CN111153797A

CN111153797A - Active substance for killing nematode and its preparing process and application

Info

Publication number: CN111153797A
Application number: CN202010029502.2A
Authority: CN
Inventors: 吴海燕; 王冬亚; 周勋波; 何琼
Original assignee: Guangxi University
Current assignee: Guangdong Zhenge Biotechnology Co ltd
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2020-05-15
Anticipated expiration: 2040-01-13
Also published as: CN111153797B

Abstract

The invention provides a nematicidal active substance prepared from a secondary metabolite product of Aspergillus japonicus ZW1, which is capable of producing nematicidal active substances through liquid fermentation culture to obtain a fermentation culture solution containing nematicidal active metabolites, then obtaining a crude extract from the fermentation culture solution, and further purifying to obtain nematicidal active substances, wherein the nematicidal active substance provided by the invention has strong stability and strong nematicidal activity on root-knot nematodes, the mortality rate of the root-knot nematodes of 48h under the concentration of 1.25mg/mL is more than 90%, and the mortality rate of the root-knot nematodes of 6h under the concentration of 2mg/mL is 100%; meanwhile, the mortality rate of the soybean cyst nematode for 48h under the concentration of 2mg/mL is 100 percent.

Description

Active substance for killing nematode and its preparing process and application

Technical Field

The invention belongs to the technical field of biology, relates to an active nematicidal substance, and particularly relates to an active nematicidal substance prepared from a secondary metabolite product of Aspergillus japonicus ZW1, a preparation method of the active nematicidal substance, and application of the active nematicidal substance in the aspect of biological pest control of plants.

Background

Root knot nematode disease is a disease distributed worldwide, is one of the most important crop diseases in China at present, and has great economic harm. Since Berkeley discovered root knot in cucumber for the first time in 1855, plant root knot nematode disease began to be valued, and it has been more than 150 years old until now. In recent years, with the rapid development of vegetable cultivation area, annual planting and remote transportation, a suitable environment is provided for the occurrence, development and propagation of root-knot nematode diseases, and the root-knot nematodes in soil are rapidly proliferated, so that the annual planting of greenhouses and greenhouse wireworms becomes a great obstacle in vegetable production. Great economic loss is caused to vegetable production in the greenhouse, and agricultural production and farmer income are seriously influenced. Once the root knot nematode disease occurs in a greenhouse, the root knot nematode disease exists for a long time and is difficult to eradicate thoroughly, which generally causes the yield reduction of 20-30 percent and the weight reduction of more than 50 percent, even the root knot nematode disease is completely harvested. The host range of the nematode is wide, which brings great difficulty to the prevention and treatment. In addition, the root-knot nematode harm also increases the occurrence probability of soil-borne fungal diseases such as root rot and blight and partial bacterial diseases, and becomes a great obstacle in the current vegetable production. The serious occurrence and prevalence of root knot nematode disease in Heilongjiang, Liaoning, Beijing, Henan, Hubei, Jiangsu, Yunnan, Hainan and the like are reported. The root-knot nematodes which are seriously harmful in China mainly include Meloidogyne incognita (Meloidogyne incognita), Meloidogyne arachidis (Meloidogyne arenaria), Meloidogyne hapla (Meloidogyne hapla) and Meloidogyne javanica (Meloidogyne javanica).

Soybean cyst nematode disease is one of the main diseases of soybean, and occurs in all soybean production areas in the world. The main soybean producing areas of northeast China and Huang-Huai-Hai in China, such as Liaoning, Jilin, Heilongjiang, Shanxi, Henan, Shandong, Anhui and other provinces, generally occur, and are seriously harmful and restrict the soybean production in China. The disease generally reduces the yield of the soybeans by 10-20 percent, the weight of the soybean can reach 30-50 percent, and certain production areas damage seeds due to large-area occurrence. The soybean cyst nematode is a fixed internal parasitic nematode with narrow host range, mainly leguminous plants such as soybean, red bean, kidney bean, mung bean, pea, etc. The eelworms have limited self-peristalsis distance and can be carried and spread mainly by farming, field water flow or wind, and can also be mixed with non-decomposed compost or seeds for carrying and spreading for a long distance.

For a long time, the prevention and control of plant nematode diseases mainly rely on chemical nematocides, but most of the nematocides are high-toxicity and high-residue medicaments, so that beneficial organisms are killed while plant parasitic nematodes in soil are killed, ecological balance is seriously damaged, the environment is polluted, and the health of human beings is threatened. Biological control is widely considered as a promising disease prevention approach, so that active substances with high toxicity to plant parasitic nematodes are obtained from biological sources, and the biological control method has very important significance for developing novel environment-friendly nematode-killing pesticides. The metabolites of the microorganism are various in types, can be produced in large scale, have short period and better economic benefit than the products of plant sources, people tend to develop and utilize the metabolites of the microorganism, the metabolites of the microorganism are widely applied to the medical field and agricultural production, and the biocontrol strain is an important treatment way for preventing and treating plant nematode diseases. At present, there are many kinds of commercial biocontrol strains, such as Paecilomyces lilacinus, Pochonia chlamydosporia, Bacillus firmus, Pseudomonas fluorescens, Streptomyces avermitilis, etc., wherein the Pochonia chlamydosporia and the Paecilomyces lilacinus are commercially produced and used in China. Therefore, the separation, extraction and identification of the metabolites of the microorganism are of great significance.

Disclosure of Invention

Against the background described above, the present invention provides a nematicidal active substance prepared from a secondary metabolite product of koji mold, a process for the preparation of such nematicidal active substance and its use in biological control of plant pests.

The inventor of the application obtains a strain of Aspergillus japonicus ZW1 through screening, the metabolite of the strain has toxic killing effect on root-knot nematodes, the applicant of the invention submits the strain to China center for type culture collection (CCTCC NO. M2014641) in 12-10 th of 2014, and applies for invention patent with application number 201510052458.6 in 30 th of 2015 1.

The invention aims to provide a nematicidal active substance extracted from Aspergillus japonicus ZW1, wherein Aspergillus japonicus ZW1 is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014641.

The active nematicidal substance is citric acid dimethyl ester hydrochloride, and the structural formula of the active nematicidal substance is shown as the following structural formula (I):

the second object of the present invention is to provide a method for preparing the nematicidal active substance, comprising the steps of:

(1) carrying out liquid fermentation culture on Aspergillus japonicus ZW1 to obtain a fermentation culture solution containing an insecticidal activity metabolite; the Aspergillus japonicus ZW1 is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014641;

(2) filtering the fermentation culture solution, concentrating to 1/16 of the original volume, extracting with n-butanol or ethyl acetate, and removing solvent n-butanol or ethyl acetate by rotary evaporation to obtain crude extract of nematicidal active substance;

the crude extract is recrystallized with alcohol such as methanol or ethanol to obtain pure product.

Wherein in the step (1), Aspergillus japonicus ZW1 is inoculated into a liquid culture medium for liquid fermentation; the components of the liquid culture medium comprise: KNO₃2g/L，KCl 0.5g/L，FeSO₄0.01g/L，K₂HPO₄1g/L，MgSO₄0.5g/L and 30g/L of cane sugar; liquid medium pH 7.

Wherein the conditions of the liquid fermentation culture are as follows: the temperature is 26-30 ℃, and the rotating speed is 130-170 r/min. Performing shake culture for 12-16 d; preferably, the conditions of the liquid fermentation culture are: the temperature is 28 ℃, the rotating speed is 150r/min, and shake culture is carried out for 14 d.

A third object of the invention provides the use of said nematicidal active substance for biological control of plant pests; wherein the plant pest is a plant parasitic nematode; further, the plant pests are root-knot nematodes and/or soybean cyst nematodes.

The invention has the advantages of

The active substance for killing nematodes provided by the invention has strong stability and strong poisoning activity on root-knot nematodes and soybean cyst nematodes, the mortality of the root-knot nematodes in 48h under the concentration of 1.25mg/mL is more than 90%, and the mortality of the nematodes in 6h under the concentration of 2mg/mL is 100%; meanwhile, the mortality rate of the soybean cyst nematode for 48h under the concentration of 2mg/mL is 100 percent.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum;

FIG. 2 is a nuclear magnetic resonance carbon spectrum.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 extraction of nematicidal active substances

1. Origin and activation of the Strain

Aspergillus japonicus ZW1 strain was obtained from the laboratory of nematology, academy of agriculture, Guangxi university, under the name Aspergillus japonicus, GenBank accession number: KR 708636.1; the strain is preserved in China center for type culture Collection with a preservation number of CCTCC NO. M2014641.

The ZW1 strain is transferred from the preserved test tube to PDA culture medium, placed in GXZ-280C-4 type constant temperature incubator, and cultured at 25 deg.C for 7 days for activation.

2. Preparation of fermentation broth

Preparing a Czapek liquid medium, wherein the liquid medium comprises the following components: KNO₃2g/L，KCl 0.5g/L，FeSO₄0.01 g/L，K₂HPO₄1g/L，MgSO₄0.5g/L and 30g/L of cane sugar; liquid medium pH 7.

Filling the Czapek liquid culture medium into a 250mL triangular flask, wherein each flask is filled with 100 mL; the bacterial cake was picked from the PDA medium cultured for 7 days in step 1 by punching with a punch of 0.6cm aperture and transferred to a triangular flask, followed by shaking culture at 28 ℃ for 14 days for a total of 8L.

3. The fermentation broth obtained in step 2 was filtered with medical gauze and then distilled with a rotary evaporator at 48 ℃ at 70r/min to remove most of the water, and the rotary evaporation was stopped when 500mL of a mixture of water and the product of ZW-1 strain remained in the distillation flask.

4. Transferring the concentrated solution to a 2L separating funnel, adding 1L of analytically pure n-butyl alcohol, covering a cover, violently shaking for 10-15 s, then placing on an iron stand, standing until the concentrated solution is layered, placing the lower layer of liquid into a 1L triangular flask, and placing the upper layer of liquid into a 2L rotary steaming bottle for rotary steaming; and pouring the lower layer liquid back to the separating funnel, adding n-butanol for analytical purification, continuing to extract, and repeating the steps for 4 times. Collecting an upper layer sample of the separating funnel, carrying out rotary evaporation to reduce the amount of liquid, then transferring to a 100mL rotary evaporation bottle, and carrying out rotary evaporation until no solvent exists in the sample to obtain a crude extract.

5. Recrystallization of the sample

Dissolving the crude extract with methanol, standing at 4 deg.C for crystallization, filtering, washing with chloroform for several times, washing with petroleum ether for several times, dissolving with methanol, and recrystallizing at 25 deg.C to obtain nematicidal active substance.

Example 2 insecticidal Activity test of nematicidal active substances

1. Collecting and hatching eggs of root-knot nematode

Root-knot nematodes were bred in the nematology laboratory of the academy of agriculture of Guangxi university, tomato (variety: Israel F1, Guangzhou horticultural seedling Co., Ltd.) as its breeding host. After 35d of inoculation of root-knot nematodes, tomato roots are taken, the tomato roots are lightly washed by tap water, then the root-knot nematode oocysts are picked under a microscope (Olympus corporation, model: SZX2-ILLT, Tokyo, Japan), the picked oocysts are placed in a small sieve of 325 meshes, the small sieve is placed in a culture dish, water is added into the culture dish until the water is in contact with or is immersed at the bottom of the sieve, a preservative film is covered on the sieve, the sieve is placed in a constant-temperature incubator without illumination at 25 ℃ for incubation, the incubated 2-instar larvae of the root-knot nematodes are collected every day, and the activity detection of poisoning the 2-instar larvae of the root-knot nematodes is carried out on the same day.

2. Collecting and hatching eggs of soybean cyst nematodes

The soybean cyst nematode was bred in the nematology laboratory of the academy of agriculture of Guangxi university, with soybean (variety: Ludou No. 4) as its breeding host. Separating cysts 35 days after inoculation, slightly taking off soybean roots after reversely buckling a flowerpot, washing away sand at the roots by running water, brushing the white cysts in a clean culture dish by using a brush, slightly picking out the white cysts to a 450-mesh sieve by using a fine needle, sterilizing for 3min by using 70% alcohol, washing for 4 times by using sterile water, crushing the cysts to release eggs, adding a little sterile water, incubating at the temperature of 25 ℃ in a dark place, and collecting incubated 2-year-old larvae for later use on the test day.

3. Insecticidal Activity test of nematicidal active substances at various concentrations

The active substance for killing the nematodes is prepared into a solution with the concentration of 0.25, 0.50, 0.75, 1.00 and 1.25mg/mL, and is added into a 96-well plate, wherein each well is 200 mu L, and then about 60 heads of 2-instar larvae of the root-knot nematodes are added into each well. The 96-well plate was placed at 25 ℃ and observed under an optical microscope (Ti-S Nikon microscope Nikon Instruments inc. tokyo, japan) at 6, 12, 24 and 48h after the treatment, respectively, and the number of deaths of 2-instar larvae of root knot nematodes was counted and photographed. Determination criteria for nematode death: the nematodes were stiff and were inactive after stimulation with a burr indicating death of the nematodes. The test was performed 3 times with sterile water as a control. The mortality rate (number of nematode deaths/total number of nematodes) × 100%, and the results of the study are shown in table 1.

Similarly, the nematode-2 nd larvae of soybean cyst nematodes were treated with nematicidal active substances of different concentrations, and the test method and nematode death judgment were the same as those of root-knot nematodes, and the results are shown in table 2.

TABLE 1 Effect of different concentrations of Meloidogyne-killing active on 2-instar larvae of Meloidogyne

Note: the same letters after the column mean indicate no significant difference at P <0.05 level as tested by duncan new double differential method.

As can be seen from table 1, there was a significant difference in the mortality rate of the different concentrations of nematicidal active substance treatment of 2 nd larvae of root-knot nematodes; at 48h, the mortality rate of the 2 nd larvae of the root-knot nematodes treated by 1.25mg/mL of the nematicidal active substances reaches 91.7 percent, which is obviously higher than that of other treatments (P < 0.05); the mortality rate of the root-knot nematode treated by 0.5mg/mL nematicidal active substance reaches 20.8% in 48h, which is obviously higher than that of a sterile water control group (P < 0.05).

TABLE 2 poisoning effect of nematicidal active substances of different concentrations on soybean cyst nematode 2-instar larvae

As can be seen from table 2, there was a significant difference in the mortality rate of the nematode 2-instar larvae of soybean cyst nematodes treated with different concentrations of the nematicidal active substance; under the condition of low concentration of 0.25-0.5 mg/mL, the soybean cyst nematode 2-instar larvae are not affected, when the concentration is 0.75mg/mL, the mortality rates of nematodes within 24h and 48h are both obviously higher than those of a control group (P <0.05), and the mortality rate of the nematode 2-instar larvae of the soybean cyst nematodes treated by 1.75mg/mL nematicidal active substances within 48h reaches 96.2%; the mortality rate of the soybean cyst nematode treated by the nematicidal active substance of 2mg/mL reaches 100 percent.

4. Insecticidal Activity test of 2mg/mL nematicidal active substance solution

Preparing the nematicidal active substance into a solution with the concentration of 2mg/mL, adding the solution into a 96-hole plate, wherein each hole is 200 mu L, and then adding about 60 root-knot nematode 2-instar larvae into each hole. The 96-well plate was placed at 25 ℃ and observed under an optical microscope (Ti-S Nikon microscope Nikon Instruments inc. tokyo, japan) at 6, 12, 24 and 48h after the treatment, respectively, to count the number of deaths of 2-instar larvae of root-knot nematodes. Determination criteria for nematode death: the nematodes were stiff and were inactive after stimulation with a burr indicating death of the nematodes. The test was performed 3 times with sterile water as a control. The mortality rate (number of nematode deaths/total number of nematodes) × 100%, and the experimental results are shown in table 3.

TABLE 32 mg/mL Effect of nematicidal actives on 2-instar larvae of root-knot nematodes

From table 3 it can be seen that the mortality rate of the nematicidal active substance at a concentration of 2mg/mL against 2 nd larvae of root-knot nematodes reached 100% at 6h, significantly higher than that of the control group (P < 0.05).

Example 3 Nuclear magnetic resonance analysis of structural formula of nematicidal active substance

And (3) determining the nuclear magnetic resonance data of the nematicidal active substance by using a high-resolution nuclear magnetic resonance spectrometer, detecting the number and the correlation of hydrogen atoms and carbon atoms, obtaining a hydrogen spectrum and a carbon spectrum, carrying out spectrum analysis, obtaining the position and the type of the object atomic nucleus, searching the molecular structure of the substance, and drawing a structural image of the substance. In order to ensure the accuracy of the structure, the relative molecular mass of the compound is detected by using an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer.

Crystalline product, colorless crystals (methanol, CD)₃OD), 1H NMR (CD3OD,400MHz) δ 3.68(each 3H, s,7,8-CH3),2.95,2.85(each 2H, AB system, d, J ═ 12.0Hz,2,4-CH 2). 13C NMR (CD3OD,100MHz) delta 175.00(s, C-6),170.46(s, C-1, C-5),72.84(s, C-3),50.78(q, C-7, C-8),42.63(t, C-2, C-4) (see Table 4, FIG. 2).

TABLE 4 correlation of hydrogen and carbon for crystalline product I

From the results of nuclear magnetic resonance, the nematicidal active substance has the following structural formula, which is named as dimethyl citrate hydrochloride:

Claims

1. a nematicidal active substance, wherein the nematicidal active substance is dimethyl citrate hydrochloride, and the structural formula of the nematicidal active substance is as follows:

2. a process for the preparation of a nematicidal active substance according to claim 1, comprising the steps of:

(1) performing liquid fermentation culture on Aspergillus japonicus ZW1 capable of producing nematicidal active substances to obtain a fermentation culture solution containing nematicidal active metabolites; the Aspergillus japonicus ZW1 strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2014641;

(2) filtering the fermentation culture solution, concentrating to 1/16 of the original volume, extracting with n-butanol or ethyl acetate, and removing solvent n-butanol or ethyl acetate by rotary evaporation to obtain crude extract of nematicidal active substance; the crude extract is recrystallized with alcohol such as methanol or ethanol to obtain pure product.

3. The method of claim 2, wherein: inoculating Aspergillus japonicus ZW1 into a liquid culture medium for liquid fermentation in the step (1); the components of the liquid culture medium comprise: KNO₃2g/L，KCl 0.5g/L，FeSO₄0.01g/L，K₂HPO₄1g/L，MgSO₄0.5g/L and 30g/L of cane sugar; liquid medium pH 7.

4. The method of claim 3, wherein: the conditions of the liquid fermentation culture are as follows: the temperature is 26-30 ℃, and the rotating speed is 130-170 r/min. And performing shake culture for 12-16 d.

5. The method of claim 3, wherein: the conditions of the liquid fermentation culture are as follows: the temperature is 28 ℃, the rotating speed is 150r/min, and shake culture is carried out for 14 d.

6. Use of a nematicidal active substance as defined in claim 1 for biological control of plant pests.

7. Use according to claim 6, characterized in that: the plant pest is a plant parasitic nematode.

8. Use according to claim 6, characterized in that: the plant pests are root-knot nematodes and/or soybean cyst nematodes.