Application of L-piperidine acid as disease-resistant activator in improving rice blast resistance of rice

Abstract

The invention relates to the technical field of plant disease prevention and treatment, in particular to application of externally sprayed L-piperidine acid as a disease-resistant activator in improving rice blast resistance, L-piperidine acid is applied to rice blast prevention and treatment for the first time, an efficient Pip treatment scheme and precise application time are established for a susceptible variety Lijiang Xingroup black grain, and the disease resistance of rice is improved. Based on a magnaporthe oryzae infection process, determining 10 hpi (after inoculation per hour) after inoculation as an optimal spraying window period; pip is a natural metabolite of plants, has no residual risk, and meets the requirements of green agriculture; and a defense signal induced by Pip can be continued to the later growth period of the rice, and the yield and the quality are not influenced.

Description

Technical Field

[0001] The present invention relates to the technical field of plant disease prevention and control, and particularly to a method for improving rice blast resistance by exogenously spraying L-pipecolic acid (Pip). The method is particularly suitable for improving the resistance of a wild-type rice variety, Lijiang Xintuan Heigu, to the rice blast fungus (Magnaporthe oryzae). Background Art

[0002] Plants have developed a complex immune system over the course of long evolution, primarily consisting of two defense mechanisms. Pathogen-associated molecular pattern-triggered immunity (PTI, PAMP-Triggered Immunity): Plants use pattern recognition receptors (PRRs) on their cell surfaces to recognize conserved molecular patterns of pathogens (such as chitin and flagellin), activating early defense responses such as calcium ion (Ca²⁺) influx, reactive oxygen species (ROS) bursts, and MAPK signaling pathways. Effector-triggered immunity (ETI, Effector-Triggered Immunity): Pathogens secrete effector proteins to suppress PTI, but some effector proteins can be recognized by plant disease resistance genes (R genes), triggering stronger immune responses such as the 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), persistence (sustainability) of resistance that can last for weeks to months (Zhao Shuqing et al., 2003), and broad-spectrum protection against fungi, bacteria, and viruses. The induction of SAR depends on multiple signaling molecules, including salicylic acid (SA), glycerol-3-phosphate (G3P), azelaic acid (AzA), and pipecolic acid (Pip). Pipecolic acid (Pip), a lysine metabolite, plays a key role in SAR.

[0003] Pipecolic acid (Pip) can activate SAR through both SA-dependent and SA-independent pathways. Exogenous Pip treatment enhances resistance to bacteria and fungi in plants such as tobacco, Arabidopsis, and cucumber. Pip promotes the accumulation of reactive oxygen species (ROS) and nitric oxide (NO), enhancing 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) D,L-piperidinic acid solution (S47167; Sigma-Aldrich) one day before bacterial inoculation. Control plants were supplemented with 10 ml of water. The results showed that exogenous application of Pip significantly enhanced tobacco resistance to the adapted pathogen Pstb or the non-adapted (hypersensitive cell death-inducing) Pseudomonas syringae pvmaculicola.

[0005] Pazarlar S et al. (2021) used D,L-piperidinic acid (CAS No. 535-75-1) and L-piperidinic acid (CAS No. 3105-95-1) purchased from TCI Chemicals (India). Piperidinic acid was dissolved in water to prepare a 20 mM stock solution and added to the hydroponic culture solution at the specified final concentration. Control plants were supplemented with an equal volume of the aqueous solution used for the piperidinic acid treatment. This was achieved by adding D,L-piperidinic acid, L-piperidinic acid, BABA (3-aminobutyric acid), and BTH (benzothiadiazole) directly to the nutrient solution 24 hours prior to inoculation. The results showed that exogenous application of D,L-piperidinic acid successfully induced systemic acquired resistance in cucumber against the powdery mildew fungus P. xanthii and Pseudomonas sp. Psl.

[0006] Hana Návarová et al. (2012) pipetted 10 mL of a 1 mM (10 μmol) solution of 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 for 1° treatment in SAR experiments. In the same manner, 10 mL of water was added to control plants. The results showed that soil drench application of exogenous Pip enhanced the immunity of Arabidopsis thaliana and was sufficient to restore the immunity of ald1 mutant plants.

[0007] Friederike Bernsdorff et al. (2015) applied 10 ml of a 1 mM (10 µmol) D,L-piperidinic acid solution (Cat. No. S47167, Sigma-Aldrich) to the soil substrate of individually cultured plants via a pipette. Control plants were treated with an equal volume (10 ml) of deionized water. This Pip irrigation treatment served as a Pip-induced defense sensitization approach. A bacterial challenge experiment was conducted one day after Pip treatment, following the systemic acquired resistance (SAR) sensitization assay. To investigate the synergistic effect of Pip and salicylic acid (SA), leaves were infiltrated with a 0.5 mM SA solution one day after Pip irrigation, and deionized water infiltration served as a mock control. Leaves from all treatments were harvested 4 hours after treatment. These results suggest that SA and Pip act both independently and synergistically in Arabidopsis thaliana's basal immunity to Pseudomonas syringae.

[0008] Currently, methods for enhancing plant disease resistance through exogenous Pip treatment primarily include soil dredging, which has drawbacks such as low soil application efficiency, Pip's susceptibility to microbial degradation, and difficulty in precisely controlling dosage. A second approach involves hydroponic addition, where a 20 mM Pip stock solution is added to cucumber hydroponic solution to induce resistance to powdery mildew. This approach, however, is only applicable to hydroponic systems, limiting its application in field settings. Furthermore, a synergistic root dredging plus foliar SA treatment, where 1 mM Pip is drenched in Arabidopsis plants followed by a foliar SA spray, aims to enhance bacterial resistance. However, this approach is complex and can potentially cause phytotoxicity to certain crops.

[0009] Therefore, the common problems of these existing technologies are the lack of Pip treatment methods for rice blast, the lack of a clear optimal application time, which affects the prevention and control effect, and the failure to evaluate the impact of Pip on rice growth and environmental safety. Summary of the Invention

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

[0011] To achieve the above object, the technical solution adopted by the present invention is: L-piperidinic acid is used as a disease resistance activator to improve rice resistance to rice blast. In the process of preparing rice resistant seeds under greenhouse conditions, when the rice seedlings grow to the three-leaf and one-heart stage, the rice is inoculated with rice blast fungus by evenly spraying a suspension of rice blast fungus spores. After 10 hpi of inoculation, the suspension is sprayed at 60 mL / m 2 The seeds were sprayed with an aqueous solution of L-piperidinic acid at a concentration of 10 μmol / L, and harvested after maturity as sowing materials.

[0012] Furthermore, the concentration of the rice blast fungus spore suspension is 1×10 5 pieces / mL.

[0013] The second object of the present invention is to provide an application of L-piperidinic acid as a disease resistance activator in the prevention and control of rice blast caused by Magnaporthe oryzae. When rice plants are infected with Magnaporthe oryzae at 10 hpi, the mixture is diluted with 60 mL / min of water. 2 Spray an aqueous solution of L-piperidinic acid at a concentration of 10 μmol / L.

[0014] The beneficial technical effects of the present invention are: in response to the deficiencies of the existing technology, the present invention provides a refined method for improving rice blast resistance by exogenously spraying L-piperidinic acid, which is applied to rice blast control for the first time; an efficient Pip treatment scheme is established for the susceptible variety Lijiang Xintuan Black Valley; precise application time: based on the infection process of rice blast fungus, 10 hpi (hours post inoculation) is determined as the optimal spraying window; low toxicity and environmental protection: Pip is a natural plant metabolite with no residue 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 stage of rice without affecting yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 This is a diagram showing the investigation of rice blast disease after the rice was inoculated with the blast fungus 10 hpi and treated with L-piperidic acid 168 h after the present invention; Figure 1 A represents the lesion statistics of LTH rice treated with water and pipecolic acid at 10 hpi after spray inoculation with rice blast fungus; Figure 1 B represents the disease index statistics of LTH in rice treated with water and pipecolic acid at 10 hpi after spray inoculation with rice blast fungus.

[0017] Figure 2 This is a diagram showing the relative growth of a single lesion fungus when the rice is inoculated with the blast fungus 10 hpi and treated with L-piperidic acid for 144 h; Figure 2 A represents the LTH lesion image of the wounded inoculated rice at 10 hpi treated with water and pipecolic acid; Figure 2 B represents the biomass of LTH lesion fungi in water-treated and pipecolic acid-treated rice at 10 hpi after injury inoculation.

[0018] Figure 3The present invention is a graph showing the number of callose in rice inoculated with rice blast fungus 10 hpi and treated with L-piperidic acid at 10 hpi, 24 hpi, 36 hpi, 48 hpi, and 72 hpi after inoculation. Figure 3 A represents the callose map in LTH of rice treated with water and pipecolic acid at 10 hpi after spray inoculation with rice blast fungus; Figure 3 B shows the statistical graph of callose deposition in LTH of rice treated with water and pipecolic acid at 10 hpi after spray inoculation with rice blast fungus.

[0019] Figure 4 This is a graph showing the number of dead cells at 10 hpi, 24 hpi, 36 hpi, 48 hpi, and 72 hpi after inoculation of rice with rice blast fungus 10 hpi and L-pipecolic acid treatment. Figure 4 A represents the dead cell images in LTH of rice treated with water and pipecolic acid at 10 hpi after spray inoculation with rice blast fungus; Figure 4 B shows the statistical graph of the number of dead LTH cells in water-treated and pipecolic acid-treated rice at 10 hpi after spray inoculation with rice blast fungus.

[0020] Figure 5 This is a view of the expression of defense-related genes (OsPR1a, OsWRKY45, OsNPR1, OsPAL1) in rice after inoculation with rice blast fungus 10 hpi and treatment with L-pipecolic acid.

[0021] Figure 6 This is a view of the rice plant height after being inoculated with rice blast fungus 10 hpi and treated with L-piperidic acid, and then grown to 60 days old.

[0022] Figure 7 The diagram shows the expression results of defense-related genes (OsCPK5, OsNPR1, and OsPAL1) in rice seeds harvested after inoculation with rice blast fungus 10 hpi and treatment with L-piperidic acid.

[0023] Figure 8 This is a diagram showing the expression results of genes (OsNPR1, OsPAL1) in rice not infected with the rice blast fungus after Pip treatment according to the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0025] Example 1

[0026] Identification of blast resistance of Lijiang Xintuan Heigu treated with exogenous Pip, with wild-type rice Lijiang Xintuan Heigu treated with water as a control.

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

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

[0029] The results are as follows Figure 1 As shown in Figure 2, compared with rice (CK) treated with equal volume of water at 10 hpi after blast inoculation, the rice leaves treated with 10 μmol / L Pip at 10 hpi after blast inoculation showed a significant difference in the growth rate. Figure 1 The rice blast symptoms shown in A are relatively mild. Figure 1 The disease index of rice treated with Pip shown in B is lower.

[0030] 1.1.2 Punch inoculation: Use a punch to inflict wounds on 40-day-old rice leaves. Use a 10 μL pipette to draw a spore suspension onto the wound site. The wound diameter is 5 mm. Keep in the dark and moisturize for 24 h. Then transfer to room temperature for moisturizing incubation. Samples are taken 144 hpi after inoculation. The length of the lesions is measured. Select the lesions and extract DNA from individual lesions using the CTAB method. Real-time fluorescence quantitative PCR is used to determine the CT values ​​of the rice gene OsUBQ and the rice blast fungus gene MoPot2. The relative growth of the fungus is calculated according to the formula = 2 [CT（MoPot2）-CT （OsUBQ）] ×100.

[0031] ① Absolute quantification of rice blast fungus DNA

[0032] Table 1 RT-PCR primer sequences

[0033]

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

[0035] Table 2 PCR reaction system composition

[0036]

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

[0038] Table 3 PCR reaction conditions

[0039]

[0040] ④ qRT-PCR data analysis of target genes

[0041] qRT-PCR data processing was performed using 2 -△△Ct The expression level of MoPot2 gene was calculated and analyzed by the method. The relative expression of gene = 2 -(Ct1-actinCt1)-(Ct2-actinCt2) The data were processed using IBM SPSS Modeler 27.0, and the graphs were drawn using Primer 9.5.1.

[0042] The results are as follows Figure 2 As shown in the figure: compared with the rice treated with equal volume of water at 10 hpi when inoculated with rice blast fungus (CK), the rice treated with Pip at 10 hpi when inoculated with rice blast fungus had a more significant anti-blast effect, with the lesion length ( Figure 2 A) and relative expression of fungi ( Figure 2 B) Both were lower when pipecolic acid was sprayed.

[0043] 1.2 Statistics of antimicrobial compounds such as callose and dead cells in rice treated with exogenous Pip

[0044] 1.2.1 Callose Observation Statistics

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

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

[0047] The results are as follows Figure 3 、 4As 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 of 75% ethanol to every 1 ml of TRIGene initially used, invert several times to mix, and wash the precipitate.

[0059] (9) Centrifuge at 12,000 × g at 4°C for 5 min and discard the supernatant.

[0060] (10) Invert at room temperature for 5-10 minutes and air dry or vacuum dry (do not use a vacuum drying centrifuge to prevent the RNA from drying out too much and becoming difficult to dissolve).

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

[0062] (12) Determine the concentration, purity, and integrity of RNA by RNA electrophoresis and UV spectrophotometry.

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

[0064] 1.3.2 RNA was reverse transcribed into cDNA, as shown in Table 4:

[0065] Table 4 RNA reverse transcription system

[0066]

[0067] Prepare the total system except RNA according to the table above, divide it into PCR tubes, add RNA one by one, mix gently, place in PCR instrument, incubate at 37℃ for 15 minutes, and then heat at 85℃ for 5 seconds.

[0068] 1.3.3 Real-time fluorescence 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 fluorescence quantitative PCR primer sequences

[0071]

[0072] (2) Prepare 20.0 μL of PCR reaction system as shown in Table 1. The reaction solution should be prepared on ice.

[0073] (3) Prepare the system according to the table above, mix gently and dispense into a 96-well PCR plate, 19 µL per well. Finally, add 1 µL of cDNA, centrifuge at 1500 rpm for 1 min, remove the plate, and perform qRT-PCR in a CFX96 quantitative PCR instrument. The reaction procedure is shown in Table 2.

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

[0075] The results are as follows Figure 5 As shown: Compared with rice treated with equal volume of water at 10 hpi (CK), the three SAR-related genes OsPR1a, OsNPR1, and OsPAL1 and OsWRKY45 in rice treated with Pip at 10 hpi were upregulated at 10-72 hpi ( Figure 5 ).

[0076] 1.4 Measuring the plant height of rice plants grown to 60 days after exogenous Pip treatment

[0077] Rice seedlings (14 days old) were inoculated with Pip-treated rice blast fungi at 10 hpi and allowed to grow for 60 days, at which time the plant height was measured.

[0078] The results are as follows Figure 6 As shown in the figure, compared with the water-treated rice inoculated with rice blast fungus at 10 hpi (CK), the plant height of the rice inoculated with rice blast fungus at 10 hpi after Pip treatment was similar to that of CK at the same time after growth for 60 days, with no significant difference, and the plant height was 60 cm ( Figure 6 ).

[0079] Example 2

[0080] 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 An aqueous solution of L-piperidinic acid at a concentration of 10 μmol / L was sprayed, and the seeds were harvested after maturity. RNA was extracted from the seeds using the RNA extraction method in Example 1 and reverse transcribed into cDNA. The expression of defense-related genes OsNPR1, OsPR1a, and OsCPK5 was detected by real-time fluorescence quantitative PCR using the primers in Table 5.

[0081] The results are as follows Figure 7 As shown: The expression levels of defense-related genes (OsNPR1, OsPR1a, OsCPK5) in rice seeds harvested from water-treated and Pip-treated rice at 10 hpi after blast inoculation were higher than those in the control group (CK), showing an up-regulated expression ( Figure 7 ).

[0082] Example 3

[0083] When rice plants were grown to the three-leaf, one-heart stage under greenhouse conditions, they were treated with equal volumes of water and 20 μmol / L L-piperidinic acid solution, respectively. Samples were collected at 0 h (immediately after L-piperidinic acid treatment) and 4 h (4 h after L-piperidinic acid treatment) and immediately placed in liquid nitrogen. RNA was extracted from the rice leaves using the RNA extraction method described in Example 1 and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect the expression of defense-related genes OsNPR1 and OsPR1a using the primers listed in Table 5.

[0084] The results are as follows Figure 8 As shown: After Pip treatment of rice not infected with rice blast fungus, the expression levels of rice defense-related genes OsNPR1 and OsPR1a were higher than those in the control group.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

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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