Plant immunity inducing composition and use thereof

Abstract

This invention discloses a plant immune-inducing composition and its applications, relating to the fields of plant protection and biopesticide technology. The plant immune-inducing composition disclosed in this invention contains the active ingredients L-histidine and quercetin. This composition enhances plant immune resistance by activating the ethylene signaling pathway, activating the MAPK signaling pathway, and increasing the expression of disease-related genes. Exogenous combined application of L-histidine and quercetin significantly reduces the accumulation of ToBRFV and decreases the area of ​​lesions caused by Botrytis cinerea infection, exhibiting broad-spectrum disease resistance activity. The active ingredients in the composition provided by this invention are natural metabolites, making it safe and environmentally friendly, and providing new candidate substances for the development of plant immune inducers and biopesticides.

Description

Technical Field

[0001] This invention relates to the fields of plant protection and biopesticide technology, and in particular to a plant immune-inducing composition and its application. Background Technology

[0002] Tomato brown wrinkled fruit virus ( tamato brown rugose fruit virus Tobacco Mosaic Virus (ToBRFV) is a newly discovered member of the genus Tobacco Mosaic Virus. This virus is highly infectious and extremely stable in the external environment, surviving in soil for months. Long-distance transmission mainly occurs through infected seeds, while in the field it spreads primarily through mechanical contact during agricultural operations.

[0003] Gray mold is a common disease of tomato crops, caused by the fungus *Botrytis cinerea* (…). Botrytis cinerea This pathogen can infect not only the fruit of tomatoes but also the leaves and stems. A grayish-white or grayish-brown mold layer will appear on the infected parts, and in severe cases, it can cause fruit and seedling rot. The occurrence of tomato gray mold can lead to a certain degree of yield reduction.

[0004] In the prior art, CN114287431A discloses a plant immune inducer containing compound sugars and compound amino acids. The compound sugars contain alginate oligosaccharides and chitosan; the compound amino acids contain arginine, phenylalanine and lysine. It can induce resistance to diseases including tomato yellow leaf curl, tomato gray mold and tomato late blight, but has no inducing resistance to tomato brown wrinkled fruit virus.

[0005] CN120210073A discloses a compound antimicrobial agent and its method for controlling tomato brown fruit wrinkling virus; the compound antimicrobial agent includes agent A: liquid inoculum of Serratia marcescens, agent B: water-soluble powder of Bacillus bellis, and agent C: polysaccharide powder of Cladosporium cicadae. This compound antimicrobial agent is used to control tomato brown fruit wrinkling virus, tomato yellow leaf curl virus, and tomato mosaic virus, but has no significant effect on gray mold.

[0006] Therefore, there is an urgent need to develop novel inducing agents that are highly efficient, broad-spectrum, and environmentally friendly. Summary of the Invention

[0007] The purpose of this invention is to provide a plant immune-inducing composition and its application to address the problems existing in the prior art. The plant immune-inducing composition of this invention provides broad-spectrum resistance. Through exogenous combined application of L-histidine and quercetin, it significantly reduces the accumulation of ToBRFV and increases gene expression in ethylene signaling, the MAPK cascade reaction, and pathogenesis-related pathways. Simultaneously, it reduces the lesion area caused by Botrytis cinerea.

[0008] To achieve the above objectives, the present invention provides the following solution: This invention provides a plant immune-inducing composition containing the active ingredients L-histidine and quercetin.

[0009] Furthermore, the plant immune-inducing composition enhances plant immune resistance by activating the plant ethylene signaling pathway; Alternatively, plant immune resistance can be enhanced by activating the MAPK signaling pathway.

[0010] Furthermore, the plant immune-inducing composition also enhances plant immune resistance by increasing the expression of pathogenesis genes; pathogenesis genes include SlPRB1-b, SlPRP4, SlGLUA, SlCHI3 and SlPR1a .

[0011] Furthermore, the mass ratio of L-histidine to quercetin is 1:10 to 10:1.

[0012] Furthermore, the mass ratio of L-histidine to quercetin is 5:1 to 8:1.

[0013] Furthermore, the plant immune-inducing composition also contains agriculturally acceptable carriers and adjuvants.

[0014] The present invention also provides the use of plant immune-inducing compositions in the preparation of plant disease-inducing drugs, wherein the drugs are drugs that enhance plant resistance to viruses and / or pathogenic fungi.

[0015] Furthermore, the virus is the tomato brown wrinkled fruit virus. tomato brown rugose fruit virus The pathogenic fungus is Botrytis cinerea. Botrytis cinerea .

[0016] Furthermore, the plant is a member of the Solanaceae family; the Solanaceae family includes the tomato.

[0017] The present invention also provides a method for enhancing plant disease resistance by applying a plant immune inducing composition to the plant leaves or roots before the plant becomes diseased; plant disease refers to infection of the plant with Tomato Brown Wrinkled Fruit Virus or infection of the plant with Botrytis cinerea.

[0018] The present invention discloses the following technical effects: The exogenous combined application of L-histidine and quercetin significantly reduced the accumulation of ToBRFV and increased the expression of genes in ethylene signaling, MAPK cascade reactions, and pathogenesis-related pathways.

[0019] The present invention utilizes L-histidine and quercetin in combination, which has a more significant anti-disease effect than using them alone. It is effective against both viruses and fungi, has a broader spectrum of action, and the two have a synergistic effect.

[0020] The L-histidine and quercetin composition of this invention enhances plant immunity and resistance by activating the ethylene signaling pathway, activating the MAPK signaling pathway, and increasing the expression of disease-related genes. Exogenous combined application of L-histidine and quercetin significantly reduces the accumulation of ToBRFV and decreases the area of ​​lesions caused by Botrytis cinerea, exhibiting broad-spectrum disease resistance activity.

[0021] The active ingredients in the composition provided by this invention are natural metabolites, which are safe and environmentally friendly. They provide new candidate substances for the research and development of plant immune inducers and biopesticides, and also provide a methodological reference for screening plant immune inducer active ingredients based on multi-omics data. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 The image shows the leaf phenotype of tomato leaves after inoculation with ToBRFV 14 days after pretreatment with water, L-histidine (His), quercetin (Que), and a combination of both (His / Que) in Example 1 of this invention. Figure 2 This is the result of RT-PCR detection of diseased leaves using ToBRFV specific detection primers in Example 1 of this invention; the target band size is 1385bp; Figure 3 This is the result of detecting the relative expression level of ToBRFV-CP in tomato leaves of each treatment group by qRT-PCR in Example 1 of the present invention; Figure 4 Tomato leaves from the water control group, quercetin-only pretreatment group (Que), L-histidine-only pretreatment group (His), and combined pretreatment group (His / Que) of Example 4 of this invention were inoculated with Botrytis cinerea (…). Botrytis cinerea B05.10 The condition of the lesions after 72 hours; Figure 5 The quantitative results of the tomato leaf lesion area in the water control group and each pretreatment group after inoculation with gray mold in Example 4 of the present invention; Figure 6 This is the result of the growth inhibition test of Botrytis cinerea B05.10 on four different PDA plates in Example 4 of the present invention; Figure 7 This is a graph showing the quantitative results of gray mold colony diameter on four different PDA plates in Example 4 of the present invention; Figure 8 for SlMAPK2, SlMAPK3, SlMAPK14, SlAC01, SlPTI5, SlPRB1-b, SlPRP4, SlGLUA, SlCHI3 and SlPR1a qRT-PCR results of relative gene expression levels; p <0.05 indicates a significant difference. p <0.01 indicates a highly significant difference. p The values ​​are already marked on the graph. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Tomato varieties: Solanum lycopersicumAC, purchased from CASMA Mall. The ToBRFV used in this invention is the ToBRFV-SD strain preserved in our laboratory (public literature, ToBRFV-GenBank accession number: Tomato brown rugosefruit virus isolate ToBRFV-SD, complete genome GenBank: OR792460.1). Botrytis cinerea B05.10 was donated by Associate Professor Chen Wenchan of the College of Plant Protection, Hebei Agricultural University (public literature: Establishment of baseline sensitivity to fluopyram and resistance risk assessment of Botrytis cinerea in strawberry in Jiangsu Province, Journal of Nanjing Agricultural University, 2015, 38(5):810-815).

[0030] The CAS number for L-histidine (abbreviated as His in this invention) is 71-00-1. The CAS number for quercetin (abbreviated as Que in this invention) is 117-39-5.

[0031] Unless otherwise specified, all chemicals and reagents used in the examples were commercially available.

[0032] Example 1 Application of L-histidine and quercetin enhances the resistance of tomatoes to ToBRFV. A. Experimental Groups: (1) L-histidine and quercetin combined pretreatment group: At the five-leaf stage of tomatoes, 10mM L-histidine and 250g / mL quercetin were mixed at a mass ratio of 5:1 and sprayed on the leaves. 72 hours later, ToBRFV was inoculated.

[0033] (2) L-histidine pretreatment group: At the five-leaf stage of tomatoes, 10mM L-histidine was sprayed on the leaves, and ToBRFV was inoculated 72 hours later.

[0034] (3) Quercetin pretreatment group: At the five-leaf stage of tomatoes, 250µg / mL of quercetin was sprayed on the leaves, and ToBRFV was inoculated 72 hours later.

[0035] (4) Water control group: At the five-leaf stage of tomatoes, the leaves were sprayed with water and ToBRFV was inoculated 72 hours later.

[0036] (5) Mock group: The negative control group was inoculated with an equal amount of 1×PBS buffer without ToBRFV at the five-leaf stage of tomatoes.

[0037] B. Test methods: Method for inoculating with ToBRFV virus: Select five-leaf stage tomato seedlings as inoculation subjects. Dilute the ToBRFV virus with 1×PBS to obtain a ToBRFV suspension. Sprinkle an appropriate amount of quartz sand evenly on the leaves as an abrasive. Then, take 30 μL of the ToBRFV suspension and apply it to the tomato leaves, gently rubbing 6-8 times with gloved fingers. After standing for 5 minutes, gently rinse off the excess quartz sand on the leaf surface with distilled water to complete the inoculation. Finally, place the inoculated plants in a greenhouse at 26℃ with a light / dark cycle of 16h / 8h for further cultivation. Observe and record the symptoms of the tomato plants daily. Results are shown in [Table missing]. Figure 1 .

[0038] Fourteen days later, the tomato brown wrinkled fruit virus coat protein gene of the above five groups of test samples was analyzed by qRT-PCR. ToBRFV-CP The relative expression levels of ) were detected.

[0039] Virus detection method: RT-PCR was performed using ToBRFV-specific detection primers ToBRFV-Test-F (SEQ ID NO.1) and ToBRFV-Test-R (SEQ ID NO.2) to amplify the 1385bp target fragment. Results are shown below. Figure 2 PCR reactions were performed using Rapid Taq Master Mix and 2× Phanta Max Master Mix (DyePlus) (Nanjing Novizan Biotechnology Co., Ltd., P222 and P525), and the presence of the target fragment in the PCR products was detected by agarose gel electrophoresis.

[0040] RNA extraction and qRT-PCR analysis: Total RNA was extracted from samples in the control and treatment groups using an RNA kit (Beijing TransGen Biotech Co., Ltd., DP452). Total RNA was reverse transcribed into cDNA using oligo(dT) primers and HiScript II Q select RTSuperMix for qPCR. qPCR reactions were performed using the ChamQSYBR qPCR Master Mix kit (Nanjing Novizan Biotechnology Co., Ltd., Q311-02) on an Applied Biosystems 7500 real-time quantitative PCR instrument. A 20 μL reaction mixture contained: 1 μL cDNA template, 10 μL of 2×ChamQ UniversalSYBR qPCR Master Mix (Q711-02, Novizan, Nanjing), and 0.4 μL each of 10 μM forward and reverse primers. The reaction program was: pre-denaturation: 95℃, 30 s, 1 cycle; cyclic amplification: 95℃, 5 s → 60℃, 30 s, 40 cycles. Melting curves: The instrument's default melting curve acquisition program was used. Use 2-ΔΔCt The relative accumulation of the virus was calculated using a method similar to GraphPad Prism software (version 8.0.1), and the results were analyzed and plotted. See [link to results]. Figure 3 The primers used in this invention are shown in Table 1.

[0041] Table 1 Primer Sequences

[0042] C. Experimental Results: like Figure 1 As shown, compared with the Mock group, the leaves of each ToBRFV-inoculated treatment group showed varying degrees of disease symptoms (leaf wrinkling, yellowing, and narrowing of the upper leaves). The combined pretreatment group (+His / Que) had the mildest symptoms, followed by the L-histidine or quercetin treatment groups, and the virus control group (+H2O) had the most severe symptoms.

[0043] Infection status in each treatment group was detected by RT-PCR using ToBRFV-specific detection primers (see Table 1). Results are as follows: Figure 2 As shown, except for the Mock group, all other treatment groups inoculated with ToBRFV showed specific amplification bands of the expected size (1385bp).

[0044] To further clarify the relative accumulation of ToBRFV in the infected leaves of each treatment group, qRT-PCR was used to analyze the five experimental samples. ToBRFV-CP The relative expression level was detected, and the results are shown in [the table]. Figure 3 .

[0045] Depend on Figure 3 It is evident that, compared with the virus control group, the expression levels of ToBRFV-CP were significantly reduced in both the L-histidine and quercetin treatment groups, while the combined pretreatment group showed the most significant reduction, with a viral accumulation level lower than either treatment group alone. This indicates that the combined pretreatment of L-histidine and quercetin has a better inhibitory effect on ToBRFV accumulation than either treatment alone.

[0046] Example 2 The only difference between Example 2 and Example 1 is that the mass ratio of L-histidine to quercetin in the combined treatment group is 8:1.

[0047] Results: The viral accumulation in the combined treatment group was significantly lower than that in the water control group, and also lower than that in either the L-histidine treatment group or the quercetin treatment group, which were treated with a single agent.

[0048] Example 3 The only difference between Example 3 and Example 1 is that the mass ratio of L-histidine to quercetin in the combined treatment group is 10:1.

[0049] Results: The viral accumulation in the combined treatment group was significantly lower than that in the water control group, and also lower than that in either the L-histidine treatment group or the quercetin treatment group, which were treated with a single agent.

[0050] Example 4 The combined application of L-histidine and quercetin enhances the resistance of tomatoes to gray mold. (1) Tomato leaf resistance test to gray mold infection A. Experimental Groups: Water control group (H2O), L-histidine pretreatment group (His), quercetin pretreatment group (Que), and L-histidine and quercetin combined pretreatment group (His / Que).

[0051] B. Test methods: Tomato leaves were pretreated with each of the above treatment groups, and inoculated with gray mold 24 hours later. Botrytis cinerea B05.10) Mycelial blocks. Leaf lesion phenotypes were observed and recorded 72 hours after inoculation, and lesion area was measured. Results are shown below. Figure 4 (lesion phenotype) and Figure 5 (Statistics on the area of ​​lesions).

[0052] C. Experimental Results: like Figure 4 and Figure 5 As shown, the lesion area in the water control group was 2.95 cm². 2 Compared to the quercetin pretreatment group (1.79 cm), 2 L-histidine pretreatment group (1.21cm) 2 ) and combined pretreatment group (0.91cm) 2 The area of ​​gray mold lesions in all groups was significantly reduced, with the combined pretreatment group showing the smallest lesion area, lower than any single treatment group. The results indicate that both L-histidine and quercetin can effectively enhance the resistance of tomatoes to gray mold infection, and their combined use has a synergistic effect.

[0053] (2) PDA plate fungal growth inhibition test A. Experimental Groups: The following four experimental groups were designed according to the different components added to the PDA plate culture medium.

[0054] PDA: Potato Dextrose Agar (PDA) medium; PDA+His: Add 2 mL of histidine stock solution (1M) to 200 mL of PDA culture medium; PDA+Que: 200mL PDA culture medium plus 2mL quercetin stock solution (25mg / mL); PDA+His / Que: 200mL PDA medium with 2mL histidine stock solution (1M) and 2mL quercetin stock solution (25mg / mL).

[0055] B. Test methods: Method for inoculating with *Botrytis cinerea* B05.10: Place *Botrytis cinerea* B05.10 mycelial discs (2mm × 2mm) in the center of the four types of plates mentioned above. After incubation at 25℃ for 3 days, measure the colony diameter using the cross-sectional method and take the average value. Three replicates were performed for each treatment. Results of the growth inhibition test of *Botrytis cinerea* B05.10 on four different PDA plates are shown below. Figure 6 The quantitative results of gray mold colony diameter on four different PDA plates are shown in the figure. Figure 7 .

[0056] C. Experimental Results: Depend on Figure 6 and Figure 7 It is evident that after 3 days of cultivation, the colony diameter of the other three treatments was significantly reduced compared to the PDA control group; the lesion area of ​​the combined treatment group (PDA+His / Que) was significantly reduced, indicating that the combination had the strongest inhibitory effect, which was significantly greater than the inhibitory effect of either L-histidine or quercetin alone, suggesting that the combined application of L-histidine and quercetin has synergistic antifungal activity.

[0057] Example 5 Mechanisms by which L-histidine and quercetin enhance plant resistance After treating tomatoes with L-histidine and quercetin, the expression of defense-related genes in tomato leaves was detected by qRT-PCR and statistically analyzed. Defense-related genes include... SlMAPK2, SlMAPK3, SlMAPK14, SlACO1, SlPTI5, SlPRB1-b, SlPRP4, SlGLUA, SlCHI3 and SlPR1a The method for detecting defense-related genes by qRT-PCR is the same as in Example 1. Two... -ΔΔCt The relative expression levels of genes were calculated using a single method, and the results were analyzed and plotted using GraphPad Prism software (version 8.0.1). See below for the results. Figure 8 .

[0058] like Figure 8 As shown, when tomatoes were treated with a combination of L-histidine and quercetin, the levels measured in tomato leaves were [data missing]. SlMAPK2, SlMAPK3, SlACO1, SlPTI5, SlPRB1-b, SlPRP4, SlGLUA, SlCHI3 and SlPR1a The transcriptional level of was significantly increased. Among them, SlAC01 It is a key enzyme in the biosynthesis of ethylene. SlPR1aIt is a typical marker of salicylic acid signaling. This indicates that the combination of L-histidine and quercetin activates the ethylene signaling pathway and the MAPK signaling pathway in plants, and increases the expression of disease-related genes. Exogenous combined application of L-histidine and quercetin effectively activates the plant's immune system. The combination of L-histidine and quercetin activates multiple defense pathways in tomatoes, enhancing their resistance to ToBRFV and Botrytis cinerea.

[0059] The exogenous combined application of L-histidine and quercetin in this invention significantly reduces the accumulation of ToBRFV and decreases the area of ​​lesions caused by Botrytis cinerea infection, demonstrating broad-spectrum disease resistance activity. The L-histidine and quercetin combination enhances plant immunity by activating the plant ethylene signaling pathway, activating the MAPK signaling pathway, and increasing the expression of disease-related genes.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A plant immune-inducing composition, characterized in that, The plant immune-inducing composition contains the active ingredients L-histidine and quercetin.

2. The plant immune-inducing composition according to claim 1, characterized in that, Plant immune-inducing composition enhances plant immune resistance by activating the plant ethylene signaling pathway; Alternatively, plant immune resistance can be enhanced by activating the MAPK signaling pathway.

3. The plant immune-inducing composition according to claim 1, characterized in that, The plant immune-inducing composition further enhances plant immune resistance by increasing the expression of pathogenesis genes; the pathogenesis genes include SlPRB1-b , SlPRP4 , SlGLUA , SlCHI3 and SlPR1a .

4. The plant immune-inducing composition according to claim 1, characterized in that, The mass ratio of L-histidine to quercetin is 1:10 to 10:

1.

5. The plant immune-inducing composition according to claim 4, characterized in that, The mass ratio of L-histidine to quercetin is 5:1 to 8:

1.

6. The plant immune-inducing composition according to any one of claims 1-5, characterized in that, The plant immune-inducing composition also contains agriculturally acceptable carriers and adjuvants.

7. The use of the plant immune-inducing composition according to any one of claims 1-5 in the preparation of plant disease-inducing drugs, characterized in that, The drug is a drug that enhances the plant's resistance to viruses and / or pathogenic fungi.

8. The application according to claim 7, characterized in that, The virus is the tomato brown wrinkled fruit virus. tomato Brown rugose fruit virus The pathogenic fungus is Botrytis cinerea. Botrytis cinerea .

9. The application according to claim 7, characterized in that, The plant belongs to the Solanaceae family; the Solanaceae family includes the tomato.

10. A method for enhancing plant disease resistance, characterized in that, Before the plant becomes diseased, the plant immune induction composition according to any one of claims 1-5 is applied to the leaves or roots of the plant; the plant disease refers to the infection of the plant with tomato brown wrinkled fruit virus or the infection of the plant with gray mold.

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