Application of 3,5-dimethylorcinol as a plant immune inducer in improving plant disease resistance

Abstract

The present invention belongs to the field of agricultural microbiology technology, and specifically relates to the use of the plant immune inducer 3,5-dimethyl orsellinic acid in improving plant disease resistance. This application determines the resistance-inducing ability of the compound 3,5-dimethyl orsellinic acid on soybeans. The results show that the compound 3,5-dimethyl orsellinic acid can induce reactive oxygen species burst in soybeans, synergistically enhance the reactive oxygen species burst triggered by chitin and XEG1, two PAMPs, and activate disease resistance-related genes. GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A The upregulation of 3,5-dimethylrucic acid activated the plant immune system and protected against infection by pathogens such as Phytophthora sojae and Fusarium graminearum. This indicates that the compound 3,5-dimethylrucic acid has strong resistance to soybeans and has great potential for development as a plant resistance agent or lead compound for plant disease prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and particularly relates to application of a plant immunity inducer, 3,5-dimethylorcinic acid, in improving plant disease resistance. Background Art

[0002] Plant immune inducers enhance disease resistance by activating the plant's innate immune system (such as PTI and ETI), and are an important green alternative to chemical pesticides. While existing chemical pesticides can quickly suppress pathogens, long-term use can lead to increased resistance, environmental pollution, and excessive residues in agricultural products. Plant immune inducers, on the other hand, achieve both broad-spectrum disease resistance and eco-friendliness by modulating plant immune signaling networks. For example, amino oligosaccharides, by activating the salicylic acid (SA) and jasmonic acid (JA) signaling pathways, increase rice's protection against rice blast by 75%, while simultaneously reducing pesticide use by 40%-60%.

[0003] However, commercialized inducers, such as amino oligosaccharides and allylbenzazole, still have significant shortcomings. They target only a single target and lack broad-spectrum activity. In particular, existing inducer production processes rely on chemical synthesis or natural extraction, resulting in low yields (e.g., chitin oligosaccharide extraction yields less than 10%), complex processes, and high costs. Producing plant immune inducers through microbial fermentation offers several advantages, including: raw materials are primarily inexpensive, renewable bioresources such as starch, sugars, corn steep liquor, and soybean flour; fermentation-based production allows for the recycling and reuse of wastewater and liquids, minimizing environmental pollution; and the same equipment can be adapted for fermentation of other bacterial species with minimal modification, resulting in relatively low investment costs.

[0004] Therefore, discovering new plant immune inducers and providing inducer dosage and production methods based on microbial fermentation will help promote plant immune inducers from laboratory to field application and promote the green transformation of agriculture. Summary of the Invention

[0005] In response to the above-mentioned problems, the present invention provides a new plant immune inducer 3,5-dimethylorcinic acid and its preparation method and application.

[0006] In one aspect, the present application provides a use of 3,5-dimethylorticic acid, wherein the use comprises one or more of the following:

[0007] A) Improve plant resistance to Phytophthora sojae, Fusarium graminearum, and Phakopsora pachyrhizi;

[0008] B) preparing a composition for improving plant resistance to Phytophthora sojae, Fusarium graminearum and Phakopsora pachyrhizi;

[0009] C) Inducing a burst of reactive oxygen species in plants;

[0010] D) preparing a composition capable of inducing a burst of active oxygen species in plants;

[0011] E) Induction of plant disease resistance-related genes GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A Upregulated expression of

[0012] F) Preparation of genes related to inducing plant disease resistance GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A Compositions that upregulate expression capacity.

[0013] Furthermore, the gene GmPR1 The NCBI accession number is: NM_001371207.1, the gene GmPR2 The NCBI accession number is: NM_001251545.1, the gene GmPR5 The NCBI accession number is: BU765509.1, the gene GmCYP93A The NCBI accession number is: NM_001254257.2.

[0014] Furthermore, the plant is soybean.

[0015] On the other hand, the present application provides a biocontrol bacterium for improving the resistance of plants to Phytophthora graminearum, Fusarium graminearum and Phakopsora pachyrhizi, the Aspergillus oryzae pTAex3-DIPKS1, classified as Aspergillus oryzae Aspergillus oryzae It was registered and deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on April 3, 2025. The deposit number is CGMCC No.41905, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0016] On the other hand, the present application also provides the application of the biocontrol bacteria, which includes the following:

[0017] A) Improve plant resistance to Phytophthora sojae, Fusarium graminearum, and Phakopsora pachyrhizi;

[0018] B) preparing a composition for improving plant resistance to Phytophthora sojae, Fusarium graminearum and Phakopsora pachyrhizi;

[0019] C) Inducing a burst of reactive oxygen species in plants;

[0020] D) preparing a composition capable of inducing a burst of active oxygen species in plants;

[0021] E) Induction of plant disease resistance-related genes GmPR1 、 GmPR2 、 GmPR5and GmCYP93A Upregulated expression of

[0022] F) Preparation of genes related to inducing plant disease resistance GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A Compositions that upregulate expression capacity.

[0023] Furthermore, the gene GmPR1 The NCBI accession number is: NM_001371207.1, the gene GmPR2 The NCBI accession number is: NM_001251545.1, the gene GmPR5 The NCBI accession number is: BU765509.1, the gene GmCYP93A The NCBI accession number is: NM_001254257.2.

[0024] In certain embodiments, the plant is soybean.

[0025] On the other hand, the present application provides a method for preparing 3,5-dimethylorcinic acid, which is obtained from the fermentation broth of Aspergillus oryzae pTAex3-DIPKS1 with a deposit number of CGMCC No. 41905.

[0026] Furthermore, the preparation method comprises the following steps:

[0027] (1) Aspergillus oryzae pTAex3-DIPKS1 with a deposit number of CGMCC No. 41905 was inoculated into CDM medium and fermented in the dark at 28-32°C for 6-8 days;

[0028] (2) Soaking the bacterial cells and solid fermentation medium obtained in step (1) in ethyl acetate, ultrasonicating twice, and concentrating the extract under reduced pressure to obtain a crude extract of the fermentation product;

[0029] (3) The crude extract of the fermentation product obtained in step (2) is subjected to gel column chromatography, and the components containing 3,5-dimethylmoseloic acid are combined by TLC detection, and separated and purified by high performance liquid chromatography to obtain 3,5-dimethylmoseloic acid.

[0030] On the other hand, the present application provides a method for improving the resistance of plants to Phytophthora graminearum, Fusarium graminearum and Phakopsora pachyrhizi and / or inducing plant active oxygen burst and / or inducing plant disease resistance related genes. GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A A composition for upregulating expression ability, wherein the main active ingredient of the composition is 3,5-dimethylorchymic acid.

[0031] Furthermore, the composition is an emulsifiable concentrate, an aqueous emulsion, a microemulsion, a wettable powder, a water-dispersible granule, or a suspension.

[0032] On the other hand, the present application provides the composition for improving the resistance of plants to Phytophthora sojae, Fusarium graminearum and Phakopsora pachyrhizi and / or inducing plant active oxygen burst and / or inducing plant disease resistance related genes. GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A Application in up-regulating expression.

[0033] Furthermore, the plant is soybean.

[0034] On the other hand, the present application provides a method for improving the resistance of plants to Phytophthora graminearum, Fusarium graminearum and Phakopsora pachyrhizi and / or inducing plant active oxygen burst and / or inducing plant disease resistance related genes. GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A The method for up-regulating expression comprises applying the composition to a soybean growth environment.

[0035] Compared with the existing technology, the present invention measures the ability of the compound 3,5-dimethyl orsellinic acid to induce resistance in soybeans. The results show that the compound 3,5-dimethyl orsellinic acid can induce reactive oxygen species burst in soybeans, synergistically enhance the reactive oxygen species burst triggered by chitin and XEG1, two PAMPs, and activate disease resistance-related genes. GmPR1 、 GmPR2 、 GmPR5 and GmCYP93A The upregulation of 3,5-dimethylrucic acid activated the plant immune system and helped protect against infection by pathogens such as Phytophthora sojae and Fusarium graminearum. This indicates that 3,5-dimethylrucic acid has strong resistance-inducing activity against soybeans and has great potential for development as a plant resistance agent or lead compound for plant disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the specific embodiments.

[0037] Figure 1 This is the recombinant plasmid map of the DIPKS1 gene cloned into the Aspergillus oryzae expression vector pTAex3.

[0038] Figure 2 This is the structural formula of 3,5-dimethylorticic acid.

[0039] Figure 3 This is the mass spectrum of 3,5-dimethylorticic acid.

[0040] Figure 4 3,5-dimethylortic acid 1 H-NMR spectrum.

[0041] Figure 5 Figure 3 shows the production of reactive oxygen species in soybean induced by 3,5-dimethylorticic acid.

[0042] Figure 6 A diagram showing that 3,5-dimethylrucic acid enhances PAMP-triggered reactive oxygen species burst.

[0043] Figure 7 This is a diagram showing the up-regulated expression of soybean disease resistance-related genes induced by 3,5-dimethylolsuccinic acid.

[0044] Figure 8 The diagram shows that 3,5-dimethylcarbohydrate inhibits soybean infection by Phytophthora sojae, Fusarium graminearum and Phakopsora pachyrhizi. DETAILED DESCRIPTION

[0045] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0046] Example 1 Preparation of 3,5-dimethylorticic acid producing strain pTAex3-DIPKS1

[0047] Will DIPKS1 The gene (PV533668) was cloned into the Aspergillus oryzae expression vector pTAex3 and obtained as Figure 1 The recombinant plasmid was transformed into the Aspergillus oryzae NSAR1 host and integrated into the genome by homologous recombination. PCR screening was used to obtain the strain pTAex3-DIPKS1 that stably expressed the DIPKS1 gene.

[0048] Example 2 Method for producing 3,5-dimethylorticic acid by strain pTAex3-DIPKS1

[0049] The strain pTAex3-DIPKS1 was inoculated into CDM solid fermentation medium and fermented in the dark at 30°C for 7 days. The bacteria and solid fermentation medium were chopped and soaked in ethyl acetate, ultrasonicated twice for 30 minutes each time, and the extract was concentrated under reduced pressure to obtain a crude extract of the fermentation product. The crude extract of the fermentation product was subjected to gel column chromatography, TLC detection and merging of the components containing 3,5-dimethyl rucic acid, and separation and purification by high performance liquid chromatography to obtain 3,5-dimethyl rucic acid. The structural formula of the compound is as follows: Figure 2 As shown, the mass spectra and 1 H-NMR spectrum Figure 3 and Figure 4The compound was analyzed to be a yellow-white solid. HR-ESI-MS. m / z: 197.0798 [M+H] + . 1 H-NMR (500 MHz, CD3OD) δ H 2.04 (3H,s), 2.09 (3H,s), 2.43 (3H,s).

[0050] The above data are consistent with the reported data of 3,5-dimethylrucic acid, thus confirming that they are the same compound.

[0051] Example 3 Experiment on the production of reactive oxygen species induced by the compound 3,5-dimethylorticic acid in soybean

[0052] 5-mm-diameter leaf discs were collected from 14-day-old soybean plants and floated overnight in 200 μl of sterile water in a 96-well plate. Sterile water was replaced with 200 μl of reaction buffer containing 35.4 μg / ml luminol, 10 μg / ml peroxidase, and various compounds (50 μg / ml chitin, 0.1 μg / ml 3,5-dimethylorchysene acid, and 1 μg / ml 3,5-dimethylorchysene acid in 0.5% DMSO). 50 μg / ml chitin served as a positive control, and 0.5% DMSO served as a negative control. Luminescence was measured using a microplate luminometer.

[0053] The results of compound 3,5-dimethylrucic acid inducing the production of reactive oxygen species in soybean are as follows Figure 5 shown.

[0054] The experimental results showed that 0.1 and 1 mg / L of 3,5-dimethylrucic acid could induce soybean to produce reactive oxygen species.

[0055] Example 4 Experiment on the Enhancement of PAMP-Triggered Reactive Oxygen Species Burst in Soybeans by 3,5-Dimethylorticoic Acid

[0056] 5-mm-diameter leaf discs were collected from 14-day-old soybean plants and floated overnight in 200 μl of sterile water in a 96-well plate. The sterile water was replaced with 200 μl of reaction buffer containing 35.4 μg / ml luminol, 10 μg / ml peroxidase, and a PAMP (50 μg / ml chitin or 1 μM XYG1), along with various compounds (0.1 μg / ml 3,5-dimethylorcinol / 1 μg / ml 3,5-dimethylorcinol / 0.5% DMSO in water). 0.5% DMSO in water served as a negative control. Luminescence was measured using a microplate luminometer.

[0057] The results of compound 3,5-dimethylrucic acid enhancing the PAMP-triggered reactive oxygen species burst are as follows Figure 6 shown.

[0058] The experimental results showed that 0.1 and 1 mg / L of 3,5-dimethylrucic acid could enhance the reactive oxygen species burst triggered by PAMP in soybeans.

[0059] Example 5 Experiment on the up-regulation of soybean disease resistance-related genes induced by the compound 3,5-dimethylorticic acid

[0060] Leaf discs with a diameter of 5 mm were collected from soybean plants grown for 14 days and placed in a 96-well plate with 200 μl of sterile water to float overnight. Sterile water was replaced with 200 μl of reaction buffer containing 1 μg / ml 3,5-dimethylorchidonic acid. A 200 μl reaction buffer containing 0.5% DMSO aqueous solution was used as a control group. Samples were taken after 6 h and 10 h of incubation in the dark. RNA of the samples was extracted and reverse transcribed into cDNA. The products were immediately used for fluorescence quantitative PCR detection or stored at -20°C. Fluorescence quantitative PCR detection used the Ct method for relative quantification, and the experimental results were expressed as 2 -ΔΔCt Statistical analysis was performed using the method of , and all samples were repeated three times. GmCYP2 (NM_001357079.1) gene was used as an internal reference to analyze disease resistance-related genes GmPR1 (NM_001371207.1), GmPR2 (NM_001251545.1), GmPR5 (BU765509.1) and GmCYP93A The relative expression levels of the genes (NM_001254257.2) were as shown in Table 1.

[0061] Table 1 Primers for gene expression detection

[0062]

[0063] The results of compound 3,5-dimethylorticic acid inducing up-regulation of soybean disease resistance-related genes are shown in Figure 2. Figure 7 shown.

[0064] The experimental results showed that the compound 3,5-dimethylrucic acid induced soybean GmPR2 and GmPR5 Up-regulation of gene expression induced soybean GmPR1 and GmCYP93A Upregulated expression of genes.

[0065] Example 6 Experiment on the Inhibition of Soybean Disease Pathogen Infection by Compound 3,5-Dimethylorticic Acid

[0066] Soybean Hefeng 47 was grown in the dark for 4 days. After cleaning the vermiculite on the surface, the roots were immersed in 1 μg / ml 3,5-dimethylornic acid or 0.5% DMSO aqueous solution in the dark for 1 h and then inoculated with soybean Phytophthora and Fusarium graminearum. The 1 μg / ml 3,5-dimethylornic acid was dissolved in 0.5% DMSO aqueous solution.

[0067] Williams 82 soybeans were grown in a greenhouse at 25°C for 14 days and sprayed with 10 ml of 1 μg / ml 3,5-dimethylornic acid or 0.5% DMSO aqueous solution. After standing for 1 h, they were inoculated with Phakopsora pachyrhizi. The 1 μg / ml 3,5-dimethylornic acid was dissolved in 0.5% DMSO aqueous solution.

[0068] The soybean Phytophthora strain P6497 was activated on 10% V8 solid medium. Mycelial blocks of 2×2 mm in size were cut from the edge of the colony and transferred to 10% V8 solid medium. After culturing in the dark at 25°C for 4 days, sterile tap water was added to the culture dish. After leaving it in the dark at 25°C for 30 minutes, the water was changed and repeated several times until a large number of zoospores were produced. The water in the culture dish was poured out, 5 ml of sterile tap water was added, and it was left in the dark for 1-2 hours until a large number of zoospores were released. The zoospores were collected and the zoospore concentration was adjusted to 10 / μl. 10 μl of soybean Phytophthora zoospores were aspirated and inoculated into the hypocotyls of the etiolated seedlings with different treatments. After culturing in the dark at 25°C for 48 hours, the lesion expansion was observed. Taking the inoculation point as the center, 3 cm above and below were taken, and the genomic DNA was extracted after grinding with liquid nitrogen. Using the genomic DNA as a template, the zoospores were respectively inoculated with soybean Phytophthora. Actin Genes and soybeans GmCYP2 The genes were detected by fluorescence quantitative PCR, and the relative biomass of soybean Phytophthora under different treatments was determined.

[0069] The PH-1 strain of Fusarium graminearum was activated on PDA solid culture medium, and 2×2 mm mycelial blocks were cut from the edge of the colony and cultured in the dark at 25°C for 3 days. The mycelial blocks were punched out with a 1.5 cm diameter puncher, and the mycelial surface was attached to the hypocotyls of the etiolated seedlings with different treatments. The hypocotyls and roots were wrapped with absorbent paper soaked in sterile water to keep them moist. After wrapping with tin foil, the cells were cultured at 25°C, 16 h light and 8 h dark for 2 days, and the expansion of the lesions was observed. Taking the inoculation point as the center, 2.5 cm above and below were taken, and the genomic DNA was extracted after grinding with liquid nitrogen. Using genomic DNA as a template, the Fusarium graminearum was used to culture the lesions. CYP51C Genes and soybeans GmCYP2 The genes were detected by fluorescence quantitative PCR, and the relative biomass of Fusarium graminearum under different treatments was determined.

[0070] Psoralea pachyrhizi strain SS4 was grown on soybean Williams82. Spores were collected and resuspended in 0.05% Tween-20 solution to adjust the spore concentration to 1.0 × 10 5 pc / ml, and sprayed on soybean leaves with different treatments. After 24 hours of moisture preservation at 25℃ in the dark, the leaves were placed in a 25℃, 16 hours light and 8 hours dark condition. After 14 days, the expansion of the lesions was checked. Soybean leaves were picked, ground with liquid nitrogen, and genomic DNA was extracted. Genomic DNA was used as a template and infected with Phakopsora pachyrhizi. Tubin Genes and soybeans GmCYP2 The genes were detected by fluorescence quantitative PCR, and the relative biomass of Phakopsora pachyrhizi under different treatments was determined.

[0071] The detection primers of the genes are shown in Table 2.

[0072] Table 2 Primers for detecting gene expression

[0073]

[0074] The results of the compound 3,5-dimethylrucic acid inhibiting the infection of soybean by Phytophthora sojae, Fusarium graminearum and Phakopsora pachyrhizi Figure 8 shown.

[0075] The experimental results showed that pretreatment of soybeans with the compound 3,5-dimethylrucic acid can significantly reduce the relative biomass of soybeans infected by Phytophthora sojae, Fusarium graminearum and Puccinia pachyrhizium, and inhibit the infection of soybeans by soybean disease pathogens. The compound 3,5-dimethylrucic acid can be used as a plant resistance agent for the prevention and control of soybean diseases.

[0076] Unless otherwise specified, the numerical value set forth in these embodiments does not limit the scope of the present invention. In all examples shown and described here, unless otherwise specified, any specific value should be interpreted as merely exemplary, rather than as restriction, and therefore, other examples of exemplary embodiments can have different values.

Claims

1. The use of 3,5-dimethyl orsellinic acid, characterized in that: The application is to improve the resistance of soybean to Phytophthora sojae, Fusarium graminearum or Phakopsora pachyrhizi.

2. Use of a composition containing 3,5-dimethylornic acid as an active ingredient in improving the resistance of soybeans to Phytophthora sojae, Fusarium graminearum or Phakopsora pachyrhizi.

3. The use according to claim 2, characterized in that The composition is emulsifiable concentrate, aqueous emulsion, microemulsion, wettable powder, water-dispersible granule or suspension.

4. A method for improving the resistance of soybean to Phytophthora sojae, Fusarium graminearum or Phakopsora pachyrhizi, characterized in that: The method comprises applying a composition containing 3,5-dimethylorticic acid as an active ingredient to a soybean growing environment.

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