Application of L-piperidic acid as anti-disease activator in improving rice resistance to rice blast

Abstract

The application relates to the technical field of plant disease prevention and treatment, and particularly relates to application of L-piperidine acid as an anti-disease activator in improving rice resistance to rice blast, L-piperidine acid is applied to rice blast prevention and treatment for the first time, an efficient Pip treatment scheme is established for the susceptible variety Lijiang Xintuanhegu, accurate application time is determined, based on the infection process of the rice blast fungus, 10 hours after inoculation (10 hpi) is determined as the best spraying window period; low toxicity and environmental protection: Pip is a natural metabolite of plants, has no residue risk, and meets the requirements of green agriculture; and long-acting prevention and control: the defense signal induced by Pip can be sustained to the late growth period of rice, and does not affect yield and quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease control, in particular to a method for improving the resistance of rice to rice blast by exogenous spraying of L-pipecolic acid (Pip), which is especially suitable for improving the resistance of wild rice variety Lijiang Xintuanheigui to Magnaporthe oryzae. BACKGROUND

[0002] Plants have developed a complex immune system during long-term evolution, mainly including two defense mechanisms. Pathogen-associated molecular pattern-triggered immunity (PTI, PAMP-Triggered Immunity): Plants recognize the conserved molecular patterns of pathogenic bacteria (such as chitin, flagellin, etc.) through cell surface pattern recognition receptors (PRRs), activate early defense responses such as calcium ion (Ca²⁺) influx, reactive oxygen species (ROS) burst, MAPK signaling pathway, etc. Effector-triggered immunity (ETI, Effector-Triggered Immunity): Pathogenic bacteria secrete effector proteins to inhibit PTI, but some effector proteins can be recognized by plant disease resistance genes (R genes), triggering stronger immune responses such as hypersensitive response (HR), salicylic acid (SA) accumulation and systemic acquired resistance (SAR). Systemic acquired resistance (SAR) is a broad-spectrum, long-lasting and systemic disease resistance mechanism, characterized by resistance signals that can be transmitted to uninfected sites (Cai Xinzhong et al., 1999) i.e. systemic; resistance can last for several weeks to several months (Zhao Shuqing et al., 2003), i.e. long-lasting, and can resist fungi, bacteria and viruses simultaneously, i.e. broad-spectrum. The induction of SAR depends on multiple signal molecules, including salicylic acid (SA), glycerol-3-phosphate (G3P), azelaic acid (AzA) and pipecolic acid (Pip) etc. Among them, pipecolic acid (Pip) as a lysine metabolite plays a key role in SAR.

[0003] Pipecolic acid (Pip) can activate SAR through SA-dependent and independent pathways. Exogenous Pip treatment can enhance the resistance of tobacco, Arabidopsis, cucumber and other plants to bacteria and fungi. Pip can promote the accumulation of reactive oxygen species (ROS) and nitric oxide (NO) and enhance plant defense responses.

[0004] Drissia Vogel-Adghough et al. (2013) supplemented the soil substrate of individually grown tobacco plants with 10 ml of a 1 mM (10 μmol) solution of D,L-piperidine acid (S47167; Sigma-Aldrich) one day before bacterial inoculation. Control plants were supplemented with 10 ml of water. Results showed that exogenous application of Pip significantly enhanced resistance of tobacco to the adapted pathogen Pstb or the non-adapted (inducing hypersensitive cell death) Pseudomonas syringae p.v. maculicola.

[0005] Pazarlar S et al. (2021) used D,L-piperidine acid (CAS number 535-75-1) and L-piperidine acid (CAS number 3105-95-1) purchased from TCI Chemicals (India). Piperidine acid was dissolved in water to make a 20 mM stock solution and added to the hydroponic solution at the indicated final concentration. Control plants were supplemented with equal amount of water solution used for piperidine acid treatment. The addition of D,L-piperidine acid, L-piperidine acid, BABA (3-aminobutyric acid) and BTH (benzothiadiazole) was performed by adding directly to the nutrient solution 24 hours before inoculation. Results showed that exogenous application of D,L-Pip successfully induced systemic acquired resistance of cucumber to powdery mildew fungus P. xanthii and Pseudomonas Psl.

[0006] Hana Návarová et al. (2012) pipetted 10 mL of a 1 mM (10 μmol) solution of d,L-Pip (S47167; Sigma-Aldrich), 10 mL of a 0.5 mM (5 μmol) solution of L-Pip (P1404; TCI Europe), 10 mL of a 0.5 mM (5 μmol) solution of D-Pip (P1830; TCI Europe), 10 mL of a 1 mM (10 μmol) solution of BABA (A44207; Sigma-Aldrich) or 10 mL of a 1 mM (10 μmol) solution of Aad (A7275; Sigma-Aldrich) onto the soil substrate of individually grown plants 1 day before bacterial inoculation or 1° treatment in the SAR experiment. Control plants were added with 10 mL of water in the same way. Results showed that soil drenching with exogenous Pip enhanced immunity of Arabidopsis sufficient to restore immunity of ald1 mutant plants.

[0007] Friederike Bernsdorff et al. (2015) applied 10 ml of 1 mM (10 µmol) D,L-piperidine acid solution (Cat. No. S47167, Sigma-Aldrich) to the substrate soil of individually cultivated plants by pipette. Control plants were applied with the same amount (10 ml) of deionized water instead. This Pip application was used as a treatment to induce defense priming. The bacterial inoculation challenge experiment was performed one day after Pip treatment according to the systemic acquired resistance (SAR) priming analysis protocol. To investigate the synergistic effect of Pip and salicylic acid (SA), one day after Pip application, leaves were infiltrated with 0.5 mM SA solution and deionized water as mock control. All treatment samples were collected 4 hours after treatment. The results showed that SA and Pip had both independent and synergistic effects on the basal immunity of Arabidopsis thaliana to Pseudomonas syringae.

[0008] Currently, the main methods of exogenous Pip treatment to improve plant disease resistance include soil irrigation, which has the disadvantages of low soil application efficiency, easy microbial degradation of Pip, and difficulty in accurately controlling the dose. Second, water culture addition method, i.e. adding 20 mM Pip mother liquor in cucumber water culture solution to induce resistance to powdery mildew, has the disadvantage of being only suitable for water culture system and limited application in the field; root irrigation + foliar SA synergistic treatment, i.e. foliar spraying of SA after irrigation of 1 mM Pip in Arabidopsis thaliana, enhances resistance to bacteria, which has the disadvantages of complex operation and SA may cause phytotoxicity to some crops.

[0009] Therefore, the common problem of the current prior art is the lack of Pip treatment method for rice blast, the unclear optimal application time affecting the control effect, and the lack of evaluation of the influence of Pip on rice growth and environmental safety. SUMMARY

[0010] The purpose of the present application is to solve the problems existing in the prior art and provide an application of L-piperidine acid as a disease resistance activator in improving the resistance of rice to blast.

[0011] To achieve the above purpose, the technical scheme adopted by the present application is: the application of L-piperidine acid as a disease resistance activator in improving the resistance of rice to blast, in the process of preparing rice resistant seeds under greenhouse conditions, when the rice seedlings grow to the three-leaf-one-heart stage, uniformly spray Magnaporthe oryzae spores on the rice to inoculate Magnaporthe oryzae, 10 hours post inoculation (hpi), apply 60 mL / m 2 Spray L-piperidine acid aqueous solution with a concentration of 10 µmol / L, and harvest the seeds after maturation as seeding materials.

[0012] Further, the concentration of the Magnaporthe spore suspension is 1×10 5 .

[0013] A second object of the present application is to provide an application of L-piperidine acid as an anti-disease activator in the prevention and treatment of rice blast caused by Magnaporthe oryzae. 2 Spray a water solution of L-piperidine acid with a concentration of 10 μmol / L.

[0014] The beneficial technical effects of the present application are: the present application provides a fine method of exogenous spraying L-piperidine acid to improve the resistance of rice to blast, which is applied to the prevention and treatment of rice blast for the first time; an efficient Pip treatment scheme is established for the susceptible variety Lijiang Xintuanheigui; precise application time: based on the infection process of Magnaporthe oryzae, 10 hpi (hours after inoculation) is determined as the best spraying window; low toxicity and environmental protection: Pip is a natural plant metabolite with no residual risk, which meets the requirements of green agriculture; and long-term prevention and control: the defense signal induced by Pip can last until the late growth period of rice, without affecting yield and quality. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 is a view of the disease investigation of rice treated with L-piperidine acid 168 hours after inoculation with Magnaporthe 10 hpi; Figure 1 A represents the lesion statistics of water-treated and pipelidine acid-treated water rice LTH 10 hpi after spraying and inoculating Magnaporthe; Figure 1 B represents the disease index statistics of water-treated and pipelidine acid-treated water rice LTH 10 hpi after spraying and inoculating Magnaporthe.

[0017] Figure 2 is a view of the relative growth of individual lesion fungi when L-piperidine acid is treated 144 hours after inoculation with Magnaporthe 10 hpi; Figure 2 A represents the lesion statistics of water-treated and pipelidine acid-treated LTH 10 hpi after wounding and inoculating water rice; Figure 2 B represents the lesion fungus biomass graph of water-treated and pipelidine acid-treated LTH 10 hpi after wounding and inoculating water rice.

[0018] Figure 3Figure 1 is a graph showing the number of callose in rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application. Figure 3 Figure 2 is a graph showing the number of callose in rice treated with water after inoculation with Magnaporthe grisea 10 hpi according to the present application. Figure 3 Figure 3 is a graph showing the number of callose in rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application.

[0019] Figure 4 Figure 4 is a graph showing the number of dead cells in rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application. Figure 4 Figure 5 is a graph showing the number of dead cells in rice treated with water after inoculation with Magnaporthe grisea 10 hpi according to the present application. Figure 4 Figure 6 is a graph showing the number of dead cells in rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application.

[0020] Figure 5 Figure 7 is a graph showing the expression of defense-related genes (OsPR1a, OsWRKY45, OsNPR1, OsPAL1) in rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application.

[0021] Figure 6 Figure 8 is a graph showing the plant height of rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application.

[0022] Figure 7 Figure 9 is a graph showing the expression of defense-related genes (OsCPK5, OsNPR1, OsPAL1) in rice seeds harvested from rice treated with L-piperidine acid after inoculation with Magnaporthe grisea 10 hpi according to the present application.

[0023] Figure 8 Figure 10 is a graph showing the expression of genes (OsNPR1, OsPAL1) in rice treated with L-piperidine acid (Pip) according to the present application. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] Example 1

[0026] The resistance of Lijiang Xintuanhegu to blast was identified by treating with exogenous Pip, and the wild type rice Lijiang Xintuanhegu treated with water was used as a control.

[0027] 1.1 Statistical analysis of rice blast symptoms after treating rice with exogenous Pip

[0028] 1.1.1 Spray inoculation: Rice seedlings growing to the three-leaf-one-heart stage (14 days) were inoculated with Magnaporthe oryzae, and the spore suspension concentration was 1×10 5 / mL. The spore suspension was uniformly sprayed on the rice, and 10 hpi after inoculation, the rice was treated with Pip at a concentration of 10 μmol / L. Samples were taken at 10 hpi, 24 hpi, 36 hpi, 48 hpi, and 72 hpi after inoculation, and disease investigation was performed at 168 hpi after inoculation. Investigation and disease index statistics were referred to Xu Zhigang (2002), and the disease index was calculated as 100×∑(number of leaves at each level × representative value at each level) / (total number of leaves investigated × highest representative value).

[0029] The results are shown in Figure 1 As shown in Figure A, the rice blast symptoms were lighter after treating rice leaves with Pip at a concentration of 10 μmol / L at 10 hpi after inoculation with Magnaporthe oryzae compared to rice (CK) treated with an equal volume of water at 10 hpi after inoculation with Magnaporthe oryzae, as shown in Figure B, the disease index of Pip-treated rice was lower. Figure 1 Figure 1

[0030] 1.1.2 punch inoculation: A puncher was used to wound the leaves of rice growing to 40 days, 10 μL of spore suspension was taken with a pipette gun on the wound site, the wound diameter was 5 mm, and the wound was treated with dark and moisture for 24 h, then transferred to room temperature for moisture culture. Samples were taken at 144 hpi after inoculation, the lesion length was measured, a single lesion was selected, the DNA of the single lesion was extracted by the CTAB method, and the CT values of the rice gene OsUBQ and the Magnaporthe oryzae gene MoPot2 were determined by real-time fluorescent quantitative PCR. The relative growth of fungi was calculated according to the formula: relative growth of fungi = 2 [CT（MoPot2）-CT （OsUBQ）] ×100.

[0031] ① Absolute quantification of Magnaporthe oryzae DNA

[0032] Table 1 RT-PCR primer sequences

[0033]

[0034] ② Preparation of PCR 20.0 μL reaction system as shown in Table 1: The reaction solution was prepared on ice.

[0035] Table 2 Composition of PCR reaction system ​​

[0036]

[0037] The reaction procedure of qRT-PCR is shown in Table 3.

[0038] Table 3 PCR reaction conditions

[0039]

[0040] Data analysis of qRT-PCR of target genes

[0041] The expression level of MoPot2 gene was calculated and analyzed by 2 -△△Ct The relative expression amount of the gene = 2 -(Ct1-actinCt1)-(Ct2-actinCt2) The data was processed by IBM SPSS Modeler 27.0, and Primer 9.5.1 was used for plotting.

[0042] The results are shown in Table 4: Figure 2 Compared with rice (CK) treated with water and inoculated with Magnaporthe oryzae 10 hpi, the effect of Pip on rice inoculated with Magnaporthe oryzae 10 hpi was more significant, and the lesion length (A) and the relative expression amount of fungi (B) were lower when Pip was sprayed. Figure 2 A) and the relative expression amount of fungi (B) were lower when Pip was sprayed. Figure 2

[0043] 1.2 Exogenous Pip treatment of rice, statistics of the number of antifungal compounds such as callose and dead cells

[0044] 1.2.1 Callose observation and statistics

[0045] Callose observation and statistics: After sampling, the rice was washed with sterile water three times, soaked in 95% ethanol for 10 minutes, and then washed three times. Then it was placed in an ethanol lactophenol solution (phenol: glycerol: lactic acid: water: ethanol = 1:1:1:1:2) prepared according to a certain volume ratio, and incubated in a 65°C constant temperature water bath until the leaves turned completely green. Then the leaves were rinsed with 50% ethanol and sterile water for 3 times each, and finally the leaves were stained with 0.1% aniline blue (aniline blue was dissolved in 150Mm K2HPO4, pH 9.5) for 1h, and observed and photographed under a fluorescence upright microscope.

[0046] 1.2.2 Dead cell observation and statistics: After sampling, the rice was washed with sterile water three times, and then stained with 1mg / ml DAB. After 24h, the number of dead cells was observed and counted under a microscope.

[0047] The results are shown in Table 4: Figure 3 , 4 ​As shown: Compared with rice treated with equal volume of water at 10 hpi (CK), the callose deposition in rice treated with Pip at 10 hpi increased from 24 to 72 hpi ( Figure 3 A. Figure 3 B), the number of dead cells increased from 10 to 72 hpi and was higher than that of the control group CK ( Figure 4 A. Figure 4 B). This indicates that pipecolic acid enhances the defense signaling during rice blast infection.

[0048] 1.3 Expression analysis of defense-related genes Ubiqutin, OsNPR1, OsPR1a, OsWRKY45, and OsPAL1 in rice after exogenous Pip treatment

[0049] 1.3.1 Extraction of total RNA from rice

[0050] Rice total RNA was extracted using the GenStar kit (Beijing Kangrun Chengrun Biotechnology Co., Ltd.)

[0051] (1) Quickly transfer the ground sample into a centrifuge tube containing 1 mL of TRIGene, quickly shake and mix on a vortexer, place on ice, centrifuge at 12,000 × g for 10 min at 4°C, and then aspirate the supernatant into a new centrifuge tube.

[0052] (2) The lysate was placed at room temperature for 5 min to allow complete separation of the nucleic acid-protein complex.

[0053] (3) Add 0.2 ml of chloroform to every 1 ml of TRIGene, cap the tube tightly, shake vigorously for 15 seconds, and let it stand at room temperature for 2-3 minutes.

[0054] (4) Centrifuge at 12,000 × g for 15 min at 4°C. The sample will separate into three layers: an orange-yellow lower organic phase, an intermediate layer, and a colorless upper aqueous phase.

[0055] (5) Pipette the upper aqueous phase containing total RNA into a new centrifuge tube. The volume of the aqueous phase should be 60% of the TRIGene reagent used.

[0056] (6) Add 0.5 ml of isopropanol to every 1 ml of TRIGene initially used, invert several times to mix, and let stand at room temperature for 10 min.

[0057] (7) Centrifuge at 12,000 × g for 10 min at 4°C and discard the supernatant. A gelatinous RNA precipitate will be visible.

[0058] (8) Add 1 ml 75% ethanol per 1 ml of TRIGene used initially, invert several times to mix, and wash the pellet.

[0059] (9) Centrifuge at 12000 x g for 5 min at 4°C, and discard the supernatant.

[0060] (10) Dry at room temperature for 5-10 min or under vacuum (do not use a vacuum centrifuge to dry the RNA, as this will make it difficult to dissolve).

[0061] (11) Add an appropriate amount (e.g., 25 μl) of DEPC-ddH2O or TE buffer, and dissolve the RNA by pipetting several times.

[0062] (12) Determine the concentration, purity, and integrity of the RNA by RNA electrophoresis and ultraviolet spectrophotometer detection.

[0063] (13) The obtained RNA should be used immediately or stored at -80°C after being aliquoted, to avoid repeated freezing and thawing.

[0064] 1.3.2 Reverse transcription of RNA to cDNA, as shown in Table 4:

[0065] Table 4 RNA reverse transcription system

[0066]

[0067] According to the above table, first prepare the total system except for RNA, aliquot it in PCR tubes, then add RNA one by one, mix gently, and place it in a PCR instrument for incubation at 37°C for 15 min, and then heat at 85°C for 5 s.

[0068] 1.3.3 Real-time fluorescent quantitative PCR detection of defense-related gene expression

[0069] (1) Design primers according to the principles of primer design, and determine the primer sequences as shown in Table 5.

[0070] Table 5 Real-time fluorescent quantitative PCR primer sequences

[0071]

[0072] (2) Prepare the PCR 20.0 μL reaction system according to Table 1: the reaction solution is prepared on ice.

[0073] (3) Prepare the system according to the above table, mix gently, and aliquot into 96-well PCR plates, 19 μL per well, finally add 1 μL of cDNA, centrifuge at 1500 rpm for 1 min, then take out, and perform qRT-PCR in a CFX96 quantitative PCR instrument, and the reaction program is shown in Table 2.

[0074] (4) Data analysis: The expression levels of the above genes were calculated and analyzed by 2 -△△Ct The relative expression of the genes = 2 -(Ct1-actinCt1)-(Ct2-actinCt2) Data were processed using IBM SPSS Modeler 27.0 and plotted using Primer 9.5.1.

[0075] The results are shown in Table 1: Figure 5 Compared with rice treated with water at 10 hpi (CK), the expression of OsPR1a, OsNPR1, OsPAL1 and OsWRKY45 was up-regulated in rice treated with Pip at 10 hpi (Pip) at 10-72 hpi (P < 0.05) (Fig. 1). Figure 5

[0076] 1.4 Measurement of plant height of rice grown for 60 days after exogenous Pip treatment

[0077] The plant height of rice seedlings treated with Pip at 10 hpi was measured when they were grown to 60 days.

[0078] The results are shown in Table 2: Figure 6 Compared with rice treated with water at 10 hpi (CK), the plant height of rice treated with Pip at 10 hpi was similar to that of CK at the same period, and the plant height was 60 cm (P < 0.05) (Fig. 2). Figure 6

[0079] Example 2

[0080] During the preparation of rice resistant seeds under greenhouse conditions, rice seedlings were inoculated with Magnaporthe grisea spores by spraying evenly on the rice at the three-leaf-one-heart stage. After 10 hpi, 60 mL / m 2 The L-piperidine acid solution was sprayed at a concentration of 10 μmol / L. After the seeds matured, the RNA in the seeds was extracted using the RNA extraction method of Example 1 and reverse transcribed into cDNA. The expression of defense-related genes OsNPR1, OsPR1a, and OsCPK5 was detected by real-time fluorescent quantitative PCR using the primers in Table 5.

[0081] The results are shown in Table 3: Figure 7 Compared with the control group (CK), the expression of defense-related genes (OsNPR1, OsPR1a, and OsCPK5) in seeds harvested from rice treated with water at 10 hpi and Pip at 10 hpi was higher, showing up-regulated expression (P < 0.05) (Fig. 3). Figure 7

[0082] Example 3 ​​​

[0083] Rice was grown in greenhouse condition to three-leaf-one-heart stage, and then treated with equal volume of water and 20 μmol / L L-piperidine acid solution, respectively. The samples were taken at 0 h (immediately after L-piperidine acid treatment) and 4 h (4 h after L-piperidine acid treatment), and immediately placed in liquid nitrogen. The RNA in rice leaves was extracted and reverse transcribed into cDNA by the RNA extraction method of Example 1. The expression of OsNPR1 and OsPR1a defense-related genes was detected by real-time fluorescent quantitative PCR using the primers in Table 5.

[0084] The results are shown in Table 2: Figure 8 After treatment, the expression levels of rice defense-related genes OsNPR1 and OsPR1a in rice not infected with Magnaporthe grisea were higher than those in the control group.

[0085] Finally, it should be explained that the above examples are only used to illustrate but not to limit the technical solutions of the present application. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or equivalently replaced without departing from the spirit and scope of the present application. Any modification or partial replacement should be covered in the scope of the claims of the present application.

Claims

1. Use of L-piperidinic acid as a disease resistance activator in improving rice resistance to rice blast, characterized in that: In the process of preparing rice resistant seeds under greenhouse conditions, when the rice seedlings grew to the three-leaf and one-heart stage, the rice blast fungus spores were suspended and evenly sprayed on the rice for rice blast inoculation. After 10 hpi, the spores were sprayed at 60 mL / m 2 The sowing material obtained by spraying an aqueous solution of L-piperidinic acid with a concentration of 10 μmol / L and harvesting the seeds after they mature is the breeding material with rice blast resistance.

2. The application according to claim 1, characterized in that The concentration of the rice blast fungus spore suspension is 1×10 5 pieces / mL.

3. Use of L-piperidinic acid as a disease resistance activator in the prevention and control of rice blast caused by the rice blast pathogen (Magnaporthe oryzae), characterized in that: When rice plants are infected with rice blast pathogen 10hpi, press 60mL / m 2 Spray an aqueous solution of L-piperidinic acid at a concentration of 10 μmol / L.

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