Application of exogenous melatonin in enhancing cowpea's resistance to Fusarium wilt

Abstract

This invention discloses the application of exogenous melatonin in improving the resistance of cowpea to Fusarium wilt, belonging to the field of crop disease control technology. The application includes spraying a melatonin solution onto cowpea leaves at a concentration of 100 μm, once daily. Using the susceptible variety "Ziqiu Cowpea 6" as material, cowpea seedlings were pretreated with melatonin solution and then inoculated with the pathogen *Fusarium oxysporum*. Treatment with 100 μm melatonin significantly reduced the disease index of cowpea wilt, improved root phenotypic indicators, increased antioxidant enzyme activity and salicylic acid accumulation, and reduced reactive oxygen species levels, thereby enhancing resistance to *Fusarium oxysporum*. This invention provides a new approach for the control of cowpea wilt.

Description

Technical Field

[0001] This invention relates to the field of crop disease control technology, and in particular to the application of exogenous melatonin in improving cowpea resistance to Fusarium wilt. Background Technology

[0002] Cowpeas (Vigna unguiculata) are highly nutritious and adaptable, making them one of the most widely cultivated legume vegetables. However, with the continuous expansion of cowpea cultivation and limited arable land, continuous cropping obstacles are becoming increasingly prominent, leading to more severe soil-borne diseases, among which Fusarium wilt is a significant one. The pathogen of Fusarium wilt primarily enters the vascular tissue through the cowpea roots, causing leaves to turn yellow, lose water, and wilt. Symptoms are particularly pronounced during the seedling, flowering, and early pod-setting stages. Fusarium wilt is highly contagious and causes severe damage, resulting in significant losses in both yield and quality. Currently, fungicides are commonly used to control Fusarium wilt, causing considerable environmental damage. Therefore, safe and effective control measures are an urgent problem to be solved in the prevention and control of cowpea Fusarium wilt.

[0003] Melatonin is a hormone widely present in organisms, playing a variety of regulatory functions in plant life activities, such as promoting root formation, fruit ripening, increasing antioxidant capacity, and promoting hormone synthesis. It plays a crucial role in responding to biotic and abiotic stresses. Melatonin is a natural free radical scavenger and a powerful antioxidant. Exogenous melatonin can enhance the resistance of apple leaves to leaf spot disease by maintaining hydrogen peroxide (H2O2) levels in apples and increasing the activity of plant defense-related enzymes. Melatonin treatment significantly inhibits the development of gray mold, enhances SA accumulation and increases the activity of its synthesis-related enzymes, and upregulates the expression of defense genes. Exogenous 0.1 mM melatonin can increase the resistance of tobacco to tobacco mosaic virus by 37.4%, by inducing an increase in NO content, promoting SA accumulation, and upregulating the expression of PR1 and PR5, thereby improving tobacco's resistance to the virus. Therefore, melatonin can enhance plant immunity and alleviate the harm of pathogens to plants.

[0004] However, wilt is a fungal disease. *Verticillium dahliae*, *Verticillium chrysogenum*, and *Fusarium oxysporum*, all belonging to the Deuteromycetes, can cause wilt in plants. These are all soil-dwelling fungi that survive for long periods in the soil as chlamydospores and mycelium. They can remain dormant in the soil, in diseased plant debris, and in uncomposted organic fertilizer, becoming the primary source of infection for the next season. Different fungicides have varying effects on different types of pathogens, and different crop species also exhibit different sensitivities and responses to the same antifungal agent. Currently, there are no research reports on the control of cowpea wilt using exogenous melatonin. Summary of the Invention

[0005] The purpose of this invention is to provide the application of exogenous melatonin in improving the resistance of cowpea to Fusarium wilt, so as to solve the problems existing in the prior art. This invention found that cowpea seedlings treated with 100μm melatonin solution can improve the resistance of cowpea to Fusarium oxysporum. This invention provides a new approach for the prevention and control of cowpea wilt.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of exogenous melatonin in improving cowpea resistance to Fusarium wilt, including the step of spraying a melatonin solution on cowpea leaves.

[0008] Furthermore, the melatonin solution has a concentration of 100 μm and is sprayed once a day.

[0009] Furthermore, the exogenous melatonin exerts resistance to cowpea wilt by reducing the disease index of cowpea wilt, improving the root traits of cowpea, enhancing the activity of antioxidant enzymes and the accumulation of salicylic acid in cowpea, and reducing the level of reactive oxygen free radicals in cowpea.

[0010] Furthermore, the pathogens causing the wilt include Fusarium oxysporum.

[0011] This invention also provides the use of melatonin in the preparation of drugs for preventing and treating cowpea wilt.

[0012] The present invention also provides a drug for preventing and treating cowpea wilt disease, the drug comprising an effective dose of melatonin.

[0013] Furthermore, the effective dose of the melatonin is 100 μm.

[0014] The present invention discloses the following technical effects:

[0015] This invention uses the susceptible variety "Ziqiu Jiang 6" as material. Cowpea seedlings were pretreated with melatonin solution and then inoculated with the pathogen *Fusarium oxysporum*. Statistical results based on disease index, plant phenotype, and root morphology showed that 100 μm melatonin treatment significantly reduced the disease index of cowpea wilt and improved root phenotypic indicators, while 200 μm and 400 μm melatonin treatments had no significant effect. Further measurements of the reactive oxygen species (O2, O2) content in cowpeas after inoculation with the pathogen following melatonin treatment were also performed. 2- Biochemical indicators such as the activity of antioxidant enzymes (CAT, GSH-PX, SOD, POD, PAL, PPO), chlorophyll content, and SA content were analyzed. It was found that 100μm melatonin can increase antioxidant enzyme activity and salicylic acid accumulation, thereby reducing reactive oxygen species levels and enhancing resistance to Fusarium oxysporum, thus alleviating the damage caused by cowpea wilt. Therefore, this invention provides a new approach for the prevention and control of cowpea wilt. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 To identify the resistance of cowpea to Fusarium wilt after pretreatment with different concentrations of melatonin; A and C are cowpea plants under different treatments 20 days after inoculation with the pathogen; B is the disease condition of each treatment group after inoculation with the pathogen.

[0018] Figure 2 The figures show the root characteristics of cowpeas inoculated with Fusarium oxysporum at different time points after melatonin pretreatment. The numbers on the horizontal axis (1, 3, 10) represent the number of days after inoculation with Fusarium oxysporum (1 day, 3 days, 10 days after inoculation), and the vertical axis represents different root characteristics, namely root length (A), average root diameter (B), root surface area (C), root volume (D), intersection point (E), and number of branches (F). Different lowercase letters indicate significant differences at the P < 0.05 level.

[0019] Figure 3 The chlorophyll content in cowpea leaves after inoculation with Fusarium oxysporum for 1, 3, and 10 days following melatonin pretreatment was measured.

[0020] Figure 4 The content of salicylic acid in cowpea roots and leaves after melatonin pretreatment and inoculation with Fusarium oxysporum for 1, 3 and 10 days was determined.

[0021] Figure 5 To investigate the levels of reactive oxygen species (H2O2 and O2) in the roots and leaves of cowpea at different growth stages after melatonin pretreatment and inoculation with Fusarium oxysporum. 2- content);

[0022] Figure 6 The activity of defense-related enzymes in the roots and leaves of cowpea inoculated with Fusarium oxysporum at different stages after melatonin pretreatment was measured; the enzymes were CAT, GSH-PX, and SOD, in that order.

[0023] Figure 7 The activity of defense-related enzymes in the roots and leaves of cowpea inoculated with Fusarium oxysporum at different stages after melatonin pretreatment was measured; the enzymes were POD, PPO, and PAL in that order. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1: Exogenous melatonin improves cowpea wilt phenotype

[0030] Preparation of melatonin solution: Weigh 0.232g of AR grade melatonin (purchased from West Asia Reagent Company), completely dissolve it in 5mL of anhydrous ethanol, add deionized water to make up to 250mL, prepare a 4mM melatonin stock solution, and dilute it according to the ratio before use.

[0031] For the susceptible variety 'Ziqiu Jiang 6', cowpea seedlings were sprayed with melatonin solutions at concentrations of 100, 200, and 400 μm (distilled water was used as a control) at 8 PM, once daily for a total of three times. The third spraying was conducted concurrently with a suspension of Fusarium oxysporum spores (spore concentration of 1×10⁻⁶). 8 / mL) root irrigation treatment, with 10 plants per batch, repeated 3 times, cultured at 25℃, RH>70%, 14 / 10h (light / dark) conditions, samples were taken at 1 day, 3 days and 10 days after inoculation, disease index was counted at 20 days, and root characteristics were counted. The roots were rinsed clean in water and root characteristics were counted using an Expression 11000XL root scanner.

[0032] The results showed that pretreatment with 100 μm melatonin significantly reduced the disease index of cowpea wilt, with the 100 μm melatonin treatment group showing the largest reduction (approximately 37%). Plant height was also increased by 53%, with fewer leaves falling and a deeper leaf color. Figure 1 Root phenotypic indicators: 1 day and 3 days after inoculation, the 100 μm and 200 μm melatonin pretreatment groups showed significantly higher root length, root surface area, root volume, forks, and crossings than other treatment groups. 10 days after inoculation, the CK group, the 100 μm and 200 μm melatonin pretreatment groups also showed significantly higher values ​​for these five traits than the Fusarium oxysporum treatment group. Figure 2 ).

[0033] Example 2: Determination of chlorophyll and salicylic acid (SA) content in cowpea chlorophyll under Fusarium oxysporum infection after melatonin pretreatment

[0034] Foliar pretreatment with 100 μM melatonin was performed. Roots and leaves of cowpea seedlings were collected at 1, 3, and 10 days after inoculation with *Fusarium oxysporum*. 0.2 g of each sample was weighed and ground in liquid nitrogen. 3 mL of 90% methanol was added and ground in an ice bath, followed by centrifugation at 10000g for 10 min. The precipitate was dissolved in 3 mL of 100% methanol, centrifuged again, and the supernatant was mixed. The mixture was centrifuged at 10000g for 10 min, and the supernatant was evaporated to dryness at 45℃. 3 mL of ddH₂O was added and dissolved at 80℃ for 10 min. Two samples were prepared for each treatment. Free and bound melatonin (SA) were determined using liquid chromatography. Chlorophyll content was also determined by acetone extraction and UV spectrophotometry.

[0035] The results showed that at 10 days, the chlorophyll content in the Fusarium oxysporum treatment group decreased by 30% compared to the control group, while the melatonin pretreatment group only decreased by 10%, and the chlorophyll content was significantly higher in the Fusarium oxysporum treatment group. Figure 3 ).

[0036] One day after root inoculation with the pathogen, the total free salicylic acid and total salicylic acid (SA) in the melatonin pretreatment group were significantly higher than those in the *Fusarium oxysporum* treatment group, at 1709 ng / g and 2637 ng / g, respectively. At 3 and 10 days, the free SA and total SA content in the *Fusarium oxysporum* treatment group were significantly higher than those in the control group (CK) and the melatonin pretreatment group. At 1 day, the free SA and total SA content in the leaves of the *Fusarium oxysporum* treatment group and the melatonin pretreatment group were significantly lower than those in the control group (CK). At 3 days, the SA content in both the *Fusarium oxysporum* treatment group and the melatonin pretreatment group significantly increased. At 10 days, there was no difference in SA content between the melatonin pretreatment group and the control group, but the SA content was significantly higher in the melatonin pretreatment group than in the *Fusarium oxysporum* treatment group. Figure 4 ).

[0037] Example 3: Determination of ROS scavenging in cowpea under Fusarium oxysporum infection after melatonin pretreatment.

[0038] Foliar pretreatment with a 100 μm melatonin solution was performed. Roots and leaves of cowpea seedlings were collected 1, 3, and 10 days after inoculation with *Fusarium oxysporum*. The ROS content of cowpea seedlings was measured 1, 3, and 10 days after inoculation, mainly including H2O2 and O2. 2- .

[0039] The results showed that, in both roots and leaves, the H2O2 content of Fusarium oxysporum and melatonin pretreatment gradually decreased over time. At 1 day and 3 days after infection, the H2O2 content of the melatonin pretreatment group was significantly lower than that of the Fusarium oxysporum treatment group. 2- The content remained basically unchanged. Figure 5 ).

[0040] Example 4: Effect of melatonin pretreatment on the activity of defense enzymes in cowpea under Fusarium oxysporum infection.

[0041] Foliar treatment with 100 μM melatonin pretreatment was performed. Roots and leaves of cowpea seedlings were collected at 1, 3, and 10 days after inoculation with *Fusarium oxysporum*. The activities of several defense-related enzymes in cowpea, including SOD, POD, CAT, GSH-PX, PAL, and PPO, were measured. Results showed that, immediately after inoculation, the activities of CAT, GSH-PX, and PAL in melatonin-pretreated seedlings were significantly higher than those in *Fusarium oxysporum*-treated seedlings in both leaves and roots. At 10 days after inoculation, the PPO activity in roots was significantly higher in the pretreated seedlings than in the *Fusarium oxysporum*-treated seedlings. POD activity in both leaves and roots gradually increased with growth and development. Inoculation with *Fusarium oxysporum* led to a decrease in POD activity. The POD activity in root tissues was most significantly affected by the pathogen at 10 days, decreasing by more than 26% compared to the control group. However, the behavior in leaves and roots was completely different. There was no significant difference in SOD activity between the *Fusarium oxysporum*-treated and melatonin-pretreated seedlings. Figure 6 and Figure 7 ).

[0042] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The use of exogenous melatonin in improving the resistance of cowpea to fusarium wilt, characterized in that, comprising the step of spraying a solution of melatonin on the leaves of the cowpea; the pathogenic fungus of the fusarium wilt is fusarium oxysporum; the inoculation is carried out by root drenching treatment; the spore concentration of the fusarium oxysporum is 1×10 8 / mL; the cowpea is Ziqiuqian No. 6; The concentration of the melatonin solution is 100 μm, and the spraying frequency is once a day.

2. Use according to claim 1, characterized in that, The exogenous melatonin plays a resistance to the fusarium wilt of cowpea by reducing the disease index of the cowpea fusarium wilt, improving the root traits of the cowpea, enhancing the antioxidant enzyme activity and salicylic acid accumulation of the cowpea, and reducing the active oxygen free radical level of the cowpea.

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