Application of ketone volatile compound in prevention and treatment of root-knot nematode

Abstract

The invention discloses application of a ketone volatile compound in prevention and treatment of root-knot nematode, and belongs to the technical field of prevention and treatment of root-knot nematode. The ketone volatile compound comprises at least one of methyl nonyl ketone, 2-nonyl ketone and 2-dodecanone. The three compounds have high-efficiency nematicidal activity when being independently or compounded, and can obviously reduce the occurrence of the plant Penniospermum erythrorhizon root knot nematode disease. Specifically, the corrected death rates of the Meloidogyne auricularia for the methyl nonyl ketone, the 2-nonyl ketone and the 2-dodecanone are 81.11%, 76.67% and 73.33% respectively, and when the methyl nonyl ketone and the 2-nonyl ketone are compounded, the corrected death rate is as high as 93.33%. Therefore, the invention provides a novel method for preventing and treating the root-knot nematode.

Description

Technical Field

[0001] The present invention relates to the technical field of nematode control, in particular to the application of ketone volatile compounds in controlling root-knot nematodes. Background Art

[0002] Root-knot nematodes belong to the genus Meloidogyne, family Meloidogynidae. Their second-instar larvae are migratory and can parasitize over 5,000 plant species. They are the second most common crop disease, after fungal diseases. After infecting plants, the larvae use their proboscises to penetrate host tissue and absorb nutrients, causing root knots to form on crop roots. This severely interferes with nutrient absorption and causes significant economic losses to agriculture.

[0003] The elephant-ear bean root-knot nematode (Meloidogyne enterolobii) is a key root-knot nematode species of concern due to its high reproductive capacity, wide host range, and ability to overcome resistance genes such as Mi, N, and RK, resulting in enhanced pathogenicity and infectivity. In recent years, this nematode has affected a large number of legume, cucurbit, solanaceae, and mulberry crops, gradually surpassing the southern root-knot nematode (Meloidogyne incognita) in infection rates and becoming the dominant species.

[0004] Currently, the primary control method for root-knot nematodes in ear beans is still chemical pesticides, while research on biocontrol bacteria is relatively limited. Long-term reliance on chemical agents not only poses safety risks but can also lead to soil compaction and environmental pollution. Therefore, developing safe, effective, and environmentally friendly biological control methods has become a research priority.

[0005] Volatile organic compounds (VOCs) have broad application prospects in plant disease control due to their excellent permeability and long-range action, enabling them to exert insecticidal and disease-suppressing effects without direct plant contact and rarely leaving residues on crop surfaces. Recent studies have shown that Bacillus sp. can release a variety of VOCs with nematicidal activity. For example, Wu et al. isolated a highly effective nematicidal strain, Bacillus velez GJ-7, from the rhizosphere soil of Panax notoginseng. Using GC-MS, they identified six VOCs, demonstrating direct contact killing and oocyst hatching inhibition against Melodogyne hapla. Furthermore, Dai et al. found that volatile gases produced by Bacillus pumilus S1-10 exhibited a significant repellent effect against Melodogyne incognita, achieving a control efficacy of up to 79% on plants. They also identified 2-(methylamino)-ethanol (2-ME), a key volatile with strong fumigant activity.

[0006] Despite this, current research on the application of volatile organic compounds in controlling root-knot nematodes in elephant ear bean remains relatively weak, and the development of related microbial agents and biofumigants is still in its infancy. Therefore, identifying volatile metabolites with insecticidal activity and developing them into highly effective and safe biological agents has become a research hotspot in the field of biocontrol and a key technical challenge that urgently needs to be overcome. Summary of the Invention

[0007] The present invention aims to provide the use of ketone volatile compounds in controlling root-knot nematodes to solve the problems existing in the above-mentioned prior art. The present invention finds that 2-nonanone, methyl nonyl ketone and 2-dodecanone all have strong nematicidal activity and can significantly inhibit the infection of elephant ear bean root-knot nematodes on plants. They have the application prospect of being developed as potential biopesticides for controlling elephant ear bean root-knot nematodes.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides application of ketone volatile compounds in preventing and controlling root-knot nematodes. The ketone volatile compounds include at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

[0010] Preferably, the ketone volatile compound is methyl nonyl ketone and 2-nonanone;

[0011] The methyl nonyl ketone and 2-nonanone are mixed in liquid form at a volume ratio of 1:1, and the concentrations of the methyl nonyl ketone and 2-nonanone are both 49.18 μL / L.

[0012] Optionally, the root-knot nematode comprises R.

[0013] The present invention also provides the use of ketone volatile compounds in preparing a medicament for controlling root-knot nematodes, wherein the ketone volatile compound comprises at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

[0014] Preferably, the volatile ketone compound is a mixture of methyl nonyl ketone and 2-nonanone;

[0015] The methyl nonyl ketone and 2-nonanone are mixed in liquid form at a volume ratio of 1:1, and the concentrations of the methyl nonyl ketone and 2-nonanone are both 49.18 μL / L.

[0016] Optionally, the root-knot nematode comprises R.

[0017] The present invention also provides a medicament for preventing and treating root-knot nematode diseases, wherein the active ingredient of the medicament comprises at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

[0018] The present invention also provides a method for controlling root-knot nematodes, comprising the step of fumigating the root-knot nematodes with at least one volatile compound selected from the group consisting of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

[0019] Preferably, the fumigation is closed fumigation.

[0020] Optionally, the root-knot nematode comprises R.

[0021] The present invention discloses the following technical effects:

[0022] The present invention found that three ketone volatile compounds, methyl nonyl ketone, 2-nonanone and 2-dodecanone, have high nematicidal activity alone or in combination, and can significantly reduce the occurrence of root-knot nematode disease in plants. Specifically, the corrected mortality rates of methyl nonyl ketone, 2-nonanone and 2-dodecanone against root-knot nematodes in ear beans were 81.11%, 76.67% and 73.33%, respectively. In potted plant efficacy verification, the control rates of root-knot nematodes in ear beans were 90.51%, 88.32% and 89.78%, respectively, which are comparable to the control efficiency of the chemical pesticide fluopyram. When the three ketone volatile compounds are compounded, the corrected mortality rate of root-knot nematodes in ear beans can reach 77.78% or more. Among them, when methyl nonyl ketone and 2-nonanone are compounded, the corrected mortality rate is as high as 93.33%. The present invention provides a new method for the prevention and control of root-knot nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 Total ion current diagram; A: total ion current diagram of control group (LB); B: total ion current diagram of GX0002980;

[0025] Figure 2 The indoor device for testing three ketone volatile compounds against the root-knot nematode R. serrata; A: 90 mm Petri dish; B: 6 mm round sterile filter paper; C: 36 mm Petri dish;

[0026] Figure 3 The insecticidal effects of three ketone volatile compounds on the root-knot nematode of elephant ear bean; A: The state of the root-knot nematode of elephant ear bean after treatment with the control group; B: The state of the root-knot nematode of elephant ear bean after treatment with methyl nonyl ketone; C: The state of the root-knot nematode of elephant ear bean after treatment with 2-nonanone; D: The state of the root-knot nematode of elephant ear bean after treatment with 2-dodecanone;

[0027] Figure 4 Diagram of the device for controlling three ketone volatile compounds in potted plants; A: soil layer at the plant roots; B: suspended layer; C: sterile cotton net;

[0028] Figure 5 The results are shown for the roots of the potted plants in the negative control group (CK); the red circle area is the concentrated area of ​​nematode root knots, and the arrow points to the root knots of the plants;

[0029] Figure 6 Results of the positive control group (fluopyram) on the root of potted plants;

[0030] Figure 7 The methyl nonyl ketone treatment group was used to control the root fruiting of the potted plants;

[0031] Figure 8 The 2-nonanone treatment group was used to control the root fruiting of the potted plants;

[0032] Figure 9 The 2-dodecanone treatment group was used to control the root fruiting of the potted plants;

[0033] Figure 10The body states of the root-knot nematode of Elephant's ear bean after treatment with a combination of three volatile ketone compounds; A: control group; B: combination of methyl nonyl ketone and 2-nonanone; C: combination of methyl nonyl ketone and 2-dodecanone; D: combination of 2-nonanone and 2-dodecanone; E: combination of methyl nonyl ketone, 2-nonanone and 2-dodecanone. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The GX0002980 used in the present invention has a deposit number of CGMCC No. 28199, which has been disclosed in patent CN117229954A - A Bacillus velezensis GX0002980 and its application.

[0040] The 2-nonanone and methyl nonyl ketone used in the present invention were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 98%. The 2-dodecanone used was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a purity of 98%.

[0041] The root knot nematodes of elephant ear bean used in the present invention are provided by Guangxi Key Laboratory of Polysaccharide Materials and Modification; the water spinach plants used are purchased from Hefei Hefeng Seed Co., Ltd.

[0042] Unless otherwise specified, materials or reagents whose sources are not specified in the examples of the present invention can be purchased through conventional channels.

[0043] Example 1 Identification of Volatile Compounds from Bacillus velezensis GX0002980

[0044] (1) Sample preparation: Sterilize the LB solid culture medium and cool it to 55°C. Pour it into a headspace bottle and place it at an angle. After it cools and solidifies, use a pipette to draw 20 μL of Bacillus velezensis GX0002980 bacterial suspension (concentration of 1×10 8 CFU / mL) were inoculated on the surface of the culture medium and cultured at 30°C for 7 days. At the same time, 20 μL of sterile water was taken as the control group. Each treatment was repeated three times.

[0045] (2) Separation and identification of volatile components using HS-SPME-GCMS: A 50 / 30 μm PDMS / CAR / DVB extraction fiber (length 2 cm) was used for sample extraction. The extraction fiber was mounted on the SPME injector and aged at 250°C in the GC-MS injection port to ensure that there was no interference from other peaks. After the sample bottle was preheated for 15 minutes, the SPME extraction head was inserted into the bottle and headspace extraction was performed for 40 minutes. The fiber was then inserted into the GC-MS injection port and desorbed at 250°C for 3 minutes before injection and analysis.

[0046] (3) Chromatographic conditions: The chromatographic column was DB-WAX (30.0 m × 250 μm, 0.25 μm). The initial temperature was set at 40°C and maintained for 5 min. The temperature was then increased to 220°C at a rate of 5°C / min, and then to 240°C at a rate of 20°C / min and maintained for 5 min. The carrier gas (He) flow rate was 1.0 mL / min, and the splitless mode was used. EI ion source conditions were: electron energy was set to 70 eV; ion temperature was 230°C; and quadrupole temperature was 150°C. The scanning mode was Scan, and the scanning mass range was 20 to 500 u.

[0047] (4) The volatile components were identified by searching the NIST 11 mass spectrometry database, and the relative content of each component was calculated using the area normalization method.

[0048] Total ion current diagram Figure 1 As shown, by comparing the control group ( Figure 1 A) and treatment group ( Figure 1 In the total ion current graph (B), changes in peak area were observed. Based on the retention time and matching degree (>80), a total of 33 volatile organic compounds were identified, including 8 alkanes, 8 benzenes, 4 alcohols, 9 ketones, 1 ether, 1 ester, 1 oxime, and 1 heterocyclic substance. In this example, three ketone compounds were selected for subsequent verification. The information of the three ketone volatile compounds is shown in Table 1.

[0049] Table 1 Three ketone volatile compounds of Bacillus velezensis GX0002980 identified by GC-MS

[0050]

[0051] Example 2 Insecticidal Effects of Volatile Substances on Root-Knot Nematodes

[0052] Three volatile ketone compounds (2-nonanone, 2-undecanone, and 2-dodecanone) were selected and their insecticidal effects against root-knot nematodes of the ear bean were verified using purchased compound standards.

[0053] Culture of second-instar larvae of the root-knot nematode: Grow water spinach plants to 5-10 cm in height and transplant them into soil infested with 4,000 root-knot nematodes. Incubate for 15 days to allow the nematodes to attach and form knots in the water spinach roots. Egg masses are collected and incubated at 30°C. J2 larvae of the root-knot nematode are then collected for bioactivity assays.

[0054] The insecticidal effects of three volatile ketone compounds on the root-knot nematode of R. Figure 2 As shown, in a 90mm culture dish (16mm high, volume about 101.67mL, Figure 2 A) Place a sterile filter paper (6 mm) in the center ( Figure 2 (B), add 5 μL of volatile compound standard (concentration of 49.18 μL / L). Pipette 200 μL of the second-instar larvae suspension of R. elegans (30) into a 36 mm small culture dish ( Figure 2(C) Place three 36 mm small Petri dishes containing nematode suspension in each 90 mm Petri dish and place them in a dark, airtight environment at 25°C (cover the 90 mm Petri dish lid and seal it with parafilm). After 72 h, observe the death of the nematode larvae and calculate the mortality rate.

[0055] Mortality rate (%) = number of dead elephant ear bean root knot nematodes / total number of elephant ear bean root knot nematodes × 100%.

[0056] Corrected mortality rate (%) = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0057] The insecticidal effects of three ketone volatile compounds on root-knot nematodes of the ear bean are as follows Figure 3 As shown in Table 2, when the volume of volatile compounds was added in the range of 5 μL, the average corrected mortality rate of methyl nonyl ketone in killing root-knot nematodes of Elephant's ear bean was 81.11%, the average corrected mortality rate of 2-nonanone in killing root-knot nematodes of Elephant's ear bean was 76.67%, and the average corrected mortality rate of 2-dodecanone in killing root-knot nematodes of Elephant's ear bean was 73.33%. All three ketones had good nematicidal activity (NA ≥ 60%).

[0058] Table 2 Preliminary test of the insecticidal effects of three ketone compounds on root-knot nematodes of the ear bean

[0059]

[0060] Example 3 Three volatile substances target LC of M. auris 50 Determination of

[0061] The toxicity regression equations of methyl nonyl ketone, 2-nonanone and 2-dodecanone to root knot nematode of ear bean are shown in Table 3. Among them, the LC of methyl nonyl ketone to root knot nematode of ear bean is 50 The concentration of 2-nonanone was 5.472 μL / L, and the LC value of 2-nonanone on R. 50 The concentration of 2-dodecanone was 13.558 μL / L, and the EC 50 The concentration is 24.712μL / L.

[0062] Table 3 Regression equations for the toxicity of three ketone compounds to root-knot nematode M.

[0063]

[0064] Example 4: Potted plant control effects of three volatile substances

[0065] Grow water spinach seedlings until they have four leaves and one heart. Mix infected soil (number of nematodes in infected soil: 4000) with ordinary nutrient matrix soil at a mass ratio of 1:1. Take 200g of the mixed soil and place it in a transparent small flower pot. Transplant water spinach seedlings with consistent growth into the flower pot filled with the mixed soil (leave 1cm of the soil and the bottom suspended). Add volatile compounds (concentration of 49.18μL / L) to the bottom for closed fumigation ( Figure 4 LB blank medium was used as a negative control, and chemical pesticide (fluopyram) was used as a positive control. Five water spinach seedlings in each group were treated with the compound on the 1st, 11th, and 21st days. The plants were harvested on the 30th day, and the root knots were observed, and the root knot control rate was calculated.

[0066] Control rate = [(number of root knots in the negative control group - number of root knots in the treatment group) / number of root knots in the negative control group] × 100%.

[0067] The control effects of three ketone compounds on potted plants are as follows Figure 5 , the root knot situation is shown in Table 4, using methyl nonyl ketone ( Figure 5 A), 2-nonanone ( Figure 5 B) and 2-dodecanone ( Figure 5 The root knot control rates of plants after treatment with medium C) can reach 90.51%, 89.78% and 88.32% respectively, which are comparable to the control rates of the chemical pesticide fluopyram, and have a good potted plant control effect.

[0068] Table 4 Potted plant control results

[0069]

[0070] Example 5 Insecticidal effect of volatile compound on root knot nematode

[0071] A preliminary study used 90 mm and 36 mm Petri dishes to test the insecticidal efficacy of three volatile ketone compounds (methyl nonyl ketone + 2-nonanone, methyl nonyl ketone + 2-dodecanone, 2-nonanone + 2-dodecanone, and methyl nonyl ketone + 2-nonanone + 2-dodecanone, each compound mixed at a volume ratio of 1:1 or 1:1:1, with each compound concentration at 49.18 μL / L) against the root-knot nematode (M. elegans). Three sterile filter paper sheets (6 mm) were placed in the center of the 90 mm Petri dishes, and 5 μL of each compound was added. A 200 μL suspension of 30 second-instar larvae of the M. elegans root-knot nematode (M. elegans) was pipetted into a 36 mm Petri dish. Three 36 mm Petri dishes containing the suspension were placed in each 90 mm Petri dish. The mixture was placed in a dark, sealed environment at 25°C (with the 90 mm Petri dish lids covered). After 72 hours, larval mortality was observed and the mortality rate was calculated.

[0072] Mortality rate (%) = number of dead elephant ear bean root knot nematodes / total number of elephant ear bean root knot nematodes × 100%.

[0073] Corrected mortality rate (%) = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0074] The insecticidal effect of the three ketone compounds on the root knot nematode of ear bean is as follows: Figure 6 The data are shown in Table 5. The mortality rate of root-knot nematodes of elephant ear bean is the highest when 2-nonanone and methyl nonyl ketone are combined at equal volumes, which can reach 93.33%. The insecticidal effect of methyl nonyl ketone combined with 2-dodecanone is lower, which is 77.78%. The effect of the combination of 2-nonanone and 2-dodecanone is equivalent to the effect of the three compounds combined at equal volumes, and the nematode mortality rate is 87.78%.

[0075] Table 5 Preliminary test of the insecticidal effect of three ketone volatile compounds on root-knot nematodes of ear bean

[0076]

[0077]

[0078] Based on the above results, the present invention found that three ketone volatile compounds, methyl nonyl ketone, 2-nonanone and 2-dodecanone, have efficient nematicidal activity alone or in combination, and can significantly reduce the occurrence of root knot nematode disease in plants. Specifically, the corrected mortality rates of methyl nonyl ketone, 2-nonanone and 2-dodecanone against root knot nematodes in ear beans were 81.11%, 76.67% and 73.33%, respectively. In the potted plant efficacy verification, the control rates of root knot nematodes in ear beans were 90.51%, 88.32% and 89.78%, respectively, which are comparable to the control efficiency of the chemical pesticide fluopyram. When the three ketone volatile compounds are compounded, the corrected mortality rate of root knot nematodes in ear beans can reach 77.78% or more. Among them, when methyl nonyl ketone + 2-nonanone are compounded, the corrected mortality rate is as high as 93.33%. The present invention provides a new method for the prevention and control of root knot nematodes.

[0079] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The use of ketone volatile compounds in controlling root-knot nematodes, characterized in that: The ketone volatile compound includes at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

2. The use according to claim 1, characterized in that The ketone volatile compounds are methyl nonyl ketone and 2-nonanone; The methyl nonyl ketone and 2-nonanone are mixed in liquid form at a volume ratio of 1:1, and the concentrations of the methyl nonyl ketone and 2-nonanone are both 49.18 μL / L.

3. The use according to claim 1, characterized in that The root-knot nematodes include the root-knot nematode R.

4. Use of ketone volatile compounds in the preparation of a medicament for controlling root-knot nematodes, characterized in that: The ketone volatile compound includes at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

5. The use according to claim 4, characterized in that The ketone volatile compound is a mixture of methyl nonyl ketone and 2-nonanone; The methyl nonyl ketone and 2-nonanone are mixed in liquid form at a volume ratio of 1:1, and the concentrations of the methyl nonyl ketone and 2-nonanone are both 49.18 μL / L.

6. The use according to claim 4, characterized in that The root-knot nematodes include the root-knot nematode R.

7. A medicament for preventing and treating root-knot nematode diseases, characterized in that: The active ingredient of the medicament includes at least one of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

8. A method for controlling root-knot nematodes, characterized in that: The method comprises the step of fumigating the root-knot nematodes by using at least one volatile compound selected from the group consisting of methyl nonyl ketone, 2-nonanone and 2-dodecanone.

9. The method according to claim 8, wherein The fumigation is closed fumigation.

10. The method according to claim 8, wherein The root-knot nematodes include the root-knot nematode R.

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