A fusarium oxysporum cell wall extract, its preparation method and use

Abstract

This invention relates to the field of plant disease biological control technology, and particularly to a Fusarium oxysporum cell wall extract, its preparation method, and its application. The extract is derived from the polysaccharide fraction of the cell wall of Fusarium oxysporum spores or hyphae, and contains at least one pathogen-associated molecular pattern (PAMP) component. This component is a non-protein, thermally stable, species-nonspecific elicitor capable of forming an aqueous dispersion system. The extract of this invention can be recognized by plant PRRs and activate PTI immunity. Rehmannia glutinosa tubers treated with the extract showed a significantly increased germination rate after inoculation with Fusarium oxysporum. It also induces superoxide anion production, upregulates the activities of superoxide dismutase, peroxidase, and catalase, and enriches differentially expressed genes in the MAPK signaling pathway, jasmonic acid metabolism pathway, and phenylpropane biosynthesis pathway. Furthermore, the extract can directly inhibit Fusarium oxysporum colony expansion, conidia production, and mycelial growth. Extracts derived from spores or hyphae show no significant difference in inducing disease resistance, and the raw materials are flexible and easy to industrialize. This extract is derived from the cell wall of the pathogen itself, making it environmentally friendly and free from the risk of chemical pesticide residues. It is suitable for the green control of root rot disease in Rehmannia glutinosa.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological control of plant diseases, and particularly relates to a cell wall extract derived from Fusarium oxysporum ( Fusarium oxysporum ), its preparation method, its application in inhibiting the growth of Fusarium oxysporum, inducing disease resistance in Rehmannia glutinosa ( Rehmannia glutinosa ), and its application in preparing a biogenic pesticide for preventing and treating Rehmannia glutinosa root rot. Background Art

[0002] Rehmannia glutinosa ( Rehmannia glutinosa ) is a perennial herbaceous plant of the genus Rehmannia in the family Scrophulariaceae, and is a traditional bulk Chinese medicinal material in China, with important nutritional, medicinal and economic values. "Huai Rehmannia glutinosa" produced in Jiaozuo, Henan, the authentic production area, has the best quality and is widely planted. However, the soil-borne diseases of Rehmannia glutinosa seriously restrict the large-scale development of its planting industry and related traditional Chinese medicine industries.

[0003] Soil-borne diseases are mostly caused by soil-borne pathogenic bacteria. The bacteria can be transmitted through seeds and soil, and the autotoxic substances secreted by roots promote the accumulation of rhizosphere pathogenic bacteria. Soil-borne pathogenic bacteria are regarded as one of the main causes of continuous cropping obstacles of Rehmannia glutinosa. The planting areas of Rehmannia glutinosa mostly belong to the temperate continental monsoon climate, with high humidity and abundant rainfall in summer, and it is easy to occur flood disasters. After the flood, the disease resistance of plants weakens, and large areas of plants are often infected and die, resulting in serious reduction in production or even crop failure.

[0004] The existing prevention and control measures mainly include crop rotation, field management and chemical pesticide treatment. However, these methods have obvious deficiencies: the efficiency of crop rotation is low; although chemical pesticide control is effective in the short term, it is easy to produce pesticide residues, which endanger food safety and the ecological environment, and long-term use can induce the generation of drug resistance in pathogenic bacteria. Although the Institute of Plant Protection of Henan Academy of Agricultural Sciences recommends the wettable powder of Bacillus subtilis for the prevention and control of root rot in the "Technical Regulations for Integrated Prevention and Control of Diseases and Pests of Huai Rehmannia glutinosa", most soil-borne diseases still rely on chemical pesticides. Biocontrol bacteria of the genus Bacillus need to colonize in a specific ecosystem, and the efficacy of their drugs is restricted by climate and soil conditions. In terms of the cultivation of resistant varieties, some resistant varieties were obtained in the 1960s of the last century, but due to long-term asexual reproduction, the varieties degenerated and the resistance gradually disappeared. Therefore, developing green and efficient biological control means is the key to promoting the sustainable development of the Rehmannia glutinosa planting industry.

[0005] Plant innate immunity depends on the recognition of pathogen-associated molecular patterns (PAMPs, also known as elicitors) on the surface of pathogens by pattern recognition receptors (PRRs) on the cell membrane, and this immune mechanism is called pattern-triggered immunity (PTI). After PTI is activated, a series of downstream reactions are triggered, including the activation of the mitogen-activated protein kinase (MAPK) cascade, callose deposition, reactive oxygen species burst, and transcription of defense genes, thereby rapidly enhancing the disease resistance of plants. Elicitors are mostly components of microbial cell walls or secreted proteins. The cell wall extract of Fusarium oxysporum belongs to a genus-nonspecific elicitor and can induce typical PTI responses in different plant systems. This extract is easy to obtain, has both water solubility and heat resistance, and has development potential in the biological control of soil-borne diseases.

[0006] However, existing PAMPs-based biocontrol products mainly focus on soluble components such as bacterial lipopolysaccharides and flagellin, and the research on the polysaccharide fraction of the cell wall of Fusarium oxysporum itself as an elicitor is not sufficient. For the soil-borne diseases of Rehmannia glutinosa, there is still a lack of systematic research on the technical solutions of using this extract to induce the disease resistance of Rehmannia glutinosa, inhibit the growth of Fusarium oxysporum, and use its polysaccharide fraction to prepare biogenic pesticides for controlling root rot. At the same time, existing chemical pesticides and biocontrol agents have defects such as residues, drug resistance, and colonization dependence on the environment. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a cell wall extract of Fusarium oxysporum ( Fusarium oxysporum ) The extract is derived from the polysaccharide fraction of the cell wall of Fusarium oxysporum spores or hyphae and contains at least one pathogen-associated molecular pattern PAMP component, which can be recognized by plant PRRs and activate PTI immunity. The Rehmannia glutinosa tuberous roots treated by soaking with the extract have a significantly increased germination rate after inoculation with Fusarium oxysporum. At the same time, it can induce the production of superoxide anions, up-regulate the activities of superoxide dismutase, peroxidase, and catalase, and enrich the differentially expressed genes in the MAPK signaling pathway, jasmonic acid metabolism pathway, and phenylpropanoid biosynthesis pathway. In addition, the extract can directly inhibit the colony expansion, conidia production, and mycelial growth of Fusarium oxysporum. There is no significant difference in the induction of disease resistance between the extracts from spores or hyphae, and the raw materials are flexible and easy for industrial production. The extract is derived from the cell wall of the pathogen itself, is environmentally friendly, has no risk of chemical pesticide residues, and is suitable for the green prevention and control of Rehmannia glutinosa root rot.

[0008] The present invention is achieved through the following technical solutions: On the one hand, a cell wall extract of Fusarium oxysporum ( Fusarium oxysporum)A cell wall extract, which is derived from the polysaccharide fraction of the cell wall of Fusarium oxysporum spores or hyphae and contains at least one pathogen-associated molecular pattern (PAMP) component, which is a non-protein, heat-stable, species-nonspecific elicitor that can form an aqueous dispersion system.

[0009] Furthermore, the active ingredient in the extract that induces disease resistance in Rehmannia glutinosa is its cell wall polysaccharide fraction, which contains β-1,3-glucan and / or chitin.

[0010] A method for preparing the above-mentioned Fusarium oxysporum cell wall extract is also provided, including the following steps:

[0011] (1) Inoculate Fusarium oxysporum into a PDA liquid medium, shake-culture at 28 °C and 220 r / min for 2 - 3 days, and collect the thalli by centrifugation; (2) Centrifuge the collected thalli at 6000 r / min, and wash the precipitate twice with a 500 mM KH2PO4 solution; (3) Wash the washed precipitate successively with a chloroform-methanol mixture (volume ratio 1:1) and acetone, and centrifuge to discard the supernatant after each washing; (4) Dry and grind the defatted precipitate to obtain the Fusarium oxysporum cell wall extract.

[0012] Furthermore, in step (1), the composition of the PDA liquid medium is: 100 g / L of potato and 10 g / L of glucose.

[0013] A method for inducing disease resistance in Rehmannia glutinosa ( Rehmannia glutinosa ) is also provided. Soak the Rehmannia glutinosa tuberous roots in an aqueous dispersion of the above-mentioned Fusarium oxysporum cell wall extract for 1 hour, the concentration of the extract is 0.1 - 10 mg / mL, and inoculate Fusarium oxysporum after treatment.

[0014] Furthermore, the extract activates pattern-triggered immunity (PTI) in Rehmannia glutinosa, including: inducing the production of superoxide anions, upregulating the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and enriching the differentially expressed genes in the mitogen-activated protein kinase (MAPK) signaling pathway, jasmonic acid metabolism pathway and phenylpropanoid biosynthesis pathway.

[0015] Furthermore, the extract is isolated from the spores or hyphae of Fusarium oxysporum.

[0016] A method for inhibiting the growth of Fusarium oxysporum is also provided. Add the above-mentioned Fusarium oxysporum cell wall extract to a PDA solid or liquid medium at a final concentration of 0.1 - 10 mg / mL, inoculate Fusarium oxysporum, and culture at 28 °C.

[0017] Finally, the application of the above extract in the preparation of a biogenic pesticide for preventing and controlling Rehmannia root rot caused by Fusarium oxysporum Fusarium oxysporum

[0018] Beneficial effects The extract of the present invention is a non-protein, heat-stable, species-nonspecific elicitor that can form an aqueous dispersion system, contains typical PAMP components such as β-1,3-glucan and / or chitin, and can be recognized by plant PRRs and activate PTI; The Rehmannia root tubers soaked in the extract at a concentration of 0.1-10 mg / mL for 1 hour showed a significant increase in germination rate after inoculation with Fusarium oxysporum. At the same time, it could induce the production of superoxide anions, up-regulate the activities of SOD, POD, and CAT, and enrich the differentially expressed genes in the MAPK signaling pathway, jasmonic acid metabolism pathway, and phenylpropanoid biosynthesis pathway; The extract could directly inhibit the radial expansion of the Fusarium oxysporum colony, the production of conidia, and the accumulation of mycelial dry weight within the same concentration range; There was no statistically significant difference in the induction of disease resistance between the extracts from spores or mycelia. The raw materials were flexible and convenient for industrial production; The extract was derived from the cell wall of the pathogen itself, was environmentally friendly, had no risk of chemical pesticide residues, and was suitable for the green prevention and control of Rehmannia root rot. Description of the drawings

[0019] Figure 1 : Photo of the dried and ground extract of Fusarium oxysporum cell wall; Figure 2 : Inhibitory effect of extracts of Fusarium oxysporum cell wall at different concentrations on the growth of Fusarium oxysporum on solid medium; Figure 3 : Effect of extracts of Fusarium oxysporum cell wall at different concentrations on the production of conidia of Fusarium oxysporum; Figure 4 : Effect of extracts of Fusarium oxysporum cell wall at different concentrations on the dry weight of mycelia of Fusarium oxysporum; Figure 5 : Statistical analysis of the germination rate of Rehmannia root tubers inoculated with Fusarium oxysporum after treatment with extracts of Fusarium oxysporum cell wall at different concentrations; Figure 6 : Statistical analysis of the germination rate of Rehmannia treated with the supernatant and precipitate parts of the Fusarium oxysporum cell wall extract and then inoculated with Fusarium oxysporum; Figure 7 : Statistical analysis of the germination rate of Rehmannia treated with cell wall extracts from Fusarium oxysporum spores and mycelia and then inoculated with Fusarium oxysporum; Figure 8: Response of superoxide anion and related antioxidant enzyme (SOD, POD, CAT) activities in Rehmannia glutinosa under different treatment times with cell wall extracts of Fusarium oxysporum; Figure 9 : Statistical chart of COG annotation classification of differentially expressed genes; Figure 10 : Bubble chart of GO enrichment of differentially expressed genes (the abscissa is GeneRatio, that is, the proportion of genes of interest annotated in this entry among all differentially expressed genes, and the ordinate is each GO annotation entry. The size of the point represents the number of differentially expressed genes annotated in this pathway, and the color of the point represents the q-value of the hypergeometric test); Figure 11 : Bubble chart of KEGG enrichment of differentially expressed genes (each circle in the figure represents a KEGG pathway, the ordinate represents the pathway name, and the abscissa is the enrichment factor (Rich factor). The color of the circle represents the qvalue; the size of the circle represents the number of genes enriched in the pathway, and the larger the circle, the more genes);

[0020] Figure 12 : Statistical analysis of the germination rate after inoculation with Fusarium oxysporum after treatment with cell wall extracts of Fusarium oxysporum and Bacillus subtilis agents. Detailed implementation manners

[0021] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions or parts are by weight.

[0023] The reagents and raw materials used in the embodiments and comparative examples of the present invention can be obtained through commercial channels without special instructions.

[0024] The Fusarium oxysporum used in this study was a strain screened by the laboratory from rotten Rehmannia glutinosa roots, and the rotten Rehmannia glutinosa materials were sourced from Wenxian Agricultural Science Research Institute. It is currently stored in Laboratory 536 of the College of Life Sciences, Henan Normal University.

[0025] The screening method for Fusarium oxysporum is as follows: Gently rinse the rotten Rehmannia glutinosa roots to remove the attached soil. Absorb the surface moisture with paper and cut off the epidermis and its two ends. Cut a 0.5 cm piece at the junction of the diseased and healthy parts. 0.5 cm The size is 0.3 cm. Soak it in 75% ethanol for 1 minute in a laminar flow hood, transfer it to 0.1%-0.2% mercury chloride for 2-3 minutes, then rinse it 4 times with sterile water and transfer it to a glucose potato medium (PDA medium) for culturing at 28 °C for 48-72 h.

[0026] Example 1: Cultivation of Fusarium oxysporum (1) Preparation of PDA solid medium: Weigh 4.6 g of PDA medium (Solarbio, batch number P8931), add 100 mL of deionized water, dissolve it and transfer it to a 150 mL conical flask, seal it with a sealing film, and sterilize it at 121 °C under high pressure for 20 min.

[0027] (2) Preparation of PDA liquid medium: Take 100 g of potatoes (peeled and cut into pieces), 10 g of glucose, add deionized water to make up to 500 mL, transfer it to 500 mL conical flasks, seal it with a sealing film, and sterilize it at 121 °C under high pressure for 20 min.

[0028] (3) Activation and cultivation of Fusarium oxysporum: Take out the Fusarium oxysporum strain from the -80 °C cryopreservation tube, evenly spread it on the surface of the PDA solid medium, and incubate it in the dark at 28 °C in an incubator for 3 days for activation. Use an inoculation loop to pick a single colony and inoculate it into a 1.5 mL centrifuge tube containing 700 μL of liquid PDA medium, and culture it on a shaker at 28 °C and 220 r / min for 2 days. Take 100 μL of the bacterial solution and inoculate it into 500 mL of PDA liquid medium, and culture it on a shaker at 28 °C and 220 r / min for 2-3 days until the spore count reaches 10

[0030] , ,

[0029] , <http: / / www.wipo.int / standards / XMLSchema / ST96 / XMLSchema / ST96-20060301 / XMLSchema / ST96-20060301- 6 , , / mL.

[0029] Method for inoculating the strain: The root-dipping method is adopted. Cut the Rehmannia glutinosa roots into sections about 3-4 cm long. To ensure the consistency of the wounds, the roots of all treatments are cut in the same way (both ends are uniformly cut off). Soak the roots in the Fusarium oxysporum bacterial solution (spore concentration 10 <http: / / www.wipo.int / standards / XMLSchema / ST96 / XMLSchema / ST96-20060301 / XMLSchema / ST96-20060301- 6 / mL) resuspended with ddH2O for 3 h. When normally planting Rehmannia glutinosa, it is customary to smear quicklime on the cut to prevent corrosion. In this experiment, quicklime is not smeared during the inoculation of the strain and different treatments to exclude the interference of lime on the pathogenic bacteria.

[0030] Example 2: Preparation of the cell wall extract of Fusarium oxysporum Take the bacterial liquid cultured for 3 days in Example 1, centrifuge it at 6000 r / min for 5 min, and collect the cell precipitate. Resuspend the precipitate with 200 mL of 500 mM KH2PO4 solution and wash it twice (centrifuge and discard the supernatant after each wash). Wash the washed precipitate successively with a chloroform-methanol mixture and acetone in a volume ratio of 1:1, and centrifuge and discard the supernatant after each wash. Place the degreased precipitate in an oven to dry, and grind it to obtain the Fusarium oxysporum cell wall extract ( Figure 1 ).

[0031] Example 3: Inhibitory effect of Fusarium oxysporum cell wall extract on the growth of Fusarium oxysporum (1) Antibacterial test on solid medium: Prepare PDA solid medium with aqueous dispersion of Fusarium oxysporum cell wall extract at 0, 0. I, 1, and 10 mg / mL respectively. Take 1.5 mL of the Fusarium oxysporum bacterial liquid activated in a centrifuge tube for 2 days in Example 1, and evenly coat it on the surface of the above medium. Incubate it in the dark in an incubator at 28°C for 48 h, and observe the colony growth ( Figure 2 ). The results show that with the increase in the extract concentration, the degree of inhibition of colony radial expansion is enhanced.

[0032] (2) Antibacterial test on liquid medium: Prepare PDA liquid medium with aqueous dispersion of Fusarium oxysporum cell wall extract at 0, 0.1, 1, and 10 mg / mL respectively. Inoculate the Fusarium oxysporum bacterial liquid activated in a 1.5 mL centrifuge tube for 2 days in Example 1 at an inoculation amount of 1% (v / v), and culture it on a shaker at 28°C and 220 r / min for 72 h. Use a hemocytometer to count the number of conidia ( Figure 3 ), and filter and collect the hyphae with four layers of gauze, dry and weigh them ( Figure 4 ). The results show that the extract treatment significantly reduces the spore yield and the dry weight of the mycelium, and shows a concentration dependence.

[0033] The above media were sterilized at 121°C for 20 - 30 min by high temperature and high pressure, and this treatment is sufficient to inactivate most proteins. However, the Fusarium oxysporum cell wall extract still retains significant antibacterial activity after sterilization, indicating that its active ingredient has thermal stability and is non-proteinaceous, presumably a cell wall structural polysaccharide (such as β-1,3-glucan and / or chitin) or its modified product.

[0034] Example 4: Induction of disease resistance in Rehmannia glutinosa by Fusarium oxysporum cell wall extract Fresh Rehmannia glutinosa roots were respectively soaked in aqueous dispersion of Fusarium oxysporum cell wall extract at 0, 0.1, 1, and 10 mg / mL for 1 h. Meanwhile, the Fusarium oxysporum bacterial liquid cultured in 500 mL PDA medium at 28 °C and 220 r / min for 3 days in Example 1 was centrifuged at 6000 r / min, and the supernatant was discarded. The bacterial cells were resuspended with ddH2O. The treated Rehmannia glutinosa roots were respectively soaked and infected with ddH2O (blank control) or the above resuspended Fusarium oxysporum bacterial liquid for 3 h, and then planted in small flower pots. The germination rate was counted on the 10th day ( Figure 5 ). The results showed that the germination rate of Rehmannia glutinosa roots pretreated with 0.1 - 10 mg / mL extract was significantly higher than that of the untreated control after inoculation with Fusarium oxysporum. Among them, the germination rate of the 1 mg / mL treatment group was the highest.

[0035] Example 5: Identification of active components To determine the effective components in the Fusarium oxysporum cell wall extract that exert induced disease resistance activity, the aqueous dispersion of 1 mg / mL extract was left standing at 4 °C to separate the supernatant and precipitate. Rehmannia glutinosa roots were respectively soaked in the supernatant (soluble part) or smeared with the precipitate (water-insoluble part) for 1 h, and then inoculated with Fusarium oxysporum according to the method in Example 4, and the germination rate on the 10th day was counted ( Figure 6 ). The results showed that the germination rates of the precipitate treatment group and the supernatant treatment group were significantly higher than those of the bacterial treatment group, but the difference between the two was not significant, indicating that both the water-soluble and water-insoluble cell wall polysaccharide fractions contain the main active components.

[0036] Example 6: Comparison of the activities of extracts from spores and hyphae Cell wall extracts were respectively extracted from the spores and hyphae of Fusarium oxysporum according to the method in Example 2. The two extracts were respectively used to treat Rehmannia glutinosa roots at a concentration of 1 mg / mL for 1 h, and then inoculated with Fusarium oxysporum according to the method in Example 4, and the germination rate on the 10th day was counted ( Figure 7 ). The results showed that the germination rates of the treatment groups with extracts from spores and hyphae were both significantly higher than those of the control, and there was no statistically significant difference between the two (P>0.05), indicating that the extracts from the two sources are equivalent in inducing disease resistance.

[0037] Example 7: Effects of extract treatment on the physiological responses of Rehmannia glutinosa Rehmannia glutinosa roots were respectively soaked in aqueous dispersion of 1 mg / mL Fusarium oxysporum cell wall extract for 0, 0.5, 1, and 3 h. 0.2 g samples were taken from each group, washed, placed in a pre-cooled mortar, added with 1.6 mL of pre-cooled 50 mmol / L phosphate buffer (pH 7.8), and ground into a homogenate in an ice bath. Then it was transferred to a 2 mL centrifuge tube and centrifuged at 4 °C and 12000 r / min for 20 min. The supernatant was the crude enzyme solution.

[0038] The content of superoxide anion was determined using a superoxide anion kit (Solarbio, BC1295).

[0039] The SOD activity was determined by the nitroblue tetrazolium photoreduction method: 162 mL of 14.5 mM methionine solution, 0.6 mL of 30 μM EDTA-Na2 solution, 5.4 mL of phosphate buffer, 6 mL of 2.25 mM NBT solution, and 6 mL of 60 μM riboflavin solution were mixed evenly to obtain a reaction mixture. 1.5 mL of the reaction mixture was added with 15 μL of crude enzyme solution, and the reaction was carried out under 4000 lux light for 20 min, and the absorbance was measured at 560 nm.

[0040] The POD activity was determined by the guaiacol method: 200 mL of phosphate buffer (0.2 M, pH 6.0) was taken, 0.076 mL of guaiacol was added, heated and stirred to dissolve, and after cooling, 0.112 mL of 30% H2O2 was added. After mixing, it was stored at 4 °C for standby. 1.5 mL of the reaction solution was added with 1.5 μL of crude enzyme solution, and the change in absorbance within 40 s was measured at 470 nm.

[0041] The CAT activity was determined: 200 mL of phosphate buffer (0.15 M, pH 7.0) was taken, 0.1 mL of crude enzyme solution and 0.1 mL of 30% H2O2 were added, and the change in absorbance within 40 s was measured at 240 nm.

[0042] The results were as Figure 8 shown: After treatment with the cell wall extract of Fusarium oxysporum, the superoxide anion level in the roots of Rehmannia glutinosa increased rapidly, and the activities of SOD, POD, and CAT enzymes were all up-regulated in a time-dependent manner, indicating that the extract activated the reactive oxygen metabolism and antioxidant defense system of Rehmannia glutinosa.

[0043] Example 8: Transcriptome analysis The control group (CK), the Fusarium oxysporum treatment group (Fo), and the cell wall extract of Fusarium oxysporum treatment group (CWE) were set respectively, with 3 biological replicates in each group. The RNA-seq technology was used to analyze the differentially expressed genes (DEGs), and the screening criteria were |log2FC|≥2 and FDR<0.05.

[0044] COG functional classification analysis ( Figure 9)Display: Among the DEGs of CK vs CWE, the highest proportion is in the signal transduction mechanism (category T), followed by the defense mechanism (category V) and carbohydrate transport and metabolism (category G); among the DEGs of CK vs Fo, the highest proportion is in carbohydrate transport and metabolism (category G), followed by the defense mechanism (category V) and biosynthesis, transport and catabolism of secondary metabolites (category Q). The results indicate that the CWE treatment mainly activates the signal transduction pathway, while the Fo infection mainly affects the metabolic pathway.

[0045] GO enrichment analysis ( Figure 10 )Display: The DEGs after the CWE treatment are significantly enriched in biological processes such as damage response, exocytosis, regulation of defense response, and regulation of stress response, and these GO terms are upregulated as a whole. Among them, exocytosis is a key cytological process for signal transmission and release of defense substances in plant disease resistance and defense, suggesting that CWE can enhance the physical barrier of the cell wall by promoting the extracellular secretion of defense-related substances.

[0046] KEGG pathway enrichment analysis ( Figure 11 )Display: The DEGs after the CWE treatment are significantly enriched in the MAPK signaling pathway, α-linolenic acid metabolism, plant hormone signal transduction, plant-pathogen interaction, and phenylpropanoid biosynthesis pathway. The MAPK signaling pathway is an early event activated by PTI; α-linolenic acid is the core precursor of the jasmonic acid signaling pathway; the phenylpropanoid biosynthesis pathway can produce lignin, phytoalexins, and phenolic acid antibacterial substances. The above results confirm that CWE induces the disease resistance of Rehmannia glutinosa by activating the PTI, jasmonic acid signal, and phenylpropanoid metabolism pathways.

[0047] Example 9: Preparation of biogenic pesticide preparation The Fusarium oxysporum cell wall extract prepared in Example 2 is prepared into a wettable powder or a water suspension agent with an agriculturally acceptable carrier and adjuvant according to a conventional method. For example: take 10 parts by weight of the extract, 15 parts by weight of silica white, 5 parts by weight of sodium lignosulfonate, 2 parts by weight of sodium dodecyl sulfate, and make up to 100 parts by weight with kaolin. After mixing evenly, it is ultrafinely pulverized to obtain a wettable powder. When applying in the field, the preparation is diluted to a final concentration of the extract of 0.1 - 10 mg / mL, and the Rehmannia glutinosa tubers or seedlings are treated by irrigation or seed soaking.

[0048] Comparative Example 10: Comparison of the induced disease resistance between the Fusarium oxysporum cell wall extract and the Bacillus subtilis bacterium agent The control group is the Rehmannia glutinosa tubers planted normally, which is used to test whether the germination rate of Rehmannia glutinosa is normal. The experimental group is divided into the following 5 groups: 1. The Rehmannia glutinosa tubers are infected with the resuspended bacterial solution of Fusarium oxysporum with a spore count of 10 6 / mL in step (3) of Example 1 for 3 h to test whether the pathogenicity of Fusarium oxysporum is normal.

[0049] 2. Soak the Rehmannia glutinosa roots in the cell wall extract of Fusarium oxysporum at a concentration of 1 mg / ml for 1 h, and then infect them with the Fusarium oxysporum resuspended bacterial solution with a spore count of 10 6 / mL for 3 h in step (3) of Example 1.

[0050] 3. Weigh 1 g of the Bacillus subtilis bactericide recommended in the "Comprehensive Prevention and Control Technology Regulations for Diseases and Pests of Rehmannia glutinosa", dilute it with 500 ml of ddH2O according to the recommended dosage of the bactericide, soak the Rehmannia glutinosa roots, and then infect them with the Fusarium oxysporum resuspended bacterial solution with a spore count of 10 6 / mL for 3 h in step (3) of Example 1.

[0051] 4. Weigh 1 g of the Bacillus subtilis bactericide recommended in the "Comprehensive Prevention and Control Technology Regulations for Diseases and Pests of Rehmannia glutinosa", dilute it with 500 ml of the cell wall extract of Fusarium oxysporum at a concentration of 1 mg / mL, soak the Rehmannia glutinosa roots, and then infect them with the Fusarium oxysporum resuspended bacterial solution with a spore count of 10 6 / mL for 3 h in step (3) of Example 1.

[0052] 5. Infect the Rehmannia glutinosa roots with the Fusarium oxysporum resuspended bacterial solution with a spore count of 10 6 / mL for 3 h and then plant them. When watering, weigh 1 g of the Bacillus subtilis bactericide recommended in the "Comprehensive Prevention and Control Technology Regulations for Diseases and Pests of Rehmannia glutinosa", dilute it with 500 ml of ddH2O and pour it.

[0053] As Figure 12 shown, it can be clearly seen from the figure that the germination rate of the roots treated with the cell wall extract of Fusarium oxysporum is much higher than that of the roots soaked and watered with the Bacillus subtilis bactericide, indicating that the cell wall extract of Fusarium oxysporum has a much better effect in inducing disease resistance in Rehmannia glutinosa than the Bacillus subtilis bactericide, and it is not suitable to be used in combination with the Bacillus subtilis bactericide.

[0054] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A type of Fusarium oxysporum ( Fusarium oxysporum Cell wall extract, characterized in that, The extract is derived from polysaccharide fractions of Fusarium oxysporum spores or hyphal cell walls and contains at least one pathogen-associated molecular pattern (PAMP) component, which is a non-protein, thermally stable, species-nonspecific elicitor capable of forming an aqueous dispersion.

2. The extract according to claim 1, characterized in that, The active ingredient in the extract that induces disease resistance in Rehmannia glutinosa is a cell wall polysaccharide fraction, which contains β-1,3-glucan and / or chitin.

3. A method for preparing the Fusarium oxysporum cell wall extract according to claim 1 or 2, characterized in that, Includes the following steps: (1) Inoculate Fusarium oxysporum into PDA liquid medium, culture at 28℃ and 220 r / min for 2-3 days with shaking, and collect the bacterial cells by centrifugation; (2) Centrifuge the collected bacterial cells at 6000 r / min and wash the precipitate twice with 500 mM KH2PO4 solution; (3) Wash the precipitate after washing with a chloroform-methanol mixture and acetone in a volume ratio of 1:1, and centrifuge and discard the supernatant after each washing. (4) Dry the defatted precipitate and grind it to obtain the Fusarium oxysporum cell wall extract.

4. The method according to claim 3, characterized in that, In step (1), the composition of the PDA liquid culture medium is: 100 g / L potato and 10 g / L glucose.

5. An induced rehmannia ( Rehmannia glutinosa The method for generating disease resistance is characterized by, The root of Rehmannia glutinosa was soaked in an aqueous dispersion of the cell wall extract of Fusarium oxysporum as described in claim 1 or 2 for 1 hour, wherein the concentration of the extract was 0.1 to 10 mg / mL, and then inoculated with Fusarium oxysporum.

6. The method according to claim 5, characterized in that, The extract activates the mode of Rehmannia glutinosa to trigger immune PTI, including: inducing the production of superoxide anions, upregulating the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and enriching differentially expressed genes in the mitogen-activated protein kinase (MAPK) signaling pathway, jasmonic acid metabolism pathway, and phenylpropane biosynthesis pathway.

7. The method according to claim 5, characterized in that, The extract was isolated from the spores or hyphae of Fusarium oxysporum.

8. A method for inhibiting the growth of Fusarium oxysporum, characterized in that, The Fusarium oxysporum cell wall extract according to claim 1 or 2 is added to PDA solid or liquid culture medium at a final concentration of 0.1 to 10 mg / mL, and then inoculated with Fusarium oxysporum and cultured at 28°C.

9. The use of the extract according to claim 1 or 2 in the preparation of a biopesticide for controlling root rot of Rehmannia glutinosa, wherein the root rot is caused by Fusarium oxysporum (… Fusarium oxysporum Caused by infection.

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