generally showed an increasing trend, and Ci and Tr showed a decreasing trend. The study found that rice infected with *Magnaporum oryzae* and grapevines infected with *Flavescence dorée* can limit the spread of the pathogen by accumulating soluble sugar content and phytoalexins such as flavonoids (Lahariat al., 2024; Cristina at al., 2024). It is evident that the soluble sugar content of the two inbred lines increased significantly and the reducing sugar content decreased significantly after exogenous ARA spraying. It is speculated that exogenous ARA spraying can limit the spread of maize white spot disease pathogens by inducing an increase in soluble sugar content and reducing the reducing sugar content.

[0117] Plant cell walls are the first line of defense against pathogens entering the cell. When pathogens invade, the plant's own defense response is rapidly activated, including PAL activity, which promotes the accumulation of lignin near the cell wall to mitigate the damage caused by pathogens. In addition to PAL, POD, LAC, and other compounds can catalyze the polymerization of lignin monomers and oxidize phenolic substrates to generate phenolic free radicals, as well as convert flavonoids into polyflavonoids, thereby enhancing plant growth, development, and antibacterial ability (You at al., 2020; Liu at al., 2020; Kong at al., 2023).

[0118] In the above experiment, under ARA treatment, lignin content increased significantly, and the activities of related enzymes such as PAL, POD, PPO, and LAC also increased significantly. Furthermore, CHI activity and soluble protein content also increased significantly, while MDA content decreased significantly. CHI contains chitin, a major component capable of degrading fungal cell walls, and is an important disease-related protein in the plant defense system. Exogenous application of benzoic acid, oxalic acid, etc., can enhance CHI activity and thus improve related resistance. However, the activities of PPO, MDA, and CHI showed a more significant effect on the highly susceptible inbred line QB2816 after 168 hours of inoculation compared to the highly resistant inbred line ZHL908. PAL activity increased significantly at 72 and 168 hours after inoculation in ZHL908, while in QB2816 it only increased significantly at 168 hours. The increase in LAC activity at 168 hours after inoculation in ZHL908 was greater than that in QB2816. These results indicate that ARA has a significant response to vitiligo resistance, but the response varies among different resistant inbred lines. ARA may induce enhanced resistance to white spot disease in maize by promoting the synthesis of immune response gene products, such as macromolecular compounds that constitute cell wall polymers and enzymes related to the synthesis of phenols and phytoalexins. It is hypothesized that exogenous ARA acts as a signaling mediator in the development and progression of maize disease resistance, triggering a broad spectrum of immune response signaling pathways.

[0119] In recent years, metabolomics technology has provided more perspectives for elucidating plant responses to various abiotic stresses. In the two maize inbred lines under ARA treatment in the above experiment, 13 shared DAMs were screened, namely Apodanthoside, 5-Hydroxy-6-methoxy-7-O-beta-D-glucosylcoumarin, Zeanoside C, etc., among which 5-Hydroxy-6-methoxy-7-O-beta-D-glucosylcoumarin was classified as coumarins and derivatives. Studies have shown that coumarins and their derivatives are precursors to lignin synthesis, possessing a wide range of biological activities. They exhibit positive effects in antibacterial, anti-inflammatory, antioxidant, antiviral, and enzyme-inhibiting properties. When used as a phytoalexin, they also possess parasitic defense characteristics, with rapid accumulation observed at the site of parasite infection. The results of this non-target metabolomics and lignin quantitative assay both indicate that 5-Hydroxy-6-methoxy-7-O-beta-D-glucosylcoumarin can serve as a candidate metabolite for resistance to maize white spot disease, warranting further investigation.

[0120] Furthermore, among the DAMs unique to the two inbred lines, the resistant inbred line ZHL908 showed the presence of the Phenols metabolite 3-Methoxytyramine-betaxanthin, while the highly susceptible inbred line QB2816 showed the presence of the Flavonoids metabolite Gomphrenol 3-methylether 4'-glucuronide. These may be key metabolite classes contributing to the differences in resistance between the two inbred lines. KEGG pathway analysis was performed on the differentially expressed metabolites, focusing on Tropane, piperidine and pyridine alkaloid biosyntheses, Phenylalanine metabolism, and the biosynthesis of various plant secondary metabolites. Whether tropane, piperidine, and pyridine alkaloid biosynthesis participates in plant stress responses remains unclear. However, phenylalanine metabolism, as a precursor to tropane, piperidine, and pyridine alkaloid biosynthesis, plays a crucial role in plant growth, development, defense, and environmental adaptation. It not only enhances the physical barrier function of cell walls by synthesizing lignin but also synthesizes phenolic and isoflavone metabolites, which are further converted into phytoalexins to inhibit pests and diseases. Furthermore, regulating phenylalanine metabolism and flavonoid biosynthesis can modulate the cold resistance of rice. Regarding the biosynthesis of various plant secondary metabolites, most secondary metabolites in plant defense systems possess antifungal properties. Among them, alkaloids, phenols, and flavonoids exhibit antiviral functions and play an important role in plant defense responses against pathogens. It is speculated that in this experiment, exogenous application of ARA induced Phenyl alanine metabolism, biosynthesis of various plant secondary metabolites, and synthesis of lignin, PAL, CHI and other defense-related substances to alleviate the accumulation of vitiligo pathogens.

[0121] The results of this experiment indicate that spraying exogenous ARA has an inducing effect on resistance to maize white spot disease, with the optimal induction effect observed at an ARA concentration of 15 μM. A 15 μM concentration of ARA increased photosynthetically effective leaf area, leaf surface area (Fm), leaf volume ratio (Fv / Fm), NPQ, soluble sugar and protein content, and the activity of immune response-related enzymes such as CHI, PAL, POD, and LAC, thereby enhancing maize's resistance to white spot disease. 3,5-Hydroxy-6-methoxy-7-O-beta-D-glucosylcoumarin may be one of the key metabolites inducing ARA-induced resistance to maize white spot disease. These results provide a theoretical basis for the application of exogenous ARA to address maize white spot disease and offer new insights for sustainable agricultural development through green regulation.

[0122] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. The application of arachidonic acid, characterized in that, The application includes any one of the following i) to iii): i) Application in inducing and enhancing resistance to white spot disease in maize; ii) Application in improving maize kernel quality under white spot disease stress; iii) Application in the preparation of agents for the prevention and control of white spot disease in maize.

2. The application according to claim 1, characterized in that, ii) To improve the quality of corn kernels under white spot disease stress, the particle size of starch granules in corn kernels is reduced.

3. A preparation for improving resistance to white spot disease in maize, characterized in that, It includes an effective amount of arachidonic acid, and the formulation has at least one of the following functions (a) to (c): a) Increase the photosynthetically effective leaf area, Fm, Fv / Fm, NPQ, and the content of soluble sugar and soluble protein in the plant; b) Increase the activity of disease-resistant defense substances; c) Catalyzes the synthesis of lignin and phenolic substances; d) Reduce the Rfd, MDA and reducing sugar content of plants to induce and improve maize white spot disease resistance.

4. The preparation for improving resistance to white spot disease of maize according to claim 3, characterized in that, Arachidonic acid is the only active ingredient.

5. A corn spray solution for improving resistance to white spot disease in corn, characterized in that, The corn spray solution includes an effective amount of arachidonic acid and a solvent.

6. The corn spray solution for improving resistance to white spot disease of corn according to claim 1, characterized in that, The solvent used is sterile water.

7. The corn spray solution for improving resistance to white spot disease of corn according to claim 1, characterized in that, The concentration of arachidonic acid is 10 μM-20 μM.

8. A spray solution for improving maize resistance to white spot disease according to claim 5, characterized in that, The concentration of arachidonic acid is 15 μM.

9. A method for planting maize to achieve at least one of the objectives of 1) to 3) below, characterized in that, When corn is planted to the four-leaf stage and / or to the large trumpet stage, spray the corn spray solution according to any one of claims 5 to 8 at least once. The objectives include: 1) Inducing and enhancing resistance to white spot disease in maize; 2) Reduce the particle size of starch granules in corn kernels under white spot disease stress; 3) Control white spot disease in corn.

10. A method for planting corn according to claim 9, characterized in that, The application method for corn spray solution is to spray the entire corn plant with a fine spray until a water film forms on the surface of the plant.

Images