Analysis method for rice leaf rhythm change after rice is infected by pyricularia grisea and application of analysis method in accurate prevention and control of rice blast

Abstract

The invention relates to the technical field of biological prevention and control, in particular to a method for analyzing rice leaf rhythm change after rice is infected by pyricularia grisea and application of the method in accurate prevention and control of rice blast. The analysis method comprises the following steps: (1) spraying a pyricularia grisea spore suspension on rice leaves; (2) carrying out whole-genome transcriptome analysis on the rice leaves, and carrying out clustering heat map; (3) analyzing expression conditions of the biological clock genes and the phytochrome related genes in the circadian rhythm pathway of the rice leaves, and analyzing the biological clock genes and the phytochrome related genes by a MetaCycle method; and (4) analyzing the deposition amount of H2O2 and callose in the rice leaves. Determining the dynamic change rule of the rice immune process caused by magnaporthe oryzae infection according to the method; in combination with the characteristics of small molecules, small-molecule pesticides are reasonably applied at a specific time point aiming at the rice blast, so that efficient and accurate prevention and control of the rice blast are realized.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, and in particular to an analytical method for the changes in the rhythm of rice leaves after rice blast fungus infection and its application in the precise control of rice blast disease. Background Technology

[0002] Rice blast is a fungal disease caused by Magnaphalthe oryzae (M. oryzae). It has a very wide distribution and can occur in all rice-growing areas of my country. This disease can occur throughout the entire rice growth cycle and infect multiple rice tissues, including leaves, stems and panicles.

[0003] Traditional methods for controlling rice blast mainly rely on the application of chemical pesticides, such as tricyclazole, isoprothiolane, and kasugamycin. However, this method has many problems, such as high pesticide usage, severe environmental pollution, and long-term use of fungicides with the same mechanism of action leading to resistance in rice blast fungi. Plant immune activators, as a new type of green and eco-friendly pesticide, have attracted widespread attention. Plant immune activators themselves or their metabolites usually do not have direct fungicidal or antiviral activity, or have very low activity. However, these inducers can activate the plant's innate immune mechanism, enabling it to effectively resist pathogen invasion. Existing research has revealed that the biological clock can control the timing and extent of glyphosate-induced plant cell death, with the effect being more significant at dawn than at dusk, which is similar to the chronotherapy used in human disease treatment. However, a deep understanding of the rhythmic changes in the immune system of crops after pathogen infection is still lacking. Based on this, the present invention proposes an immune activator application scheme based on the principle of biological clock, which aims to achieve efficient and precise control of rice blast by accurately grasping the application time and optimizing the application strategy. Previously, there has been no systematic immune activator application scheme based on the principle of chronopharmacology. Summary of the Invention

[0004] The purpose of this invention is to deeply analyze the rhythmic changes occurring within rice leaves during the two days following infection with rice blast fungus, clearly revealing that infection by rice blast fungus induces dynamic changes in the rice's immune process. Secondly, it precisely identifies specific application points for immune activators, enabling rational application operations against rice blast. At key time points, the immune activator can achieve better synergistic effects with the rice's immune mechanism, thereby maximizing the control efficacy of the immune activator, significantly improving the control effect against rice blast, and providing a more solid guarantee for the healthy growth of rice.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for analyzing the changes in the rhythm of rice leaves after infection with rice blast fungus, comprising the following steps:

[0007] (1) Spraying rice blast fungus spore suspension onto rice leaves as the rice blast group, and rice without spraying rice blast fungus spore suspension as the control, and cultured day and night alternately.

[0008] (2) Collect rice leaves from different groups and cultured for different times for gene transcription analysis. After standardizing the analysis results, perform cluster heatmap to obtain the diurnal rhythm variation of rice leaf genes after rice blast infection.

[0009] (3) Collect rice leaves from different groups and cultured for different times, analyze the expression of clock genes and phytochrome-related genes in the diurnal rhythm pathway, analyze the rhythm significance p-value, amplitude, period and phase of clock genes and phytochrome-related genes using MetaCycle method, and obtain the diurnal rhythm change pattern of clock genes and phytochrome-related genes after rice blast infection.

[0010] (4) Collect rice leaves from different groups and cultured for different times, observe the amount of H2O2 and callose deposited in the rice leaves, and obtain the changes in immune-related physiological indicators in rice leaves after infection with rice blast fungus.

[0011] Preferably, the number of conidia in the *Magnaporthe oryzae* spore suspension in step (1) is 3–8 × 10⁻⁶. 5 per mL.

[0012] Preferably, the circadian rhythm genes include OsFTL1, OsFTL7, OsFTL12, OsFTL3, OSCKA2, OsTOC1, OsGl, OsCK2a3, OsCK2b3, OsTCP7, OsTCP9, OsCKA2-2, OsTCP25, OsCKA2-4, OsTCP19, OsFTL6, OsPCF2, OsELF3b, OsZTL2, OsCHS1, OsELF3a, OsZTL1, OsCOP1, OsSPA1, OsLHY, OsTCP6, OsFTL8, OsTCP11, OsPRR95, OsPRR73, and OsPRR37.

[0013] Preferably, the photopigment-related genes include OsbZIP01, OsbZIP15, OsbZIP20, OsbZIP58, OsCRYD, OsbZIP04, OsPIF3, OsbZIP87, OsHY5L3, OsHY5L1, OsPHOT2, OsCRY1a, OsbZIP44, OsPHOT1b, OsbZIP65, OsbZIP60, and OsPIF4. , OsbZIP88, OsCRY2, OsPIF14, OsbZIP73, OsPIL13, OsPIL12, OsbZIP52, OsbZIP53, OsCOP1, OsSP A1, OsbZIP38, OsHY5L2, OsSPA3 / 4, OsPHOT1a, OsZTL2, OsbZIP22, OsbZIP39, OsbZIP33, OsCRY1b.

[0014] This invention also provides the application of the aforementioned analytical method in detecting the diurnal rhythm changes in rice after rice blast infection.

[0015] This invention also provides the application of the aforementioned analytical method in the precise application of pesticides to control rice blast disease.

[0016] This invention also provides the application of cyclic (valine-proline) in the prevention and / or treatment of rice blast.

[0017] This invention also provides a method for controlling rice blast disease using a cyclic (valine-proline) mechanism based on the principle of biological clock, comprising the following steps:

[0018] Rice blast pathogens were inoculated at different time points after the application of cyclic (valine-proline) 10–14 h after inoculation. The deposition of H2O2 and callose and the expression level of disease resistance genes were measured. The lesion area of ​​rice leaves was observed 6–8 days after inoculation. The optimal time for the application of cyclic (valine-proline) to control rice blast was determined by selecting the time point with the smallest lesion area, the highest deposition of H2O2 and callose, and the highest expression level of disease resistance genes.

[0019] The optimal time determined by the above screening was used to spray the ring (valine-proline) to control rice blast.

[0020] The best time is between 13:00 and 15:00.

[0021] This invention also provides the use of jinggangmycin in the prevention and / or treatment of rice blast.

[0022] This invention also provides a method for controlling rice blast disease using jinggangmycin based on the principle of biological clock, comprising the following steps:

[0023] Jinggangmycin was applied to rice leaves infected with rice blast fungus at different time points. The area of ​​lesions on the rice leaves was observed 6-8 days after application. The time point with the smallest lesion area was selected to determine the optimal time for Jinggangmycin to control rice blast.

[0024] The optimal time determined by the above screening was used to spray Jinggangmycin to control rice blast.

[0025] The best time is between 19:00 and 21:00.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] Jinggangmycin (VMA) and small molecule active peptide rings (valine-proline) do not have antifungal activity, do not have direct toxic effects on rice, and do not have a negative impact on the ecological environment.

[0028] By activating the rice's own immune system, its resistance to diseases is enhanced, rather than directly killing pathogens. This immune activation allows rice to maintain its defense against diseases for a longer period, reducing the occurrence and spread of diseases.

[0029] The pesticide is applied to rice via spraying, making it simple and convenient to operate and easy to promote in agricultural production. Furthermore, application plans can be tailored to the control effects at different time points after rice blast infection, offering strong targeting and flexibility.

[0030] By analyzing the rhythmic changes within rice leaves after infection with rice blast fungus, the dynamic changes in the rice immune process caused by rice blast fungus infection were determined. Combining the characteristics of small molecules, small molecule agents were rationally applied at specific time points to maximize the effect of small molecule agents and achieve efficient and precise control of rice blast. Attached Figure Description

[0031] Figure 1 This diagram illustrates the impact of rice blast infection on the overall diurnal rhythm of rice leaves.

[0032] Figure 2 The diagram shows the rhythm analysis of genes related to the biological clock and phytochrome; A) Diurnal rhythm variations of core biological clock genes; B) Rhythmic significance, amplitude, period, and phase variations of core biological clock genes; C) Rhythmic variations of phytochrome-related genes; D) Rhythmic significance, amplitude, period, and phase variations of phytochrome-related genes; The smaller the p-value, the more the data conforms to a periodic distribution, and p < 0.05 indicates that the gene has rhythmic significance.

[0033] Figure 3Validation diagrams for circadian rhythm changes and phytochrome-related genes; A. RT-qPCR validation of expression of core circadian rhythm genes and phytochrome-related genes; B. Amplitude changes in the control group and rice blast group; C. Phase changes in the control group and rice blast group; D. Periodic changes in the control group and rice blast group; Data are expressed as mean ± standard error (SEM), n = 5 for each group in each independent experiment, *p < 0.05, **p < 0.01 vs. Control.

[0034] Figure 4 The diagram shows the changes in immune indicators during rice blast infection of rice leaves; A) the process of rice blast fungus spores infecting rice leaves; B) the amount of H2O2 produced in rice leaf cells at different time points after rice blast infection; C) the amount of callose produced in rice leaf cells at different time points after rice blast infection.

[0035] Figure 5 Figure 1 shows the optimal application stage of cyclophosphamide (valine-proline); A. Phenotypic changes of rice leaves 7 days after infection with rice blast fungus in detached rice leaves; B. Phenotypic changes of rice leaves 7 days after infection with rice blast fungus in live rice leaves; C. Quantitative analysis of lesion area in live rice leaves (data are expressed as mean ± standard error n = 8, *p < 0.05, **p < 0.01 indicates significant difference between rice blast groups; #p < 0.05, ##p < 0.01 indicates significant difference between two groups).

[0036] Figure 6 Figure 1 shows the optimal application time for cyclophosphamide (valine-proline); A. Phenotypic changes in rice leaves 7 days after infection with rice blast fungus; B. Quantitative analysis of lesion area (data expressed as mean ± standard error n = 5, *p < 0.05, **p < 0.01 indicates significant difference between rice blast groups; #p < 0.05, ##p < 0.01 indicates significant difference between two groups); C. Accumulation of H2O2 in detached rice leaves; D. Deposition of callose in detached rice leaves, scale bar 150μm; E. Callose... Quantitative results of callose (data are expressed as mean ± standard error n = 8, *p < 0.05, **p < 0.01 indicates a significant difference compared with the rice blast group; #p < 0.05, ##p < 0.01 indicates a significant difference between the two groups); F, expression levels of rice-related immune genes mRNA (data are expressed as mean ± standard error n = 5, *p < 0.05, **p < 0.01 indicates a significant difference compared with the control group; #p < 0.05, ##p < 0.01 indicates a significant difference between the two groups).

[0037] Figure 7To investigate the gene map of how cyclic (valine-proline) alleviates rice blast symptoms and affects the biological clock: A. Phenotypic changes in rice leaves 7 days after infection with rice blast fungus; B. Quantitative analysis of lesion area; C. qPCR validation of core biological clock genes and immune genes (data are expressed as mean ± standard error n = 5, *p < 0.05, **p < 0.01 indicates significant difference compared to the control group; #p < 0.05, ##p < 0.01 indicates significant difference compared to the rice blast group); D. Phase and amplitude changes in the rice blast group and the 100 μg / mL cyclic (valine-proline) group (data are expressed as mean ± standard error n = 5; *p < 0.05, **p < 0.01 indicates significant difference compared to the control group; #p < 0.05, ##p < 0.01 indicates significant difference between the two groups).

[0038] Figure 8 The diagram shows the effect of jinggangmycin on activating the immune response; A. Changes in rice husk ketone content; B. Changes in salicylic acid content (data are expressed as mean ± standard error n = 5, *p < 0.05, **p < 0.01 indicates a significant difference between the two groups).

[0039] Figure 9 Figure 1 shows the optimal application time of jinggangmycin; A. Phenotypic changes in rice leaves 7 days after infection with rice blast fungus; B. Quantitative analysis of lesion area (data are expressed as mean ± standard error n = 5, *p < 0.05, **p < 0.01 indicates a significant difference between the two groups). Detailed Implementation

[0040] The following detailed description of the solutions provided by the present invention, in conjunction with embodiments, should not be construed as limiting the scope of protection of the present invention.

[0041] The reagents in this embodiment of the invention are sourced from the following sources:

[0042] Diaminobenzidine (DAB), catalog number 0430, was purchased from Beijing Jintai Hongda Biotechnology Co., Ltd.

[0043] Aniline blue, product number S19056, was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0044] The reverse transcription kit, catalog number R223-01, was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0045] The PCR kit, catalog number Q312-03, was purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0046] Trypan Blue, product number 72-57-1, purchased from Hefei Lanjieke Technology Co., Ltd., brand: Biosharp;

[0047] Cyclic (valine-proline), catalog number G4730.0250, purchased from GLPBIO.

[0048] Example 1

[0049] The diurnal rhythm changes of rice leaf genes affected by rice blast fungus infection

[0050] 1. Test crop: Indica rice plants (9311).

[0051] 2. Test strain: Rice blast fungus strain (Guy11), donated by Professor Zhou Mingguo of Nanjing Agricultural University.

[0052] 3. Planting methods

[0053] The soil for rice cultivation came from a rice paddy in Hangzhou. After being air-dried, it was broken up and settled at 1 dm³. 3 In black plastic boxes, rice seedlings were germinated until the sprouts reached about 1 cm in length, then planted in the boxes, with an average of 15 seedlings per box. The experimental rice was grown in a constant-temperature plant room with a 12h / 12h day-night cycle (lights were turned on at 8:00 am, defined as timekeeping time Zeitgeber 0 (ZT0); lights were turned off at 8:00 pm, defined as timekeeping time ZT (-12)). The ambient temperature was controlled at 28±2℃, and the humidity was controlled at 35%. Sterile water was used for irrigation daily to ensure a stable water supply for the rice.

[0054] 4. Cultivation and Sporulation of Rice Blast Strains

[0055] The rice blast fungus strain (Guy11) was removed from a -80℃ freezer and inoculated onto CM medium. The medium was then placed in a 28℃ incubator to ensure uniform growth. The inoculated petri dishes were then incubated at 28℃ for 7 days until colonies were clearly visible and growing vigorously. Next, well-grown colonies were selected from the CM medium and incubated at 28℃ for another 7 days. Once the surface of the SDC medium was fully covered with hyphae, the hyphae were gently scraped off using a sterile pipette tip, ensuring aseptic technique. The scraped hyphae were placed in unsealed petri dishes and incubated under black light to induce spore formation for 6 days. The culture media used in the experiment are shown in Tables 1-5.

[0056] Table 1 lists the formulations for CM culture medium (1L).

[0057] Formula composition content 20×NitrateSalts(I) 50mL TraceElements(II) 1mL Vitamin solution (III) 1mL Dextrorotatory glucose 10g peptone 2g Yeast paste 1g Casein amino acids 1g Agar 15g

[0058] Table 2 Nutritional Information for 20× Nitrate Salts (I) (1L)

[0059] Nutritional components content <![CDATA[NaNO3]]> 120g KCl 10.4g <![CDATA[MgSO4·7H20]]> 10.4g <![CDATA[KH2PO4]]> 30.4g

[0060] Table 3. Nutritional Information for Trace Elements (II) (100mL)

[0061] Nutritional components content <![CDATA[ddH2O]]> 80mL <![CDATA[MnCL2·4H20]]> 0.5g <![CDATA[ZnSO4·7H20]]> 2.2g <![CDATA[H3BO3]]> 1.1g <![CDATA[FeSO4·7H20]]> 0.5g <![CDATA[CoCL2·6H20]]> 0.17g <![CDATA[CuSO4·5H2O]]> 0.16g <![CDATA[Na2MoO4·2H20]]> 0.15g <![CDATA[Na4EDTA·2H20]]> 5.5g

[0062] Table 4. Nutritional composition list of Vitamin solution (III)

[0063]

[0064]

[0065] Table 5. SDC medium (1L) formulation list

[0066] Formula composition content rice straw 100g cornmeal 40g Agar powder 15g

[0067] 5. Preparation of rice blast strain spore suspension

[0068] After the SDC medium has fully grown under black light, add an appropriate amount of sterile water to the SDC plate. Spores are scraped off using a blue pipette tip, and the spore suspension is aspirated using a cut blue pipette tip. Filter the suspension through a layer of clean paper into a 50mL centrifuge tube. Rinse the SDC plate and filter paper with sterile water. Finally, adjust the conidia concentration to 5 × 10⁻⁶ under a 100x microscope using a micro-counting chamber. 5 The concentration of rice blast fungus spores was increased to 1 / mL to obtain a suspension of rice blast fungus spores.

[0069] 6. Treatment of the test group

[0070] Two-week-old indica rice variety 9311, grown to the two-leaf-one-heart stage, was used in the experiment. It was placed in a 24℃ incubator with 12h / 12h alternating day and night incubation for one day to acclimatize, with humidity maintained at 90%. On the second day, a concentration of 5×10⁻⁶ was prepared. 5 A conidial suspension of 10 mL / mL (with 0.05% Tween solution added) was used to infect rice (i.e., ZT(-12)) at 20:00 in the evening. The suspension was evenly sprayed onto the rice leaves using a spray bottle, with each container containing 10 mL of the *Magnapordica oryzae* conidial suspension. The containers were then placed in the aforementioned incubator for cultivation. Two groups were set up in the experiment: a control group and a *Magnapordica oryzae* group. The control group served as a blank control, without *Magnapordica oryzae* infection.

[0071] Starting from 0h after rice blast infection, rice leaf samples from the control group and the rice blast group were collected every 4 hours until 48h.

[0072] 7. Experimental Results

[0073] RNA samples from rice leaves at different time points (0–48 h) in the control group and the rice blast group were sent to Ling'en Biotechnology for eukaryotic transcriptome sequencing (gene transcription analysis). Cluster heatmap analysis of the obtained genes showed that the genes in the control group and the rice blast group were divided into two parts (separated by a white line, the upper part being the upper half and the lower part being the lower half), and both groups showed obvious rhythmic patterns. Figure 1 The upper half of the genes peaked during the light-dark cycle, while the lower half peaked primarily during the dark period. This pattern was also verified by cosine fitting. Figure 1 Furthermore, compared to the control group, the amplitude of the upper half of the genes in the rice blast infection group showed a decreasing trend, with an overall amplitude reduction of 20%; in contrast, the expression of the lower half of the genes was significantly upregulated 24 hours after rice blast infection. Figure 1 This indicates that rice blast infection disrupts the original diurnal rhythm pattern of rice leaves, and its impact is more pronounced on the second day.

[0074] Example 2

[0075] The effects of rice blast fungus infection on rice circadian rhythm genes

[0076] After Z-score normalization of circadian rhythm pathway genes in the transcriptome data, cluster heatmaps were generated. Heatmap analysis showed that the expression of circadian rhythm genes and photosensitivity genes exhibited a clear circadian rhythm expression pattern in the control group, while after rice blast infection, the expression of most genes was downregulated, especially on the second day. Figure 2 Next, MetaCycle specifically analyzed the significance p-values, amplitudes, periods, and phases of the circadian rhythm and phytochrome genes. The results showed that most genes in both groups exhibited significant rhythmicity, and the periods were mostly 24 hours in both the control and blast groups. Figure 2 Although there were no significant differences in the phases of these genes between the two groups, the overall amplitude of the blast rice group decreased compared to the control group. For example, the amplitudes of the core circadian rhythm genes OsLHY and OsTOC1 decreased significantly. Figure 2 This result indicates that the influence of rice blast fungus infection on the rice biological clock is mainly achieved through the regulation of amplitude.

[0077] Example 3

[0078] Verification of circadian rhythm changes in biological clock and phytochrome genes

[0079] 1. RNA extraction from rice leaves

[0080] 1) Liquid nitrogen grinding: Add 800 μL of Trizol to a centrifuge tube and place it on ice. Cool the mortar and iron spoon, put the blade directly into the mortar, add liquid nitrogen, grind quickly, add a small amount of liquid nitrogen, grind again, and repeat this process 3 times. Pour the ground powder into a centrifuge tube and shake to mix.

[0081] 2) Transfer the supernatant to a new centrifuge tube;

[0082] 3) Add 1 / 4 volume of chloroform (200 μL) to the centrifuge tube, shake to mix, emulsify the solution until it turns milky white, and let it stand at room temperature for 5 min;

[0083] 4) Centrifuge at 12000 rpm at 4℃ for 15 min; at this time, the homogenate will be divided into three layers: a colorless supernatant (containing RNA), a middle white protein layer (mostly DNA), and a lower milky white organic phase;

[0084] 5) Transfer the supernatant (300 μL) to another new centrifuge tube (do not aspirate the middle layer);

[0085] 6) Add an equal volume of isopropanol (300 μL) to the supernatant, mix thoroughly by inverting the container, and let stand at room temperature for 10 min.

[0086] 7) Centrifuge at 12000 rpm at 4℃ for 10 min. At this time, a small amount of white RNA precipitate can be seen at the bottom of the centrifuge tube.

[0087] 8) Carefully discard the supernatant, avoiding contact with the precipitate. Slowly add an equal volume of 75% ethanol (800 μL) to the Trizol, gently invert the centrifuge tube to wash the walls, centrifuge at 7500g at 4℃ for 5 min, discard the supernatant, and repeat the washing and centrifugation steps twice more;

[0088] 9) After discarding excess ethanol, let the centrifuge tube stand at room temperature for about 3-5 minutes with the cap open. After the precipitate dries (do not over-dry, otherwise the RNA will be difficult to dissolve), add an appropriate amount (15 μL) of RNase-free water to fully dissolve the RNA precipitate, and store at -80℃ for later use.

[0089] 2. Reverse transcription and RT-qPCR

[0090] RNA concentration and quality were measured by taking 2 μL of thawed RNA stock solution and determining the OD values ​​and concentrations at 280 nm and 260 nm. An OD260 / 280 value between 1.8 and 2.0 met the concentration requirements for total RNA during reverse transcription. Then, the detected mRNA was used to prepare the reverse transcription reaction system according to the reverse transcription kit instructions (Table 6). After thorough mixing, the mixture was incubated at 37°C for 15 min, followed by reverse transcription at 98°C for 5 min. The prepared cDNA template was stored at -20°C for later use. After reverse transcription, RT-PCR was performed according to the ChamQBlue Universal SYBR qPCR MasterMix kit. The PCR reaction was conducted in a real-time PCR system. The cDNA, primers (Table 8), and PCR reagents were prepared according to Table 7 and added to a 96-well plate for the experiment. The two-step reaction conditions were as follows: Step 1: Pre-denaturation, 95°C for 2 min; Step 2: PCR reaction, 95°C for 15 s, 60°C for 30 s, 40 cycles. Relative quantification of genes is achieved by using 2– ΔΔCt Analysis was performed using a comparative threshold cycle number (Ct). The amount of cDNA in each sample was normalized by calibration using the housekeeping gene Ubiquitin.

[0091] Table 6 RNA reverse transcription system

[0092]

[0093]

[0094] Table 7 RT-qPCR reaction system

[0095]

[0096] Table 8 Primer sequences

[0097]

[0098] 3. Experimental Results

[0099] The expression of core circadian rhythm genes was verified by RT-qPCR. In the control group, the mRNA expression of OsLHY and rice OsTOC1 showed a rhythmic pattern, with peak times at photoperiods ZT(0) / ZT(24) and ZT(12) / ZT(36), respectively. Figure 3 Consistent with the results of transcriptome analysis, mRNA levels were significantly downregulated at multiple time points, especially at the peak time point. Figure 3Similarly, rhythmic expression of representative photosensitizing factors was observed in both groups, such as the rice long hypocotyl 5-like 2 gene (OsHY5L2) and its downstream gene, rice phytochrome A-105 suppressor 1 (OsSPA1). The mRNA levels in the rice blast infection group were significantly lower than those in the control group, with the changes being more pronounced on the second day. Figure 3 Furthermore, compared to the control group, the amplitudes of both the circadian rhythm gene and the phytochrome gene were significantly reduced in the rice blast infection group. Figure 3 The phase of the OsLHY gene was slightly delayed, while the phases of other genes remained unchanged. Figure 3 The cycles of the OsLHY and OsSPA1 genes were slightly prolonged, while other genes remained unchanged. Figure 3 ).

[0100] Example 4

[0101] Analysis of immune-related physiological indicators at different time points after rice infection by rice blast fungus

[0102] 1. H2O2 determination in rice leaves

[0103] Rice leaves were stained with diaminobenzidine (DAB) to observe cell death. A DAB solution was prepared by dissolving 1 mg of DAB in 1 mL of water and adjusting the pH to 5.8. The samples were rinsed twice with sterile water and then placed in the DAB solution in the dark for 24 hours. They were then soaked in 100% ethanol:acetic acid (3:1 ratio) for 24 hours until the leaves completely lost their green color. The destained leaves were then directly prepared as slides, and cell death was observed under an inverted microscope.

[0104] 2. Callosity assay

[0105] Aniline blue was used to detect callose deposition. Aniline blue binds to callose and fluoresces under ultraviolet excitation. Leaves to be tested were immersed in a destaining solution (phenol:glycerol:lactic acid:water:100% ethanol = 1:1:1:1:2). After adding the destaining solution, two times the volume of ethanol was added. After destaining, the leaves were gently rinsed with sterile water, then stained in 4 mL of 150 mM aniline blue solution in the dark for 24 hours. Subsequently, slides were prepared with glycerol, and after gently rinsing the leaves with water, they were observed and photographed under ultraviolet excitation using a fluorescence microscope.

[0106] 3. Staining process of rice leaf infection by rice blast fungus

[0107] Rice blast fungus was stained with trypan blue. The specific preparation method of trypan blue staining solution is as follows: 30 mL ethanol, 10 g phenol, 10 mL water, 10 mL glycerol, 10 mL lactic acid, and 10 mg trypan blue are mixed thoroughly. During the experiment, the leaves to be observed were completely immersed in the prepared trypan blue staining solution, and then heated in boiling water for 2.5 min. After boiling, the leaves were allowed to stand in the trypan blue staining solution at room temperature for 1 h. Then, the leaves were transferred to a solution of ethanol and acetic acid in a volume ratio of 3:1 for decolorization. Subsequently, slides were prepared with glycerol, and the leaves were gently rinsed with water before being observed and photographed under a microscope.

[0108] 4. Experimental Results

[0109] Leaf samples from the infected group were collected at 12-hour intervals, 48 ​​hours post-infection. Within 72 hours post-infection, the infection process of *Blastoma oryzae* spores on rice leaves was identified by trypan blue staining at 12-hour intervals. As the infection progressed, *Blastoma oryzae* gradually produced adherent spores, which penetrated the rice leaves, and its mycelium gradually spread and elongated. Figure 4 The production of intracellular H2O2 and callose deposition in rice leaves after rice blast infection were studied at 12-hour intervals. Between 12 h ZT(0) and 24 h ZT(12) post-infection, H2O2 and callose deposition began to appear on the leaves. Figure 4 As the disease progressed, from ZT(24) to ZT(36), the production of H2O2 and the deposition of callosity in the leaves increased significantly, while from ZT(48) to ZT(60), they tended to decrease. Figure 4 This indicates that the immune process in rice caused by rice blast infection is dynamic.

[0110] Example 5

[0111] Investigation into the optimal application stage of cyclic (valine-proline)

[0112] Three treatment methods were designed: valine-proline pretreatment, simultaneous treatment with the fungus, and post-infection treatment. Two methods were implemented: spraying with live rice and dripping with detached rice. One day before, simultaneously with, and one day after inoculation with *Pseudomonas oryzae*, rice was sprayed with valine-proline at an initial concentration of 100 μg / mL. A control group treated with sterile water and a rice blast model group (rice blast group) inoculated only with *Pseudomonas oryzae* were also included. The valine-proline treatment on detached rice leaves was replaced with dripping, and mycelial blocks of equal size were inoculated with *Pseudomonas oryzae*. Seven days later, the lesions on the rice leaves of each treatment group were observed and compared. The results showed that, in both in vitro and live experiments, the lesions on the rice leaves of the valine-proline pretreatment group were significantly less severe than those in the control group. Figure 5Rice leaves pretreated with a cyclic (valine-proline) solution before inoculation with rice blast fungus showed the least severity of disease and the smallest lesion area. Figure 5 In contrast, rice leaves treated with cyclic (valine-proline) simultaneously with rice blast fungus and those treated one day after inoculation showed some reduction in lesions, but the difference in lesion area between the two methods was not significant. This indicates that cyclic (valine-proline) pretreatment is the optimal way to alleviate rice blast symptoms.

[0113] Example 6

[0114] Investigation into the optimal timing of cyclic (valine-proline) administration

[0115] Based on the optimal application method, rice was treated with cyclic (valine-proline) at different time points. DAB staining, callose staining, and RT-qPCR were used to detect disease resistance indicators and the expression levels of immune-related genes in rice. The experimental results will provide a time-based basis for the precise application of cyclic (valine-proline) treatment. The experimental arrangement was as follows: cyclic (valine-proline) treatment was performed at 20:00 the night before inoculation, and at 8:00, 14:00, and 20:00 on the day of inoculation. Rice leaves were collected 12 hours after inoculation with rice blast fungus for DAB staining, callose staining, and analysis of disease resistance gene expression levels. The results showed that the pretreatment at 14:00 significantly reduced the lesion area compared to treatments at other time points. Figure 6 This increases the accumulation of hydrogen peroxide and the amount of callosity deposited. Figure 6 Meanwhile, the expression level of the disease resistance gene PR1 was also highest at this treatment point. Figure 6 The results indicate that the pretreatment with cyclo(valine-proline) 14:00 has the best effect on the resistance of rice to rice blast.

[0116] Example 7

[0117] Cyclic (valine-proline) effects on the expression of circadian clock genes

[0118] To clarify whether cyclic (valine-proline) treatment affects the circadian rhythm of rice infected with rice blast, thereby regulating its immune function, three groups were set up: a control group, a rice blast group, and a rice blast group + cyclic (valine-proline) group. The control group was sprayed with 10 mL of sterile water at ZT(-12); the rice blast group was sprayed with 10 mL of 5×10⁻⁶ water at ZT(-12). 5 Rice blast fungus conidia suspension (spores / mL); the rice blast group + cyclic (valine-proline) group was sprayed with 10 mL of 100 μg / mL cyclic (valine-proline) solution at ZT(-18) 6 hours before rice blast infection, and then sprayed with 10 mL of 5×10⁻⁶ solution at ZT(-12) at the time of rice blast infection. 5A suspension of *Magnapordia oryzae* conidia was collected at a concentration of [number] cells / mL. Control and infected leaves, as well as infected leaves treated with a valine-proline ring, were collected every 6 hours post-infection. Plants were photographed to record the phenotypic pattern on day 7 after *Magnapordia oryzae* infection. Figure 7 The rhythmic expression of core circadian clock genes was verified by RT-qPCR. Results showed that in the control group, the mRNA expression of rice circadian clock genes OsLHY and OsTOC1 exhibited a clear diurnal rhythm, with peak expression occurring at 12h / 36h and 24h / 48h after rice blast infection (ZT(0) / ZT(24) and ZT(12) / ZT(36), respectively). In contrast, the disease resistance gene OsPR1 showed an overall increasing trend. Figure 7 In the rice blast infection group, mRNA levels were significantly downregulated at multiple time points, especially at the peak time point. The cyclic (valine-proline) group partially recovered the mRNA level reduction caused by rice blast infection. Analysis of the amplitude and phase of these genes using MetaCycle revealed no significant changes in the phases of the OsLHY and OsTOC1 genes after rice blast infection in rice infected with rice blast spores and in rice pretreated with cyclic (valine-proline) before infection with rice blast spores. Compared to the control group, the amplitude of circadian clock genes in rice infected with rice blast spores was significantly reduced, while the amplitude in the cyclic (valine-proline) group was slightly increased, but still lower than that in the control group. Figure 7 This indicates that the cyclic (valine-proline) mechanism alleviates rice blast symptoms while affecting changes in the amplitude of circadian rhythm genes.

[0119] Example 8

[0120] Image showing the effect of Jinggangmycin activating the immune response

[0121] The contents of rice ketone and salicylic acid in the bacterial contamination group and the bacterial contamination group plus jinggangmycin (initial concentration 10 μg / mL) were investigated. The results showed that the contents of rice ketone and salicylic acid in the bacterial contamination group were not significantly different from those in the control group, but the contents of rice ketone and salicylic acid increased significantly after the addition of jinggangmycin. Figure 8 This study confirmed that jinggangmycin has a certain immune-activating effect.

[0122] Example 9

[0123] Research on the optimal timing of jinggangmycin administration

[0124] Four groups were set up: a control group, a rice blast fungus infection group, a rice blast infection group + jinggangmycin application at 8:00 AM, and a rice blast infection group + jinggangmycin application at 8:00 PM. Rice blast fungus infection was performed at 8:00 PM in all cases. The experimental results showed that applying jinggangmycin at either 8:00 AM or 8:00 PM had a significant control effect on rice blast fungus infection, and the control effect of jinggangmycin applied at 8:00 PM was significantly better than that applied at 8:00 AM. Figure 9 ).

[0125] As demonstrated by the above embodiments, this invention aims to effectively improve the control of rice blast disease through an innovative application scheme based on the principle of biological clock. Specifically, this invention, through in-depth research on the rhythmic changes within the leaves of rice plants during the early stages of rice blast fungus infection, reveals the dynamic changes in the immune response triggered by rice blast fungus infection. Based on this discovery, this invention further precisely determines the optimal application time according to the characteristics of different immune activators. At these key time points, the immune activators can synergistically interact with the rice's own immune mechanism, thereby maximizing the control efficacy of the immune activators, significantly improving the control effect of rice blast disease, and providing a stronger guarantee for the healthy growth of rice.

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for analyzing the changes in the rhythm of rice leaves after infection with *Magnaportis oryzae*, characterized in that, Includes the following steps: (1) Spraying rice blast fungus spore suspension onto rice leaves as the rice blast group, and rice without spraying rice blast fungus spore suspension as the control, and cultured day and night alternately. (2) Collect rice leaves from different groups and cultured for different times for gene transcription analysis. After standardizing the analysis results, perform cluster heatmap to obtain the diurnal rhythm variation of rice leaf genes after rice blast infection. (3) Collect rice leaves from different groups and cultured for different times, analyze the expression of clock genes and phytochrome-related genes in the diurnal rhythm pathway, analyze the rhythm significance p-value, amplitude, period and phase of clock genes and phytochrome-related genes using MetaCycle method, and obtain the diurnal rhythm change pattern of clock genes and phytochrome-related genes after rice blast infection. (4) Collect rice leaves from different groups and cultured for different times, observe the amount of H2O2 and callose deposited in the rice leaves, and obtain the changes in immune-related physiological indicators in rice leaves after infection with rice blast fungus.

2. The analytical method according to claim 1, characterized in that, The number of conidia in the *Magnaporthe oryzae* spore suspension described in step (1) is 3–8 × 10⁻⁶. 5 per mL.

3. The analytical method according to claim 2, characterized in that, The biological clock genes include OsFTL1, OsFTL7, OsFTL12, OsFTL3, OSCKA2, OsTOC1, OsGl, OsCK2a3, OsCK2b3, OsTCP7, OsTCP9, OsCKA2-2, OsTCP25, OsCKA2-4, OsTCP19, OsFTL6, OsPCF2, OsELF3b, OsZTL2, OsCHS1, OsELF3a, OsZTL1, OsCOP1, OsSPA1, OsLHY, OsTCP6, OsFTL8, OsTCP11, OsPRR95, OsPRR73, and OsPRR37.

4. The analytical method according to claim 3, characterized in that, The photopigment-related genes include OsbZIP01, OsbZIP15, OsbZIP20, OsbZIP58, OsCRYD, OsbZIP04, OsPIF3, OsbZIP87, OsHY5L3, OsHY5L1, OsPHOT2, OsCRY1a, OsbZIP44, OsPHOT1b, OsbZIP65, OsbZIP60, OsPIF4, O sbZIP88, OsCRY2, OsPIF14, OsbZIP73, OsPIL13, OsPIL12, OsbZIP52, OsbZIP53, OsCOP1, OsSPA 1. OsbZIP38, OsHY5L2, OsSPA3 / 4, OsPHOT1a, OsZTL2, OsbZIP22, OsbZIP39, OsbZIP33, OsCRY1b.

5. The application of the analytical method according to any one of claims 1 to 4 in detecting the diurnal rhythm changes of rice after rice blast infection.

6. The application of the analytical method according to any one of claims 1 to 4 in the precise application of pesticides to control rice blast disease.

7. Application of cyclic (valine-proline) in the prevention and / or treatment of rice blast.

8. A method for controlling rice blast disease using a cyclic (valine-proline) mechanism based on the principle of biological clock, characterized in that... Includes the following steps: Rice blast pathogens were inoculated at different time points after the application of cyclic (valine-proline) 10–14 h after inoculation. The deposition of H2O2 and callose and the expression level of disease resistance genes were measured. The lesion area of ​​rice leaves was observed 6–8 days after inoculation. The optimal time for the application of cyclic (valine-proline) to control rice blast was determined by selecting the time point with the smallest lesion area, the highest deposition of H2O2 and callose, and the highest expression level of disease resistance genes. The optimal time determined by the above screening was used to spray the ring (valine-proline) to control rice blast. The best time is between 13:00 and 15:

00.

9. Application of Jinggangmycin in the prevention and / or treatment of rice blast.

10. A method for controlling rice blast disease using jinggangmycin based on the principle of biological clock, characterized in that, Includes the following steps: Jinggangmycin was applied to rice leaves infected with rice blast fungus at different time points. The area of ​​lesions on the rice leaves was observed 6-8 days after application. The time point with the smallest lesion area was selected to determine the optimal time for Jinggangmycin to control rice blast. The optimal time determined by the above screening was used to spray Jinggangmycin to control rice blast. The best time is between 19:00 and 21:00.

Images