Use of n-feruloyl-1,4-butanediamine in stimulating egg hatching of globodera pallida

Abstract

The application discloses application of N-vanillyl-1,4-butanediamine in stimulating egg hatching of Globodera rostochiensis, and belongs to the field of nematode control. The N-vanillyl-1,4-butanediamine for stimulating egg hatching of Globodera rostochiensis is screened from root exudates of potatoes. Experimental results show that the N-vanillyl-1,4-butanediamine can effectively stimulate egg hatching of Globodera rostochiensis; the N-vanillyl-1,4-butanediamine applied to soil can induce egg hatching of Globodera rostochiensis in advance in non-cultivation seasons, so that the larvae die due to lack of hosts, and a control effect is achieved. The Globodera rostochiensis control method based on the N-vanillyl-1,4-butanediamine has the advantages of simple operation, low cost and environmental friendliness, and the application has important significance for green control of Globodera rostochiensis and protection of host crop production safety.

Description

Technical Field

[0001] This invention relates to the field of nematode control, and in particular to the application of N-feruloyl-1,4-butanediamine in stimulating the hatching of potato golden nematode eggs. Background Technology

[0002] Potato nematode ( Globodera rostochiensis Potato nematode (Golden Potato Nematode) is an internationally quarantined plant parasitic nematode that causes disease in various Solanaceae plants, with potatoes being an important crop host. A single female Potato nematode can lay 200-500 eggs and survive in the soil for up to 25 years. Currently, control mainly relies on chemical fumigants such as dazomet, which is not only costly but also disrupts soil microbial communities and causes environmental pollution. Therefore, the development of new green control technologies is urgently needed.

[0003] The egg soaking method can directly demonstrate the stimulating effect of compounds on egg hatching, thus enabling the screening of effective hatching stimulants. Currently, solanoeclepin A, α-solanoine, and α-carboxine, found in root exudates of Solanaceae plants, have been reported to effectively stimulate the hatching of potato nematode eggs. However, these substances suffer from problems such as near-insolubility in water, high synthesis difficulty, and high cost, making them difficult to apply in practical production. Therefore, finding cost-effective compounds that can effectively stimulate potato nematode egg hatching is an important means of controlling the potato nematode through "suicide hatching." Summary of the Invention

[0004] The purpose of this invention is to provide the application of N-feruloyl-1,4-butanediamine in stimulating the hatching of potato nematode eggs, thereby solving the problems existing in the prior art. This invention obtains N-feruloyl-1,4-butanediamine, which stimulates the hatching of potato nematode eggs, from potato root exudates. Applying N-feruloyl-1,4-butanediamine to the soil can induce potato nematode eggs to hatch prematurely during the non-planting season, causing the larvae to die due to lack of host, thus playing a control role. This invention is of great significance for carrying out green control of potato nematode and ensuring the safety of host crop production.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of N-feruloyl-1,4-butanediamine in stimulating the hatching of nematode eggs.

[0006] This invention also provides the use of N-feruloyl-1,4-butanediamine in the preparation of formulations that stimulate the hatching of nematode eggs.

[0007] Furthermore, the nematode is the potato nematode.

[0008] The present invention also provides a product for stimulating the hatching of potato nematode eggs, with N-feruloyl-1,4-butanediamine as the main active ingredient.

[0009] Furthermore, in the product, the concentration of N-feruloyl-1,4-butanediamine is 8 μM.

[0010] This invention also provides the application of N-feruloyl-1,4-butanediamine in the control of potato nematode.

[0011] The present invention also provides the application of N-feruloyl-1,4-butanediamine in the preparation of formulations for controlling potato nematodes.

[0012] The present invention also provides a formulation for controlling potato nematode, with N-feruloyl-1,4-butanediamine as the main active ingredient.

[0013] Furthermore, in the formulation, the content of N-feruloyl-1,4-butanediamine is 1~2.5 ppm.

[0014] The present invention also provides a method for controlling potato nematode, comprising the step of applying the above-mentioned preparation.

[0015] The present invention discloses the following technical effects: This invention screened N-feruloyl-1,4-butanediamine, a substance that stimulates the hatching of potato nematode eggs, from potato root exudates. Experimental results showed that N-feruloyl-1,4-butanediamine effectively stimulates the hatching of potato nematode eggs; applying an aqueous solution of N-feruloyl-1,4-butanediamine to the soil can induce the premature hatching of potato nematode eggs outside the planting season, causing the larvae to die due to lack of a host, thus exerting a control effect. The potato nematode control method based on N-feruloyl-1,4-butanediamine has significant advantages such as simple operation, low cost, and environmental friendliness. This invention is of great significance for carrying out green control of potato nematodes and ensuring the safety of host crop production. 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 Figure 1 shows the results of the experiment on hatching of potato nematode eggs stimulated by N-feruloyl-1,4-butanediamine; where a is the screening results of the optimal stimulation concentration of N-feruloyl-1,4-butanediamine; and b is a comparison of the stimulation effects of different compounds. Figure 2 Figure 1 shows the results of an indoor pot experiment on the control of potato golden nematode by N-feruloyl-1,4-butanediamine; where a represents the nematode infection reduction rate during the potato seedling stage and b represents the sporangium reproduction coefficient during the potato harvest period. Detailed Implementation

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] 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.

[0023] Example 1: Analysis of root exudates from resistant potato varieties 1. Experimental Methods 1.1 Potato Varieties Qingshu No. 9 and Yunshu 505 were harvested from Zhaoyang District, Zhaotong City, Yunnan Province.

[0024] 1.2 Collection of potato root exudates Wash the roots of potatoes that have grown for 5 weeks thoroughly with tap water. Using 3 potatoes as one sample, immerse the roots in a 250 mL Erlenmeyer flask, add 75 mL of ddH2O to soak all roots, and repeat this process 3 times for each sample. After the roots are kept in the dark and placed in a greenhouse at 18-25℃ for 24 hours, collect the liquid and filter it using a bacterial filter.

[0025] 1.3 Extraction of substances from root exudates Take 1 mL of root exudate, concentrate it under vacuum to a solid, add 20 μL of 50% methanol aqueous solution, vortex for 3 min at 4℃ and 2000 rpm to dissolve the substance, centrifuge for 15 min at 4℃ and 12000 rpm, and collect the supernatant for immediate sample analysis.

[0026] 1.4 LC-MS Sample processing was performed using an ultra-high performance liquid chromatography (UHPLC) system. Samples were delivered via an autosampler at 8°C with an injection volume of 2 μL. Separation was achieved using an HSS T3 column at 40°C and a flow rate of 0.3 mL / min. QC samples were inserted into the sample queue to monitor and evaluate system stability and the reliability of experimental data.

[0027] Each sample was detected using electrospray ionization (ESI) in both positive and negative ion modes. Samples were separated by UHPLC and then analyzed by mass spectrometry using a Thermo QE HF-X mass spectrometer.

[0028] The raw data obtained from mass spectrometry analysis were processed using the Compound Discovery program for peak alignment, retention time correction, and peak area extraction. The obtained metabolites were structurally identified using the Compound Discovery program through precise mass number matching (<10 ppm) and secondary spectrum matching, and then searched in the database.

[0029] 2. Experimental Results The main components of root exudates from Qingshu No. 9 and Yunshu No. 505 were identified by total ion mass spectrometry. Differential metabolites between the root exudates of Qingshu No. 9 and Yunshu No. 505 were analyzed using fold change (FC, log2FC), t-test score (t), significance P.Value, adj.P.Val, and log-odds (B). A fold change |FC| ≥ 2 and P.Value < 0.05 were used as screening criteria, resulting in 30 main differential components in both positive and negative ion modes of root exudates from Qingshu No. 9 and Yunshu No. 505. Further, based on factors such as chemical structure, metabolic pathway, solubility, function, CAS number, and price, the affordable and easily marketable natural product N-feruloyl-1,4-butanediamine was selected for incubation experiments.

[0030] Example 2: Effect of root exudates on the hatching of potato nematodes In this embodiment, 2, 4, 6, 8, and 10 μM N-feruloyl-1,4-butanediamine were used as treatment solutions, with distilled water as a negative control. This has been publicly demonstrated to effectively stimulate the potato nematode ( ). Globodera rostochiensis The 5 μM α-solanine and 5 μM α-carboxine used as positive controls were used to demonstrate the use of N-feruloyl-1,4-butanediamine as the active ingredient in stimulating the hatching of potato nematodes through a test of its effect on potato nematode hatching.

[0031] 1. Experimental Methods 1.1 Collection of Potato nematode cysts Diseased soil was collected in Zhaoyang District, Zhaotong City, Yunnan Province. The soil and sporangia were initially separated by washing and sieving. Under a microscope, plump, dark brown sporangia of the potato nematode were collected with tweezers and stored in a refrigerator at 4°C for more than 4 months before being used in subsequent experiments.

[0032] 1.2 Preparation of treatment solution α-Solanine, α-carbocyanine, and N-feruloyl-1,4-butanediamine standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd. 5 μM α-solanine aqueous solution, 5 μM α-carbocyanine aqueous solution, and 2, 4, 6, 8, and 10 μM N-feruloyl-1,4-butanediamine aqueous solutions were prepared and stored in sterile glass bottles at 4°C, protected from light.

[0033] 1.3 Nematode hatching Prepare 24-well cell culture plates, placing 10 sporangia and 1 mL of treatment solution in each well, with each treatment repeated 3 times. Incubate in a dark incubator at 18°C, recording the number of newly appearing J2 cells every 5 days for 6 consecutive days. After each count, transfer the sporangia to a new culture plate and replace the treatment solution with fresh solution.

[0034] 2. Experimental Results like Figure 1 As shown in Figure a, the results indicate that 8 μM N-feruloyl-1,4-butanediamine has the strongest stimulating effect on the hatching of potato nematode eggs.

[0035] like Figure 1 As shown in Figure b, 5 μM α-solanine and 5 μM α-carboxine have been reported as effective stimulants for the hatching of potato nematode eggs. 8 μM N-feruloyl-1,4-butanediamine showed significantly better stimulant effects on potato nematode egg hatching than 5 μM α-solanine and 5 μM α-carboxine.

[0036] Example 3: Indoor pot experiment on hatching of potato nematode eggs stimulated by N-feruloyl-1,4-butanediamine. Based on molar mass conversion, the optimal stimulating concentration (8 μM) of N-feruloyl-1,4-butanediamine determined in Example 2 corresponds to a treatment solution concentration of approximately 2 ppm. Therefore, in this example, 1 ppm, 1.5 ppm, and 2.5 ppm of N-feruloyl-1,4-butanediamine were used as treatment solutions, with distilled water as a control. An indoor pot experiment was conducted to demonstrate that N-feruloyl-1,4-butanediamine, as an active ingredient, is effective against the potato nematode (Gynostemma pentaphyllum). Globodera rostochiensis Its uses in prevention and control.

[0037] 1. Experimental Methods 1.1 Potato Varieties Green potato variety No. 9 was harvested from Zhaoyang District, Zhaotong City, Yunnan Province.

[0038] 1.2 Collection of Potato nematode cysts Diseased soil was collected in Zhaoyang District, Zhaotong City, Yunnan Province. The soil and sporangia were initially separated by washing and sieving. Under a microscope, plump, dark brown sporangia of the potato nematode were collected with tweezers and stored in a refrigerator at 4°C for more than 4 months before being used in subsequent experiments.

[0039] 1.3 Preparation of Treatment Fluid N-feruloyl-1,4-butanediamine was prepared in distilled water to obtain 1 ppm, 1.5 ppm, and 2.5 ppm solutions. These solutions were then stored in sterile glass bottles protected from light at 4°C.

[0040] 1.4 Potato potted plant setup Each pot contained 800 mL of moist, sterilized sand, with 80 potato nematode cysts evenly mixed into each pot of sterile sand. Potatoes were disinfected with 1% sodium hypochlorite, cut into pieces centered around a single bud, and placed in seedling trays. The surface was covered with moist fine sand and kept in a greenhouse at 18-25℃ to maintain humidity. After sprouting, the potatoes were transplanted. Following the experimental protocol, the sprouted potato pieces were transplanted into flowerpots and placed in a greenhouse at 18-25℃ with 16 / 8 hours of light and 200 mL of water every 2 days. Specific experimental protocols are shown in Table 1.

[0041] Table 1 Experimental Scheme 1.5 root tissue staining Root tissue staining was performed on potato seedlings to observe the infection status of the potato root system. Clean root tissue was soaked in 50 mL ddH2O and 10 mL 5.25% NaClO solution, stirred with a glass rod, and allowed to stand for 5 min. The NaClO was then rinsed off with running water. The root tissue was transferred to another beaker containing 50 mL ddH2O and 1 mL acidic fuchsin staining solution and boiled for 1 min. After cooling, the tissue was rinsed with water and then placed in a clean beaker with 30 mL acidic glycerol added. The mixture was boiled until the roots lost their color.

[0042] 1.6 Sporangium Isolation and Counting During the potato harvest season, the soil from the potted plants is poured into a clean bucket of water, and the soil and sporangia are initially separated by washing and sieving. The number of sporangia is then recorded under a microscope using a counter.

[0043] 1.7 Data Processing During the potato seedling stage, the root system weight was measured, and root tissue staining was used to investigate the amount of potato golden nematode infection. The nematode infection reduction rate per unit weight of root system was calculated using the following formula: .

[0044] During potato harvest, the number of potato nematode cysts was investigated by isolating and counting them. Based on the initial number of potato nematode cysts, the cyst reproduction coefficient was calculated using the following formula: .

[0045] 2. Experimental Results like Figure 2 As shown in Figures a and b, when potato seedlings were transplanted 20 days and 30 days after applying N-feruloyl-1,4-butanediamine solution at different concentrations and depths, the combination of 1 ppm application, 0-7 cm application depth, and transplanting potato seedlings 30 days after application showed the highest reduction rate of nematode infection per unit weight of root system during the potato seedling stage (57.3%), and the lowest sporangium reproduction coefficient at the potato harvest period (3.95).

[0046] Example 4: Field efficacy trial of N-feruloyl-1,4-butanediamine stimulating the hatching of potato nematode eggs. This example uses 1 ppm, 1.5 ppm, and 2.5 ppm N-feruloyl-1,4-butanediamine as treatment solutions, distilled water as a negative control, and 0.15 ppm 41.7% fluopyram suspension and 0.15 ppm 30% prothioconazole·fluopyram suspension as positive controls. The aim is to determine the optimal application concentration of N-feruloyl-1,4-butanediamine through field trials, investigate its effect on the suicide hatching of potato nematodes, and provide a better basis for controlling potato nematodes (…).Globodera rostochiensis This provides a basis for identifying and protecting the safety of potato production.

[0047] 1. Experimental Methods 1.1 Potato Varieties Green potato variety No. 9 was harvested from Zhaoyang District, Zhaotong City, Yunnan Province.

[0048] 1.2 Preparation of treatment solution N-feruloyl-1,4-butanediamine standard was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Aqueous solutions of N-feruloyl-1,4-butanediamine at concentrations of 1 ppm, 1.5 ppm, and 2.5 ppm were prepared and used immediately.

[0049] 1.3 Experimental Field Located in Xikuipingzi, Songshan Village, Jing'an Town, Zhaoyang District, Zhaotong City, Yunnan Province. Before planting potatoes, the soil was tilled, and approximately 2 kg of soil was collected from 5 points in each plot. After mixing, 200 g of soil was collected to count the initial cysts of potato nematodes in the field plots.

[0050] 1.4 Experimental Field Plot Treatment Plant spacing was 40 cm, row spacing was 50 cm, randomized block design, and replicated 4 times.

[0051] Fifteen seed potatoes (approximately 73 g each) were used per plot, totaling approximately 1100 g of seed potatoes per plot. A total of 24 plots were used (4 replicates, 6 treatments per replicate), for a total of 360 seed potatoes and approximately 26.5 kg of seed potatoes. Each potato plant was treated with 300 mL of treatment solution. N-feruloyl-1,4-butanediamine was applied 30 days before potato planting, while 41.7% fluopyram suspension and 30% prothioconazole·fluopyram suspension were applied at planting time, as shown in Table 2. The water volume per plot was 5 L, and the total water volume for the four replicates was 20 L.

[0052] Table 2 Dosing Regimen 1.5 root tissue staining Root tissue staining was performed on potato seedlings to observe the infection status of the potato root system. Clean root tissue was soaked in 50 mL ddH2O and 10 mL 5.25% NaClO solution, stirred with a glass rod, and allowed to stand for 5 min. The NaClO was then rinsed off with running water. The root tissue was transferred to another beaker containing 50 mL ddH2O and 1 mL acidic fuchsin staining solution and boiled for 1 min. After cooling, the tissue was rinsed with water and then placed in a clean beaker with 30 mL acidic glycerol added. The mixture was boiled until the roots lost their color.

[0053] 1.6 Sporangium Isolation and Counting During the potato harvest season, the soil from the potted plants is poured into a clean bucket of water, and the soil and sporangia are initially separated by washing and sieving. The number of sporangia is then recorded under a microscope using a counter.

[0054] 1.7 Data Processing During the potato seedling stage, a five-point sampling method was used to weigh the roots and investigate the amount of potato golden nematode infection by staining the root tissue. The nematode infection reduction rate per unit weight of roots was calculated according to the following formula: .

[0055] During the potato harvest period, a five-point sampling method was used to investigate the amount of potato nematode cysts through cyst isolation and counting. Based on the initial amount of potato nematode cysts in the field plots, the cyst reproduction coefficient was calculated using the following formula: ; Weigh the potatoes and calculate the yield increase rate: .

[0056] 2. Experimental Results Before potato planting, irrigating the ridges with 1 ppm N-feruloyl-1,4-butanediamine resulted in a 41.34% reduction in nematode infection during the potato seedling stage, which was about 20 percentage points higher than the reduction rate achieved by irrigating the ridges with 0.15 ppm 41.7% fluopyram suspension or 30% prothioconazole·fluopyram suspension after planting. The sporangium reproduction coefficient at harvest was only 1.77, about one unit lower than the sporangium reproduction coefficient at harvest when treated with water or irrigated with 0.15 ppm 41.7% fluopyram suspension and 30% prothioconazole·fluopyram suspension after planting. The yield increase was best at harvest, with a rate as high as 57.53%.

[0057] Table 3. Control and yield-increasing effects of different compounds on potato nematode. 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. A product that stimulates the hatching of potato nematode eggs, characterized in that, The main active ingredient is N-feruloyl-1,4-butanediamine. The concentration of the N-feruloyl-1,4-butanediamine was 8 μM.

2. A method for controlling potato nematodes, characterized in that, The steps include applying a formulation containing N-feruloyl-1,4-butanediamine as the active ingredient; The formulation is applied at a concentration of 1 ppm at a depth of 0-7 cm, and potato seedlings are transplanted 30 days after application.

Images