Method for preventing and treating vegetable root knot nematode by using spirodiclofen and synthetic gene thereof and application thereof

Abstract

The application discloses a method for preventing and treating vegetable root-knot nematodes by using spiro-ranaculatone and a synthetic gene thereof and application thereof, and belongs to the field of agricultural biotechnology and plant protection. The application finds that spiro-ranaculatone has a significant repellent effect on southern root-knot nematodes when the concentration is higher than 0.1 mg / L, and can effectively inhibit the infestation of the nematodes on the roots of tomatoes at a concentration of 5-10 mg / L. Meanwhile, the application identifies key genes in a synthetic pathway of spiro-ranaculatone K15472 and K14182 Overexpression of the genes can significantly increase the content of spiro-ranaculatone in plants and enhance the resistance of the plants to the nematodes. The application provides two prevention and treatment strategies of exogenous application of spiro-ranaculatone and planting of transgenic resistant plants, and has the advantages of high efficiency, environmental protection, strong specificity and the like, and provides a new technical scheme for green prevention and control of vegetable root-knot nematodes.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and plant protection, specifically relating to the application of a plant secondary metabolite, solavetivone, in the control of plant parasitic nematodes, and the application of a gene encoding a key enzyme in the solavetivone synthesis pathway in the cultivation of nematode-resistant transgenic plants. More specifically, it relates to the utilization of solavetivone and its synthesis gene. K15472 , K14182 Control of root-knot nematodes in vegetables, especially southern root-knot nematodes ( Meloidogyne incognita The method. Background Technology

[0002] Vegetable root-knot nematode disease is one of the most destructive plant diseases worldwide, among which the southern root-knot nematode ( Meloidogyne incognita Due to its wide host range, high reproductive rate, and difficulty in control, root-knot nematodes are considered one of the most important plant parasitic nematodes. After infecting plant roots, the nematodes form root knots, hindering the absorption of water and nutrients, leading to stunted growth, yellowing, and even death of the plant, causing severe economic losses. It is estimated that global crop yield losses due to root-knot nematode diseases reach more than 5% annually.

[0003] Current pest control measures mainly include chemical control, biological control, and agricultural control. While chemical control is fast-acting, long-term and excessive use of nematicides (such as methyl bromide and aldicarb) can lead to soil pollution, excessive pesticide residues, ecological damage, and the development of nematode resistance. Many traditional chemical agents have been banned or restricted. Biological control utilizes natural enemies or microbial metabolites, which is environmentally friendly, but its effectiveness is often unstable and greatly affected by environmental factors. Agricultural control, such as crop rotation and soil disinfection, is costly and complex to implement, making it difficult to popularize in large-scale planting.

[0004] Secondary metabolites produced by plants are an important component of plant defense systems. In recent years, the use of plant-derived bioactive substances to control pests and diseases has become a research hotspot. Spirophyllone is a sesquiterpene compound known to have antibacterial activity, but its mechanism of action and application potential in nematode resistance have not been fully explored. Current technologies lack specific reports on the effects of spirophyllone on the chemotaxis of root-knot nematodes, and also lack technical solutions for enhancing nematode resistance by regulating the spirophyllone synthesis pathway in plants through genetic engineering.

[0005] Furthermore, flg22 (a conserved epitope peptide of bacterial flagellin) is known to induce plant defense responses as a plant immune elicitor, but its specific downstream metabolites and nematode-resistant effects remain unclear. Therefore, developing a highly efficient, environmentally friendly, and specific root-knot nematode control method based on an immune elicitor to induce the plant resistance substance spirochetone and utilize its biosynthetic gene is of significant practical importance for reducing chemical pesticide use and ensuring safe vegetable production. Currently, there is an urgent need for a technical solution that can definitively repel nematodes and achieve durable resistance through genetic modification. Summary of the Invention

[0006] To address the problems of environmental pollution from chemical pesticides, the easy development of pesticide resistance in nematodes, and the unstable effectiveness of existing biological control methods, this invention provides a method and application for controlling root-knot nematodes in vegetables using spirochetone and its synthetic gene. This invention aims to utilize the repellent properties of spirochetone and genetic engineering techniques to enhance endogenous plant resistance, achieving efficient and environmentally friendly nematode control.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of spirochetone in the preparation of formulations for controlling plant parasitic nematodes. Preferably, the plant parasitic nematode is the southern root-knot nematode (…). Meloidogyne incognita ).

[0008] Furthermore, the concentration of spirochetone in the formulation is higher than 0.1 mg / L, which makes the chemotaxis index of the plant parasitic nematode lower than 1.0, thereby producing a repellent effect on the plant parasitic nematode.

[0009] Furthermore, the concentration of spirocarpus in the formulation is 5 mg / L to 10 mg / L, used to inhibit the infection of plant roots by the plant parasitic nematodes.

[0010] Secondly, this invention provides the application of a gene encoding a key enzyme in the spirochetone synthesis pathway in the cultivation of nematode-resistant plants. The gene is... premnaspirodiene oxygenase ( K15472 ) or genes vetispiradiene synthase ( K14182 ).

[0011] Furthermore, the gene K15472 The nucleotide sequence of the gene is shown in SEQ ID NO.1. K14182 The nucleotide sequence is shown in SEQ ID NO.2.

[0012] Furthermore, overexpression of the gene increases the content of spirochetone in the plant, thereby repelling plant parasitic nematodes.

[0013] Thirdly, the present invention provides a transgenic plant that overexpresses the aforementioned gene. K15472 or genes K14182 This increases the content of spirochetone in the plant. Preferably, the plant is a tomato.

[0014] Fourthly, the present invention provides a method for controlling plant parasitic nematodes, comprising applying spirochetone to the rhizosphere soil of plants and / or the aforementioned transgenic plants.

[0015] Furthermore, the concentration of the spirochetone is 5 mg / L to 10 mg / L, and the application time is within 12 hours before nematode inoculation. Beneficial effects

[0016] (1) Clear repellency mechanism: This invention confirms that spirocarpus has a significant repellency effect on southern root-knot nematodes when the concentration is higher than 0.1 mg / L, and the chemotactic index (CI) drops to below 1.0 and is positively correlated with concentration.

[0017] (2) Significantly enhanced resistance: This invention achieves this by overexpressing key genes. K15472 and K14182 The content of spirochetone in the roots of tomato plants increased by 6.21 times and 4.57 times, respectively. The attractiveness of transgenic plants to nematodes decreased by 60%-63%, and the number of nematodes infecting the roots was significantly reduced.

[0018] (3) Excellent control effect: The pot experiment of this invention shows that the exogenous application of 5 mg / L and 10 mg / L spirochete ketone can reduce the number of nematodes in tomato roots by 56% and 65%, respectively.

[0019] (4) Green and environmentally friendly: Utilizing plant-derived metabolites and genetic modification to reduce the use of chemical pesticides is in line with the direction of sustainable agricultural development.

[0020] (5) Flexible application: It provides two strategies: exogenous application and endogenous improvement, and the appropriate scheme can be selected according to the actual planting conditions. Attached Figure Description

[0021] Figure 1 To excite the flagellin polypeptide flg22 pf Transcriptome analysis and determination of the tomato-induced resistance to southern root-knot nematode via the spirochetal pathway; among which, (a) is flagellin polypeptide flg22. pf Tomato sample category treated; (b) flagellin polypeptide flg22 pf (c) Synthetic pathway of spirotropin in tomato plants (red box indicates significant upregulation of the gene after flg22pf stimulation); (d) Genes regulating spirotropin synthesis. K15472Transcriptome RT-qPCR results of its expression level; (e) is the gene regulating the synthesis of spirochete ketone. K14182 The expression levels were determined by transcriptome RT-qPCR; data are mean ± standard deviation (ns ≥ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, analyzed by one-way ANOVA).

[0022] Figure 2 This study aimed to construct hairy roots overexpressing the gene regulating the synthesis of spirochetin in tomato and to determine the gene expression level. (a) shows the results of transiently overexpressing hairy roots verified by confocal microscopy (ZEISS LSM 900); (b) shows the relative fluorescence intensity of the overexpressing hairy roots; and (c) shows the relative expression level of the gene regulating the spirochetin synthesis pathway in the transiently overexpressing hairy roots verified by RT-qPCR. Data are presented as mean ± standard deviation (ns ≥ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, one-way ANOVA).

[0023] Figure 3 To enhance the resistance of tomatoes overexpressing the spirochetin synthesis regulatory gene to southern root-knot nematodes, the study found that (a) the relative content of spirochetin in stably overexpressing plants; (b) the resistance of stably overexpressing plants to southern root-knot nematodes; (c) the root attraction of fluorescent hairy roots overexpressing the spirochetin synthesis regulatory gene to southern root-knot nematodes; and (d) the state of nematodes touched after 10 hours of microscopic observation, with images showing partial root segments and arrows indicating the location of southern root-knot nematodes attached to the roots. Data are presented as mean ± standard deviation (**P ≤ 0.01, ****P ≤ 0.0001, one-way ANOVA).

[0024] Figure 4 To determine the repellent activity of spirotropin against southern root-knot nematodes and its interference with their infection in tomatoes using Pluronic gel, the following data were presented: (a) the repellent effect of different concentrations of spirotropin on southern root-knot nematodes in the Pluronic F-127 repellency model; (b) the interference effect of 5 mg / L and 10 mg / L spirotropin on the infection of southern root-knot nematodes into tomato roots; and (c) microscopic observation of the nematode status of southern root-knot nematodes exposed to tomato root tips after 10 hours at concentrations of 5 mg / L and 10 mg / L spirotropin. Phloroglucinol, a repellent compound against root-knot nematodes, was also used. Data are presented as mean ± standard deviation (**P ≤ 0.01; ***P ≤ 0.001, determined by one-way ANOVA). Figure 5This study investigated the control effect of spirotropin on tomato plants infected with southern root-knot nematodes in potted plants. (a) Acid fuchsin staining was used to observe the infection status of tomato plants after different treatments; (b) The number of southern root-knot nematodes infected in tomatoes after spirotropin treatment was statistically analyzed; (c) Spectroscopic observation of southern root-knot nematode infection on tomato roots was performed, with arrows pointing to the nematodes after staining. Phloroglucinol is a root-knot nematode repellent compound. Data are expressed as mean ± standard deviation (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001, ****P ≤ 0.0001, analyzed by one-way ANOVA). Detailed Implementation

[0025] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0026] Example 1: Transcriptome Prediction of the Spirodela polyrhizone Synthesis Pathway in Tomato Resistance to Southern Root-Knot Nematode To investigate the bacterial flagellin polypeptide flg22 pf Inducing tomatoes to produce resistance to southern root-knot nematodes ( Meloidigyne incognita The molecular mechanism of resistance is described in this invention, which sets up four treatment groups ( Figure 1 a): Sterile water treatment (control group, CK), sterile water treatment followed by nematode inoculation (Mi group), flg22 pf Peptide solution treatment (flg22 group) and flg22 pf After treatment with peptide solution, nematodes (flg22-Mi group) were inoculated. Specifically, sterile water or 5 μmol / L flg22 was used. pf The tomato roots were rinsed with the solution, and after 12 hours of treatment, 800 second-instar larvae (J2s) of the southern root-knot nematode were inoculated into each plant. Tomato root samples were collected from each group on day 3 after inoculation for transcriptome sequencing (RNA-seq) analysis.

[0027] Using a false discovery rate (FDR) < 0.05 and a fold change in gene expression (|log2FoldChange|) > 2 as screening criteria, multiple groups of differentially expressed genes (DEGs) were identified. Specifically, 274 DEGs were identified between the CK group and the flg22 group; and 182 DEGs were identified between the Mi group and the flg22-Mi group. Venn diagram analysis showed that these two groups shared a total of 49 DEGs (…). Figure 1 b), indicating that these genes are flg22 pfInduced stable response genes whose expression is unaffected by nematode inoculation. Functional annotation and pathway enrichment analysis of these 49 core DEGs revealed that two genes were annotated as key enzymes involved in the biosynthesis of solavetivone. Figure 1 c).

[0028] The transcriptome sequencing results were validated using RT-qPCR technology, and the results showed that these two candidate genes ( K15472 and K14182 ) in flg22 pf The expression levels in the treated groups (flg22 group and flg22-Mi group) were significantly higher than those in the untreated groups (CK group and Mi group), and their expression trends were highly consistent with RNA-seq data. Figure 1 d, e). This result suggests that flg22 pf It is possible that the resistance of tomatoes to southern root-knot nematodes is enhanced by upregulating the expression of genes related to the spirochetal ketone synthesis pathway.

[0029] Example 2: Overexpression of key genes for spirulina ketone synthesis in tomatoes enhances resistance to southern root-knot nematodes. To directly verify the above two candidate genes K15472 (SEQ ID NO.1) and K14182 (SEQ ID NO.2) Function in the process of tomato resistance to nematodes. This invention utilizes Agrobacterium-mediated transformation technology to construct [a specific product / process] using Agrobacterium rhizogenes MSU440. K15472 and K14182 A transient overexpression system of hairy roots in tomatoes was established. The reporter gene green fluorescent protein (GFP) in the hairy roots was observed using a confocal laser scanning microscope (Zeiss LSM 900). The results showed that, compared with the control group transfected with empty vector, K15472 and K14182 Strong green fluorescence signals were observed in all overexpressed hairy roots. Figure 2 a). Quantitative analysis using ImageJ software further confirmed that the average fluorescence intensity of the overexpressing hairy roots was significantly higher than that of the control group (a). Figure 2 b). Meanwhile, RT-qPCR results showed that in the corresponding overexpressing hairy roots, K15472 and K14182 The transcriptional level was significantly upregulated, confirming the successful overexpression of the target gene. Figure 2 c).

[0030] Based on this, to further investigate the in vivo insecticidal effect of spirochetone, a method was successfully constructed by Wuhan Boyuan Biotechnology Co., Ltd. K15472 andK14182 Stable overexpression tomato plants were obtained. The content of spirochetin in the roots of the stable overexpression plants was detected by liquid chromatography-mass spectrometry (LC-MS). The results showed that compared with wild-type (WT) plants, K15472 and K14182 The relative content of spirochetone in the roots of overexpressing plants increased significantly by 6.21 times and 4.57 times, respectively. Figure 3 a). Experiments on resistance to nematode infection showed that after inoculation with nematodes, K15472 and K14182 The number of nematodes in the roots of stably overexpressing plants was significantly lower than that in wild-type controls. Figure 3 (b) This demonstrates that the in vivo accumulation of spirochetone can effectively improve the overall resistance of tomatoes.

[0031] Finally, using the Pluronic F-127 transparent gel system, the attraction and infection of *J2s* from transiently overexpressed hairy roots and control roots were visually compared. After 10 hours of experimentation, microscopic observation and statistical results showed that contact... K15472 and K14182 The number of periapical nematodes in the hairy roots was significantly reduced in the transient overexpression group compared with the control group. Figure 3 d), its attractiveness to J2s decreased by 63% and 60% respectively ( Figure 3 c). This result is directly confirmed at the organizational level by... K15472 and K14182 Gene-mediated synthesis and accumulation of spirochetone can effectively repel southern root-knot nematodes, thereby interfering with their infection process on tomato roots.

[0032] Example 3: Gel system detection of the inhibition of southern root-knot nematode infection in tomato host by spirochete salsa. To evaluate the effects of spirochete ketone on southern root-knot nematodes ( Meloidogyne incognita To investigate the effect of the ability of the virus to infect the tomato host, this invention uses Pluronic F-127 gel as a medium to conduct chemotaxis and infection experiments.

[0033] Different concentrations of spirotropin (0.01 mg / L, 0.1 mg / L, 1 mg / L, 5 mg / L, and 10 mg / L) were used to treat nematodes in the Pluronic F-127 repellency model, and the chemotaxis index (CI) of nematodes was measured. The results showed that when the concentration of spirotropin was 0.01 mg / L, the chemotaxis index (CI) was 1.51 ± 0.15, indicating that 0.01 mg / L of spirotropin had an attractive effect on nematodes; when the concentration of spirotropin was higher than 0.1 mg / L, the CI significantly decreased to below 1.0. Spirotropin exhibited a repellent effect on nematodes at concentrations higher than 0.1 mg / L, indicating that spirotropin has a repellent effect on nematodes at concentrations higher than 0.1 mg / L, and this repellent effect was positively correlated with increasing spirotropin concentration. Figure 4 a).

[0034] To further verify its effect on nematode infestation behavior in tomato roots, tomato seedling roots were buried in Pluronic F-127 gel containing different concentrations of spirogellone (5 mg / L and 10 mg / L), and the number of nematodes gathered around the root tips was counted 10 hours after treatment. The results showed that the number of nematodes at the root tips was significantly reduced in the spirogellone-treated groups compared to the control group, with a reduction of approximately 53% in the 5 mg / L treatment group and approximately 59% in the 10 mg / L treatment group. Figure 4 b). Meanwhile, microscopic observation further confirmed that the attachment and infection behavior of nematodes at the root tips was significantly disrupted in the treatment group. Figure 4 c).

[0035] In summary, experiments using the Pluronic F-127 gel system confirmed that spirocarpus syringone has a significant repellent effect on southern root-knot nematodes at concentrations above 0.1 mg / L, and can effectively inhibit its infection of tomato roots, with this effect being concentration-dependent.

[0036] Example 4: Pot experiment to detect the inhibition of southern root-knot nematode infection in tomato host by spirochetone To further verify the actual control efficacy of spirochetone in the soil environment, this invention evaluated its effect on southern root-knot nematodes (Symplocos spp.) through pot experiments. Meloidogyne incognitaThe inhibitory effect of spirochetin on tomato host infection was investigated. The experiment included a control group and 5 mg / L and 10 mg / L spirochetin treatment groups. Spirochlein solutions of corresponding concentrations were applied to the rhizosphere soil of potted tomato plants. Twelve hours after treatment, each plant was inoculated with 500 second-instar larvae (J2s) of the southern root-knot nematode. On day 7 post-inoculation, tomato plant roots were collected, and the nematodes in the root tissue were stained using a modified acid fuchsin staining-decolorization method. The number of infected nematodes was counted under a stereomicroscope. The staining results showed that the number of nematodes in the roots of the spirochetin-treated groups was significantly reduced compared to the control group, with a 56% reduction in the 5 mg / L treatment group and a 65% reduction in the 10 mg / L treatment group. Figure 5 (a, b) indicates that treatment with spirochetone effectively inhibits nematode infection of tomato roots. Microscopic observation further confirmed that the number of nematodes invading the root tissue of the treated group was significantly reduced. Figure 5 c). In summary, exogenous application of spirochetone to the soil can significantly reduce the infection of tomato plants by southern root-knot nematodes, and the inhibitory effect increases with increasing concentration, demonstrating its potential for field control.

Claims

1. The application of spirochetone in the preparation of formulations for controlling plant parasitic nematodes, characterized in that, The plant parasitic nematode is the southern root-knot nematode. Meloidogyne incognita .

2. The application according to claim 1, characterized in that, The concentration of spirocarpus in the formulation is higher than 0.1 mg / L, which makes the chemotaxis index of the plant parasitic nematodes lower than 1.0, thereby repelling the plant parasitic nematodes.

3. The application according to claim 1, characterized in that, The concentration of spirochete ketone in the formulation is 5 mg / L to 10 mg / L, used to inhibit the infection of plant roots by the plant parasitic nematodes.

4. The application of genes encoding key enzymes in the spirochetone synthesis pathway in the cultivation of nematode-resistant plants, characterized by: The gene is a gene. K15472 or genes K14182 ; wherein, the gene K15472 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. K14182 The nucleotide sequence is shown in SEQ ID NO.

2.

5. The application according to claim 4, characterized in that, Overexpression of the gene increases the content of spirochetone in plants, thereby repelling plant parasitic nematodes.

6. A transgenic plant, characterized in that, Including overexpression of the gene of claim 4 K15472 or genes K14182 This increases the content of spirochetone in plants.

7. The transgenic plant according to claim 6, characterized in that, The plant in question is a tomato.

8. A method for controlling plant parasitic nematodes, characterized in that, This includes applying spirochetone to the rhizosphere soil of plants and / or planting the transgenic plants as described in claim 6 or 7.

9. The method according to claim 8, characterized in that, The concentration of the spirochetone is 5 mg / L to 10 mg / L, and it is applied within 12 hours before nematode inoculation.

Images