Application method for preventing and treating cotton verticillium wilt by utilizing terbinafine targeted sterol binding protein VdPBP1

Abstract

The invention discloses an application method for controlling cotton verticillium wilt by utilizing terbinafine to target verticillium dahliae sterol binding protein VdPBP1, and relates to the technical field of cotton control, and the method comprises the following application steps: S1, confirming the characteristics of a terbinafine medicament; s2, formulating a terbinafine application scheme; and S3, verification and guarantee of prevention and control effects. According to the method, theoretical support is provided for precise prevention and control; the EC50 of the terbinafine is 10.8 ng / mL, the dosage of the medicament can be greatly reduced, the monovalence of the terbinafine is 30.4 yuan / g, so that the large-scale application cost is lower, the terbinafine is only combined with verticillium dahliae VdPBP1 and blocks mediated electron transfer of the verticillium dahliae VdPBP1, and the existing medicaments such as phenazino-1-carboxylic acid can inhibit histone acetylation and benomyl has wide action targets of interfering microtubulin, so that the application range of the medicament is widened. Non-target organisms are easily influenced; moreover, an ergosterol synthesis pathway is an essential pathway for fungi, and the VdERG1 is a single copy gene in verticillium dahliae, so that pathogenic bacteria are difficult to generate drug resistance through gene mutation, and prevention and control nodes widely cover the whole growth period.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of cotton prevention and treatment, and particularly relates to an application method for preventing and treating cotton verticillium wilt by using terbinafine targeted sterol binding protein VdPBP1. BACKGROUND

[0002] Cotton verticillium wilt is caused by the soil-borne pathogenic fungus Verticillium dahliae (Vd) and is a devastating disease in global cotton production, causing economic losses of up to 10 billion US dollars per year and seriously restricting the yield and quality of agricultural and forestry crops. Verticillium dahliae The V. dahliae has a special infection mode (such as invading through root wounds) and a dormancy mechanism (producing microsclerotia with strong stress resistance), which makes it extremely difficult to prevent and control cotton verticillium wilt. Among them, ergosterol is a key component of the fungal cell membrane, directly regulates fungal polarity development, material metabolism and pathogenicity, and its synthesis pathway is an important target for antifungal agents. Azole compounds: by inhibiting the ergosterol 14alpha-demethylase (CYP51), the accumulation of toxic intermediates in the synthesis of ergosterol is caused, which is mainly used for treating pneumonia caused by Aspergillus fumigatus and Candida albicans in clinical treatment; it has been developed as a commercial pesticide in the agricultural field, such as triadimefon for regulating melanin synthesis of Colletotrichum gloeosporioides. Propylene amine compounds: it is known to act on squalene epoxidase (ERG1) in the ergosterol synthesis pathway, and is widely used in the medical field to treat fungal tinea, candidaemia and other diseases, such as oral or external use of terbinafine, and has good drug efficacy and no obvious toxic side effects, and has in vitro inhibitory effect on plant pathogenic fungi under experimental conditions, but has not been used in agricultural production.

[0003] However, the existing cotton verticillium wilt prevention and treatment technology has the following defects: High concentration of drug application: the half-inhibitory concentration (EC 50 ) of the main commercial agents such as fenpropimorph and benomyl is 328.1 ng / mL and 496.6 ng / mL respectively, and high-dose application is required to achieve the prevention and control effect; Poor sustained effect: the effect of the existing agents depends on the concentration, and the V. dahliae can produce drug resistance through the repair system, resulting in a short prevention and control period; Unclear mechanism: the target and bactericidal mechanism of propylene amine compounds in the agricultural field have not been analyzed, and precise targeted prevention and control cannot be achieved; High cost: the unit price of the main component of fenpropimorph, phenazine-1-carboxylic acid, is 452 yuan / g, and the unit price of benomyl is 62 yuan / g, so the cost of large-scale application is high. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides an application method for preventing and treating cotton verticillium wilt by targeting sterol binding protein VdPBP1 with terbinafine, which solves the problems in the above background art.

[0005] To achieve the above object, the application is implemented by the following technical scheme: an application method for preventing and treating cotton verticillium wilt by targeting sterol binding protein VdPBP1 with terbinafine, comprising the following application steps: S1, confirming the characteristics of terbinafine agent: Through the plate antibacterial experiment, the inhibition effect of the commercially available bactericides on the mycelial growth of verticillium dahliae is compared, and the half-inhibitory concentration EC of terbinafine on the mycelial growth of verticillium dahliae is determined. 50 is 10.8 ng / mL, which is significantly lower than 328.1 ng / mL of shenqinmycin and 496.6 ng / mL of benomyl, thereby clearly determining the high bacteriostatic property thereof; S2, formulating a terbinafine application scheme: For different stages of cotton verticillium wilt, two types of application modes are set: one is the prevention stage, and terbinafine with a concentration of 50 ng / mL is applied by root irrigation 7 days before cotton is inoculated with verticillium dahliae or at the same time as the inoculation; the other is the treatment stage, and terbinafine with a concentration of 20 ng / mL is applied by leaf root irrigation after cotton is infected with verticillium dahliae for 7 days, that is, when the pathogen completes the infection and colonization; S3, verification and guarantee of the prevention and treatment effect: After application, the residual amount of terbinafine in cotton is confirmed to be 7.52-8.96 ng / g by high performance liquid chromatography (HPLC) detection, and does not affect the normal growth of cotton; at the same time, the cotton verticillium wilt disease symptoms are counted, the biomass of verticillium dahliae in cotton is detected by real-time fluorescent quantitative PCR (RT-qPCR), and the disease index is calculated, to verify the prevention and treatment effect of terbinafine on cotton verticillium wilt, and finally to achieve high-efficiency prevention and control of cotton verticillium wilt.

[0006] Further, in the step S3, the statistical standard of the disease index is: 0 level, no disease spot, 1 level, disease spot accounting for ≤25% of the leaf area, 2 level, disease spot accounting for 26%-50% of the leaf area, 3 level, disease spot accounting for 51%-75% of the leaf area, 4 level, disease spot accounting for >75% of the leaf area or leaf wilting, and the disease index = Σ (disease level leaf number x corresponding disease level value) ÷ (total leaf number x highest disease level value) x 100.

[0007] Further, it further comprises the following terbinafine and VdPBP1 action mechanism analysis process: S4, VdPBP1 target identification: The terbinafine sensitive mutant is screened from verticillium dahliae, and the mutation site is located in the VdPBP1 gene by isothermal asymmetric PCR. VDAG_08647Gene, the gene encodes a protein containing cytochrome b5 hormone and sterol binding domain and is conserved in pathogenic fungi, while VdPBP1 is located in the cytoplasm of spores and hyphae of Verticillium dahliae by green fluorescent protein GFP labeling, and VdPBP1 is preliminarily identified as a potential target of terbinafine; The coding gene of VdPBP1 VDAG_08647 has been registered in GeneBank, and the cytochrome b5 hormone and sterol binding domain of the encoded protein are key functional regions involved in electron transfer.

[0008] Further, the following terbinafine and VdPBP1 action mechanism analysis process is also included: S5, VdPBP1 function verification: Construction VdPBP1 Knockout strain Δ VdPBP1 and complemented strain EC ΔVdPBP1 , the growth phenotype of wild type strain WT, Δ VdPBP1 and EC ΔVdPBP1 was compared, and it was found that Δ VdPBP1 The amount of growth, spore production decreased significantly, the hyphal tip bifurcation increased, the chitin synthesis was damaged, and it was only sensitive to terbinafine and had no response to voriconazole, and it was also found that the ergosterol content in Δ VdPBP1 Decreased, accumulation of toxic intermediate squalene and cell membrane rupture, confirming that VdPBP1 is involved in the growth and development of Verticillium dahliae, ergosterol metabolism and membrane homeostasis regulation; Among them, when verifying the sensitivity of VdPBP1 to terbinafine, each strain was cultured in Czapek medium, and the colony diameter was measured after 7 days of culture at 25 DEG C. The inhibition rate was calculated to distinguish the response difference of the strain to terbinafine and voriconazole.

[0009] Further, the following terbinafine and VdPBP1 action mechanism analysis process is also included: S6, VdPBP1 and VdERG1 interaction verification: Through fluorescence co-localization, it was observed that VdPBP1-GFP and VdERG1-mCherry, a key enzyme for ergosterol synthesis in Verticillium dahliae, were co-localized near the lipid droplets of spores and hyphae. Further through yeast two-hybrid Y2H and BiFC experiments, it was confirmed that VdPBP1 and VdERG1 directly interacted in vivo and in vitro, and the interaction site was located on the outer layer of the lipid droplets; At the same time, the expression level of Δ VdPBP1 VdERG1 It was found that the mRNA level had no significant change but the protein abundance decreased significantly, which confirmed that VdPBP1 regulated VdERG1 protein expression through interaction; Among them, VdERG1 is a squalene epoxidase. ​

[0010] Further, the following terbinafine and VdPBP1 mechanism of action analysis process is also included: S7, VdPBP1 electron transfer function verification: Based on the cytochrome b5 domain of VdPBP1, exogenous addition of electron transfer carrier heme, it is observed that ΔV dPBP1 growth is restored; in Δ VdPBP1 overexpression of electron transfer compensation gene VdPBP1 , it is found that the sensitivity of the strain to terbinafine decreases from 40% to 25% ± 2%, and both the above treatments can increase the VdERG1 protein abundance and restore the ergosterol and squalene content, confirming that VdPBP1 activates VdERG1 expression by transferring electrons.

[0011] Further, the following terbinafine and VdPBP1 mechanism of action analysis process is also included: S8, terbinafine and VdPBP1 binding mechanism analysis: Using molecular docking technology, it is found that the docking score of terbinafine and VdPBP1 is -6.565 kcal / mol, the binding region is the heme binding hydrophobic pocket of VdPBP1, forming 14 hydrogen bonds, with a binding distance of 1.5-3.9 Å, and the active center of terbinafine (-N(CH3)) is close to the 95th aspartic acid of VdPBP1, namely D95; further mutating the heme binding site D63, Y104 of VdPBP1 to glycine (G) and phenylalanine (F) to construct a complementation strain, it is found that the strain cannot restore the developmental defects and terbinafine tolerance of ΔVdPBP1, and the VdERG1 activation is impaired, finally confirming that terbinafine blocks electron transfer by occupying the heme binding domain of VdPBP1.

[0012] Further, in step S6, the fluorescence co-localization experiment of VdPBP1 and VdERG1 is observed by laser confocal microscope, and the nucleus is labeled by DAPI staining, with a scale of 10 μm.

[0013] Further, in step S7, the concentration of exogenous heme added is 20 μg / mL, and after addition, the growth of the strain on the medium containing terbinafine is determined to evaluate the recovery effect of electron transfer function.

[0014] Further, in step S8, the molecular docking experiment uses computer modeling technology to simulate the three-dimensional binding structure of terbinafine and VdPBP1, where D63, Y104 are the core heme binding sites of VdPBP1, which are the key action sites of terbinafine.

[0015] This invention provides a method for controlling cotton Verticillium wilt by using terbinafine to target the sterol-binding protein VdPBP1, which has the following beneficial effects: 1. This method of using terbinafine to target the sterol-binding protein VdPBP1 to control cotton Verticillium wilt elucidates the mechanism of action of acrylamide compounds in agriculture, providing theoretical support for precision control; while the EC50 of terbinafine... 50 The concentration is 10.8 ng / mL, which is much lower than that of shenqinmycin and benomyl, significantly reducing the dosage. Terbinafine's unit price is 30.4 yuan / g, which is also much lower than that of shenqinmycin and benomyl, making it more cost-effective for large-scale application. Moreover, terbinafine only binds to VdPBP1 in Verticillium dahliae, blocking its mediated electron transport. Existing agents such as shenqinmycin inhibit histone acetylation, and benomyl interferes with a wide range of microtubule targets, easily affecting non-target organisms. Furthermore, the ergosterol synthesis pathway is an essential pathway for fungi, and VdERG1 is a single-copy gene in Verticillium dahliae, making it difficult for pathogens to develop drug resistance through gene mutation. The control points are broad, covering the entire growth period. Attached Figure Description

[0016] VdCPRA This diagram illustrates the identification and developmental functional analysis of terbinafine-sensitive mutants in an application method of terbinafine targeting the sterol-binding protein VdPBP1 for the prevention and control of cotton Verticillium wilt according to the present invention. Figure 1 This is a diagram illustrating the ergosterol metabolism function of VdPBP1 and the terbinafine response pattern identification of an application method for controlling cotton Verticillium wilt using terbinafine targeting the sterol-binding protein VdPBP1. Figure 2 This diagram illustrates the interaction and expression analysis of VdPBP1 and VdERG1 in an application method of terbinafine targeting the sterol-binding protein VdPBP1 to control cotton Verticillium wilt according to the present invention. Figure 3 This is a diagram showing the electron transport function analysis of VdPBP1 in an application method of terbinafine targeting sterol-binding protein VdPBP1 to control cotton Verticillium wilt according to the present invention. Figure 4 This is a functional analysis diagram of the VdPBP1 hemoglobin binding site in an application method of terbinafine targeting the sterol-binding protein VdPBP1 to control cotton Verticillium wilt according to the present invention. Figure 5 This is a graph showing the pathogenicity of VdPBP1 and the control effect of terbinafine in an application method of terbinafine targeting sterol-binding protein VdPBP1 to control cotton Verticillium wilt according to the present invention. Figure 6 This is a schematic diagram of the steps in the application method of the present invention for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1. DETAILED DESCRIPTION

[0017] The embodiments of the present application will be further described in details below with reference to the accompanying drawings and examples. The following examples are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0018] As shown in Figure 7 The present application provides a technical solution: a method for preventing and treating cotton verticillium wilt by using terbinafine to target sterol binding protein VdPBP1, comprising the following application steps: S1, confirming the characteristics of terbinafine agent: Through plate antibacterial experiment, the inhibitory effect of commercially available fungicides on verticillium dahliae was compared to determine the half-inhibitory concentration EC 50 of terbinafine on the mycelial growth of verticillium dahliae, which is 10.8 ng / mL, significantly lower than 328.1 ng / mL of shenqinmycin and 496.6 ng / mL of benomyl, thereby clearly determining its high bacteriostatic property; S2, developing a terbinafine application scheme: For different stages of cotton verticillium wilt, two types of application methods are set: one is the prevention stage, 50 ng / mL of terbinafine is applied by root irrigation 7 days before cotton is inoculated with verticillium dahliae or at the same time as inoculation; the other is the treatment stage, 20 ng / mL of terbinafine is applied by root irrigation 7 days after cotton is infected with verticillium dahliae, that is, when the pathogen completes infection and colonization; S3, verification and guarantee of prevention and treatment effect: After application, high performance liquid chromatography (HPLC) detection is performed to confirm that the residual amount of terbinafine in cotton is 7.52~8.96 ng / g and does not affect the normal growth of cotton; at the same time, the symptoms of cotton verticillium wilt are counted, real-time fluorescent quantitative PCR (RT-qPCR) is used to detect the biomass of verticillium dahliae in cotton and calculate the disease index, the prevention and treatment effect of terbinafine on cotton verticillium wilt is verified, and finally the efficient prevention and control of cotton verticillium wilt is realized; The statistical standard of disease index is: 0 level, no disease spot, 1 level, disease spot accounts for ≤25% of leaf area, 2 level, disease spot accounts for 26%~50% of leaf area, 3 level, disease spot accounts for 51%~75% of leaf area, 4 level, disease spot accounts for >75% of leaf area or leaf wilting, disease index = Σ (disease level leaf number x corresponding disease level value) ÷ (total leaf number x highest disease level value) x 100; It also includes the following terbinafine and VdPBP1 action mechanism analysis process: S4, VdPBP1 target identification: Terbinafine-sensitive mutants are screened from verticillium dahliae, and isothermal asymmetric PCR is used to determine that the mutation site is located in Figures 1-7The gene was analyzed, and the protein encoded by the gene contained cytochrome b5 hormone and sterol binding domains and was conserved in pathogenic fungi. At the same time, the location of VdPBP1 in the cytoplasm of Verticillium dahliae spores and hyphae was confirmed by green fluorescent protein (GFP) labeling. VdPBP1 was preliminarily identified as a potential target of terbinafine. The gene encoding VdPBP1 VDAG_08647 Registered in GeneBank, its encoded protein's cytochrome b5 hormone and sterol-binding domains are key functional regions involved in electron transport; S5 and VdPBP1 Function Verification: Build VDAG_08647 Knockout strain Δ VdPBP1 and replenishment strain EC ΔVdPBP1 Compared with wild-type strains WT and Δ VdPBP1 and EC ΔVdPBP1 The growth phenotype revealed Δ VdPBP1 Growth and sporulation were significantly reduced, mycelial branching increased, chitin synthesis was impaired, and the fungus was only sensitive to terbinafine, showing no response to voriconazole. Δ was also detected. VdPBP1 The decrease in ergosterol content, the accumulation of the toxic intermediate squalene, and the rupture of cell membranes confirm that VdPBP1 is involved in the growth and development of Verticillium dahliae, ergosterol metabolism, and membrane homeostasis regulation. In verifying the sensitivity of VdPBP1 to terbinafine, each strain was cultured in Czapek medium. After culturing at 25 °C for 7 days, the colony diameter was measured and the inhibition rate was calculated to distinguish the differences in the response of the strains to terbinafine and voriconazole. Verification of S6, VdPBP1, and VdERG1: Fluorescent colocalization revealed that VdPBP1-GFP and VdERG1-mCherry, a key enzyme in ergosterol synthesis from Verticillium dahliae, colocalized near lipid droplets in spores and hyphae. Further experiments using yeast two-hybrid Y2H and bimolecular fluorescence complementary BiFC assays confirmed that VdPBP1 and VdERG1 directly interact in vitro and in vivo, with the interaction site located on the outer layer of lipid droplets. Simultaneously, Δ... VdPBP1 middle VdPBP1 The expression level of VdPBP1 was investigated, and it was found that the mRNA level did not change significantly, but the protein abundance decreased significantly, which clarified that VdPBP1 regulates the expression of VdERG1 protein through interaction. The fluorescence colocalization experiment of VdPBP1 and VdERG1 was observed using a laser confocal microscope, and the cell nuclei were labeled with DAPI staining. The scale bar was set to 10 μm. VdERG1 is squalene epoxidase; S7, VdPBP1 electronic transfer function verification: Based on the cytochrome b5 domain of VdPBP1, the exogenous addition of electron transport carrier ferriheme, observed Δ VdERG1 growth to resume; in Δ VdPBP1 overexpressing electron transport compensation genes VdPBP1 , found that the strain's sensitivity to terbinafine decreased from 40% to 25% ± 2%, and both treatments could increase the VdERG1 protein abundance and restore the ergosterol and squalene content, confirming that VdPBP1 activates VdERG1 expression by transferring electrons; , where the exogenous addition of ferriheme concentration is 20 μg / mL, after adding by measuring the growth of the strain in the medium containing terbinafine, to evaluate the effect of electron transport function recovery; S8, terbinafine and VdPBP1 binding mechanism analysis: Using molecular docking technology, it was found that the docking score of terbinafine and VdPBP1 was -6.565 kcal / mol, the binding region was the heme binding hydrophobic pocket of VdPBP1, forming 14 hydrogen bonds, with a binding distance of 1.5-3.9 Å, and the active center of terbinafine, methylamino (-N(CH3)), was close to the 95th aspartic acid of VdPBP1, namely D95; further mutating the heme binding site D63, Y104 of VdPBP1 to glycine (G) and phenylalanine (F) to construct a complementation strain, it was found that the strain could not restore the developmental defects and terbinafine resistance of Δ VdPBP1 , and the activation of VdERG1 was impaired, ultimately confirming that terbinafine blocks electron transport by occupying the heme binding domain of VdPBP1; , where the molecular docking experiment uses computer modeling technology to simulate the three-dimensional binding structure of terbinafine and VdPBP1, where D63 and Y104 are the core heme binding sites of VdPBP1 and are the key action sites of terbinafine; In this paper, VdPBP1: a L. digitatum progesterone binding protein, the corresponding encoding gene is L. digitatum VdPBP1 08647 , contains cytochrome b5 hormone and sterol binding domain, involved in pathogenic fungus ergosterol synthesis and electron transport; italicized represents the gene, and the positive body represents the protein; ERG1: squalene epoxidase, a key enzyme in the ergosterol synthesis pathway, catalyzes the conversion of squalene to squalene epoxide; italicized represents the gene, and the positive body represents the protein; EC 50 : half inhibitory concentration, refers to the concentration of the drug required to inhibit 50% of the growth of the pathogenic fungus, the lower the value, the stronger the antibacterial activity of the drug; Y2H: yeast two-hybrid, an experimental technique for detecting in vitro protein-protein interactions; BiFC: Bimolecular fluorescence complementation, an experimental technique used to detect protein-protein interactions in vivo; RT-qPCR: Real-time fluorescence quantitative polymerase chain reaction, used to quantitatively detect the transcription level of a gene; HPLC: High performance liquid chromatography, used to detect the content of compounds and metabolites in plants; Based on the above, the terbinafine and VdPBP1 action mechanism analysis process of steps S4-S8 is illustrated by examples: 1) Terbinafine is a potential high-efficiency compound for inhibiting L. maculans: Comparing the inhibition of L. maculans on the plate by commercially available control agents and terbinafine, it can be seen that the common pesticides, such as zineb and benomyl, show strong inhibitory ability at low concentrations, with EC 50 values of 328.1 and 496.6 ng / mL, respectively. Terbinafine has a more significant effect, with a half-inhibition rate of only 10.8 ng / mL. This result confirms that ergosterol synthesis inhibitors can be used as potential control agents for Verticillium wilt, and further exploring their targets and mechanisms is crucial. The specific details are shown in the following table, which shows the virulence regression equation and EC 50 values of common fungicides on the mycelial growth of L. maculans: 2) Development-related sterol-binding protein VdPBP1 responds to terbinafine: A sensitive mutant (#79, VDAG_ A-B) was obtained by screening the mutant library of L. maculans in response to terbinafine, a drug that blocks ergosterol synthesis. Isothermal asymmetric PCR technology was used to determine that the resistant hygromycin fragment was inserted at the 51st and 52nd bases of Figure 1 (GeneBank accession number); analysis of the protein encoded by this gene showed that it contains a cytochrome b5 hormone / sterol binding domain ( VDAG_08647 C), which is conserved in pathogenic fungi ( Figure 1 D), and its functional annotation is progesterone binding protein (PBP); using green fluorescent protein (GFP) labeling, it was observed that VdPBP1-GFP was located in the cytoplasm of spores and hyphae ( Figure 1 E); site-directed knockout (Δ Figure 1 ) and random insertion complementation (EC ΔVdPBP1 ) were performed on this gene, and the growth indicators of the resulting transformants were determined compared with the wild type. The results showed that the deletion of VdPBP1 led to a significant decrease in growth and sporulation ( VdPBP1F-H); hyphal microscopic structure observation and chitin staining results showed that the mutant had increased apical branching, significantly reduced normal smooth hyphae, and impaired chitin synthesis Figure 1 I-J), the above results showed that VdPBP1 was involved in the growth and development of V. dahliae and was a potential target of terbinafine; Figure 1 In the meantime, A. T-DNA mutant mutant#79 and wild type WT strain were tested for terbinafine sensitivity; B. Terbinafine sensitivity statistics; C. VdPBP1 domain disassembly; D. Phylogenetic tree of VdPBP1 and its fungal homologs based on protein sequence; E. VdPBP1-GFP subcellular localization observation, DAPI for nuclear staining, scale = 10 μm; F-H. WT, Figure 1 Knockout and complementation strains in PDA plate growth phenotype (F), colony diameter (G) and spore production statistics (H); I and J. WT, VdPBP1 Knockout and complementation strains hyphal morphology, chitin-calcium fluorescent white staining and normal development hyphae ratio, scale = 10 μm; 3) The expression of ergosterol synthesis positive regulator VdPBP1 was inhibited by terbinafine: To determine whether VdPBP1 specifically responds to terbinafine, another ergosterol synthesis inhibitor, voriconazole, was introduced, and the results of plate phenotype and inhibition rate statistics showed that, VdPBP1 The mutant was only sensitive to terbinafine ( VdPBP1 A-2C); by testing WT, Figure 2 The content of ergosterol metabolites (ergosterol) and intermediates (squalene) in knockout and complementation strains was found that, VdPBP1 The deletion of VdPBP1 led to a decrease in ergosterol and an increase in toxic intermediates ( VdPBP1 D and 2E); the changes in membrane components were Figure 2 The cell membrane of the mutant was ruptured, and the cytoplasm flowed out, producing a large number of vacuolar structures ( VdPBP1 F), the above results showed that VdPBP1 was a key factor in regulating ergosterol metabolism and membrane homeostasis of V. dahliae, to explore the response mechanism of VdPBP1, the transcription ( Figure 2 G) and protein level changes ( Figure 2 H) before and after terbinafine treatment were detected, the results showed that the inhibition level of VdPBP1 protein abundance was positively correlated with the concentration of terbinafine, in addition, overexpression Figure 2 Could not improve the tolerance of V. dahliae to terbinafine ( VdPBP1 I), indicating that the response of VdPBP1 to the compound depended on normal expression; Figure 2 In the meantime, A-C. Wild type WT, Figure 2Sensitivity determination of mutant and complemented strains to ergosterol synthesis inhibitors terbinafine and voriconazole; D and E. Detection of ergosterol and intermediate squalene content in the above strains; F. Transmission electron microscopy observation of hyphal structure and cell membrane of the above strains; scale bar = 500 nm; G and H. RT-qPCR and western blot analysis of VdPBP1 response patterns to terbinafine at the transcriptional and translational levels; I. VdPBP1 Sensitivity assay of overexpression strains to terbinafine; 4) VdPBP1 interacts with VdERG1 and regulates its expression: Since terbinafine is an inhibitor of fungal squalene epoxidase (ERG1), this study investigated the interaction and functional relationship between VdPBP1 and VdERG1. Fungal ERG1 proteins are mainly localized in lipid droplets (intracellular spherical energy storage structures of varying sizes) and the endoplasmic reticulum (extranuclear ring structures). Co-localization of these two proteins was observed using fluorescent labeling. The results showed that VdPBP1-GFP and VdERG1-mCherry were located near irregularly shaped spherical lipid droplets in spores and hyphae, with a small overlap in lipid droplets within hyphae. VdPBP1 A); Yeast two-hybrid (Y2H) and bimolecular fluorescence complementary (BiFC) assays confirmed that VdPBP1 and VdERG1 interact directly in vitro and in vivo, and the interaction site between Verticillium dahliae spores and hyphae is the outer layer of lipid droplets ( Figure 3 (B and 3C), therefore, the function of ERG1 is independent of its transcriptional level, and this is detected in the test. Figure 3 In mutants VdPBP1 The mRNA levels confirmed this. VdERG1 D); Further testing Figure 3 Changes in VdERG1 protein levels in the mutant strain showed that the protein abundance in the mutant strain was significantly lower than that in the wild-type WT strain after terbinafine treatment. VdPBP1 E), these results indicate that VdPBP1 mediates ergosterol synthesis by interacting with VdERG1 and regulating its protein expression; Figure 3 In the middle section, A. Microscopic observation of the fluorescence localization of the VdPBP1-GFP and VdERG1-mCherry fluorescent protein fusion strain, scale bar = 10 μm; B and C. Functional analysis of yeast two-hybrid and bimolecular fluorescence complementary interactions of VdPBP1 and VdERG1; D and E. Figure 3 Analysis of the impact of deletion on VdERG1 at the transcriptional and translational levels; 5) VdPBP1 transfers electrons to activate VdERG1 expression: Domain display VdPBP1 contains cytochrome b5-like domain, which is involved in electron transfer process of cytochrome b5 reductase system in eukaryotes, to verify its electron transfer function, first exogenous add electron transfer carrier-haematin (20 μg / mL), plate phenotype can be seen WT, Δ VdPBP1 and EC ΔVdPBP1 strains can improve the growth after adding haematin, while adding haematin and terbinafine can significantly reduce the sensitivity of Δ VdPBP1 mutant (restored to wild type WT and complemented EC ΔVdPBP1 strain level; VdPBP1 A and 4B), in yeast and aspergillus, the activity of ERG1 is directly regulated by terbinafine, when electron transfer is blocked, cytochrome P450 reductase cprA will partially compensate for the defect ( Figure 4 C); RT-qPCR detection shows, Figure 4 unrelated to VdPBP1 ( VdCPRA D), to determine that the decline in protein expression caused by the blockage of ERG1 electron transfer, followed by overexpression of Figure 4 (Δ VdCPRA _OE VdCPRA ) in the mutant, the results show that the overexpression strain reduces the sensitivity to terbinafine from 40% to about 25% ( VdPBP1 E and 4F); further through western blot detection of protein expression, the results show that adding haematin and overexpression Figure 4 can improve the abundance of VdERG1 ( VdCPRA G), similarly, the two methods of promoting electron transfer basically restore the content of ergosterol synthesis intermediates and products ( Figure 4 H and 4I), the above results show that VdPBP1 is activated by transferring electrons to VdERG1 to activate its expression and participate in the regulation of ergosterol synthesis; Figure 4 A and B. Wild type WT, Figure 4 mutant and complemented strains were determined for their sensitivity to terbinafine in the presence of the electron transfer carrier-haematin; C. Schematic diagram of fungal ergosterol synthesis pathway electron transfer and terbinafine action; D. Gene expression detection in wild type WT, VdPBP1 mutant and complemented strains VdPBP1 E and F. VdCPRA overexpression of VdPBP1 strains for their sensitivity to terbinafine; G. Protein abundance of VdERG1 in wild type WT and VdCPRA mutant when adding haematin and overexpression VdCPRA H and I. Adding haematin and overexpressionVdPBP1 Ergosterol and squalene content determination of strains; 6) Terbinafine targets the heme-binding domain of VdPBP1 to inhibit electron transport: To explore the physical space binding ability of VdPBP1 and terbinafine, computer modeling was used to fit the molecular binding effect of the two. The docking score was -6.565 kcal / mol, indicating strong interaction. The docking region of the compound was mainly concentrated in the hydrophobic pocket of VdPBP1 used for binding heme molecules, forming 14 hydrogen bond forces with a binding distance of 1.5-3.9 Å (Fig. VdCPRA A), in which the naphthyl (-C10H8) and methyl (-CH3) at both ends of the compound were close to the two heme binding sites of VdPBP1 at 63 aspartic acid (D63) and 104 tyrosine (Y104); the active center of terbinafine, methylamino (-N(CH3)), was next to 95 aspartic acid (D95, distance 3.5 Å) (Fig. Figure 5 A). To determine the effects of the two heme binding sites on the expression of VdPBP1, interaction with VdERG1, and electron transport functions, glycine G and phenylalanine F were used to substitute D63 and Y104 to construct back-up strains. Subcellular localization results showed that key site mutations did not change the cytoplasmic localization of VdPBP1 (Fig. Figure 5 B). Further verification of the effects of site mutations on the interaction of VdPBP1 with VdERG1 showed that in vivo and in vitro tests (Y2H and BiFC) also indicated that VdPBP1 could still directly interact with VdERG1 after site mutation (Fig. Figure 5 C and 5D). Growth phenotypes and sensitivity to terbinafine of back-up strains showed that the developmental and terbinafine tolerance defects of the Figure 5 mutants could not be restored VdPBP1 (Fig. VdPBP1 E). The heme-binding pocket is a key region for cytochrome b5-like protein electron transport, and western blot detection confirmed that VdPBP1 with site mutations damaged the electron transport of VdERG1 (Fig. Figure 5 F). The above results indicate that terbinafine inhibits the activity and electron transport function of VdPBP1 by occupying its heme-binding region; Figure 5 A. Construction of a three-dimensional molecular docking model of VdPBP1 and terbinafine (the heme-binding sites of D63 and Y104 are marked with stars; the yellow dashed line is the binding distance; D95 is the active group binding site of terbinafine); B. Subcellular localization observation of VdPBP1 with heme-binding site mutations, scale bar = 10 μm; C and D. Yeast two-hybrid and bimolecular fluorescence complementation interaction function analysis of VdPBP1 with heme-binding site mutations and VdERG1; E. Growth phenotype of VdPBP1 with heme-binding site mutations; F. Sensitivity of VdPBP1 with heme-binding site mutations to terbinafine.Figure 5 Terbinafine response phenotype of the complement strain; F. western blot detection of hemoglobin binding site mutations VdPBP1 VdERG1 protein levels in the reinjected strains; 7) VdPBP1 is a potential target for the control of Verticillium dahliae (corresponding to steps S1-S3 above): Based on the above analysis of the inhibitory mechanism of terbinafine on VdPBP1 in Verticillium dahliae, and to explore its application prospects, the pathogenic function of VdPBP1 was first tested. Wild-type, knockout, and complement strains were simultaneously inoculated. On day 21, VdPBP1 The mutant showed only mild Verticillium wilt symptoms on a few leaves, while the wild-type and the replacement strains were severely affected and even died. VdPBP1 A) The biomass of pathogens in cotton is consistent with the plant phenotype. Figure 6 The level of the mutant in cotton was significantly lower than that in the wild-type and complement strain inoculation groups. VdPBP1 B), to determine the role of VdPBP1-mediated electron transport in the pathogenesis process, a hypothesis was established. Figure 6 The mutant was supplemented with heme and inoculated with an overexpressing strain (Δ). VdPBP1 _OE VdCPRA In both treatment groups, the cotton phenotype confirmed that electron transport compensation could be partially restored. VdPBP1 The virulence of mutants ( VdPBP1 C and 6D), and then the efficacy of the compounds against cotton Verticillium wilt was identified. The control group was divided into two groups: a prevention group (50 ng / mL) treated with terbinafine inoculated with *Verticillium dahliae* 7 days in advance (Terbinafine 7 dpi + WT) and a treatment group (Terbinafine 0 dpi + WT) treated 7 days after *Verticillium dahliae* inoculation (WT 7 dpi + Terbinafine, concentration 20 ng / mL). Pathogenicity was observed. The results showed that terbinafine could be absorbed by cotton and retained in the body, with concentrations ranging from 7.52 to 8.96 ng / g cotton. Figure 6 E), and the drug treatment does not affect cotton growth. In terms of disease severity, terbinafine has a good control effect, and both early prevention and treatment after infection significantly reduce the symptoms. Figure 6 F and 6G); pathogen biomass detection and disease index statistics also conformed to the pathogenesis phenotype (F and 6G); Figure 6 (H and 6I), the above results indicate that terbinafine can be used as a VdPBP1 electron transport inhibitor in the development of a cotton Verticillium wilt control technology system; Among them, A and B are wild-type WT, Figure 6 Determination of pathogenicity phenotypes and pathogen colonization biomass in cotton by mutant and complement strains; C and D. VdPBP1 The mutant was supplemented with heme and overexpressed.VdPBP1 VdCPRA Pathogenicity recovery phenotype analysis; E. HPLC detection of terbinafine content in cotton in vivo; F and G. Analysis of the preventive and therapeutic effects of terbinafine on cotton Verticillium wilt; H and I. Statistics of pathogenic colonization biomass, disease occurrence grade and disease index of cotton inoculated with the above strains; Based on the above description, the application analyzes the mechanism of action of propylene amine compounds in the field of agriculture, providing theoretical support for precise prevention and control; and the EC 50 is 10.8 ng / mL, which is much lower than that of shenzixin (328.1 ng / mL) and benomyl (496.6 ng / mL), can greatly reduce the dosage of the pesticide, and the unit price of terbinafine is 30.4 yuan / g (152 yuan / 5g), which is much lower than that of shenzixin (452 yuan / g) and benomyl (62 yuan / g), and the cost of large-scale application is lower. Moreover, terbinafine only binds to VdPBP1 of L. digitata, blocks the electron transfer mediated by it, while existing pesticides such as shenzixin and benomyl have a wide range of targets for inhibiting histone acetylation and interfering with microtubulin protein, respectively, which can easily affect non-target organisms. In addition, the ergosterol synthesis pathway is an essential pathway for fungi, and VdERG1 is a single copy gene in L. digitata, so it is difficult for the pathogen to develop drug resistance through gene mutation, and the prevention and control node covers the whole growth period.

[0019] The embodiments of the present application are given for the purpose of illustration and description, and are not exhaustive or limit the present application to the disclosed form. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are chosen and described in order to better illustrate the principles and practical application of the present application, and to enable those of ordinary skill in the art to understand the present application so as to design various embodiments with various modifications for specific uses.

Claims

1. A method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1, characterized in that: The following application steps are included: S1. Confirmation of Terbinafine's pharmaceutical properties: By conducting plate inhibition experiments, the inhibitory effects of commercially available fungicides on *Verticillium dahliae* were compared, and the half-maximal inhibitory concentration (EC5) of terbinafine on *Verticillium dahliae* mycelial growth was determined. 50 The concentration was 10.8 ng / mL, significantly lower than that of shenqinmycin (328.1 ng / mL) and benomyl (496.6 ng / mL), thus confirming its highly effective antibacterial properties; S2. Develop a terbinafine administration plan: Two application methods were set for different stages of cotton Verticillium wilt: the first is the prevention stage, in which terbinafine at a concentration of 50 ng / mL is applied by root drenching 7 days before or at the same time as the inoculation of cotton with Verticillium dahliae; the second is the treatment stage, in which terbinafine at a concentration of 20 ng / mL is applied by root drenching 7 days after cotton is infected with Verticillium dahliae, that is, when the pathogen has completed infection and colonization. S3. Verification and assurance of prevention and control effectiveness: After application, high-performance liquid chromatography (HPLC) confirmed that the residual amount of terbinafine in cotton was 7.52~8.96 ng / g, and it did not affect the normal growth of cotton. At the same time, the symptoms of cotton Verticillium wilt were counted, and the biomass of Verticillium dahliae in cotton was detected by real-time quantitative PCR (RT-qPCR) and the disease index was calculated to verify the control effect of terbinafine on cotton Verticillium wilt, and finally achieve efficient control of cotton Verticillium wilt.

2. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 1, characterized in that: In step S3, the statistical standards for the disease index are as follows: Level 0, no lesions; Level 1, lesions covering ≤25% of the leaf area; Level 2, lesions covering 26%~50% of the leaf area; Level 3, lesions covering 51%~75% of the leaf area; Level 4, lesions covering >75% of the leaf area or leaf wilting. The disease index is calculated as Σ(number of diseased leaves × corresponding disease level value) ÷ (total number of leaves × highest disease level value) × 100.

3. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 1, characterized in that: This also includes the following mechanism of action analysis of terbinafine and VdPBP1: S4 and VdPBP1 target identification: Terbinafine-sensitive mutants were screened from *Verticillium dahliae*, and the mutation site was determined by isothermal asymmetric PCR. VDAG_08647 The gene was analyzed, and the protein encoded by the gene contained cytochrome b5 hormone and sterol binding domains and was conserved in pathogenic fungi. At the same time, the location of VdPBP1 in the cytoplasm of Verticillium dahliae spores and hyphae was confirmed by green fluorescent protein (GFP) labeling. VdPBP1 was preliminarily identified as a potential target of terbinafine. The gene encoding VdPBP1 VDAG_08647 Registered in GeneBank, its cytochrome b5 hormone and sterol-binding domains, which encode proteins, are key functional regions involved in electron transport.

4. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 3, characterized in that: This also includes the following mechanism of action analysis of terbinafine and VdPBP1: S5 and VdPBP1 Function Verification: Build VdPBP1 Knockout strain Δ VdPBP1 and replenishment strain EC ΔVdPBP1 Compared with wild-type strains WT and Δ VdPBP1 and EC ΔVdPBP1 The growth phenotype revealed Δ VdPBP1 Growth and sporulation were significantly reduced, mycelial branching increased, chitin synthesis was impaired, and the fungus was only sensitive to terbinafine, showing no response to voriconazole. Δ was also detected. VdPBP1 The decrease in ergosterol content, the accumulation of the toxic intermediate squalene, and the rupture of cell membranes confirm that VdPBP1 is involved in the growth and development of Verticillium dahliae, ergosterol metabolism, and membrane homeostasis regulation. In verifying the sensitivity of VdPBP1 to terbinafine, each strain was cultured in Czapek medium. After culturing at 25 °C for 7 days, the colony diameter was measured and the inhibition rate was calculated to distinguish the differences in the response of the strains to terbinafine and voriconazole.

5. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 4, characterized in that: This also includes the following mechanism of action analysis of terbinafine and VdPBP1: Verification of S6, VdPBP1, and VdERG1: Fluorescent colocalization revealed that VdPBP1-GFP and VdERG1-mCherry, a key enzyme in ergosterol synthesis from Verticillium dahliae, colocalized near lipid droplets in spores and hyphae. Further experiments using yeast two-hybrid Y2H and bimolecular fluorescence complementary BiFC assays confirmed that VdPBP1 and VdERG1 directly interact in vitro and in vivo, with the interaction site located on the outer layer of lipid droplets. Simultaneously, Δ... VdPBP1 middle VdERG1 The expression level of VdPBP1 was investigated, and it was found that the mRNA level did not change significantly, but the protein abundance decreased significantly, which clarified that VdPBP1 regulates the expression of VdERG1 protein through interaction. VdERG1 is squalene epoxidase.

6. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 5, characterized in that: This also includes the following mechanism of action analysis of terbinafine and VdPBP1: S7, VdPBP1 electronic transfer function verification: Based on the cytochrome b5 domain of VdPBP1, the exogenous addition of the electron transport carrier heme resulted in the observation of Δ VdPBP1 Growth was restored; in Δ VdPBP1 Overexpression of electron transport compensation gene VdCPRA The results showed that the sensitivity of the strain to terbinafine decreased from 40% to 25% ± 2%, and both treatments could increase the abundance of VdERG1 protein and restore the content of ergosterol and squalene, confirming that VdPBP1 activates VdERG1 expression by transferring electrons.

7. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 6, characterized in that: This also includes the following mechanism of action analysis of terbinafine and VdPBP1: Analysis of the binding mechanism between S8, terbinafine, and VdPBP1: Using molecular docking technology, the docking score of terbinafine with VdPBP1 was found to be -6.565 kcal / mol. The binding region is the heme-binding hydrophobic pocket of VdPBP1, forming 14 hydrogen bonds with a binding distance of 1.5–3.9 Å. Furthermore, the methylamino group (-N(CH3)) at the active site of terbinafine is close to the aspartic acid at position 95 of VdPBP1, i.e., D95. Further mutation of the heme-binding sites D63 and Y104 of VdPBP1 to glycine (G) and phenylalanine (F) to construct a complement strain revealed that this strain could not restore the Δ... VdPBP1 The developmental defects and tolerance to terbinafine, as well as impaired VdERG1 activation, ultimately clarified that terbinafine blocks electron transport by occupying the heme-binding domain of VdPBP1.

8. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 5, characterized in that: In step S6, the fluorescence colocalization experiment of VdPBP1 and VdERG1 was observed using a laser confocal microscope, and the cell nuclei were stained with DAPI. The scale bar was set to 10 μm.

9. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 6, characterized in that: In step S7, the concentration of exogenously added heme is 20 μg / mL. After addition, the growth of the strain on a culture medium containing terbinafine is measured to evaluate the recovery effect of electron transport function.

10. The method for controlling cotton Verticillium wilt by using terbinafine to target sterol-binding protein VdPBP1 according to claim 7, characterized in that: In step S8, the molecular docking experiment uses computer modeling technology to simulate the three-dimensional binding structure of terbinafine and VdPBP1, where D63 and Y104 are the core heme binding sites of VdPBP1, which are the key sites of action of terbinafine.

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