Method for preventing and treating plant fusarium wilt by scopolin and antibacterial protein ckpgip1

Abstract

The application discloses a new method for preventing and treating plant verticillium wilt by scopolamine and bacteriostatic protein CkPGIP1. The method adopts scopolamine and / or protein CkPGIP1 as bacteriostatic agents, and is applied to plants. The nucleotide sequence of the protein CkPGIP1 is shown as SEQ ID No. 1, and the amino acid sequence of the protein CkPGIP1 is shown as SEQ ID No. 2. Scopolamine and / or bacteriostatic protein CkPGIP1 can inhibit the growth of verticillium dahliae in culture medium and gossypium hirsutum plants. Especially, the combination of scopolamine and CkPGIP1 bacteriostatic protein has a very significant inhibitory effect on cotton verticillium wilt, and is expected to become a safe, green and efficient bactericide.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically involving a new method for controlling Verticillium wilt in plants using scopolamine and the antibacterial protein CkPGIP1. Background Technology

[0002] Cotton is an important economic crop in my country. Verticillium dahliae Kleb., a fungus causing cotton wilt, is known as "cotton cancer." Severe cases can lead to complete leaf drop, resulting in decreased cotton quality and yield. Verticillium dahliae is a semi-living, soil-borne pathogen that infects the host through the roots, proliferates, and colonizes the plant's vascular system, causing symptoms such as wilting, yellowing leaves, cell death, stunted growth, and premature aging. Many crops, including cotton, sunflower, eggplant, pepper, and tomato, are susceptible to Verticillium dahliae infection, leading to Verticillium wilt and significant economic losses. Therefore, there is an urgent need for new, residue-free, safe, and environmentally friendly methods for controlling Verticillium dahliae.

[0003] Cynanchum komarovii is a desert plant that has survived for a long time by adapting to extremely arid and barren desert environments. In traditional Chinese medicine, it is used for disease and insect control in farmland and also has analgesic, anti-inflammatory, antitussive, and expectorant effects. Domestic and international research papers have reported that the total alkaloids in Cynanchum komarovii have insecticidal and antibacterial activities, but there are few reports on its antibacterial proteins. Polygalacturonase inhibitors (PGIPs) are proteins rich in leucine repeats, belonging to plant defense proteins, and inhibit polygalacturonase (PG), a key virulence factor in pathogens. CkPGIP1 protein from the desert plant Cynanchum komarovii was isolated and purified to obtain an inhibitor of the pathogen, CkPGIP1, which significantly inhibited the pathogen PG (Liu N, Ma X, Zhou S, Wang P, Sun Y, Li X, Hou Y. Molecular and Functional Characterization of a Polygalacturonase-Inhibiting Protein from Cynanchum komarovii That ConfersFungal Resistance in Arabidopsis. PLoS One. 2016 Jan 11;11(1):e0146959.).

[0004] Scopolamine has a coumarin core structure of benzopyranone, and its chemical name is 7-hydroxy-6-methoxycoumarin. Its chemical structural formula is shown below. Figure 1Scopolamine is synthesized via a phenylpropane metabolic pathway and is found in the roots, stems, and leaves of higher plants such as Scopolia japonica Maxim and Herba Atropea Belladonnae, belonging to the Apiaceae, Rutaceae, Moraceae, Thymelaeaceae, and Solanaceae families (Antika LD, Tasfiyati AN, Hikmat H, Septama AW. Scopolamine: a review of its source, biosynthesis, methods of extraction, and pharmacochemical activities[J]. Zeitschrift fur Naturforschung.C, Journal of biosciences, 2022, 77(7-8):303-316). Scopolamine possesses various pharmacological activities, including regulating blood pressure, exhibiting antibacterial activity, and being used to treat bronchial diseases and asthma, as well as regulating hormone balance. Currently, it has attracted attention both domestically and internationally (He BT, Liu ZH, Li BZ, Yuan YJ. Advances in biosynthesis of scopoletin[J]. Microbial Cell Factories, 2022, 21(1):152). In recent years, studies have found that scopoletin not only has wide applications in medicine, but also plays a positive role in the control of pests, which is reflected in its agricultural biological activity. Scopoletin not only has insecticidal and acaricidal activities, but also has significant antibacterial activity.Scopolamine has a significant inhibitory effect on Fusobacterium fusiformis, Fusobacterium semitectum, and Alternaria alternata; 0.5 mg / mL of scopolamine can completely inhibit the spore germination and mycelial growth of Botrytis cinerea (El Oirdi M, Trapani A, Bouarab K. The nature of tobacco resistance against Botrytis cinerea depends on the infection structures of the pathogen[J]. Environmental microbiology, 2010, 12(1):239-253), and effectively inhibit the mycelial growth of Rhizopus stolonifer and Lasiodiplodia theobromae (Peterson JK, Harrison HF, Jackson DM. Biological activities and contents of scopolin and scopolamine). in sweetpotato clones[J].Hort Science,2003,38(6):1129-1133).

[0005] While the aforementioned existing technologies have disclosed that scopolamine and protein CkPGIP1 have inhibitory effects on certain plant pathogens, the inhibitory activity of scopolamine and CkPGIP1 against *Verticillium dahliae*, which causes Verticillium wilt, is unpredictable due to the unique biological species of *Verticillium dahliae*. Therefore, further research and demonstration are needed on how to apply scopolamine and CkPGIP1 to the control of *Verticillium wilt*. Summary of the Invention

[0006] The purpose of this invention is to provide a new method for controlling plant Verticillium wilt by using scopolamine and the antibacterial protein CkPGIP1.

[0007] According to a first aspect of the present invention, a method for controlling Verticillium wilt in plants is provided, comprising applying scopolamine and / or protein CkPGIP1 as antifungal agents to the plants. The nucleotide sequence of the protein CkPGIP1 is shown in SEQ ID No. 1, and the amino acid sequence of the protein CkPGIP1 is shown in SEQ ID No. 2.

[0008] Specifically, scopolamine and / or protein CkPGIP1 are prepared into a solution and sprayed onto the plants. Methanol is preferred as the solvent. The concentration of scopolamine is 10-100 μg / mL, and the concentration of protein CkPGIP1 is 50-200 μg / mL.

[0009] Preferably, a combination of scopolamine and protein CkPGIP1 is used as the antibacterial agent. Further, in the composition, the concentration of scopolamine is 50-100 μg / mL, and the concentration of protein CkPGIP1 is 50-100 μg / mL.

[0010] In specific cases, the plants mentioned include, but are not limited to, cotton, sunflower, eggplant, pepper, and tomato.

[0011] According to a second aspect of the present invention, the present invention provides a fungicide for controlling Verticillium wilt in plants, wherein the active ingredient of the fungicide is scopolamine and / or protein CkPGIP1, the nucleotide sequence of protein CkPGIP1 is shown in SEQ ID No. 1, and the amino acid sequence of protein CkPGIP1 is shown in SEQ ID No. 2.

[0012] Specifically, the preparation method of the protein CkPGIP1 includes the following steps:

[0013] (1) Construct a recombinant prokaryotic expression vector, wherein the recombinant prokaryotic expression vector contains the sequence shown in SEQ ID No. 1;

[0014] (2) Transform the recombinant prokaryotic expression vector into bacteria to prepare the CkPGIP1 expression system;

[0015] (3) Inducing the expression of the antibacterial protein CkPGIP1 in the CkPGIP1 expression system.

[0016] This invention first discovered that scopolamine, or the antibacterial protein CkPGIP1 derived from *Cynanchum komarovii*, can inhibit the mycelial growth and spore germination of *Verticillium wilt*, exhibiting significant antibacterial activity. The protein CkPGIP1 significantly inhibits the activity of polygalacturonase (PG) in *Verticillium wilt*, a major enzyme that degrades pectin in plant cell walls and is an important pathogenic factor. CkPGIP1 limits the proliferation and spread of the pathogen by inhibiting the key virulence factor PG. Scopolamine, as a plant-secreted phytoalexin, rapidly accumulates at the infection site when infected by *Verticillium wilt*, disrupting the cell membrane structure of the pathogen, causing cellular energy metabolism disorders, inhibiting the growth and germination of the pathogen, and playing a role in chemical defense. This invention further discovers that the antibacterial effect of the combination of scopolamine and the antibacterial protein CkPGIP1 is stronger than that of the monomer. Through the synergistic effect of their antibacterial mechanisms, a new economical and efficient method is provided for the prevention and control of cotton Verticillium wilt.

[0017] This invention provides a novel method for controlling Verticillium wilt in plants using scopolamine and the antibacterial protein CkPGIP1. Scopolamine and / or the antibacterial protein CkPGIP1 inhibit the growth of Verticillium wilt pathogens in both culture media and upland cotton plants. In particular, the combination of scopolamine and the CkPGIP1 antibacterial protein exhibits a highly significant inhibitory effect on Verticillium wilt, making it an effective control method for the disease. Furthermore, scopolamine in this composition is abundant in nature, present in high concentrations in plants, has low toxicity, is easily degraded, and is safe for the ecological environment. It can also be synthesized artificially, making it low-cost and easily applicable for large-scale industrial production. This has profound significance for the long-term development of the ecological environment and the integrated management of diseases. Attached Figure Description

[0018] Figure 1 The structural formula of scopolamine is given.

[0019] Figure 2 A schematic diagram of the construction of the prokaryotic expression vector pET-32a-CkPGIP1.

[0020] Figure 3 SDS-PAGE electrophoresis pattern for purifying prokaryotic expression of CkPGIP1.

[0021] Figure 4 Photographs showing the inhibitory activity of CKPGIP1 measured by agarose diffusion assay.

[0022] Figure 5 Photographs showing the effects of scopolamine, antimicrobial protein CkPGIP1, and a combination of scopolamine and antimicrobial protein CkPGIP1 on the mycelial growth of Verticillium wilt in culture medium.

[0023] Figure 6 Photographs showing the effects of scopolamine, antibacterial protein CkPGIP1, and a combination of scopolamine and antibacterial protein CkPGIP1 on the germination of Verticillium wilt spores.

[0024] Figure 7 This is a statistical chart showing the inhibition rate of scopolamine, antibacterial protein CkPGIP1, and their combination on the mycelial growth of Verticillium wilt in cotton in culture medium.

[0025] Figure 8 Photographs taken on day 10 of an experiment on upland cotton plants to inhibit Verticillium wilt, an antibacterial protein CkPGIP1, and their combination.

[0026] Figure 9 A statistical chart showing the disease index of scopolamine, antibacterial protein CkPGIP1 and their combination on upland cotton plants inhibiting Verticillium wilt. Specific Implementation

[0027] The technical solution of the present invention is illustrated below through specific embodiments. The embodiments described in this art are helpful in understanding the present invention and should not be considered as specific limitations thereof.

[0028] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0029] The terms “comprising” and “having”, and any variations thereof, are intended to cover non-exclusive inclusion.

[0030] For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products, or devices.

[0031] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0032] Unless otherwise stated, the experimental methods used in the embodiments of the present invention are all traditional methods such as plant protection, botany, microbiology and plant pathology, and all reagents and consumables used in the embodiments are commercially available products.

[0033] The following are specific examples.

[0034] Example 1: Construction of a prokaryotic expression vector to prepare CkPGIP1 protein

[0035] Design upstream and downstream primers based on the target gene sequence CkPGIP1:

[0036] CkPGIP-F:5`-CGCGGATCCTGCCACTAAAAAAGAAAAGTGC-3`

[0037] CkPGIP-R:5`-TCGCTCGAGCTTGCAAGAAGGCAAAGGA-3`

[0038] The prokaryotic expression vector pET-32a and the CkPGIP1 gene with restriction sites were double-digested with BamHI and XhoI, respectively. The resulting DNA fragments were ligated using T4 ligase. The ligation products were transformed into E. coli strain DH5α, and recombinants were screened using LB plates containing Amp (50 μg / mL). The vector construction procedure is described below. Figure 2 After plasmid digestion and PCR identification, the correctly ligated recombinants were sequenced, confirming that the DNA sequence of the inserted vector was in the correct reading frame. The expression vector containing the CkPGIP1 gene was constructed and named pET-32a-CkPGIP1. The pET-32a-CkPGIP1 plasmid was extracted and transformed into the BL21(DE3) expression strain to obtain pET-

[0039] BL21(DE3) strain containing plasmid pET-32a-CkPGIP1 was inoculated into 3 mL of LB medium containing Amp (50 μg / mL) and cultured overnight. 1% of the overnight culture was then inoculated into 100 mL of LB medium containing Amp (50 μg / mL) and cultured at 37°C for at least 2 hours until mid-logarithmic growth (OD50). 600=0.6). Add 500 μL of 0.1 M IPTG solution to the culture to a final concentration of 0.5 mmol / L, and incubate overnight at 16°C and 220 rpm. Centrifuge the overnight culture at 7500 rpm for 10 min to collect the cells, and resuspend the cells in 20 mL of PBS (pH 7.5) buffer. Disrupt the cells using an ultrasonic cell disruptor with the following program: 3 sec on, 3 sec off, 100 W, 120 cycles. Centrifuge the disrupted cell suspension at 10000 rpm at 4°C for 15 min, and collect the supernatant as the crude protein enzyme solution. Then, obtain purified protein by His-Tag affinity chromatography. Dilute the bacterial lysate with Binding Buffer and load it onto the column, collecting the flow-through. Wash the column with 15 column volumes of Binding Buffer to remove contaminating proteins. Elute with an appropriate amount of Elution Buffer and collect the elution peak. Protein concentration was determined spectrophotometrically, and the purity and size of the protein were detected by SDS-PAGE (polyacrylamide gel electrophoresis). Figure 3 The results showed that the purified fusion protein band was single, consistent with the theoretical size of 35.2 kDa. The purified CkPGIP1 protein was as follows: Figure 3 As shown, M is the protein molecular weight standard marker, lane 1 is the total protein from recombinant CkPGIP1 expression, lane 2 is the protein eluted from the Ni-IDA column, and lane 3 is the tag-removed purified CkPGIP1.

[0040] Example 2: Inhibitory activity of antibacterial protein CkPGIP1 and agarose plate diffusion experiment

[0041] PGIP activity was determined using DNS reagent by reducing the terminal groups. The determination was performed using a reaction mixture containing 0.8% agarose and 0.5% polygalacturonic acid (Wang X, Zhu X, Tooley P, Zhang X. Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens[J]. Plant molecular biology, 2013, 81(4-5):379-400). After adding VdPG1 to agarose plates, PGIP was optionally added or omitted. The mixture was incubated at 30°C for 12 h, then the agarose plates were stained with 0.05% ruthenium red and finally thoroughly washed with sterile water. PG activity was expressed in agarose diffusion units, with a ring of 0.5 cm radius defined as one agarose diffusion unit. One unit of PGIP activity was defined as the amount of PGIP required to reduce PG activity by 50%. The inhibitory activity of PGIP was determined using a mixture containing 0.0011 reducing units. The agarose diffusion of polygalacturonase from Verticillium wilt after the addition of purified CkPGIP1 protein is shown in the following figures: a, 25 μL enzyme; b, 25 μL enzyme + 15 μg CkPGIP1; c, 25 μL enzyme + 25 μL phosphate-buffered saline; d, 25 μL enzyme + 15 μg heat-denatured CkPGIP1. (See...) Figure 4 Inhibitory activity is inversely proportional to ring size, and the degree of inhibition is indicated by the reduction in ring size. The results show that CkPGIP1 can effectively inhibit the activity of VdPG1, with an IC50 concentration of [missing value]. 50 It was 145.53 μg / mL.

[0042] Example 3: Test on the inhibition of Verticillium wilt of cotton by scopolamine, antibacterial protein CkPGIP1 and their combination in PDA medium.

[0043] 1. The reagents used in the experiment, methanol and scopolamine, were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0044] 2. Preparation of culture medium:

[0045] (1) The culture medium used in the experiment was potato glucose agar medium. The preparation method was as follows: 200g of peeled potatoes were cut into pieces, boiled in water until soft, filtered through gauze, and 20g of agar powder and 8g of glucose were added to the filtrate. The volume was adjusted to 1000mL, sterilized at high temperature, cooled to 50℃, poured into petri dishes, and cooled and solidified into plates for later use.

[0046] (2) Methanol (negative control group), scopolamine, antimicrobial protein CkPGIP1, and a combination of scopolamine and antimicrobial protein CkPGIP1 were added to potato dextrose agar medium, respectively. The scopolamine used was dissolved in methanol, so methanol was used as a negative control.

[0047] The working concentrations of each additive in the potato dextrose agar medium were as follows: Negative control: methanol, scopolamine (100 μg / mL), antimicrobial protein CkPGIP1 (100 μg / mL), scopolamine (50 μg / mL) + antimicrobial protein CkPGIP1 (50 μg / mL).

[0048] 3. Antibacterial test in culture medium: 4 mm cotton Verticillium wilt pathogen blocks were inoculated into the prepared culture medium. Inhibition rates were continuously recorded and calculated from day 0 to day 5 post-inoculation. The inhibition rate was used to evaluate the inhibitory activity of each drug group against cotton Verticillium wilt.

[0049] This experiment set up 3 biological replicates, and each replicate contained 3 parallel experiments as technical replicates. The growth of cotton Verticillium wilt pathogens in each experimental group and control group was randomly selected for photographing.

[0050] The results are as follows Figure 5 , 6 As shown in the figure, both scopolamine and the antibacterial protein CkPGIP1 can inhibit the growth of Verticillium wilt in cotton. However, the combination of scopolamine and CkPGIP1 has a better inhibitory effect on the growth of Verticillium wilt, and the amount of Verticillium wilt plaque is reduced. This indicates that the combination of scopolamine and CkPGIP1 can inhibit the mycelial growth of Verticillium wilt in cotton in the culture medium, and the inhibitory effect of this combination is significantly higher than that of scopolamine and CkPGIP1 alone. Figure 7 ).

[0051] Example 4: Application of scopolamine, antibacterial protein CkPGIP1, and their combination in inhibiting Verticillium wilt in upland cotton plants.

[0052] The upland cotton material used in the experiment was "Zhongzhimian No. 2". A suspension of Verticillium wilt spores was inoculated onto the cotton plants using the stem-piercing method, with a spore concentration of 1×10⁻⁶. 6Spray the cotton plants inoculated with Verticillium wilt spores at a concentration of 1 spore / mL with the following solutions: Negative control: methanol, scopolamine (100 μg / mL), antimicrobial protein CkPGIP1 (100 μg / mL), and a combination of scopolamine (50 μg / mL) + antimicrobial protein CkPGIP1 (50 μg / mL). Each group consisted of 60 cotton seedlings, and 10 mL of solution was sprayed until the plants were completely moistened. The disease index of each group of cotton seedlings was recorded. The severity of disease symptoms on each cotton seedling was assessed using a 0-4 scale. Grade 0: Healthy plant, no wilting or yellowing symptoms observed; Grade 1: One or two cotyledons yellow and wilted; Grade 2: One true leaf wilted or fell off; Grade 3: Two true leaves wilted or fell off; Grade 4: Entire plant dead or all true leaves fallen off.

[0053] Disease Index (DI) = [∑(Disease grade × Number of diseased plants at each grade) / (Total number of surveyed plants × Highest disease grade)] × 100.

[0054] Three biological replicates were set up, and three technical replicates were set up in each biological replicate. Twenty cotton seedlings were set up in each experimental group and control group in each technical replicate. Cotton seedlings were randomly selected for photography.

[0055] Disease incidence in cotton plants as follows Figure 8 As shown, the negative control group exhibited more severe yellowing and wilting symptoms, while plants treated with scopolamine and the antibacterial protein CkPGIP1 showed only a few lesions. Plants treated with the combination of scopolamine and the antibacterial protein CkPGIP1 showed good growth. Statistical analysis of the disease index revealed that the disease index of cotton seedlings treated with scopolamine, the antibacterial protein CkPGIP1, and the combination of scopolamine and the antibacterial protein CkPGIP1 was lower than that of the negative control group. Furthermore, the treatment group treated with the combination of scopolamine and the antibacterial protein CkPGIP1 showed the best effect, with a significantly lower disease index than the positive control group. Figure 9 The results indicate that the combination of scopolamine and the antibacterial protein CkPGIP1 has a significant inhibitory effect on the growth of Verticillium wilt in upland cotton plants. The antibacterial effect of this combination is significantly higher than that of scopolamine or the antibacterial protein CkPGIP1 alone. This combination can be used as or prepared as a drug for the prevention and control of Verticillium wilt in cotton. This invention provides a new method for the prevention and control of Verticillium wilt in cotton by combining scopolamine and the antibacterial protein CkPGIP1.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. As long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for controlling cotton Verticillium wilt, characterized in that, The active ingredients are scopolamine and protein CkPGIP1, which are applied to the plants as antibacterial agents; the nucleotide sequence of protein CkPGIP1 is shown in SEQ ID No. 1, and the amino acid sequence of protein CkPGIP1 is shown in SEQ ID No.

2.

2. The method according to claim 1, characterized in that: in, The application concentration of scopolamine is 10-100 μg / mL, and the application concentration of protein CkPGIP1 is 50-200 μg / mL.

3. The method according to claim 1, characterized in that: The preparation method of the protein CkPGIP1 includes the following steps: (1) Construct a recombinant prokaryotic expression vector, wherein the recombinant prokaryotic expression vector contains the sequence shown in SEQ ID No. 1; (2) Transform the recombinant prokaryotic expression vector into bacteria to prepare the CkPGIP1 expression system; (3) Inducing the expression of the antibacterial protein CkPGIP1 in the CkPGIP1 expression system.

4. A fungicide for controlling cotton Verticillium wilt, characterized in that, The active ingredients of the bactericide are scopolamine and protein CkPGIP1. The nucleotide sequence of protein CkPGIP1 is shown in SEQ ID No. 1, and the amino acid sequence of protein CkPGIP1 is shown in SEQ ID No. 2.

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