Use of L-glutamyl glycine in preventing and treating plant root knot nematodes

Abstract

This invention discloses the application of L-glutamylglycine in the control of plant root-knot nematodes, belonging to the field of biopesticide technology. The L-glutamylglycine effectively inhibits the infectivity of root-knot nematodes, reducing their harm to host plants. The L-glutamylglycine provided by this invention is highly efficient, green, and environmentally friendly, safe for agricultural products, humans, livestock, and soil microecology, avoiding the residue and pollution risks of traditional chemical pesticides. It provides a novel, sustainable, and green control strategy for plant root-knot nematode disease, and has broad prospects for industrial application in the field of biopesticides.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically the application of L-glutamylglycine in the control of plant root-knot nematodes. Background Technology

[0002] Plant-parasitic nematodes (PPNs) are widely distributed and cause significant direct damage. Among them, root-knot nematodes (PPNs) are particularly prevalent. Meloidogyne spp. As the most widespread and damaging plant endoparasitic nematode, especially the southern root-knot nematode (… M. incognita The southern root-knot nematode can parasitize more than 3,000 kinds of crops. Plants severely affected by nematodes exhibit stunted growth and curled leaves, which many researchers attribute to insufficient root nutrition and water supply. Root-knot nematodes grow and reproduce rapidly with crop planting, leading to a dramatic increase in their population density in the soil, ultimately resulting in declining crop yields from generation to generation. This phenomenon is quite common in modern greenhouse agriculture, and root-knot nematode disease is often accompanied by fungal diseases, seriously hindering the rapid modernization of agriculture.

[0003] In recent years, exploring natural small-molecule compounds with nematode-resistant activity, starting from the plant's own defense system, has become a promising research direction. To resist nematode infestation, especially soil-borne nematodes, plants synthesize and secrete various nematode-resistant chemicals, such as phenols, terpenes, glucosinolates, and indole derivatives. Among these, flavonoids, as the largest family of phenolic secondary metabolites, have received widespread attention in nematode-resistant research. In vitro experiments have shown that certain flavonoid compounds (such as kaempferol and quercetin) have repulsive or inhibitory effects on southern root-knot nematodes; furthermore, glycerol I in soybeans has also been confirmed to be associated with nematode-resistant traits, with its content in the roots of resistant varieties being significantly higher than that of susceptible varieties. Currently, the main methods for controlling root-knot nematodes include plant quarantine, screening of resistant varieties, agricultural control, chemical control, and biological control. However, these methods have obvious limitations: plant quarantine has limited effectiveness against root-knot nematodes; the breeding of resistant varieties has problems such as incompatibility in distant hybridization, long cycle, and unstable expression of antigen genes; agricultural control (such as crop rotation) is limited by the wide host adaptability of nematodes; although chemical control is fast-acting, it is prone to causing environmental pollution, food safety risks, and nematode resistance, and has been restricted or banned in many places.

[0004] Therefore, developing efficient, green, and sustainable root-knot nematode control strategies has become a key issue that urgently needs to be addressed in current agricultural production. Summary of the Invention

[0005] To address the problem of root-knot nematode disease, the present invention aims to provide an application of L-glutamylglycine in the control of plant root-knot nematodes, wherein the structural formula of the L-glutamylglycine is as follows:

[0006] .

[0007] Another object of the present invention is to provide the application of L-glutamylglycine in the preparation of root-knot nematode infection inhibitors, wherein the structural formula of the L-glutamylglycine is as follows:

[0008] .

[0009] Preferably, the insecticide of the present invention is in the form of an aqueous solution, an emulsion, a wettable powder, a soluble powder, a soluble granule, a water-dispersible granule, a suspension concentrate, an oil suspension concentrate, a tablet, or an effervescent granule.

[0010] This invention provides an application of L-glutamylglycine in the control of plant root-knot nematodes, which has the following beneficial effects:

[0011] (1) This invention treats root-knot nematodes with L-glutamyl glycine, which effectively inhibits the infectivity of root-knot nematodes, reduces their harm to plants, and has the characteristics of rapid action and high efficacy, which is conducive to the rapid control of nematode diseases in actual production.

[0012] (2) The L-glutamyl glycine in this invention does not pollute agricultural products, humans, livestock and the environment, meets the requirements of green agriculture and sustainable development, avoids the residual risks and ecotoxicity problems of traditional chemical pesticides, and is friendly to soil microecology.

[0013] (3) This invention provides a novel control strategy for root-knot nematodes, a soil-borne disease that is difficult to control. L-glutamylglycine, as a small molecule compound, has advantages such as ease of synthesis and has good prospects for industrialization in the fields of green plant protection and biopesticides. Attached Figure Description

[0014] Figure 1 This is a graph showing the relationship between the concentration of L-glutamylglycine and the disease status of tomato seedlings caused by the southern root-knot nematode in Example 1 and Comparative Example 1 of the present invention.

[0015] Figure 2 This is a graph showing the relationship between L-glutamyl glycine concentration and the effects of Javan root-knot nematode on tomato seedling disease in Example 2 and Comparative Example 2 of the present invention.

[0016] Figure 3 This is a graph showing the relationship between the disease index of Javan root-knot nematode and the concentration of L-glutamyl glycine in tomato seedlings in Example 3 and Comparative Example 3 of the present invention.

[0017] Figure 4 These are root system diagrams of tomato seedlings after being infected by Javan root-knot nematodes in Example 3 and Comparative Example 3 of the present invention; (a) is the control group, without the addition of L-glutamylglycine; (b) L-glutamylglycine concentration is 75 μg / mL; (c) L-glutamylglycine concentration is 50 μg / mL; (d) L-glutamylglycine concentration is 25 μg / mL; (e) L-glutamylglycine concentration is 12.5 μg / mL; (f) L-glutamylglycine concentration is 6.25 μg / mL. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The 23% Prönnicke solution used in the embodiments and comparative examples of this invention is specifically an aqueous solution of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer with a mass percentage concentration of 23%. The tested pathogenic nematodes were *N. southernus* and *N. Javanese*. *N. southernus* was obtained through laboratory propagation, while *N. Javanese* was obtained from soil samples collected in Yuanmou County, after separation, identification, and purification. It was identified as *N. Javanese*. Vigorous second-instar larvae were used for testing. L-Glutamylglycine was purchased from Shanghai Leyan Biotechnology Co., Ltd., with a purity of 97%, CAS number 13716-89-7, and batch number 1281281.

[0020] Example 1

[0021] The specific steps for determining the inhibitory effect of L-glutamylglycine on the infection of southern root-knot nematodes are as follows:

[0022] 1. Cultivating tomato seedlings:

[0023] (1) Soak tomato seeds in a 9 cm plate filled with warm water (the volume ratio of boiling water to cold water is 3:2) to submerge the tomato seeds and prevent them from clumping together. Place the plate in a constant temperature incubator at 28°C for 24 hours. After 24 hours, remove the plate and wash the tomato seeds thoroughly with sterile water 10 times to obtain tomato seeds that have been thoroughly washed with sterile water.

[0024] (2) Use sterile tweezers to lay the filter paper flat on a 9 cm culture plate and add 3 ml of sterile water to it; after thoroughly washing the tomato seeds with sterile water, place them one by one onto the moistened filter paper and place them in a 28℃ constant temperature incubator for 72 hours; after 72 hours, inoculate tomato seedlings with roots of 1 cm into a culture plate containing 2 ml of 23% Prönkel solution (24 wells), with one tomato seedling inoculated in each well, and 8 seedlings per group, for a total of 3 groups.

[0025] 2. Preparation of L-glutamylglycine working solution:

[0026] (1) Add 19.6 mL of pure water to 0.4 mL of methanol (analytical grade) to prepare a methanol dilution with a volume percentage concentration of 2%.

[0027] (2) Weigh 0.5 mg of L-glutamylglycine and place it in a 1.5 mL centrifuge tube. Use a pipette to transfer 1 mL of 2% methanol dilution to the 1.5 mL centrifuge tube containing L-glutamylglycine. Mix thoroughly and then transfer it to a 15 mL centrifuge tube. Add 4 mL of 2% methanol dilution to prepare a stock solution with a concentration of 100 μg / mL.

[0028] (3) Dilute the 100 μg / mL mother solution with 2% methanol diluent to prepare 2 mL of working solutions with concentrations of 75 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively. The dilution methods are as follows: 75 μg / mL (volume ratio of mother solution to diluent is 1.5:0.5); 50 μg / mL (volume ratio of mother solution to diluent is 1:1); 25 μg / mL (volume ratio of mother solution to diluent is 0.5:1.5); 12.5 μg / mL (volume ratio of mother solution to diluent is 0.25:1.75). Add the different concentrations of L-glutamylglycine working solutions to 6-well plates, and set 2% methanol diluent as a blank control.

[0029] 3. Southern root-knot nematode feeds on L-glutamylglycine and its infectivity was tested:

[0030] (1) Collect freshly hatched second-instar larvae of the southern species into a 15mL centrifuge tube, centrifuge at 3500rpm for 3min, discard the supernatant, add sterile water and centrifuge at 3500rpm for 3min. Repeat the centrifugation operation three times to obtain washed second-instar larvae of the southern species.

[0031] (2) Dilute the standard M9 buffer solution 4 times with sterile deionized water to obtain 0.25×M9 working solution; rinse the pipette tip with 0.25×M9 working solution, then aspirate the washed second instar nematodes and count the number of nematodes under a stereomicroscope.

[0032] (3) Add about 3,500 washed second-instar larvae of Southern Asia to working solutions of L-glutamyl glycine with concentrations of 75 μg / mL, 50 μg / mL, 25 μg / mL and 12.5 μg / mL, and then place them in a constant temperature incubator at 28°C for 48 hours.

[0033] (4) The nematodes in the working solution of L-glutamyl glycine of different concentrations were extracted, and the nematodes were washed three times with sterile water. The nematodes were then inoculated into the root tips of tomato seedlings planted in 23% Prönkel solution. The inoculation amount of each root tip was 100 nematodes within 0.5 cm of the outer periphery. The seedlings were left to stand in the dark at 28℃ for 48 hours to obtain tomato seedlings infected with Southern root-knot nematodes.

[0034] (5) Place the 24-well cell culture plate on ice for 10 minutes. Soak the tomato seedlings infected with southern root-knot nematodes in a 5% sodium hypochlorite aqueous solution for 13 minutes. Then rinse the seedlings with sterile water until odorless. Stain the roots of the tomato seedlings with a 13% (w / v) acidic fuchsin aqueous solution. Microwave for 2 minutes. Count the number of nematodes in each tomato root. The results are as follows: Figure 1 As shown.

[0035] Comparative Example 1

[0036] The difference between this comparative example and Example 1 is that the culture medium for culturing nematodes is L-glutamylglycine working solution (concentration of 6.25 μg / mL), sterile water control group and 2% methanol dilution control group (volume percentage concentration), while the other steps are the same.

[0037] The results of the infection ability of southern root-knot nematodes on tomato seedlings are as follows: Figure 1 As shown, the concentration of L-glutamylglycine in the southern root-knot nematode infection inhibitor was 12.5-75 μg / mL, which inhibited the infection of the southern root-knot nematode. At a lower concentration (6.25 μg / mL), the compound's effective dose was insufficient and failed to effectively inhibit the nematode's infection ability. Furthermore, using a 2% methanol dilution as a control, the results showed that its infection level was basically consistent with that of the sterile water control group, indicating that this solvent concentration did not affect the normal infection behavior of the nematodes in this system, thus eliminating solvent interference.

[0038] Example 2

[0039] The specific steps for determining the inhibitory effect of L-glutamylglycine on Javan root-knot nematode infection are as follows:

[0040] 1. Cultivating tomato seedlings:

[0041] (1) Soak tomato seeds in a 9 cm plate filled with warm water (the volume ratio of boiling water to cold water is 3:2) to submerge the tomato seeds and prevent them from clumping together. Place the plate in a constant temperature incubator at 28°C for 24 hours. After 24 hours, remove the plate and wash the tomato seeds thoroughly with sterile water 10 times to obtain tomato seeds that have been thoroughly washed with sterile water.

[0042] (2) Use sterile tweezers to lay the filter paper flat on a 9 cm culture plate and add 3 ml of sterile water to it; after thoroughly washing the tomato seeds with sterile water, place them one by one onto the moistened filter paper and place them in a 28℃ constant temperature incubator for 72 hours; after 72 hours, inoculate tomato seedlings with roots of 1 cm into cell culture plates (24 wells) containing 2 ml of 23% Prönkel solution, with one tomato seedling inoculated in each well, and 8 seedlings per group, for a total of 3 groups.

[0043] 2. Preparation of L-glutamylglycine working solution:

[0044] (1) Add 19.6 mL of pure water to 0.4 mL of methanol (analytical grade) to prepare a methanol dilution with a volume percentage concentration of 2%.

[0045] (2) Weigh 0.5 mg of L-glutamylglycine and place it in a 1.5 mL centrifuge tube. Use a pipette to transfer 1 mL of 2% methanol dilution to the 1.5 mL centrifuge tube containing L-glutamylglycine. Mix thoroughly and then transfer it to a 15 mL centrifuge tube. Add 4 mL of 2% methanol dilution to prepare a stock solution with a concentration of 100 μg / mL.

[0046] (3) Dilute the 100 μg / mL mother solution with 2% methanol diluent to prepare 2 mL working solutions with concentrations of 75 μg / mL, 50 μg / mL, and 25 μg / mL, respectively. The dilution methods are as follows: 75 μg / mL (volume ratio of mother solution to diluent is 1.5:0.5); 50 μg / mL (volume ratio of mother solution to diluent is 1:1); 25 μg / mL (volume ratio of mother solution to diluent is 0.5:1.5). Add the different concentrations of L-glutamylglycine working solutions to 6-well plates, and set 2% methanol diluent as a blank control.

[0047] 3. Javan root-knot nematode feeds on L-glutamylglycine and its infectivity was tested:

[0048] (1) Collect freshly hatched second-instar Javan larvae into a 15mL centrifuge tube, centrifuge at 3500rpm for 3min, discard the supernatant, add sterile water and centrifuge at 3500rpm for 3min. Repeat the centrifugation operation three times to obtain washed second-instar Javan larvae.

[0049] (2) Dilute the standard M9 buffer solution 4 times with sterile deionized water to obtain 0.25×M9 working solution; rinse the pipette tip with 0.25×M9 working solution, then aspirate the washed second instar Java larvae and count the number of nematodes under a stereomicroscope.

[0050] (3) Add about 3,500 washed second-instar Javan larvae to working solutions of L-glutamyl glycine with concentrations of 75 μg / mL, 50 μg / mL and 25 μg / mL, and then place them in a constant temperature incubator at 28°C for 48 hours.

[0051] (4) The nematodes in the working solution of L-glutamyl glycine of different concentrations were extracted, and the nematodes were washed three times with sterile water. The nematodes were then inoculated into the root tips of tomato seedlings planted in 23% Prönkel solution. The inoculation amount of each root tip was 100 nematodes within 0.5 cm of the outer periphery. The seedlings were left to stand in the dark at 28℃ for 48 hours to obtain tomato seedlings infected with Javan root-knot nematodes.

[0052] (5) Place the 24-well cell culture plate on ice for 10 minutes. Soak the tomato seedlings infected with Javan root-knot nematodes in a 5% sodium hypochlorite aqueous solution for 13 minutes. Then rinse the seedlings with sterile water until odorless. Stain the roots of the tomato seedlings with a 13% (w / v) acid fuchsin aqueous solution, microwave for 2 minutes, prepare slides, and observe under a microscope. Count the number of nematodes in each tomato root. The results are as follows: Figure 2 As shown.

[0053] Comparative Example 2

[0054] The difference between this comparative example and Example 2 is that the culture medium for culturing nematodes is L-glutamylglycine working solution (concentration of 12.5 μg / mL), sterile water control group and 2% methanol dilution control group (volume percentage concentration), while the other steps are the same.

[0055] The results of the infectivity of Javan root-knot nematode on tomato seedlings are as follows: Figure 2 As shown, L-glutamyl glycine in the Javan root-knot nematode infection inhibitor exhibited a significant inhibitory effect on Javan root-knot nematode infection at concentrations of 25-75 μg / mL; however, a concentration of 12.5 μg / mL was insufficient to effectively interfere with the nematode's infection ability. Furthermore, using a 2% methanol dilution as a control, the results showed that its infection level was essentially consistent with that of the sterile water control group, indicating that this solvent concentration did not affect the normal infection behavior of the nematodes in this system, thus eliminating solvent interference.

[0056] Example 3

[0057] The inhibitory effect of L-glutamylglycine on Javan root-knot nematode infection was determined in a pot experiment. The specific steps are as follows:

[0058] 1. Cultivating tomato seedlings:

[0059] (1) Soak the seeds of infected tomato 903 in a 9 cm warm water plate (the volume ratio of boiling water to cold water is 3:2) to cultivate the seeds, so that the warm water submerges the seeds and prevents the seeds from clumping together. Place the plate in a constant temperature incubator at 28°C for 24 hours. After 24 hours, take out the plate and wash the tomato seeds thoroughly with sterile water 10 times to obtain the tomato seeds after being thoroughly washed with sterile water.

[0060] (2) Use sterile tweezers to lay the filter paper flat on a 9 cm culture plate and add 3 ml of sterile water to it; after thoroughly washing the tomato seeds with sterile water, place them one by one onto the moistened filter paper and place them in a 28℃ constant temperature incubator for 24 hours; after the seeds sprout white buds, place the seeds into a 50-well seedling tray, put two seeds in each well, gently cover the surface of the seeds with a layer of soil, and water them regularly. After 20 days, transplant the seedlings into a large pot and allow the seedlings to adapt for 5 days.

[0061] 2. Preparation of L-glutamylglycine working solution:

[0062] (1) Add 19.6 mL of pure water to 0.4 mL of methanol (analytical grade) to prepare a methanol dilution with a volume percentage concentration of 2%.

[0063] (2) Weigh 0.5 mg of L-glutamylglycine and place it in a 1.5 mL centrifuge tube. Use a pipette to transfer 1 mL of 2% methanol dilution to the 1.5 mL centrifuge tube containing L-glutamylglycine. Mix thoroughly and then transfer it to a 15 mL centrifuge tube. Add 4 mL of 2% methanol dilution to prepare a stock solution with a concentration of 100 μg / mL.

[0064] (3) Dilute the 100 μg / mL mother solution with 2% methanol diluent to prepare 2 mL working solutions with concentrations of 75 μg / mL, 50 μg / mL, and 25 μg / mL, respectively. The dilution methods are as follows: 75 μg / mL (volume ratio of mother solution to diluent is 1.5:0.5); 50 μg / mL (volume ratio of mother solution to diluent is 1:1); 25 μg / mL (volume ratio of mother solution to diluent is 0.5:1.5). Add the different concentrations of L-glutamylglycine working solutions to 6-well plates, and set 2% methanol diluent as a blank control.

[0065] 3. Javan root-knot nematode feeds on L-glutamylglycine and its infectivity was tested:

[0066] (1) Collect freshly hatched second-instar Javan larvae into a 15mL centrifuge tube, centrifuge at 3500rpm for 3min, discard the supernatant, add sterile water and centrifuge at 3500rpm for 3min. Repeat the centrifugation operation three times to obtain washed second-instar Javan larvae.

[0067] (2) Dilute the standard M9 buffer solution 4 times with sterile deionized water to obtain 0.25×M9 working solution; rinse the pipette tip with 0.25×M9 working solution, then aspirate the washed second instar Java larvae and count the number of nematodes under a stereomicroscope.

[0068] (3) Add about 20,000 washed second-instar Javan larvae to working solutions of L-glutamyl glycine with concentrations of 75 μg / mL, 50 μg / mL and 25 μg / mL, and then place them in a constant temperature incubator at 28°C for 48 hours.

[0069] (4) Extract the nematodes from the working solutions of L-glutamylglycine at different concentrations, wash the nematodes three times with sterile water, add 2500 nematodes to each pot of tomatoes, add an appropriate amount of water to flush the nematodes to the middle of the soil layer, and continue cultivation for 45 days; after 45 days, pull out the tomatoes, wash the soil off the roots with clean water, and soak the tomato seedlings infected with Javan root-knot nematodes in a 5% sodium hypochlorite aqueous solution for 13 minutes to make the roots appear whiter. Figure 4 As shown.

[0070] (5) The root knot grade was determined using the grading standard of Garabedian and Van Grundy (1983): Disease index = ∑(number of diseased plants at each level × representative value at each level) / (total number of plants surveyed × highest representative value) × 100, where the representative values ​​for each level are 0, 1, 2, 3, 4, and 5. The number of root knots and roots of Javan root-knot nematodes treated with different concentrations of L-glutamyl glycine were counted, and their disease grade indices were calculated. The results are as follows: Figure 3 As shown.

[0071] Comparative Example 3

[0072] The difference between this comparative example and Example 3 is that the culture medium for nematodes was L-glutamylglycine working solution (concentrations of 6.25 μg / mL and 12.5 μg / mL) and a control group with a 2% (v / v) methanol dilution. All other steps were the same. The results of the infection ability of the southern root-knot nematode on tomato seedlings are as follows: Figure 3 and Figure 4 As shown.

[0073] Combination Figure 3 and Figure 4Analysis showed that when the concentration of L-glutamylglycine was 25-75 μg / mL (Example 3), it had a significant effect on inhibiting nematode infection, especially when the concentration of L-glutamylglycine was 50 μg / mL, the disease index dropped to below 50; when the concentration of L-glutamylglycine was reduced to 6.25-12.5 μg / mL (Comparative Example 3), the inhibitory effect on nematode infection was not obvious, and the disease index was similar to that of the control group. Figure 4 (a) is the control group, i.e., no L-glutamylglycine was added; Figure 4 In the tomato roots of (a), (e) and (f), the number of root knots was the largest, and the roots were blackened and deformed, showing severe symptoms of nematode infestation. Figure 4 In (b) and (c), when the concentration of L-glutamylglycine was 75 μg / mL and 50 μg / mL, the disease severity in tomatoes decreased by about 50%.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of L-glutamylglycine in the control of plant root-knot nematodes, wherein, The structural formula of the L-glutamylglycine is as follows: 。 2. The application of L-glutamylglycine in the preparation of root-knot nematode infection inhibitors, wherein, The structural formula of the L-glutamylglycine is as follows: 。

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