Use of the compound in the tropism of root-knot nematodes

Abstract

The present invention provides the use of a compound in the tropism of root-knot nematodes. The compound is at least one of phloroglucinol, diethyl phosphite, geraniol, 4-ethylaniline, methyl thiophene-2-carboxylate, or p-anisidine, and the application of the compound in the preparation of a root-knot nematode repellent; the compound is at least one of p-anisidine, 4-hydroxy-5,6-dimethylpyran-2-one, racemic camphorsulfonic acid, potassium sodium tartrate tetrahydrate, or p-butylaniline, and the application of the compound in the preparation of a root-knot nematode attractant. The present invention designs a model based on P glue to quantify the tropism index (including positive and negative tropisms) of root-knot nematodes to a test substance, and finds that phloroglucinol exhibits the strongest repellent activity against root-knot nematodes, while 5 μg / mL and 10 μg / mL of p-anisidine exhibit the strongest attractant activity against root-knot nematodes, and a repellent activity appears when the concentration is increased to 25 μg / mL and above.

Description

Technical Field

[0001] The present invention relates to the field of nematode chemotaxis, and in particular to the use of compounds in root-knot nematode chemotaxis. Background Art

[0002] Root-knot nematodes (Meloidogyne spp) are parasitic nematodes that live in plant roots and can harm most flowering plants. They mainly rely on their mouth needles to injure the parasites and induce giant cells at the feeding sites to affect plant health. Crops infected with root-knot nematodes have a 40%-80% reduction in yield. In severe cases, large tracts of crops in continuously cropped fields may even fail to produce a harvest. Their populations are widely distributed around the world, causing crop losses of more than $100 billion each year. As a major agricultural country, my country's main root-knot nematode species that harms production is the southern root-knot nematode. Developing green control methods to reduce the economic losses caused by root-knot nematodes has become a very important topic at present.

[0003] After the root-knot nematode develops from an egg into a second-instar larva, using its limited energy to move to the root of the host plant and complete the infection is the most important part of its life history. This process is inseparable from the directional effect of the chemical receptors in the nematode's body on the movement of the nematode. The head sensor, as the main chemical sensor in the nematode, can instruct the nematode to distinguish the secretions of the plant root system. Studies have shown that plant-specific metabolites such as tomatine, zeatin, and limonene can effectively guide the directional movement of nematodes and have a concentration-dependent effect. This indicates that after the nematode head sensor is stimulated by specific secretions, it moves from the low-concentration area to the high-concentration area and finally reaches the plant root for infection. In addition to substances that attract nematodes, the soil environment also contains substances that can have a negative impact on nematodes, inhibit their life activities or repel them. Some of these substances come from the soil, such as inorganic salt potassium nitrate, and some come from microorganisms in the soil, such as benzothiazole, 2-octanone, etc. There are also self-defense active substances such as thymol and dihydrocapsaicin that are secreted by plants after being stimulated by disease infection. These substances can repel the second-instar larvae of root-knot nematodes, indicating that the root-knot nematode chemoreceptors can warn them to escape harmful substances. By taking advantage of this characteristic, if substances with repellent activity are applied near the roots of crops in advance, it will be possible to prevent infection and reduce diseases. Therefore, developing specific compounds that affect the tropism of root-knot nematodes and applying them to crop root protection is a new strategy for root-knot nematode control.

[0004] In order to effectively test the chemotaxis of root-knot nematodes to substances, many research methods have been applied and verified. For example, the agarose method, the sand method, the microfluidic chip method, Pluronic F-127 (hereinafter referred to as "P gel"), etc. Among them, P gel is a new type of high molecular weight non-ionic surfactant, which is a polyoxyethylene polyoxypropylene ether block copolymer. P gel with a concentration of 23% has the reversible property of being a liquid at 4°C and condensing into a solid gel at room temperature. It has been widely used in the field of nematode behavior research because it is easy to operate and root-knot nematodes can move three-dimensionally in it and be observed in real time. At present, the behavioral research of P gel on root-knot nematodes mainly focuses on the qualitative screening of attracting active substances. Many scholars have used this material to complete the research on the chemotaxis of root-knot nematodes to plant host secretions and found many attracting substances such as methyl salicylate, rhamnogalacturonan-I, cadaverine, and putrescine. However, there are still many compounds used for the chemotaxis of root-knot nematodes. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to propose the use of compounds in the chemotaxis of root-knot nematodes, to provide compounds including phloroglucinol that exhibit strong repellent activity against Meloidogyne incognita, and to provide a repellent activity model designed using P gel for the qualitative and quantitative analysis of chemotactic compounds, laying a foundation for the rapid screening of nematode chemotactic compounds.

[0006] The technical solution of the present invention is realized as follows:

[0007] Use of a compound in the chemotaxis of root-knot nematodes, wherein the compound is at least one of phloroglucinol, diethyl phosphite, geraniol, 4-ethylaniline, methyl thiophene-2-carboxylate, or p-anisidine, and the application of the compound in the preparation of a root-knot nematode repellent.

[0008] Furthermore, the use concentration of the compound is 5 - 200 μg / mL.

[0009] Furthermore, when the compound is p-anisidine, the use concentration of p-anisidine is 25 - 200 μg / mL.

[0010] Use of a compound in the chemotaxis of root-knot nematodes, wherein the compound is at least one of p-anisidine, 4-hydroxy-5,6-dimethylpyran-2-one, racemic camphorsulfonic acid, potassium sodium tartrate tetrahydrate, or p-butylaniline, and the application of the compound in the preparation of a root-knot nematode attractant.

[0011] Furthermore, the use concentration of the compound is 5 - 10 μg / mL.

[0012] Furthermore, the root-knot nematode is Meloidogyne incognita.

[0013] Further, the present invention provides a root-knot nematode repellent or attractant. The active ingredient of the repellent includes at least one of phloroglucinol, diethyl phosphite, geraniol, 4-ethylaniline, methyl thiophene-2-carboxylate or anisidine; the active ingredient of the attractant includes at least one of p-anisidine, 4-hydroxy-5,6-dimethylpyran-2-one, racemic camphorsulfonic acid, potassium sodium tartrate tetrahydrate or p-butylaniline.

[0014] The present invention also provides a chemotaxis model for quantitatively evaluating root-knot nematodes. Based on the P gel, a model for quantifying the chemotaxis index (including positive and negative chemotaxis) of root-knot nematodes to test substances is designed. The evaluation method for quantitatively screening test compounds using the chemotaxis model includes the following:

[0015] S1 Design of the model: Place a silica gel mold at the bottom of a petri dish. Divide the silica gel mold into two regions with equal areas. Region 1 is filled with a Pluronic F-127 gel colloid containing the test compound, and Region 2 is filled with a Pluronic F-127 gel colloid containing the control solvent. Scrape off the solidified colloid at the junction of Region 1 and Region 2 to form a blank region, and add a Pluronic F-127 gel colloid to the blank region as a buffer region.

[0016] S2 Trajectory processing: Inject root-knot nematode larvae into the center of the buffer region and place it in a dark environment. After the root-knot nematodes move in the gel to form a trajectory, use a stereomicroscope and a camera to take pictures of the trajectory.

[0017] S3 Chemotaxis calculation: Process the photographed trajectory picture into a black-and-white image. After processing and calculating the number of white pixels corresponding to the trajectory, the percentage of the nematode trajectory in the total number of pixels in the whole image can be obtained, and then the chemotaxis index CI is calculated. The formula is as follows:

[0018] Chemotaxis index CI = (Percentage of the number of white pixels corresponding to the trajectory in Region 1 in the total number of pixels in the whole image) / (Percentage of the number of white pixels corresponding to the trajectory in Region 1 in the total number of pixels in the whole image + Percentage of the number of white pixels corresponding to the trajectory in Region 1 in the total number of pixels in the whole image) × 100% / 50%.

[0019] Further, the temperature in the dark environment is 24 - 26 °C, and the time in the dark environment is 7 - 9 h; the black-and-white image is an 8-bit image.

[0020] Further, it is stipulated that when the chemotaxis index CI is 0.95 - 1.05, it is considered that the test compound has no obvious effect on the chemotaxis of root-knot nematodes. When it is higher than 1.05, it is determined that the test compound has an attracting activity on root-knot nematodes. When it is lower than 0.95, it is determined that the test compound has a repellent activity on root-knot nematodes.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention finds that compounds such as phloroglucinol, diethyl phosphite, geraniol, 4-ethylaniline or thiophene-2-carboxylic acid methyl ester have repellent activity against root-knot nematodes, among which phloroglucinol exhibits the strongest repellent activity; it is also found that compounds such as p-anisidine, 4-hydroxy-5,6-dimethylpyran-2-one, chromatic camphorsulfonic acid, potassium sodium tartrate tetrahydrate or p-n-butylaniline have attractive activity against root-knot nematodes, among which p-anisidine exhibits the strongest attractive activity.

[0023] (2) Based on P glue, the present invention designs a model that can quantify the tropism index (including positive and negative tropism) of root-knot nematodes to test substances. The model can be used to quantitatively evaluate the tropism index of the test compound to southern root-knot nematodes, and can quickly screen root-knot nematode tropism compounds, providing a theoretical basis and foundation for the quantitative evaluation of the activity of root-knot nematode tropism substances.

[0024] (3) The model provided by the present invention was used to detect the concentration gradient response of southern root-knot nematodes to p-anisidine and phloroglucinol. The results showed that within the concentration range of 5, 10, 25, 50, 100, and 200 μg / mL, p-anisidine had a tropism activity of attracting the second-instar larvae of southern root-knot nematodes at low concentrations and repelling them at high concentrations; while the repellent activity of phloroglucinol on the second-instar larvae of southern root-knot nematodes increased with increasing concentration, and no lethal effect was observed on the nematodes within this range.

[0025] (4) The attraction activity of p-anisidine to nematodes increased with the increase of concentration at 5 μg / mL and 10 μg / mL; and repellent activity appeared when the concentration was increased to 25 μg / mL and above; phloroglucinol showed repellent activity at 5 μg / mL, and the repellent activity gradually increased with the increase of concentration, and the tropism activity was concentration-dependent. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the model design of Example 1 of the present invention, A is the test compound side, and B is the solvent side;

[0027] Figure 2 Comparison between the tridecane-treated side and the solvent side, A is the tridecane side, and B is the solvent side;

[0028] Figure 3 Comparison between the benzothiazole-treated side and the solvent side, A is the benzothiazole side, and B is the solvent side;

[0029] Figure 4 For comparison between the p-anisidine-treated side and the solvent side, A is the p-anisidine side and B is the solvent side;

[0030] Figure 5For the comparison between the phloroglucinol-treated side and the solvent side, A is the phloroglucinol side and B is the solvent side. Detailed implementation manners

[0031] To better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0032] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0033] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can all be obtained from commercial channels.

[0034] Example 1 - Design a chemotaxis model for quantitatively evaluating root-knot nematodes using P glue as a material

[0035] (1) Preparation of P glue colloid

[0036] Take 23 g of P glue colloid powder, stir and dissolve it in 80 mL of distilled water at 4°C for 24 h to obtain 100 mL of P glue colloid. When making P glue colloid containing the test compound or the control solvent, add the liquid medicine containing the test drug to the distilled water and stir to dissolve the P glue colloid powder to prepare the P glue colloid containing the test compound at the corresponding concentration. For the solvent control, add the same amount of solvent as the liquid medicine. The prepared colloids are all stored in a 4°C refrigerator for later use.

[0037] (2) Design and production of the model

[0038] Place a silica gel mold with an internal length of 40 mm, width of 20 mm, and height of 5 mm at the bottom of a 60-mm-diameter glass petri dish. Place a silica gel spacer in the center of the mold to divide the area into two equal-sized square areas. Add 1 mL of P glue colloid containing the test compound to the left as the treatment side ( Figure 1 A), and add 1 mL of P glue colloid containing the control solvent to the right as the solvent side ( Figure 1 B). After placing it at room temperature and waiting for it to solidify, remove the central silica gel spacer, and scrape off 8 mm wide of the colloid, 4 mm on each side at the junction. Add 0.5 mL of blank P glue colloid to the remaining blank area as the buffer area. After all the colloids solidify, inject 10 μL of about 150 second-stage larvae of Meloidogyne incognita into the center of the colloid buffer area, keep it moist and place it in a 25°C dark environment for 8 h. After the root-knot nematodes move in the colloid to form a track, take pictures of the treatment side and the solvent side of the colloid plane at a fixed magnification and parameters using a NIKON SMZ745T stereomicroscope and a supporting digital camera.

[0039] (3) Calculation method of chemotaxis index

[0040] Processed into a black-and-white 8-bit image using the image processing software ImageJ. After processing and calculating the number of white pixels corresponding to the track, the percentage of the nematode track in the total number of pixels in the whole image can be obtained, and then the chemotaxis index (CI) can be calculated.

[0041]

[0042] The calculation method is as shown in formula (1), where A is the percentage of the treated side and B is the percentage of the solvent side. It is stipulated that the chemotaxis index of the blank control with 50% for both the left and right percentages is 1.00. When the chemotaxis index is in the range of 1.00 ± 0.05, it is considered that the treatment has no obvious effect on nematode chemotaxis. When it is higher than 1.05, it is determined to have attracting activity, and when it is lower than 0.95, it has repellent activity. That is, the sparser the track in the treatment area and the denser the track in the solvent area, the better the repellent effect of the drug and the lower the chemotaxis index. On the contrary, the better the attracting effect and the higher the chemotaxis index.

[0043] Example 2 - Verification of the chemotaxis model

[0044] The reliability of the model was verified using 10 reported compounds with chemotactic activity. The verification results are shown in Table 1. The results show that the chemotaxis results measured by this model are all consistent with the literature reports, proving the rationality of the design and statistical methods of this model. Meloidogyne incognita is more sensitive to attracting substances such as methyl salicylate and tridecane, and obvious attracting activity can be detected at 1 or 10 μg / mL; for compounds with repellent activity, generally a concentration of 50 μg / mL or higher is required to effectively make Meloidogyne incognita avoid. In the repellent activity test of this model, it can be clearly observed that the track distribution of Meloidogyne incognita in the treated side is relatively sparse, proving that the repellent substance makes the nematodes give up entering the treated side and turn to move in the solvent side.

[0045] Table 1 Chemotactic activity of verified compounds against Meloidogyne incognita

[0046]

[0047] Example 3 - Chemotactic activity of Meloidogyne incognita against 40 compounds

[0048] (1) Chemotactic activity of Meloidogyne incognita against 40 compounds

[0049] As shown in Table 2 and Figure 4 、 Figure 5As shown, the present invention has completed the determination of the tropism indices of root-knot nematodes towards 40 untested compounds. Among them, 20 have attracting activity and 13 have repellent activity. Among the 40 tested compounds, p-anisidine shows the strongest attracting activity with a tropism index of 1.53 ± 0.09, while phloroglucinol shows the strongest repellent activity with a tropism index of 0.69 ± 0.06.

[0050] Table 2 Tropism activities of tested compounds against Meloidogyne incognita

[0051]

[0052]

[0053] (2) Concentration gradient tropism activities of Meloidogyne incognita towards p-anisidine and phloroglucinol

[0054] The model of Example 1 was used to detect the concentration gradient responses of Meloidogyne incognita towards p-anisidine and phloroglucinol. The results showed that the attracting activity of p-anisidine towards nematodes increased with the increase in concentration at 5 μg / mL and 10 μg / mL; while repellent activity appeared when the concentration increased to 25 μg / mL and above, indicating that it attracts nematodes at low concentrations and produces a repellent effect when the concentration is too high. Phloroglucinol showed repellent activity at 5 μg / mL, and the repellent activity gradually increased with the increase in concentration, and the tropism activity was concentration-dependent. When the concentration increased to 50, 100, and 200 μg / mL, the change in the tropism activity of nematodes tended to be flat, and there was no obvious change in the tropism index when the concentration was further increased; no obvious death of nematodes was found at the measured concentrations.

[0055] Table 3 Tropism activities of concentration gradients of p-anisidine and phloroglucinol against Meloidogyne incognita

[0056]

[0057]

[0058] As can be seen from the above examples, the quantitative evaluation method for the tropism model of Meloidogyne incognita obtained in the present invention is reasonable in design and calculation. Through this model, the tropism activities of 40 compounds were analyzed, and p-anisidine with the strongest attracting activity and phloroglucinol with the strongest repellent activity were screened out, which are expected to be developed into potential nematode attractants and repellents in the field.

[0059] The description of the above examples is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a compound in the tropism of root-knot nematodes, characterized in that, The compound is phloroglucinol, and its application in preparing a root-knot nematode repellent. The usage concentration of the compound is 5 - 200 μg / mL, and the root-knot nematode is Meloidogyne incognita.

2. A method for evaluating the use of the compound of claim 1 in the tropism of root-knot nematodes, characterized in that, The evaluation method using the tropism model includes the following: Design of the S1 model: Place a silicone mold at the bottom of a Petri dish. Divide the silicone mold into two regions with equal areas. Add Pluronic F-127 colloidal solution containing the test compound to region 1, and add Pluronic F-127 colloidal solution containing the control solvent to region 2. Scrape off the solidified colloidal solution at the junction of region 1 and region 2 to form a blank region, and add Pluronic F-127 colloidal solution to the blank region as a buffer region. Treatment of the S2 trajectory: Inject the root-knot nematode larvae into the center of the buffer region and place it in a dark environment. After the root-knot nematodes move in the colloid to form a trajectory, use a stereomicroscope and a camera to take pictures of the trajectory. The temperature in the dark environment is 24 - 26 °C, and the time in the dark environment is 7 - 9 h. Calculation of tropism: Process the photographed trajectory image into a black-and-white image. After processing and calculating the number of white pixels corresponding to the trajectory, the percentage of the nematode trajectory in the total number of pixels in the whole image can be obtained, and then the tropism index CI is calculated. The formula is as follows: Tropism index CI = (Percentage of the number of white pixels corresponding to the trajectory in region 1 in the total number of pixels in the whole image) / (Percentage of the number of white pixels corresponding to the trajectory in region 1 in the total number of pixels in the whole image + Percentage of the number of white pixels corresponding to the trajectory in region 1 in the total number of pixels in the whole image) × 100% / 50%; The black-and-white image is an 8-bit image. It is stipulated that when the tropism index CI is 0.95 - 1.05, it is determined that the test compound has no obvious effect on the tropism of root-knot nematodes. When it is higher than 1.05, it is determined that the test compound has an attracting activity on root-knot nematodes. When it is lower than 0.95, it is determined that the test compound has a repellent activity on root-knot nematodes.

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