CN115067337B - Application of novel pheromone in online behavior research and reagent for inducing fungi to trap and kill nematodes - Google Patents

Abstract

The invention discloses application of a novel pheromone in online nematode behavior research and a reagent for inducing fungi to trap and kill nematodes, and relates to the technical field of nematode behavior research and trapping and killing. The invention discovers two glucopyranosides, namely ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1), from a nematode culture for the first time, and utilizes the two glucopyranosides to carry out verification tests of nematode behaviors and induction of fungi to trap and kill nematodes, thereby proving the novel pheromones of the two glucopyranosides for determining the nematode, providing substantial progress evidence for a novel nematode 'pheromone system' theory, and solving the technical problem that the prior art lacks practical research evidence for the novel nematode 'pheromone system' theory.

Description

Application of novel pheromone in online behavior research and reagent for inducing fungi to trap and kill nematodes

Technical Field

The invention relates to the technical field of nematode behavior research and trapping and killing, in particular to application of a novel pheromone in nematode behavior research and a reagent for inducing fungi to trap and kill nematodes.

Background

The root knot nematode disease causes huge loss to grain crops all over the world, and abundant nematode populations coexist in the soil habitat. The green and safe prevention and control of the root-knot nematodes are an important aspect of maintaining the ecological stability of the whole soil, and it is a new direction for people to mobilize biological groups in soil ecological niches to deal with the root-knot nematodes. "things-by-things and people-by-groups" are very common human behaviors, and we believe that the phenomenon of 'insects-by-groups' also exists in soil. Ascaroside systems (ascarosides systems) are currently recognized as nematode-secreted pheromone systems, which can help nematodes sense the environment and realize communication among nematode individuals, and have a regulating effect on the physiology, development and behavior of nematodes. Ascaroside is a specific signal molecule produced by nematodes, and the skeleton is mainly: fatty acid side chains with different lengths and the like are connected to the dideoxy ascaridyl, and can be used as signals for the communication between nematodes to regulate the aggregation, mating, goldol formation and the like of the nematodes. Nematodes are among the most strategic animals on earth, although more than about 100 ascarosides have been isolated and identified from nematodes and have been shown to contain more than about 200 ascarosides by instrumental detection.

However, various studies have shown that nematodes may contain an additional set of "pheromone systems" that can provide long-term protection of nematodes, small animals, throughout the ecosystem. A novel pheromone system for deeply exploring nematodes is used for performing population distribution intervention on the nematodes in soil, and is an effective means for controlling the number of root-knot nematodes. However, the theory of the new type "pheromone system" in the prior art lacks practical research evidence, and for the new type "pheromone system", we need to find out specific pheromone substances and provide research data to obtain further development of the theory.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of a novel pheromone in the research of nematode behaviors and the reagent for inducing fungi to trap and kill nematodes, two glucopyranosides, namely ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1), are found from nematode cultures for the first time, and the two glucopyranosides are utilized to carry out verification tests in the nematode behaviors and the induction of fungi to trap and kill nematodes, so that the novel pheromones of the two glucopyranosides in definite lines of nematodes are proved, substantial progress evidences are provided for the novel nematode pheromone system theory, and the technical problem that the novel nematode pheromone system theory in the prior art lacks practical research evidences is solved.

The technical scheme adopted by the invention is as follows:

the application of a novel pheromone in researching on-line nematode behaviors and inducing fungi to trap and kill nematodes is characterized in that the novel pheromone is two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside, and the structures of the novel pheromone are respectively shown as formula 1 and formula 2:

preferably, the two glucopyranosides are separated from the culture solution of the whole tooth revival nematodes.

Preferably, the nematode behaviour is nematode chemotaxis.

In summary, compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention discovers two glucopyranosides, namely ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1), from a nematode culture for the first time, and utilizes the two glucopyranosides to carry out verification tests of nematode behaviors and induction of fungi to trap and kill nematodes, thereby proving the novel pheromones of the two glucopyranosides for determining the nematode, providing substantial progress evidence for a novel nematode 'pheromone system' theory, and solving the technical problem that the prior art lacks practical research evidence for the novel nematode 'pheromone system' theory.

2. The invention discovers that two glucopyranosides, namely ethyl-beta-D-glucopyranoside (wp-2) and butyl-beta-D-glucopyranoside (wp-1), have remarkable and broad-spectrum trap induction activity;

3. the root knot nematode disease causes huge loss to grain crops in the world, and biological control is a relatively green method for controlling the nematode disease at present. A large amount of fungi and nematodes exist in soil, 2 glucopyranosides in the invention can obviously regulate and control the behavior of the nematodes and induce the fungi to produce traps at the nM concentration, the compound does not cause pollution, and the nematology can be developed into a nematode biocontrol preparation, thus having better application prospect.

Drawings

FIG. 1 is a schematic diagram of the chemotactic behavior of a compound towards nematodes;

FIG. 2 is a statistical chart of chemotaxis results of compound wp-1 on caenorhabditis elegans;

FIG. 3 is a statistic diagram of chemotaxis results of compound wp-1 on Meloidogyne incognita;

FIG. 4 is a statistical chart of chemotactic results of compound wp-1 on flower rootworm and knot nematode;

FIG. 5 is a statistical chart of chemotaxis results of compound wp-2 on C.elegans;

FIG. 6 is a statistical chart of chemotaxis results of compound wp-2 on Meloidogyne incognita;

FIG. 7 is a statistical chart of chemotactic results of compound wp-2 on flower rootworm and knot nematode;

FIG. 8 is a graph showing the results of the induction of three-dimensional bacterial reticulum of Arthrobotrys by compounds wp-1 and wp-2;

FIG. 9 is a graph showing the results of induction of myxosphere of interdigital spores by compounds wp-1 and wp-2;

FIG. 10 is a graph showing the results of the induction of the contractile loops of Acremonium monospora and Dinophora septaeformans by compounds wp-1 and wp-2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The preparation process of the compounds wp-1 and wp-2 used in the invention comprises the following steps:

(1) Biological culture: inoculating the whole tooth revival nematodes into a sterile oat culture medium, and culturing at 28 ℃ for 8-15 days;

(2) Crude extraction of products: adding sterile ddH into the culture medium when the nematode climbs the bottle wall ₂ O, softly washing out the nematodes, transferring the nematodes to a separating funnel, discarding the supernatant after the nematodes settle, repeating the steps for 3-5 times to sufficiently remove the oat culture medium, and collecting the nematodes; sterile ddH was added in a volume of 3 times the nematode volume ₂ O, culturing at 20 ℃ and 180rpm for 6-10 h to secrete signal substances in the nematodes, centrifuging at 4000rpm for 10-30 min to remove nematode bodies and leave supernatant, adding 3 times of volume of absolute ethyl alcohol into the supernatant for leaching, leaching for 48h each time, performing ultrasonic crushing midway, repeating for 3 times, and concentrating by a rotary evaporator to obtain crude extract;

(3) And (3) product purification: separating by column chromatography to obtain two glucopyranosides, namely ethyl-beta-D-glucopyranoside and butyl-beta-D-glucopyranoside.

The conditions of the reagents used in the preparation processes of wp-1 and wp-2 of the invention are as follows:

oat culture medium: 100g oatmeal, 100mL ddH ₂ Soaking oatmeal in water, and sterilizing at 121 ℃ for later use;

and (3) testing nematodes: the holoodonte revival nematodes are cultured in an oat culture medium.

Absolute ethanol as a solvent for the nematode extract (analytical purity, west longa science ltd); petroleum ether, ethyl acetate, acetone and methanol used for separating and purifying the compound are all redistilled industrial pure solvents; a GF254 thin-layer chromatography silica gel plate and 200-300 mesh column chromatography silica gel are used in China Qingdao oceanic plant, and column chromatography methanol gel Sephadex LH-20 is a Pharmacia product; the nuclear magnetic resonance instrument is a Bruker DRX-500 NMR superconducting nuclear magnetic resonance instrument and a Bruker Avance III-600NMR superconducting nuclear magnetic resonance instrument; the mass spectrometer was Finnigan LCQ-Advantage.

The following examples use the reagents:

culture medium

NGM culture medium: naCl 3g, peptone 2.5g, peptone 15g agar, ddH ₂ O constant volume is 1000mL, after sterilization, when the temperature is reduced to 45-55 ℃, 25mL 1M phosphate buffer solution and 1mL 1M CaCl are added ₂ Solution, 1mL 1M MgSO ₄ Mixing the solution and 1mL of cholesterol solution of 5mg/mL, and pouring the mixture into a flat plate; pouring the plate, completely cooling, adding the cultured Escherichia coli OP50, and culturing at 37 ℃ for 12h for culturing caenorhabditis elegans N2;

PDA culture medium: boiling potato 200g in boiling water for 20min, filtering with three layers of gauze, adding glucose 20g and agar 15g, and adding ddH ₂ O is constant volume to 1000mL; PDA culture medium is used for culturing fungal spores;

WA medium: agar 15g, ddH ₂ O is subjected to constant volume to 1000mL, and is sterilized at 121 ℃ for standby; WA medium is used for chemotaxis experiment of compound to nematode and induction of fungus predator by compound.

Experimental nematodes and induced strains

Nematodes for experiments: caenorhabditis elegans N2, meloidogyne incognita, and meloidogyne arachidis; caenorhabditis elegans N2 is cultured in an NGM culture medium, meloidogyne incognita is used after eggs are picked from the root of a tomato and incubated for 3 days, and meloidogyne arachidis is used after eggs are picked from the root of an eggplant and incubated for 3 days;

trap induced strains: the Arthrobotrys oligospora YMF1.01883, arthrobotrys guizhou YMF1.00014, arthrobotrys conoides YMF1.01879, alternaria longipes YMF1.01460, trichosporon monterellis YMF1.01427 and Acremonium ovale YMF1.00032, and the strains are stored in Yunnan university Yunnan biological resource protection and utilization national key laboratory strain bank-China southwest wild germplasm resource bank.

Example 1

This example examines the chemotaxis of compounds wp-1 and wp-2 towards nematodes, and experiments with nematodes: caenorhabditis elegans N2, meloidogyne incognita and meloidogyne arachidis, the test process is as follows:

pouring the sterilized WA culture medium into a culture dish with the diameter of 60mm, cooling, dividing the culture dish into three areas A, B and C (as shown in figure 1, showing a specific chemotaxis experiment process of a compound for nematodes), adding 10 mu L of each concentration compound at a position 5mm away from the edge of the area A, adding 10 mu L of sterile water at a position 5mm away from the edge of the area B as a control, adding nematodes (about 150 nematodes) at the center of the area C after the liquid is air-dried, and timing when the nematodes start to climb around; the number of nematode in the area A and the number of nematode in the area B are respectively counted after the caenorhabditis elegans N2 crawls for 1h, the number of nematode in the area A and the number of nematode in the area B are respectively counted after the caenorhabditis elegans and the caenorhabditis elegans crawl for 2h, the chemotaxis index is calculated through the following formula, 5 times of repetition are set in each experiment, and the experiment is repeated for 3 times. The effect of compounds wp-1 and wp-2 as pheromones on nematode behaviour was evaluated by the flooding index.

Flooding index (CI) = (number of experimental nematodes-number of control nematodes)/(number of experimental nematodes + number of control nematodes)

Note: CI is greater than 0.2, which indicates that the compound has stronger attraction effect on the nematode; CI < -0.2 indicates that the compound has stronger avoidance effect on nematodes; 0-straw CI-straw 0.2 shows that the compound has weak attraction effect on the nematodes; -0.2-woven CI-woven 0 means that the compound has weak avoidance effect on nematodes.

Through chemotaxis experiments, the chemotaxis index situations of the finally obtained compounds wp-1 and wp-2 on three nematodes, namely caenorhabditis elegans N2, meloidogyne incognita and meloidogyne arachidis, are shown in figures 2-7, and figure 2 shows that the compound wp-1 has stronger attractive activity on caenorhabditis elegans at nM concentration; FIG. 3 shows that compound wp-1 has strong evasion activity to Meloidogyne incognita at nM concentration; FIG. 4 shows that compound wp-1 has strong attractive activity to floral root nodulation nematode at high concentration (μ M-nM grade) and strong avoidance activity at low concentration (pM grade); FIG. 5 shows that compound wp-2 has weak avoidance activity on C.elegans at concentrations on the order of μ M, and has strong attraction activity at concentrations on the order of nM; FIG. 6 shows that compound wp-2 has a strong attractive activity against Meloidogyne incognita at a concentration of 10 nM; FIG. 7 shows that compound wp-2 has a strong evasive activity against floral root nodulation nematode at high concentration (μ M-nM scale) and a strong attractive activity at low concentration (pM scale).

To sum up, at nM concentration, compound wp-1 has very strong attractive activity against C.elegans N2 and G.arachidicola, and strong avoidance activity against G.aminovorans; the compound wp-2 has strong attractive activity to caenorhabditis elegans N2 and meloidogyne incognita, and has obvious avoidance activity to floral meloidogyne.

Example 2

In this example, the induction of Arthrospora (Arthrospora oligospora, arthrospora guizhouensis, arthrospora conoides), acremonium (Acremonium vesiculosum, acremonium ellipsoidea) and Acremonium septorium (Acremonium septorium) trap by compounds wp-1 and wp-2 was examined, and the specific operation was as follows: inoculating fungi on a PDA culture medium, culturing for 10 days, washing fungal spores with sterile water, counting, coating 2000 spores on a poured WA culture medium, culturing at 25 ℃ for 36h, respectively adding 1.5mL of compounds wp-1 and wp-2 with different concentrations, adding sterile water with the same volume into a control group, observing formation of a trap after 48h, counting, setting 5 repeats for each group, and repeating the experiment for 3 times.

Through activity induction, nM concentration is the optimal induction concentration of the compounds wp-1 and wp-2, and the compounds can induce the fungi to generate three traps of three-dimensional bacterial net, sticky ball and contraction ring, which shows that the compounds wp-1 and wp-2 can effectively induce the fungi to generate various traps, and the compounds wp-1 and wp-2 are shown in Table 1.

TABLE 1 Compounds wp2-1 and wp2-2 induce different kinds of traps

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Note: v. means a trap capable of inducing generation; and x represents the failure to induce the trap.

After inducing 48h by adding 10nM of compounds wp-1 and wp-2, the compounds wp-1 and wp-2 were found to induce Arthrobotrys oligosporus YMF1.01883, arthrobotrys guizhouensis YMF1.00014 and Arthrobotrys conoti YMF1.01879 to generate three-dimensional bacterial networks, and the control group generated a small amount of traps, and the results are shown in FIG. 8, wherein A and B are the three-dimensional bacterial network structures induced by the compounds wp-1 and wp-2 on YMF 1.01883: * P <0.05; * P <0.01; * P <0.001, figure 8 shows that compounds wp-1 and wp-2 are able to significantly induce arthrobotrys to produce three-dimensional reticulum.

After inducing 48h by adding 10nM of compounds wp-1 and wp-2, it was found that compounds wp-1 and wp-2 induced the production of cosmids by YMF1.00032, which is Acremonium ovale, and YMF1.01460, which is Erythrophlospora elongata, and that the control group spontaneously produced only a small amount of cosmids. In contrast, compound wp-1 induces stronger activity than compound wp-2, and the results are shown in FIG. 9, wherein A and B are myxosphere structures induced by compounds wp-1 and wp-2 on YMF1.00032, and FIG. 9 shows that compounds wp-1 and wp-2 can significantly induce myxosphere generation by Acremonium monospora and Dietzia septoria.

After adding 10nM of compounds wp-1 and wp-2 to induce 48h, compound wp-1 was found to induce contraction rings of the Trichosporon monochoridium YMF1.01427 and Alternaria elongata YMF1.01460, while compound wp-2 induced contraction rings of only Alternaria elongata YMF1.01460, and the control group induced or not induced contraction rings spontaneously, and the results are shown in FIG. 10, wherein A and B are the contraction rings and viscose structures induced by wp-1 on YMF1.01460, and C is the contraction ring structure induced by wp-2 on YMF1.01460, and FIG. 10 shows that compounds wp-1 and wp-2 can significantly induce contraction rings of Trichosporon monochoria and Alternaria elongata.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

Claims (2)

1. The application of the pheromone in the nematode behavior research and the reagent for inducing fungi to trap and kill nematodes is characterized in that the pheromone is ethyl-beta-D-glucopyranoside or butyl-beta-D-glucopyranoside, and the structures of the pheromone are respectively shown as formula 1 and formula 2:

wherein the nematode behavior is: the butyl-beta-D-glucopyranoside has attractive activity to caenorhabditis elegans at nM concentration and has evasion activity to Meloidogyne incognita at nM concentration; the attractive activity to the peanut root-knot nematode in the mu M-nM level and the avoidance activity to the peanut root-knot nematode in the pM level; the ethylene-beta-D-glucopyranoside has the evasion activity to caenorhabditis elegans at the mu M level concentration and has the attraction activity to the caenorhabditis elegans at the nM level concentration; attractive activity to meloidogyne incognita at a concentration of 10 nM; the evasion activity to the peanut root-knot nematode at the mu M-nM concentration and the attraction activity to the peanut root-knot nematode at the pM concentration;

the fungi adopted by the ethyl-beta-D-glucopyranoside for inducing and killing the nematodes are one of Arthrobotrya oligospora, arthrobotrya guizhou, acremonium ovale, alternaria longissima and Arthrobotrya conoides; the fungi adopted by the butyl-beta-D-glucopyranoside for inducing and killing the nematodes are one of Arthrobotrya oligospora, arthrobotrya guizhou, acremonium ovale, alternaria longissima, arthrobotrya conoides and Acremonium vesiculosum.

2. The use of a pheromone as defined in claim 1 in a reagent for studying nematode behavior and inducing fungi to trap and kill nematodes, wherein said two glucopyranosides are isolated from the culture of whole tooth nematodes.

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