Streptomyces gracilis, Streptomyces gracilis oil suspension concentrate, and preparation method and application thereof

Abstract

The present invention provides a Streptomyces gracilis, a Streptomyces gracilis oil suspension, and a preparation method and application thereof. The Streptomyces gracilis oil suspension of the present invention comprises Streptomyces gracilis conidia powder, an emulsifier, a stabilizer, a wetting dispersant, an antioxidant, a thickener, a UV protectant, a preservative, and a carrier oil. The carrier oil is used as a dispersion medium, which is environmentally friendly and has good permeability and adhesion, and is resistant to rain erosion. The carrier oil has a synergistic effect on the germination of Streptomyces gracilis spores. It also has low application cost, slow spore sedimentation rate, is easy to shake during use, has stable control effect, long storage time, is suitable for various spraying technologies, and has a simple preparation method. The Streptomyces gracilis oil suspension of the present invention has a long spore survival period, with a spore germination rate of up to 85% after 18 months of storage. It has good control effect on yam, peanut, forest nematodes, etc., such as yam coffee nematode, peanut root-knot nematode, tomato southern root-knot nematode, pine wood nematode, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial pesticide preparation and processing, in particular to a Streptomyces glabrata, a Streptomyces glabrata oil suspension concentrate and a preparation method and application thereof. Background Art

[0002] Plant nematode diseases cause at least $157 billion in global crop losses annually, and are poised to become the world's leading crop disease. In my country, nematode diseases cause an average annual yield loss of 10%-15% for all crops. Furthermore, the severe outbreak of pine wilt disease in forestry not only poses a significant threat to my country's pine forest ecosystems, but also results in billions of yuan in annual economic losses.

[0003] Currently, chemical control methods are primarily used worldwide to control plant nematodes, such as soil fumigation (dazomet, chloropicrin, and methamphetamine) and the use of chemical nematicides (fosfothiazolin and carbosulfan). Chemical control effectively suppresses the release of nematodes and reduces agricultural and forestry losses. However, the extensive use of chemical pesticides not only pollutes the environment but also causes severe soil degradation, leading to a continuous decline in crop yields. To address these issues, the development of highly effective and environmentally friendly bionematicides is imperative.

[0004] Actinomycetes are ubiquitous in soil environments, with a vast array of species and numbers. In recent years, they have been recognized as a promising area of ​​research for their secondary metabolites, with promising prospects. According to incomplete statistics, over 8,600 active metabolites have been isolated from actinomycetes, representing a diverse range of structural types. The number of active metabolites has increased exponentially, from just over ten compounds in 1940 to over 22,000 in 2002. Furthermore, recent trends indicate that the proportion of secondary metabolites produced by actinomycetes has continued to expand. As a member of the actinomycetes family, Streptomyces metabolites offer novel chemical structures, unique targets, broad spectrum, low toxicity, low residue, and minimal environmental impact. Therefore, the development of pesticides derived from live Streptomyces bacteria is of great significance not only for the ecological environment but also for social and economic development.

[0005] However, there is currently no technical solution for preparing nematicides using Streptomyces. Summary of the Invention

[0006] In view of this, the present invention proposes a Streptomyces gracilis, a Streptomyces gracilis oil suspension concentrate, and a preparation method and application thereof, in order to solve or at least partially solve the technical problems existing in the prior art.

[0007] In a first aspect, the present invention provides a Streptomyces graminearus, the classification name of the Streptomyces graminearus is Streptomyces graminearus NBERC-VWS578, and the deposit number of the Streptomyces graminearus is CCTCC NO: M2022512.

[0008] In a second aspect, the present invention further provides a conidia powder of Streptomyces praecox. The preparation method of the conidia powder of Streptomyces praecox is as follows: the Streptomyces praecox is activated and cultured until spores are produced, thereby obtaining the conidia powder of Streptomyces praecox.

[0009] Preferably, the conidia powder of Streptomyces praecox, and the preparation method of the conidia powder of Streptomyces praecox, comprise the following steps:

[0010] The Streptomyces raphis strain was inoculated onto a PDA culture plate and activated and cultured until spores were produced. Spore powder was collected from the colonies cultured on the PDA culture plate and prepared into a concentration of (2-4) × 10 7 spores / mL suspension;

[0011] Add 0.5-2 mL of the suspension to the PD culture medium to make the spore concentration in the culture medium (0.5-2) × 10 6 spores / mL, shake culture, transfer to PDA culture medium, and continue shaking culture to obtain the inoculum;

[0012] adding rice to the inoculum, culturing to produce spores, and obtaining a rice culture;

[0013] The rice culture is subjected to freeze vacuum drying treatment to separate the spore powder from the rice matrix, thereby obtaining the conidia powder of Streptomyces praecox.

[0014] In a third aspect, the present invention further provides a Streptomyces oxalis oil suspension concentrate comprising the Streptomyces oxalis conidia powder.

[0015] Preferably, the Streptomyces oxalis oil suspension comprises the following raw materials by mass fraction: 3-25% of Streptomyces oxalis conidia powder, 5-10% of emulsifier, 0.5-3% of stabilizer, 3-8% of wetting and dispersing agent, 0.1-3% of antioxidant, 3-8% of thickener, 0.1-0.5% of UV protectant, 0.1-3% of preservative, and 39.5-85.2% of carrier oil.

[0016] Preferably, in the Streptomyces ovale oil suspension concentrate, the carrier oil comprises at least one of rosin-based vegetable oil, soybean oil, sunflower oil, and corn oil;

[0017] and / or, the emulsifier comprises at least one of SP-OF3472B, DR7792, SP-OF3477, TERMUL2500, TERMUL3015, LT-F192, and OP-10;

[0018] And / or, the stabilizer includes at least one of CMC-Na, BHA, and epoxidized soybean oil;

[0019] and / or, the wetting and dispersing agent comprises at least one of Morwet EFW, TERWET 1010, MF-5, Morwet D425, calcium lignin sulfonate, TERSPERSE 2020, and NNO;

[0020] And / or, the antioxidant includes at least one of vitamin C, vitamin E, and BHA;

[0021] And / or, the thickener includes at least one of white carbon black and organic bentonite;

[0022] And / or, the UV protectant includes at least one of dextrin, xanthan gum, sodium fluorescein, vitamin C, and nano zinc oxide;

[0023] And / or, the preservative includes at least one of sodium diacetate and calcium propionate.

[0024] Preferably, the Streptomyces oxalis oil suspension concentrate contains spores of ≥7×10 9 The mass water content is 2-5%.

[0025] In a fourth aspect, the present invention further provides a method for preparing the Streptomyces oxyphylla oil suspension concentrate, comprising the following steps:

[0026] Streptomyces facilis conidia powder, emulsifier, stabilizer, wetting dispersant, antioxidant, thickener, UV protectant, preservative and carrier oil are mixed and stirred to obtain Streptomyces facilis oil suspension.

[0027] Preferably, the preparation method of the Streptomyces phalerata oil suspension comprises mixing an emulsifier, a stabilizer, a wetting and dispersing agent, an antioxidant, a thickener, a UV protectant, a preservative, and a carrier oil, and shearing the mixture at 8000 to 10000 r / min to obtain a mixture;

[0028] The mixed material and the conidia powder of Streptomyces praecox are mixed and ground to obtain the Streptomyces praecox oil suspension.

[0029] In a fifth aspect, the present invention further provides a use of the Streptomyces gracilis or the Streptomyces gracilis conidia powder or the Streptomyces gracilis oil suspension or the Streptomyces gracilis oil suspension prepared by the preparation method in the preparation of a nematode-killing agent.

[0030] Compared with the prior art, the Streptomyces gracilis, the Streptomyces gracilis oil suspension concentrate and the preparation method and application thereof of the present invention have the following advantages:

[0031] Beneficial effects:

[0032] The Streptomyces gracilis oil suspension of the present invention comprises Streptomyces gracilis conidia powder, an emulsifier, a stabilizer, a wetting and dispersing agent, an antioxidant, a thickener, a UV protectant, a preservative, and a carrier oil. The carrier oil is used as a dispersion medium, which is environmentally friendly, has good permeability and adhesion, and is resistant to rain erosion. The carrier oil also has a synergistic effect on the germination of Streptomyces gracilis spores. Furthermore, the carrier oil has low application costs, slow spore settling rates, is easy to shake during use, has stable control effects, a long storage life, is suitable for various spraying techniques, and is simple to prepare. The Streptomyces gracilis oil suspension of the present invention has a long spore survival period, with a spore germination rate of up to 85% after 18 months of storage. It has good control effects on yam, peanut, and forest nematodes, such as the coffee nematode of yam, the root-knot nematode of peanut, the southern root-knot nematode of tomato, and the pine wood nematode. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0034] Figure 1 This is a photo of Streptomyces graminearus NBERC-VWS578 of the present invention under an optical microscope (1000X);

[0035] Figure 2 This is a front plate photograph of Streptomyces graminearus NBERC-VWS578 of the present invention grown on ISP2 (Streptomyces culture medium No. 2);

[0036] Figure 3 This is a photograph of the reverse side of a plate of Streptomyces graminearus NBERC-VWS578 of the present invention grown on ISP2 (Streptomyces culture medium No. 2);

[0037] Figure 4This is a front plate photograph of Streptomyces graminearus NBERC-VWS578 of the present invention grown on Gao's No. 1 medium;

[0038] Figure 5 This is a photograph of the reverse side of a plate of Streptomyces graminearus NBERC-VWS578 of the present invention grown on Gao's No. 1 medium;

[0039] Figure 6 This is a phylogenetic tree diagram of the Streptomyces graminearus NBERC-VWS578 of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be understood as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0042] The classification name of Streptomyces graminearus provided in the examples of the present application is Streptomyces graminearus NBERC-VWS578, and the deposit number of Streptomyces graminearus is CCTCC NO: M 2022512. The Streptomyces graminearus is deposited with the China Center for Type Culture Collection, Wuhan University, Wuhan, China; the deposit date is May 5, 2022.

[0043] Specifically, the Streptomyces graminearus NBERC-VWS578 of the present invention is shown in the optical microscope photo (1000X) as follows: Figure 1 shown.

[0044] The present invention streptomyces graminearus (Streptomyces graminearus NBERC-VWS578) is placed on the front plate of ISP2 (Streptomyces culture medium No. 2) as shown in FIG. Figure 2 The present invention is shown in Figure 1. The Streptomyces graminearus NBERC-VWS578 is grown on the reverse side of ISP2 (Streptomyces culture medium No. 2). Figure 3 The front plate of the Streptomyces graminearus NBERC-VWS578 of the present invention on Gao's No. 1 medium is as shown. Figure 4 The reverse side of the plate of Streptomyces graminearus NBERC-VWS578 on Gao's No. 1 medium is shown in FIG. Figure 5 shown.

[0045] The morphological characteristics of the Streptomyces graminearus NBERC-VWS578 of the present invention are as follows: the strain grows normally on Gao's medium No. 1, the colonies are off-white, without soluble pigments, the colonies are concentric circles in the middle, and the edges are irregular; under optical microscopy, the spore chains are curled, and the spores are ball-shaped or nearly ellipsoidal.

[0046] The phylogenetic tree of Streptomyces graminearus NBERC-VWS578 of the present invention is as follows: Figure 6 Neighbor-Joining phylogenetic tree of Streptomyces graminearus NBERC-VWS578 constructed as an outbranch of Actinomyces naeslundii DSM 43013 (X53226) based on 16S rRNA gene sequence alignment.

[0047] The 16S rRNA gene sequence of Streptomyces graminearus NBERC-VWS578 of the present invention is (see sequence listing):

[0048]

[0049] The BLAST results of the 16S rRNA gene sequencing of Streptomyces graminearus NBERC-VWS578 of the present invention are shown in Table 1 below.

[0050] Table 1 - BLAST results of 16S rRNA gene sequencing of Streptomyces ovale

[0051]

[0052]

[0053] The physico-biochemical characteristics - carbon source utilization of Streptomyces graminearus NBERC-VWS578 of the present invention are shown in Table 2 below.

[0054] Table 2 - Physicochemical characteristics of Streptomyces ovale

[0055]

[0056]

[0057] In Table 2 above, +: positive reaction; -: negative reaction; W: weak positive reaction;

[0058] The above results indicate that the strain was identified as strain NBERC-VWS578: Streptomyces graminearus.

[0059] Based on the same inventive concept, the present invention also provides a conidia powder of Streptomyces gracilis. The preparation method of the conidia powder of Streptomyces gracilis is as follows: activating and culturing the Streptomyces gracilis until spores are produced, thereby obtaining the conidia powder of Streptomyces gracilis.

[0060] In some embodiments, the method for preparing conidia powder of Streptomyces herba comprises the following steps:

[0061] S1. Inoculate Streptomyces raphis strains onto PDA culture plates and activate the culture until spores are produced. Take spore powder from the cultured colonies on the PDA culture plates and prepare a concentration of (2-4) × 10 7 spores / mL suspension;

[0062] S2. Add 0.5-2 mL of the suspension to the PD culture medium to make the spore concentration in the culture medium (0.5-2) × 10 6 spores / mL, shake culture, transfer to PDA culture medium, and continue shaking culture to obtain the inoculum;

[0063] S3, adding rice to the inoculum, cultivating spores, and obtaining a rice culture;

[0064] S4. The rice culture is subjected to freeze vacuum drying to separate the spore powder from the rice matrix to obtain conidia powder of Streptomyces praecox.

[0065] Specifically, the method for preparing conidia powder of Streptomyces herba includes the following steps:

[0066] S1. Inoculate Streptomyces raphis onto a PDA culture plate and activate the culture at 28°C and 12L:12D until spores are produced. Spore powder is collected from the colonies cultured on the PDA culture plate to prepare a concentration of (2-4)×10 7 spores / mL suspension;

[0067] S2. Add 0.5-2 mL of the suspension to the PD culture medium to make the spore concentration in the culture medium (0.5-2) × 10 6 spores / mL, shake culture (28±1℃, 180r / min) for 48h, transfer to 1000mL PDA culture medium (PDA is a standard fungal culture medium), and continue shaking culture for 24h to prepare the inoculum;

[0068] S3, 100g rice is placed in a beaker, and water is added to soak for 30min by rice (g): water (mL)=1:1.5 (i.e. every g rice adds 1.5mL water). After washing and draining, sterilize. The sterilization conditions are: 121 ℃, 0.1MPa sterilization for 30 minutes; After cooling, the inoculum size of 10% volume is mixed into the inoculum in step S2, and the rice material after the inoculation is poured into a culture dish with a diameter of 15cm and covered with plastic wrap; Then the inoculated rice material is cultivated and spore-forming under the conditions of 28 ± 1 ℃ and 12L:12D, and then cultivated for 3-5d after removing the plastic wrap;

[0069] S4, rice culture is processed through freeze vacuum drying, its moisture content is reduced to below 4%, utilize vibrating sieve, spore powder is separated from rice matrix, promptly obtains streptomyces grass conidia powder; Measure its spore content, viable spore rate, place in 4 ℃ refrigerator for standby use.

[0070] Among them, PDA medium is potato dextrose agar medium, PD culture medium is pd liquid medium, and the PDA medium and PD culture medium used in this application are both commercially available culture medium / culture medium; 12L:12D refers to the photoperiod, 12 hours of light and then 12 hours of darkness.

[0071] Based on the same inventive concept, the present invention also provides a Streptomyces oxalis oil suspension, comprising the above-mentioned Streptomyces oxalis conidia powder.

[0072] In some embodiments, the Streptomyces oxalis oil suspension comprises the following raw materials by mass fraction: 3-25% of Streptomyces oxalis conidia powder, 5-10% of emulsifier, 0.5-3% of stabilizer, 3-8% of wetting and dispersing agent, 0.1-3% of antioxidant, 3-8% of thickener, 0.1-0.5% of UV protectant, 0.1-3% of preservative, and 39.5-85.2% of carrier oil.

[0073] In some embodiments, the carrier oil comprises at least one of rosin-based vegetable oil, soybean oil, sunflower oil, and corn oil;

[0074] In some embodiments, the emulsifier includes at least one of SP-OF3472B (produced by Nanjing Qingyu Chemical Research Co., Ltd.), DR7792, SP-OF3477, TERMUL2500, TERMUL3015, LT-F192 (i.e., Langtai LT-F192 emulsifier), and OP-10;

[0075] In some embodiments, the stabilizer includes at least one of CMC-Na (sodium carboxymethylcellulose), BHA (butylated hydroxyanisole), and epoxidized soybean oil.

[0076] In some embodiments, the wetting and dispersing agent includes at least one of Morwet EFW (Akzo Nobel Morwet series dispersants), TERWET 1010, MF-5, Morwet D425 (Akzo Nobel dispersant), calcium lignin sulfonate, TERSPERSE 2020 (produced by Huntsman, USA, alkyl naphthalene sulfonate), and NNO (sodium salt of 2-naphthalenesulfonic acid formaldehyde polymer).

[0077] In some embodiments, the antioxidant comprises at least one of vitamin C, vitamin E, and BHA;

[0078] In some embodiments, the thickener includes at least one of white carbon and organic bentonite.

[0079] In some embodiments, the UV protectant includes at least one of dextrin, xanthan gum, sodium fluorescein, vitamin C, and nano zinc oxide.

[0080] In some embodiments, the preservative comprises at least one of sodium diacetate and calcium propionate.

[0081] In some embodiments, the conidia of Streptomyces herba contain ≥7×10 9 The mass water content is 2-5%.

[0082] Preferably, the Streptomyces oxalis oil suspension comprises the following raw materials by mass fraction: 5% Streptomyces oxalis conidia powder, 8% SP-OF3472B, 0.5% epoxidized soybean oil, 10% Morwet EFW and calcium lignin sulfonate, 0.3% vitamin C, 3% organic bentonite, 0.4% nano zinc oxide, 0.5% calcium propionate, and 72.3% rosin-based vegetable oil (which can be OD-1 rosin-based vegetable oil solvent produced by Fujian Nord Biotechnology Co., Ltd.).

[0083] Preferably, the Streptomyces oxalis oil suspension comprises the following raw materials by mass fraction: 15% Streptomyces oxalis conidia powder, 17% DR7792 (Shenzhen Langtai Biotechnology Co., Ltd.), 1.2% VE (vitamin E), 10% Morwet D425, 0.3% vitamin C, 8% white carbon black, 0.4% vitamin C, 0.5% calcium propionate, and 47.6% corn oil.

[0084] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned Streptomyces ossifolia oil suspension concentrate, comprising the following steps:

[0085] Streptomyces facilis conidia powder, emulsifier, stabilizer, wetting dispersant, antioxidant, thickener, UV protectant, preservative and carrier oil are mixed and stirred to obtain Streptomyces facilis oil suspension.

[0086] In some embodiments, an emulsifier, a stabilizer, a wetting and dispersing agent, an antioxidant, a thickener, a UV protectant, a preservative, and a carrier oil are mixed and sheared at 8000 to 10000 r / min to obtain a mixture;

[0087] The mixed material and the conidia powder of Streptomyces praecox are mixed and ground to obtain the Streptomyces praecox oil suspension.

[0088] Specifically, the above substances (except the conidia powder of Streptomyces gracilis) are added to a magnetic stirrer for premixing, and then the mixture is sheared at a high speed of 8000-1200 bar in a Canadian homogenizer or a high-speed shearing machine at 8000-10000 r / min for 10-30 minutes to obtain a mixture; at room temperature, the mixture and the conidia powder of Streptomyces gracilis are added to a sand mill according to a proportion, ground for 10-60 minutes, and mixed evenly to obtain a dispersible oil suspension concentrate of Streptomyces gracilis.

[0089] The Streptomyces gracilis oil suspension concentrate of the present invention uses a carrier oil as a dispersion medium, is environmentally friendly, has good permeability and adhesion, and is resistant to rain erosion. The vegetable oil has a synergistic effect on the germination of Streptomyces gracilis spores. Furthermore, it has low application costs, slow spore settling rates, is easy to shake evenly during use, has stable control effects, and has a long storage life. It is suitable for use with various spraying techniques and has a simple preparation method. The Streptomyces gracilis oil suspension concentrate of the present invention has a long spore survival period, with a spore germination rate of up to 85% after 18 months of storage. It has good control effects on yam, peanut, and forest nematodes, such as the coffee nematode of yam, the root-knot nematode of peanut, the southern root-knot nematode of tomato, and the pine wood nematode.

[0090] Based on the same inventive concept, the present invention also provides a use of Streptomyces gracilis or Streptomyces gracilis conidia powder or Streptomyces gracilis oil suspension or the Streptomyces gracilis oil suspension prepared by the above preparation method in preparing nematode killing agents.

[0091] The present invention is further described below with specific examples of Streptomyces gracilis oil suspension concentrate and its preparation method. This section further illustrates the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. If not otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this area.

[0092] Example 1

[0093] The present application provides an oil suspension concentrate of Streptomyces gracilis, comprising, by mass fraction, 5% conidia powder of Streptomyces gracilis, 8% SP-OF3472B, 0.5% BHA, 0.5% CMC-Na, 0.3% calcium propionate, 3% organic bentonite, 8% wetting and dispersing agent mixed with Morwet EFW and NNO, 0.2% xanthan gum, and 74.5% rosin-based vegetable oil (OD-1 rosin-based vegetable oil solvent produced by Fujian Nord Biotechnology Co., Ltd.); the mass ratio of Morwet EFW to NNO is 1:1;

[0094] The preparation method of Streptomyces herbicides conidia powder comprises the following steps:

[0095] S1. Inoculate Streptomyces raphis strains onto PDA culture plates and activate and culture them at 28°C and 12L:12D until spores are produced. Spore powder is collected from the colonies cultured on the PDA culture plates to prepare a concentration of 3×10 7 spores / mL suspension;

[0096] S2. Add 1 mL of the suspension to 30 mL of PD culture medium to make the spore concentration in the culture medium 1×10 6spores / mL, shake culture (28±1℃, 180r / min) for 48h, transfer to 1000mL PDA culture medium (PDA is a standard fungal culture medium), and continue shaking culture for 24h to prepare the inoculum;

[0097] S3, 100g rice is placed in a beaker, and water is added to soak for 30min by rice (g): water (mL)=1:1.5 (i.e. every g rice adds 1.5mL water). After washing and draining, sterilize. The sterilization conditions are: 121 ℃, 0.1MPa sterilization for 30 minutes; After cooling, the inoculum size of 10% volume is mixed into the inoculum in step S2, and the rice material after the inoculation is poured into a culture dish with a diameter of 15cm and covered with plastic wrap; Then the inoculated rice material is cultivated and spore-forming under the conditions of 28 ± 1 ℃ and 12L:12D, and then cultivated for 3-5d after removing the plastic wrap;

[0098] S4, with rice culture after freeze vacuum drying, make its moisture content be reduced to below 4%, utilize vibrating screen, spore powder and rice matrix are separated, promptly obtain streptomyces grass conidia powder; Measure its spore amount, viable spore rate, place in 4 ℃ refrigerator for standby use;

[0099] The preparation method of the Streptomyces oxyphylla oil suspension concentrate of the present embodiment comprises the following steps:

[0100] S1. Add the raw materials other than the conidia powder of Streptomyces herbicides into a magnetic stirrer and stir at 1500 rpm for 10 min to obtain a mixture; then place the mixture in a Canada homogenizer (working pressure of 800-1000 bar) and homogenize and mix until the particle size of the mixture is controlled to be about 25 μm, and obtain a mixture after homogenization;

[0101] S2. Add the mixture and Streptomyces facilis conidia powder into a sand mill at room temperature, grind for 20 minutes, and mix well to obtain a Streptomyces facilis dispersible oil suspension concentrate.

[0102] Example 2

[0103] The present application provides an oil suspension concentrate of Streptomyces facilis, comprising, by mass fraction, 10% conidia powder of Streptomyces facilis, 9% DR7792, 0.5% VC (vitamin C), 1.2% BHA, 0.5% calcium propionate, 5% white carbon black, 12% MF-5 (Hubei Shangzhu Chemical Co., Ltd.) and a wetting and dispersing agent mixed with NNO, 0.3% dextrin (corn dextrin), and 61.5% sunflower oil; the mass ratio of MF-5 to NNO is 1:1;

[0104] The preparation method of Streptomyces herbicides conidia powder is the same as that in Example 1;

[0105] The preparation method of Streptomyces oxalis oil suspension is the same as that in Example 1.

[0106] Example 3

[0107] The present application provides an oil suspension concentrate of Streptomyces gracilis, which comprises, by mass fraction, 15% of Streptomyces gracilis conidia powder, 17% of DR7792, 1.2% of VE (vitamin E), 2.5% of epoxidized soybean oil (Qilu Petrochemical), 1.2% of sodium diacetate, 7% of white carbon black, 15% of a wetting and dispersing agent composed of Morwet EFW and TERSPERSE 2020, 0.5% of nano zinc oxide, and 40.6% of corn oil; the mass ratio of Morwet EFW to TERSPERSE 2020 is 1:1;

[0108] The preparation method of Streptomyces herbicides conidia powder is the same as that in Example 1;

[0109] The preparation method of Streptomyces oxalis oil suspension is the same as that in Example 1.

[0110] Example 4

[0111] The present application provides an oil suspension concentrate of Streptomyces gracilis, which comprises, by mass fraction: 20% conidia powder of Streptomyces gracilis, 18% SP-OF3477 (produced by Nanjing Qingyu Chemical Research Co., Ltd.), 2% BHA, 1.5% CMC-Na, 1.5% sodium diacetate, 5% organic bentonite, 15% wetting and dispersing agent composed of Morwet EFW and TERSPERSE 2020, 0.6% nano zinc oxide, and 36.4% sunflower oil; the mass ratio of Morwet EFW to TERSPERSE 2020 is 1:1;

[0112] The preparation method of Streptomyces herbicides conidia powder is the same as that in Example 1;

[0113] The preparation method of Streptomyces oxalis oil suspension is the same as that in Example 1.

[0114] Performance Testing

[0115] Effects of adjuvants on spore and preparation performance

[0116] (a) Screening of additives

[0117] Add various additives (emulsifier, suspension stabilizer, antioxidant, preservative, thickener, and wetting and dispersing agent) to a temporary suspension of spore powder (50g:1L) of solvent oil. Shake to evenly disperse the suspension to create an oil. Dispense into vials and store sealed at room temperature, away from light. Test the spore germination rate every 30 days.

[0118] Biocompatibility: Before sampling, make it uniform on an oscillator, then take the above oil solutions of different concentrations to prepare 10 6 After incubation at 28±1°C, 180 rpm, and shaking for 16 hours, samples were taken for microscopic examination of the germination rate (viable spore rate). The effect of each adjuvant was determined by the germination rate. This experiment was repeated three times, with an aqueous spore suspension serving as a control. All spores with a germ tube length equal to or greater than the spore diameter were considered germinated.

[0119] (b) Screening of important indicators of preparations

[0120] The main indicators for evaluating the quality of oil suspension concentrates include stability, suspension rate, redispersibility, flash point, viscosity, fluidity, etc. The most intuitive indicators are suspension rate and redispersibility.

[0121] Suspension rate: The property of the initial state of solute dissolved or dispersed in the solvent in an oil suspension, which is related to fluidity, flash point and viscosity.

[0122] Redispersibility: refers to the property of the oil suspension to return to its original fully dispersed state after sedimentation, which is related to fluidity, flash point, viscosity, etc.

[0123] The selection should be made based on the biocompatibility of the adjuvant and the suspension rate and redispersibility of the adjuvant to the oil suspension concentrate.

[0124] (c) Quality testing of preparations:

[0125] The Streptomyces raphis dispersible oil suspension prepared in Example 1 had a viable spore rate >80.00%, a moisture content ≤4.0, a pH value of 5.6, an open flash point >150°C, 98% of the particles passed a 25 μm test sieve, a viscosity (20°C) of 0.42 Pa·s, and acceptable heat storage stability (30°C, 2 months) and low-temperature stability.

[0126] (3) Efficacy test

[0127] Pot test: After the peanut seeds were surface disinfected with 75% alcohol, they were rinsed three times with sterile water, dried naturally, and coated with a 20-fold diluted solution. Sterile water was used as a blank control. Sow seeds in plastic pots filled with sterilized soil, with 3 seeds sown in each pot, so that the seedlings with poor growth can be pulled out after germination to ensure that there is a peanut seedling in each pot. Culture them at room temperature under natural light. After four cotyledons have grown, each pot is inoculated with 2 ml of a suspension of 500 / ml second-instar larvae of the northern root-knot nematode. Each treatment was repeated three times and arranged randomly. The northern root-knot nematodes were inoculated with sterile water as a control, and the number of oocysts was investigated 50 days after the root-knot nematodes were inoculated. The root knot reduction rate was calculated, and the control effect was expressed as the oocyst reduction rate:

[0128] Control effect (%) = oocyst reduction rate (%) = control oocysts - number of treated oocysts / number of control oocysts × 100%

[0129] To evaluate the growth-promoting and yield-increasing effects, the fresh weight of different treatments was investigated.

[0130] Field trials:

[0131] The experiment was conducted in a continuously cropped field of Buddha's hand yam. Treatments involved root irrigation with diluted water, while the blank control received pure water. Each treatment consisted of three plots, replicated three times, with each plot measuring 10 square meters, arranged in randomized blocks. Application was via root irrigation with diluted water, using 300 mL of liquid per plant. Three applications were performed throughout the growing season, the first one week after yam emergence, followed by 20-day intervals thereafter. The effects on yam growth were investigated after harvest.

[0132] Table 3 below shows the classification criteria for plant tuber disease index

[0133] Table 3 - Tuber disease index classification standards

[0134]

[0135] The disease index (DI) was calculated according to the formula.

[0136] DI = 100×Σ(Ni ×i) / (Nt ×9) (1)

[0137] Among them: DI is the disease index, Ni is the number of diseased plants at each level, i is the corresponding level value, and Nt is the total number of plants surveyed.

[0138] (4) Pine wood nematode efficacy test:

[0139] When most of the nematodes grown on the Botrytis cinerea culture medium were in the gestation stage (J4 / adult), the nematodes were homogenized (growing period) to make all the nematodes at the same growth level. The nematodes were collected into a centrifuge tube after being left to stand for 12 h using the Baermann funnel method and centrifuged at 3500 r / min. -1 Centrifuge for 4 minutes, rinse three times with 0.9% saline, and prepare 100 mL -1 In a 24-well plate, 0.5 mL of nematode suspension was added to each well. An equal volume of the drug solution diluted to a desired concentration was then added. A background solution was used as a blank control. Three parallel experiments were performed. Nematodes were observed for survival under a stereomicroscope 36, 48, and 72 hours after treatment. Nematodes were considered alive if they moved and exhibited a spiral shape, while those that were motionless and rigid were considered dead. Mortality and adjusted mortality were calculated using the following formulas.

[0140] Mortality rate = (number of dead insects / number of test insects) × 100%;

[0141] Adjusted mortality rate = [(mortality rate of treatment group - mortality rate of control group) / (1 - mortality rate of control group)] × 100%.

[0142] The results of the pot test of the Streptomyces ossifolia oil suspension prepared in Example 1 are shown in Table 4 below.

[0143] Table 4- Example 1 Potted test of Streptomyces oxalis oil suspension against peanut root knot nematodes

[0144]

[0145] Table 4 shows the results of a potted plant test using the 5% Streptomyces ossi oil suspension formulation from Example 1. As shown in Table 4, the Streptomyces ossi oil suspension formulation prepared in Example 1 achieved over 70% control efficacy against peanut root-knot nematodes, while the control agents, avermectin and Paecilomyces lilacinus, achieved only 58.47% and 47.21%, respectively. In terms of yield and growth promotion, the 5% Streptomyces ossi oil suspension formulation increased yield by over 25%, while the other control agents also achieved a modest yield-increasing effect of around 10%.

[0146] The results of the field test of the Streptomyces ossifolia oil suspension prepared in Example 4 are shown in Table 5 below.

[0147] Table 5-Example 4 Field test of Streptomyces oxalis oil suspension against Chinese yam root knot nematodes

[0148]

[0149] As shown in Table 5, the three concentrations of the Streptomyces oxalis oil suspension prepared in Example 4 showed significant differences in their efficacy against Chinese yam root-knot nematodes. The control efficacy was ranked as follows: 20% Streptomyces oxalis diluted 200 times > 20% Streptomyces oxalis diluted 500 times > 20% Streptomyces oxalis diluted 1000 times. The three concentrations of the Streptomyces oxalis dispersible oil suspension significantly increased lateral yield, with the 200-fold dilution treatment increasing yield by 22.36%.

[0150] The Streptomyces oxalis oil suspension prepared in Example 4 was tested for efficacy against pine wood nematodes. The nematicidal activity of the Streptomyces oxalis oil suspension prepared in Example 4 after 500-fold dilution against pine wood nematodes is shown in Table 6 below.

[0151] Table 6 - Nematicidal activity of 500-fold diluted Streptomyces oxysporum oil suspension against pine wood nematodes

[0152] Time (hours) Adjusted mortality rate / % 36 32.51±1.36 48 66.33±2.56 72 100±0.00

[0153] As shown in Table 6, the activity of the 20% Streptomyces oxalis oil suspension prepared in Example 4 at a 500-fold dilution against pine wood nematodes gradually increased with time, and after 72 hours, the corrected mortality of the 20% Streptomyces oxalis dispersible oil suspension at a 500-fold dilution against pine wood nematodes was as high as 100%.

[0154] The storage stability of the Streptomyces ossifolia oil suspension prepared in Example 1 at room temperature (25° C.) was tested, and the results are shown in Table 7 below.

[0155] Table 7 - Storage stability of Streptomyces oxyphylla oil suspension at room temperature

[0156] Storage time at room temperature (months) 0 3 6 18 24 Viable spore rate (%) 97.00 91.29 88.10 85.64 70.91

[0157] As can be seen from Table 1, the spore survival period of the dispersible oil suspension concentrate of Streptomyces facilis of the present invention is long, and the spore germination rate is as high as 85% after storage for 18 months.

[0158] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a Streptomyces herba oil suspension concentrate in the preparation of a nematode-killing agent; The nematode is peanut root knot nematode; The Streptomyces oxalis oil suspension comprises the following raw materials by mass fraction: 5% Streptomyces oxalis conidia powder, 8% emulsifier, 0.5% stabilizer, 8% wetting and dispersing agent, 0.5% antioxidant, 3% thickener, 0.2% UV protectant, 0.3% preservative, and 74.5% carrier oil; The carrier oil is rosin-based vegetable oil; The emulsifier is SP-OF3472B; The stabilizer is CMC-Na; The wetting and dispersing agent includes Morwet EFW and NNO; the mass ratio of Morwet EFW and NNO is 1:1; The antioxidant is BHA; The thickener is organic bentonite; The ultraviolet protection agent is xanthan gum; The preservative is calcium propionate; The preparation method of the conidia powder of Streptomyces herba comprises the following steps: The Streptomyces raphis strain was inoculated onto a PDA medium plate and activated and cultured until spores were produced. Spore powder was collected from the colonies cultured on the PDA medium plate and prepared into a concentration of 3×10 7 spores / mL suspension; 1 mL of the suspension was added to the PD culture medium to make the spore concentration in the culture medium 1×10 6 spores / mL, shake culture, transfer to PDA culture medium, and continue shaking culture to obtain the inoculum; Add rice to the inoculum and culture to produce spores to obtain a rice culture; add 1.5 mL of water per gram of rice; The rice culture is subjected to freeze vacuum drying to separate the spore powder from the rice matrix, thereby obtaining conidia powder of Streptomyces herba; The classification of the streptomyces is named as: streptomyces Streptomyces graminearus ), the preservation number of the Streptomyces praecox is CCTCC NO: M 2022512.

2. The use according to claim 1, characterized in that The conidia content of the Streptomyces herba conidia powder is ≥7×10 9 The mass water content is 2~5%.

3. The use according to claim 1, characterized in that The preparation method of the Streptomyces rapa oil suspension concentrate is characterized by comprising the following steps: Streptomyces facilis conidia powder, emulsifier, stabilizer, wetting dispersant, antioxidant, thickener, UV protectant, preservative and carrier oil are mixed and stirred to obtain Streptomyces facilis oil suspension.

4. The use according to claim 3, characterized in that After mixing an emulsifier, a stabilizer, a wetting and dispersing agent, an antioxidant, a thickener, an ultraviolet protective agent, a preservative, and a carrier oil, the mixture is sheared at 8000-10000 r / min to obtain a mixture; The mixed material and the conidia powder of Streptomyces praecox are mixed and ground to obtain the Streptomyces praecox oil suspension.

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