Application of streptomyces ahygroscopicus secondary metabolite in prevention and treatment of pine wood nematode disease and preparation method of streptomyces ahygroscopicus secondary metabolite

Abstract

The invention relates to the field of agricultural microorganisms, in particular to application of a streptomyces ahygroscopicus secondary metabolite in prevention and treatment of pine wood nematode diseases and a preparation method of the streptomyces ahygroscopicus secondary metabolite. According to the invention, the streptomyces ahygroscopicus secondary metabolite is applied to prevention and treatment of pine wood nematode disease. The streptomyces ahygroscopicus secondary metabolite can achieve a good pine wood nematode disease prevention and treatment effect, does not pollute the environment during prevention and treatment, and is a green and environment-friendly prevention and treatment mode.

Description

Technical Field

[0001] The invention relates to the field of agricultural microorganisms, and in particular to an application and a preparation method of secondary metabolites of Streptomyces hygrophila in preventing and treating pine wood nematode disease. Background Art

[0002] Pine wood nematodes are the pathogen that causes pine wood nematode disease. North America is the main place where pine wood nematodes are spread, and they have now spread to East Asia and Europe. Since they were first discovered in China in 1982, pine wood nematodes have spread to 19 provinces in China. When pine wood nematodes invade pine trees, they will multiply in large numbers, reduce the secretion of pine resin, and cause the pine trees to die quickly within 1-3 months.

[0003] There are many ways to prevent and control pine wood nematodes. Quarantine is an effective way to prevent pine wood nematodes in unaffected areas. Chemical methods are often used in infected areas to curb the infection and spread of pine wood nematodes. Chemical agents such as thiacloprid, cypermethrin and thiamethoxam are used for aerial spraying to control vector insects, but they cannot directly kill pine wood nematodes and will cause air pollution. Agents such as avermectin, emamectin benzoate and fluopyram are often used as tree trunk injection agents to prevent and control pine wood nematode disease. Due to the long-term use of a single agent to control pine wood nematodes, the resistance of pine wood nematodes has increased, which ultimately causes problems such as unsatisfactory efficacy. Therefore, it is urgent to find a new type of nematicide that is highly efficient, highly water-soluble, environmentally safe, low-toxic, and has independent intellectual property rights.

[0004] Streptomyces ahgroscopicus subsp. Wuzhoensis 11371 strain is a biocontrol actinomycete with the functions of promoting plant growth and preventing and controlling plant diseases. It was isolated and screened from soil samples collected in Wuzhou, Guangxi. The secondary metabolites of non-hygroscopic Streptomyces wuzhou subspecies can inhibit the growth of many pathogenic microorganisms such as Rhizoctonia solani, poplar blister canker, apple tree rot, tobacco black shank, apple tree rot, tomato leaf mold, wheat fusarium wilt, wheat stem base rot, bitter melon wilt, melon wilt, watermelon wilt, gray mold, tobacco black root rot, cucumber target spot pathogen, vegetable soft rot pathogen, rice white leaf blight pathogen, and rice streak pathogen. Its "tetramycin" component has now been officially registered by Micro-Tech Biotechnology as tetramycin mother drug and aqueous solution with the Ministry of Agriculture and Rural Affairs of the People's Republic of China, and the registered objects are: rice blast, rice bacterial streak disease, rice damping-off disease, peanut root rot, cucumber bacterial angular spot disease, wheat powdery mildew, wheat fusarium wilt, poplar canker, and corn head smut. However, the secondary metabolites of Streptomyces hygroscopicus subsp. wuzhouensis have not been registered in China for the control of pine wood nematodes, and there are no reports on their nematicidal activity.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a new application and preparation method of the secondary metabolites of Streptomyces hygroscopicus in preventing and treating pine wood nematode disease for the first time. The secondary metabolites of Streptomyces hygroscopicus can effectively prevent and treat pine wood nematode disease and are environmentally friendly.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] One aspect of the present invention relates to the use of secondary metabolites of Streptomyces hygroscopicus in the prevention and treatment of pine wood nematode disease.

[0009] The secondary metabolites of non-hygroscopic Streptomyces can achieve better prevention and control effects of pine wood nematode disease without causing pollution to the environment. It is a green and environmentally friendly prevention and control method.

[0010] Furthermore, the non-hygroscopic Streptomyces is non-hygroscopic Streptomyces ahgroscopicus subsp. Wuzhoensis 11371.

[0011] Furthermore, the Streptomyces ahygroscopicus subsp. wuzhouensisn 11371 was disclosed in a published paper (Acta Microbiologica Sinica, 1988, Vol. 28 (1): pp. 85-87). The strain is preserved in the Liaoning Provincial Microbiological Culture Collection Center and has now been transferred to Liaoning Weike Bioengineering Co., Ltd. The applicant promises to release it to the public for verification of the technical effects of the present invention within 20 years from the date of application of the present invention.

[0012] This strain was isolated from the soil in Wuzhou, Guangxi, and has been shown to have preventive and therapeutic effects on a variety of plant diseases, but there have been no reports on the effectiveness of its secondary metabolites in preventing and controlling pine wood nematode disease.

[0013] Another aspect of the present invention relates to a method for preparing the non-hygroscopic Streptomyces secondary metabolite, comprising the following steps:

[0014] (a) inoculating spores of Streptomyces immobilis into a seed culture medium for seed culture to obtain a seed propagation solution;

[0015] (b) inoculating the seed propagation liquid into a fermentation medium for liquid fermentation culture, and performing solid-liquid separation after the culture is completed to obtain a secondary metabolite of Streptomyces hygroscopicus.

[0016] The preparation method of the non-hygroscopic Streptomyces secondary metabolite has a simple preparation process and does not require complicated processes and equipment. The prepared secondary metabolite has the effect of preventing and treating pine wood nematode disease and inducing pine wood to produce disease resistance, which is conducive to promoting the development of biological control of pine wood nematode disease.

[0017] Furthermore, the non-hygroscopic Streptomyces spores are added in the form of a bacterial suspension, and the inoculation amount is 1wt% to 3wt%, including but not limited to any point value of 1wt%, 1.5wt%, 2wt%, 2.5wt% or 3wt% or any range value between the two. When the addition amount of the spore suspension is within a certain range, the non-hygroscopic Streptomyces can grow rapidly in the seed culture medium.

[0018] Furthermore, the seed culture temperature is 25-35°C, including but not limited to any point value of 25°C, 27°C, 29°C, 31°C, 33°C or 35°C or a range value between any two of them.

[0019] Furthermore, the seed culture time is 20 to 24 hours, including but not limited to any point value of 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, or a range value between any two of them.

[0020] Furthermore, the ventilation volume of the seed culture is 0.5 to 1 vvm, including but not limited to any one of 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm or 1 vvm or a range between any two of them.

[0021] Furthermore, the seed culture speed is 150 to 300 rpm, including but not limited to any one of 150 rpm, 170 rpm, 190 rpm, 210 rpm, 230 rpm, 250 rpm, 270 rpm, 290 rpm or 300 rpm or a range between any two of them.

[0022] Limiting the seed culture conditions within a reasonable range can accelerate the growth of non-hygroscopic Streptomyces and reduce the culture time.

[0023] Furthermore, the inoculation amount of the seed propagation fluid is 5wt% to 20wt%, including but not limited to any point value of 5wt%, 8wt%, 10wt%, 13wt%, 15wt%, 18wt% or 20wt% or a range value between any two of them.

[0024] Furthermore, the temperature of the liquid fermentation culture is 25-35°C, including but not limited to any point value of 25°C, 27°C, 29°C, 31°C, 33°C or 35°C, or a range value between any two of them.

[0025] Furthermore, the liquid fermentation culture time is 64 to 72 hours, including but not limited to any point value of 64 hours, 66 hours, 68 hours, 70 hours or 72 hours, or a range value between any two of them.

[0026] Furthermore, the speed of the liquid fermentation culture is 150 to 300 rpm, including but not limited to any one of 150 rpm, 170 rpm, 190 rpm, 210 rpm, 230 rpm, 250 rpm, 270 rpm, 290 rpm or 300 rpm or a range between any two of them.

[0027] Furthermore, the pH of the liquid fermentation culture is 6.0-7.0, including but not limited to any point value of 6.0, 6.2, 6.4, 6.6, 6.8 or 7.0 or a range value between any two of them.

[0028] When the conditions of liquid fermentation culture are limited to a certain range, the non-hygroscopic Streptomyces can produce a large amount of secondary metabolites and improve the ability of the secondary metabolites to prevent and treat pine wood nematode disease.

[0029] Furthermore, the liquid fermentation culture includes: a first fermentation culture, a second fermentation culture and a third fermentation culture.

[0030] Furthermore, the first fermentation culture is carried out from 0 to 12 hours of the liquid fermentation culture.

[0031] Furthermore, the second fermentation culture is carried out from 12 to 40 hours of the liquid fermentation culture.

[0032] Furthermore, the third fermentation culture is carried out at 40 to 72 hours of the liquid fermentation culture.

[0033] Furthermore, the ventilation rate of the first fermentation culture is 0.5-1 vvm, including but not limited to any point value of 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm or 1 vvm or any range value between two of them. This is the period of massive growth and reproduction of mycelium, and a large ventilation rate is not required. A small ventilation rate protects the mycelium from being damaged and can also save energy costs.

[0034] Furthermore, the ventilation rate of the second fermentation culture is 1.0 to 1.5 vvm, including but not limited to any point value of 1.0 vvm, 1.1 vvm, 1.2 vvm, 1.3 vvm, 1.4 vvm or 1.5 vvm or any range value between two of them. This is the period of large-scale metabolism of mycelium, and a large ventilation rate is required to meet the demand for metabolites.

[0035] Furthermore, the ventilation rate of the third fermentation culture is 0.5-1 vvm, including but not limited to any point value of 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm or 1 vvm or any range value between two of them. At this time, the mycelium enters a stable metabolic period, and the mycelium metabolizes while growing.

[0036] Furthermore, during the second fermentation culture, the fermentation medium is added to the reaction system in an amount of 10 vol% to 20 vol% of the total fermentation amount. Because the second stage is a period of large-scale mycelial metabolism, insufficient energy will affect the production of metabolites, so the medium should be added at this time.

[0037] Further, the seed culture medium comprises the following components per 1000 mL of seed culture medium:

[0038] 10-25g soybean cake powder, 5-10g starch, 10-20g glucose, 2-14g peptone, 0.1-0.5g magnesium sulfate, 1-5g ammonium sulfate, 0.1-0.5g dipotassium hydrogen phosphate, 5-20g calcium carbonate, 0.5-2g sodium chloride, 2-5g soybean oil or vegetable oil and water, pH 7.0-8.0 (for example, but not limited to, any one of 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0 or a range between any two of them).

[0039] In some specific embodiments, per 1000mL seed culture medium, soybean cake powder can be, for example, but not limited to any one of 10g, 15g, 20g or 25g or a range value between any two; starch can be, for example, but not limited to any one of 5g, 6g, 7g, 8g, 9g or 10g or a range value between any two; glucose can be, for example, but not limited to any one of 10g, 12g, 14g, 16g, 18g or 20g or a range value between any two; peptone can be, for example, but not limited to any one of 2g, 4g, 6g, 8g, 10g, 12g or 14g or a range value between any two; magnesium sulfate can be, for example, but not limited to 0.1g, 0.2g, 0.3g, 0.4g or ammonium sulfate, for example, may be, but not limited to, 1g, 2g, 3g, 4g or 5g, or a range between any two of them; dipotassium hydrogen phosphate, for example, may be, but not limited to, 0.1g, 0.2g, 0.3g, 0.4g or 0.5g, or a range between any two of them; calcium carbonate, for example, may be, but not limited to, 5g, 10g, 15g or 20g, or a range between any two of them; sodium chloride, for example, may be, but not limited to, 0.5g, 1g, 1.5g or 2g, or a range between any two of them; soybean oil, for example, may be, but not limited to, 2g, 3g, 4g or 5g, or a range between any two of them.

[0040] Further, the fermentation medium comprises the following components per 1000 mL of fermentation medium:

[0041] 10-30g soybean cake powder, 8-25g starch, 10-25g glucose, 5-20g cornmeal, 2-8g peptone, 0.1-0.5g magnesium sulfate, 1-5g ammonium sulfate, 0.1-0.5g dipotassium hydrogen phosphate, 5-20g calcium carbonate, 0.5-2g sodium chloride, 2-10g vegetable oil, 1-5g amylase and water, pH 7.0-8.0 (for example, it can be, but not limited to, any one of 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0 or a range between any two of them).

[0042] In some specific embodiments, per 1000mL fermentation medium, soybean cake powder can be, for example, but not limited to any one of 10g, 15g, 20g, 25g or 30g or a range between any two of them; starch can be, for example, but not limited to any one of 8g, 10g, 13g, 15g, 18g, 20g or 25g or a range between any two of them; glucose can be, for example, but not limited to any one of 10g, 15g, 20g or 25g or a range between any two of them; cornmeal can be, for example, but not limited to any one of 5g, 10g, 15g or 20g or a range between any two of them; peptone can be, for example, but not limited to any one of 2g, 4g, 6g or 8g or a range between any two of them; magnesium sulfate can be, for example, but not limited to any one of 0.1g, 0.2g, 0.3g, 0.4g or 0.5g. ammonium sulfate may be, for example, but not limited to, any one of 1g, 2g, 3g, 4g or 5g or a range between two of them; dipotassium hydrogen phosphate may be, for example, but not limited to, any one of 0.1g, 0.2g, 0.3g, 0.4g or 0.5g or a range between two of them; calcium carbonate may be, for example, but not limited to, any one of 5g, 10g, 15g or 20g or a range between two of them; sodium chloride may be, for example, but not limited to, any one of 0.5g, 1.0g, 1.5g or 2g or a range between two of them; vegetable oil may be, for example, but not limited to, any one of 2g, 4g, 6g, 8g or 10g or a range between two of them; amylase may be, for example, but not limited to, any one of 1g, 2g, 3g, 4g or 5g or a range between two of them.

[0043] Another aspect of the present invention also relates to the non-hygroscopic Streptomyces secondary metabolite prepared by the preparation method of the non-hygroscopic Streptomyces secondary metabolite.

[0044] Another aspect of the present invention also relates to an agent for preventing and treating pine wood nematode disease, comprising the secondary metabolite of the non-hygroscopic Streptomyces.

[0045] The main active ingredient of the agent for preventing and controlling pine wood nematode disease is the secondary metabolite of non-hygroscopic Streptomyces, and it can also include auxiliary materials of conventional agents in the field of microbial pesticides, and has the advantage of being green and environmentally friendly.

[0046] Another aspect of the present invention also relates to the use of the agent for preventing and controlling pine wood nematode disease in preventing and controlling pine wood nematode disease.

[0047] Another aspect of the present invention also relates to a method for preventing and treating pine wood nematode disease, which comprises injecting the secondary metabolite of non-hygroscopic Streptomyces or the agent for preventing and treating pine wood nematode disease into the trunk of pine wood.

[0048] The method for preventing and controlling pine wood nematode disease adopts the trunk injection method to inject the agent into the trunk, and the drug solution diffuses to various parts of the crown with transpiration. On the one hand, it has the effect of killing pine wood nematodes, and on the other hand, it can induce the pine tree's own resistance.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The present invention uses the secondary metabolites of Streptomyces hygroscopicus for the prevention and treatment of pine wood nematode disease. Compared with existing chemical agents, the secondary metabolites of Streptomyces hygroscopicus have better prevention and treatment effects and are environmentally friendly.

[0051] (2) The method for preparing the secondary metabolites of Streptomyces hygroscopicus provided by the present invention can obtain a large amount of secondary metabolites of Streptomyces hygroscopicus in a short period of time. At the same time, the method can also ensure that the obtained secondary metabolites of Streptomyces hygroscopicus have a good control effect on pine wood nematode disease.

[0052] (3) The method for preventing and controlling pine wood nematode disease provided by the present invention comprises injecting the secondary metabolites of non-hygroscopic Streptomyces or the drug for preventing and controlling pine wood nematode disease into the trunk of pine wood. The drug solution diffuses to various parts of the tree crown due to transpiration. On the one hand, it has the effect of killing pine wood nematodes, and on the other hand, it can induce the pine tree's own resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 The effect of the secondary metabolites of Streptomyces hygroscopicus provided in the embodiment of the present invention on the feeding ability and reproduction rate of pine wood nematodes, A is the feeding ability of pine wood nematodes, B is the growth rate of pine wood nematodes, and C is the correlation analysis between the concentration of the secondary metabolites of Streptomyces hygroscopicus and the growth rate. Different letters indicate that there are significant differences between the Tukey data after treatment with different concentrations (P<0.05);

[0055] Figure 2Effect of the secondary metabolites of Streptomyces hygrophila provided in the embodiment of the present invention on the activity of pine wood nematode detoxification enzymes, A is green, representing the production of reactive oxygen species (ROS), and red represents lipid accumulation. As the treatment concentration increases, the production of ROS and lipid accumulation increase, B is the relative fluorescence intensity of ROS and lipids, C is the activity of glutathione transferase (GST), D is the level of cytochrome P450 (CYP450), e is the activity of superoxide dismutase (SOD), and F is the activity of peroxidase (POD);

[0056] Figure 3 This is a study on the prevention and control effect of the secondary metabolites of Streptomyces hygroscopicus on pine wood nematode disease provided in an embodiment of the present invention. A is the safety evaluation of the secondary metabolites of Streptomyces hygroscopicus on pine trees, B is the evaluation of the effect of the secondary metabolites of Streptomyces hygroscopicus on the prevention and control of pine wood nematode disease, and C is the enzyme activity and chlorophyll content. DETAILED DESCRIPTION

[0057] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0058] The non-hygroscopic Streptomyces involved in the following examples is non-hygroscopic Streptomyces ahgroscopicus subsp. Wuzhouensis 11371, which was publicly published in a paper (Acta Microbiologica Sinica, 1988, Vol. 28 (1) pp. 85-87). The strain is preserved in the Liaoning Provincial Microbiological Culture Collection Center and has now been transferred to Liaoning Micro-Tech Biotechnology Co., Ltd. It was isolated from the soil in Wuzhou, Guangxi.

[0059] Example 1

[0060] The method for preparing the secondary metabolites of Streptomyces hygroscopicus provided in this embodiment comprises the following steps:

[0061] 1. Preparation of seed culture medium:

[0062] Each 1000 ml contains: soybean meal 17g, starch 8g, glucose 15g, peptone 8g, magnesium sulfate 0.3g, ammonium sulfate 3g, dipotassium hydrogen phosphate 0.3g, calcium carbonate 13g, sodium chloride 1.2g and soybean oil 4g, pH 7.5;

[0063] 2. Seed culture conditions and methods:

[0064] A spore suspension of non-hygroscopic Streptomyces wuzhou subspecies 11371 was taken at an inoculum volume of 2%, and cultured in a seed culture medium at 30° C. for 22 hours, with a ventilation volume of 0.8 vvm and a rotation speed of 200 rpm. After the hyphae were observed to be a large web under a microscope, a seed propagation solution was prepared for use;

[0065] 3. Preparation of fermentation medium:

[0066] Each 1000 ml contains: 20g glucose, 18g starch, 13g cornmeal, 15g soybean meal, 5g peptone, 3g ammonium sulfate, 0.3g magnesium sulfate, 0.3g dipotassium hydrogen phosphate, 12g calcium carbonate, 1.1g sodium chloride, 6g vegetable oil and 3g amylase, pH 7.5;

[0067] 4. Liquid fermentation culture conditions:

[0068] The seed propagation liquid is taken at an inoculum amount of 12% and liquid fermentation culture is carried out at 30°C for 68 hours at a rotation speed of 150-300 rpm; the ventilation volume is 0.5vvm from 0 to 12 hours; the ventilation volume is 1.5vvm from 12 to 40 hours, and 20% of the fermentation medium of equal concentration is added at the same time; the ventilation volume is 1.0vvm from 40 to 68 hours; the pH value is controlled at 6.5 until the end of the fermentation, and the reaction liquid is filtered after the liquid fermentation culture is completed to obtain the secondary metabolites of non-hygroscopic Streptomyces wuzhou subspecies.

[0069] Example 2

[0070] The method for preparing the secondary metabolites of Streptomyces hygroscopicus provided in this embodiment comprises the following steps:

[0071] 1. Preparation of seed culture medium:

[0072] Each 1000 ml contains: soybean meal 10g, starch 5g, glucose 10g, peptone 2g, magnesium sulfate 0.1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 0.1g, calcium carbonate 5g, sodium chloride 0.5g and soybean oil 2g, pH 7.0;

[0073] 2. Seed culture conditions and methods:

[0074] A spore suspension of non-hygroscopic Streptomyces wuzhouensis 11371 was taken at an inoculation rate of 1%, and cultured in a seed culture medium at 25° C. for 24 hours, with a ventilation rate of 0.5 vvm and a rotation speed of 150 rpm. After the hyphae were observed to be a large web under a microscope, a seed propagation solution was prepared for use;

[0075] 3. Preparation of fermentation medium:

[0076] Each 1000 ml contains: glucose 10g, starch 8g, cornmeal 5g, soybean meal 10g, peptone 2g, ammonium sulfate 1g, magnesium sulfate 0.1g, dipotassium hydrogen phosphate 0.1g, calcium carbonate 5g, sodium chloride 0.5g, vegetable oil 2g and amylase 1g, pH 7.0;

[0077] 4. Liquid fermentation culture conditions:

[0078] The seed propagation liquid is taken at an inoculum amount of 5% and liquid fermentation culture is carried out at 25°C for 72 hours at a rotation speed of 150 rpm; the ventilation volume is 0.8vvm from 0 to 12 hours; the ventilation volume is 1.5vvm from 12 to 48 hours, and 15% of the fermentation medium of equal concentration is added at the same time; the ventilation volume is 1.0vvm from 48 to 72 hours; the pH value is controlled at 6.0 until the end of the fermentation, and the reaction liquid is filtered after the liquid fermentation culture is completed to obtain the secondary metabolites of non-hygroscopic Streptomyces wuzhou subspecies.

[0079] Example 3

[0080] The method for preparing the secondary metabolites of Streptomyces hygroscopicus provided in this embodiment comprises the following steps:

[0081] 1. Preparation of seed culture medium:

[0082] Each 1000 ml contains: 25g soybean cake powder, 10g starch, 20g glucose, 14g peptone, 0.5g magnesium sulfate, 5g ammonium sulfate, 0.5g dipotassium hydrogen phosphate, 20g calcium carbonate, 2g sodium chloride and 5g soybean oil, pH 8.0;

[0083] 2. Seed culture conditions and methods:

[0084] A spore suspension of non-hygroscopic Streptomyces wuzhou subspecies 11371 was taken at an inoculum volume of 3%, and cultured in a seed culture medium at 35° C. for 20 hours, with a ventilation volume of 0.8 vvm and a rotation speed of 300 rpm. After the hyphae were observed to be a large web under a microscope, a seed propagation solution was prepared for use;

[0085] 3. Preparation of fermentation medium:

[0086] Each 1000 ml contains: 25g glucose, 25g starch, 20g cornmeal, 30g soybean meal, 8g peptone, 5g ammonium sulfate, 0.5g magnesium sulfate, 0.5g dipotassium hydrogen phosphate, 20g calcium carbonate, 2g sodium chloride, 10g vegetable oil and 5g amylase, pH 8.0;

[0087] 4. Liquid fermentation culture conditions:

[0088] The seed propagation liquid is taken at an inoculum amount of 20% and liquid fermentation culture is carried out at 35°C for 64 hours at a rotation speed of 150-300 rpm; the ventilation volume is 0.5vvm from 0 to 12 hours; the ventilation volume is 1.5vvm from 12 to 40 hours, and 25% of the fermentation medium of equal concentration is added at the same time; the ventilation volume is 1.0vvm from 40 to 64 hours; the pH value is controlled at 7.0 until the end of the fermentation, and the reaction liquid is filtered after the liquid fermentation culture is completed to obtain the secondary metabolites of non-hygroscopic Streptomyces wuzhou subspecies.

[0089] Experimental Example 1 Nematode lethality test of secondary metabolites of Streptomyces hygroscopicus Wuzhou subspecies

[0090] 1. Test reagents

[0091] Agent 1: a secondary metabolite of Streptomyces wuzhouensis subspecies with a mass concentration of 15%, obtained by concentrating and extracting the secondary metabolite of Streptomyces wuzhouensis subspecies prepared in Example 1;

[0092] Agent 2: a secondary metabolite of Streptomyces wuzhouensis subspecies with a mass concentration of 0.3%, the secondary metabolite of Streptomyces wuzhouensis subspecies with no hygroscopicity prepared in Example 1;

[0093] Agent 3: Emamectin benzoate with a mass concentration of 2%.

[0094] 2. Research Methods

[0095] Take a 24-well plate and add about 100 pine wood nematodes, add 500μL of the agent, and use sterile water as a control to correct the mortality rate. Agent 1 was diluted to 30μg / mL, 60μg / mL, 90μg / mL, 120μg / mL, and 150μg / mL, agent 2 was diluted to 15μg / mL, 20μg / mL, 25μg / mL, 30μg / mL, and 35μg / mL, and agent 3 was diluted to 15μg / mL, 30μg / mL, 45μg / mL, 60μg / mL, and 75μg / mL. The nematodes were treated and placed in a 25℃ incubator in the dark for 24h and 72h. Each test was repeated in three groups, and the experiment was repeated twice. The nematodes were touched with platinum wire at least three times under a stereoscope. The nematodes were judged to be dead if they did not swing, and the nematode mortality rate was counted.

[0096] Corrected mortality rate (%) = (mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group) × 100%.

[0097] 3. Test results

[0098] As can be seen from Table 1, with the extension of treatment time, the LC of the pesticide on pine wood nematodes 50 The values ​​gradually decreased. LC values ​​of 24-hour treatment with Agent 1, Agent 2, and Agent 3 50 The values ​​were: 93.0594μg / ml, 26.4945μg / ml and 60.8187μg / ml. LC after 48 hours of treatment 50 The values ​​were: 20.1398, 23.3799 and 29.2185 μg / ml. LC after 72 hours of treatment 50 The values ​​were: 11.43441, 12.7949 and 16.5556 μg / ml, respectively. Compared with Agent 3, Agent 1 and Agent 2 had slightly better nematode lethal activity.

[0099] Table 1 Effects of different treatment times on the activity of pine wood nematodes

[0100]

[0101] Experimental Example 2 Effects of secondary metabolites of Streptomyces hygrophila subsp. wuzhouensis on the feeding ability and reproduction rate of pine wood nematodes

[0102] 1. Test methods

[0103] With sterile water treatment as a control, 400 adults were picked and immersed in three different concentrations (12, 16, 19 μg / ml) of aqueous solutions of secondary metabolites of Streptomyces wuzhouensis prepared in Example 1, and placed in a dark incubator at 25°C for 24h. The adults were washed three times with sterile water, transferred to a culture medium covered with Botrytis cinerea for 3d, and the feeding situation was observed. After the nematodes ate the Botrytis cinerea clean, the culture medium was soaked by the Baermann funnel method to obtain pine wood nematodes and the total number of worms was counted. The nematode reproduction rate was calculated according to Nt=N0λt. The activities of superoxide dismutase (SOD), glutathione transferase (GST) and peroxidase (POD) and cytochrome P450 (CYP450) were used to determine the effects of secondary metabolites of Streptomyces wuzhouensis and emamectin benzoate on the detoxification substances and defense enzyme activities in nematodes.

[0104] 2. Test results

[0105] like Figure 1As shown: the nematodes survive in a medium full of gray mold with no natural enemies, sufficient food and suitable climate, which conforms to the growth mode of the J-type survival curve. Therefore, Nt=N0λt is used to calculate the nematode reproduction rate. Under the treatment of 0μg / ml, 12μg / ml, 16μg / ml, and 19μg / ml of the secondary metabolites of non-hygroscopic Streptomyces wuzhouensis, the reproduction rates of pine wood nematodes are 2.13, 1.91, 1.77, and 1.63. At the same time, the regression curve equation is calculated based on the relationship between the daily concentration and the reproduction rate, R 2 =0.9535.

[0106] like Figure 2 As shown: Cytochrome P450 is an important detoxification substance in the body, which can promote drug metabolism in the body. The secondary metabolites of non-hygroscopic Streptomyces wuzhou subspecies at 19 μg / mL can significantly reduce its content compared with emamectin benzoate, thereby inhibiting the detoxification metabolism process in the nematode. Among the three protective enzymes GST, SOD and POD, GST can reduce the toxic effects of chemical drugs by catalyzing the combination of GSH and chemical drugs. SOD has antioxidant and aging effects and can remove internal O 2- And generate H2O2 and O2. POD can oxidize H2O2 into phenols and ammonia substances, and cooperate with SOD to remove excess O 2- Compared with emamectin benzoate, the secondary metabolites of Streptomyces hydratus subsp. wuzhouensis could inhibit the increase of SOD, GST and POD activities at lower concentrations, and reduce the activities of detoxification enzymes at lower concentrations.

[0107] Experimental Example 3 Effect of secondary metabolites of Streptomyces hygrophila subspecies Wuzhou on the prevention and control of pine wood nematode disease

[0108] 1. Test methods

[0109] The micro-experimental material used was cultivated two-year-old pine seedlings, with a plant height of about 50-60 cm and a diameter of about 2 cm.

[0110] Control A group: Drill a hole about 0.5 cm at the junction of the trunk and the treetop and inject about 1% clean water as a control.

[0111] Control B group: A hole of about 0.5 cm was drilled at the junction of the trunk and the treetop and about 2,000 pine wood nematodes were injected.

[0112] Prevention group: According to the application standard of 1.5 cm from the soil, 45° tilt, and 1 cm diameter injection of 1 mL, each plant was injected with 2 mL of the preparation of the non-hygroscopic Streptomyces wuzhou subspecies secondary metabolite of Example 1, and then a hole of about 0.5 cm was drilled at the junction of the trunk and the treetop and about 2000 pine nematodes were injected.

[0113] Fifteen and 30 days after inoculation with pine wood nematodes, pine leaves were collected to determine the contents of chlorophyll and cytochrome P450 (CYP450), as well as the activities of superoxide dismutase (SOD), glutathione transferase (GST) and peroxidase (POD) and other plant defense substances.

[0114] 2. Test results

[0115] like Figure 3 As shown in A, after the pine trees were treated with the secondary metabolites of Streptomyces hygroscopicus for 30 days, there was no drug damage to the pine trees. This shows that the secondary metabolites of Streptomyces hygroscopicus are safe for pine trees. Figure 3 As shown in Figure B, during the 30-day observation, there was no change in the pine trees in the Control A group. On the 15th day, the pine needles in the Control B group clearly began to turn yellow. By the 30th day, all the pine needles had withered. In the Prevention group, the pine trees showed mild signs of disease on the 15th day. Until the 30th day, the severity of the disease was significantly lower than that in the Control B group. SOD and APX can work synergistically to eliminate O in plants. 2- , enhancing the disease resistance of plants. The activity of CYP450 is an important indicator of plant response to external stress. Within 20 days, the chlorophyll content and SOD and APX activities were significantly higher than those on day 0, and the CYP450 content increased slightly.

[0116] The above experiments show that the secondary metabolites of Streptomyces wuzhouensis can prevent the occurrence of pine wood nematode disease.

[0117] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. Application of secondary metabolites of Streptomyces hygrophila in the prevention and control of pine wood nematode disease.

2. The use according to claim 1, characterized in that: The non-hygroscopic streptomyces is non-hygroscopic streptomyces ahgroscopicus subsp. Wuzhoensis 11371.

3. The method for preparing the secondary metabolite of non-hygroscopic Streptomyces according to claim 1 or 2, characterized in that: The following steps are involved: (a) inoculating spores of Streptomyces immobilis into a seed culture medium for seed culture to obtain a seed propagation solution; (b) inoculating the seed propagation liquid into a fermentation medium for liquid fermentation culture, and performing solid-liquid separation after the culture is completed to obtain a secondary metabolite of Streptomyces hygroscopicus.

4. The method for preparing the secondary metabolite of non-hygroscopic Streptomyces according to claim 3, characterized in that: Includes at least one of the following technical features: (1) The temperature of the seed culture is 25 to 35° C.; (2) The seed culture time is 20 to 24 hours; (3) The ventilation rate of the seed culture is 0.5 to 1 vvm; (4) The seed culture speed is 150 to 300 revolutions per minute.

5. The method for preparing the secondary metabolite of non-hygroscopic Streptomyces according to claim 3, characterized in that: Includes at least one of the following technical features: (1) The temperature of the liquid fermentation culture is 25 to 35° C.; (2) The liquid fermentation culture time is 64 to 72 hours; (3) The speed of the liquid fermentation culture is 150 to 300 revolutions per minute; (4) The pH of the liquid fermentation culture is 6.0 to 7.0; (5) The liquid fermentation culture includes: a first fermentation culture, a second fermentation culture and a third fermentation culture.

6. The method for preparing the secondary metabolite of non-hygroscopic Streptomyces according to claim 5, characterized in that: Includes at least one of the following technical features: (1) performing the first fermentation culture at hour 0 to hour 12 of the liquid fermentation culture; (2) performing the second fermentation culture at 12 to 40 hours in the liquid fermentation culture; (3) performing the third fermentation culture at 40 to 72 hours of the liquid fermentation culture; (4) the ventilation rate of the first fermentation culture is 0.5 to 1 vvm; (5) The ventilation rate of the second fermentation culture is 1.0 to 1.5 vvm; (6) The ventilation rate of the third fermentation culture is 0.5 to 1 vvm. 7 . The non-hygroscopic Streptomyces secondary metabolite prepared by the method for preparing the non-hygroscopic Streptomyces secondary metabolite according to any one of claims 3 to 6 .

8. A medicament for preventing and treating pine wood nematode disease, characterized in that: Including the non-hygroscopic Streptomyces secondary metabolite according to claim 7.

9. Use of the agent for preventing and controlling pine wood nematode disease as claimed in claim 8 in preventing and controlling pine wood nematode disease.

10. A method for preventing and controlling pine wood nematode disease, characterized in that: The secondary metabolite of the non-hygroscopic Streptomyces according to claim 7 or the agent for preventing and controlling pine wood nematode disease according to claim 9 is injected into the trunk of the pine wood.

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