Co-culture agent of saccharopolyspora spinosa d2302 and paenibacillus polymyxa d2303, and preparation method and application thereof

Abstract

The application discloses a Saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303 co-cultured microbial agent and a preparation method and application thereof. The application provides a composite bacterium composed of Saccharopolyspora spinosa D2302 (CGMCC No. 26388) and Paenibacillus polymyxa D2303 (CGMCC No. 26387). The Saccharopolyspora spinosa D2302 and the Paenibacillus polymyxa D2303 are co-cultured, so that the yield of the former producing polymyxin can be significantly improved, and the yield of the latter producing indole acetic acid is not affected. The application has important significance for improving the stress resistance performance of crops, such as disease and pest resistance, and promoting the growth and development of crops.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a co-culture agent of Polysaccharidobacterium sarcodactylus D2302 and Bacillus polymyxa D2303, its preparation method, and its application. Background Technology

[0002] *Saccharopolyspora spinosa* is a widely studied actinomycete species within the genus *Saccharopolyspora*. *Saccharopolyspora spinosa* was initially isolated by a chemist from an abandoned brewery (Mertz FP, Yao RC. *Saccharopolyspora spinosa sp. nov. isolated from soil collected in a sugarmill rum still. Int J System Bacteriol. 1990; 40(1):34-39. doi:10.1099 / 00207713-40-1-34.). Later studies revealed that *Saccharopolyspora spinosa* is widely distributed in various soil environments. Some strains of *Saccharopolyspora spinosa* can produce polyfungicides during fermentation under aerobic conditions. Spinosad belongs to a class of macrolide biological insecticides (Baker DR, Fenyes JG, Steffens JJ, et al. Synthesis and Chemistry of Agrochemicals III [M]. Washington DC: American Chemical Society, 1992. 214-225; Kirst H, Michel KH, Martin JW, et al. A83543A-D, unique fermentation-derived tetracyclic acrolides [J]. Tetrahedron Lett, 1991, 32(37): 4839-4842; Boeck LD, Chio H, Eaton TE, et al. Macrolide compounds. EP: 375316, 19906 27; Anastas P, Kirchchoff M, Williamson T. Spinosad - a new natural product for insect control. Green Chemistry. 1999; 1(4): G88.).Spinosad is hailed as a highly effective biological insecticide, mainly used for the control of agricultural and forestry pests, stored grain pests, sanitary pests, and livestock parasites. Its products are currently primarily used on crops such as cotton, fruits and vegetables, tea, tobacco, medicinal herbs, and grains (Salgado VL, Sheers JJ, Watson GB, et al. Studies on the mode of action of spinosad: the internal effective concentration and the concentration dependence on neural excitation[J]. Pestic Biochem Physoil, 1998, 60: 103-110). Due to its advantages as a biological pesticide, including high specificity, high activity, and environmental safety, spinosad is widely used in many countries. It has the characteristics of broad insecticidal spectrum, high biological activity, low toxicity and low residue, and has the advantages of extremely high safety limits for mammals, fish, birds and most beneficial insects. It won the US Presidential Green Chemicals Challenge Award. In 2005, the US Environmental Protection Agency approved spinosad as a grain storage protectant (Thompson GD, Dutton R, Sparks TC. Spinosad-a case study: an example from a natural products discovery programme[J]. PestManag Sci, 2000, 56(8): 696-702; Wu Xia. Spinosad-a case study: an example from a natural products discovery programme[J]. World Pesticides, 2004, 1: 24-28; Xu Zhihong, Jiang Zhisheng. Symptoms of poisoning and mechanism of action of the biological insecticide spinosad[J]. Pesticide Science and Management, 2004, 25(2): 25-28). Despite the widespread application of synthetic biology, the commercially available biopesticide spinosad still relies on fermentation by *Saccharomyces cerevisiae*. Therefore, research into identifying high-quality wild-type *Saccharomyces cerevisiae* resources and improving the efficiency of spinosad production by *Saccharomyces cerevisiae* is of great significance.

[0003] Spinosyns, a secondary metabolite produced by the fermentation of *Spinosyns spinosad*, contains a mixture of two structurally similar compounds: Spinosyns A and Spinosyns D (Gary D, Thomas C. Recent advance in the chemistry of spinosyns[J]. Pest Management Sci, 2001, 57(2):177-185; Thompson GD, Dutton R, Sparks T C. Spinosyns - a case study: an example from a natural products discovery programme[J]. Pest Management Sci, 2000, 56(8):696-702.). Currently, the wild-type strain of *Spinosyns spinosad*, NRRL18395, which is being studied as a model, produces 78.7 mg / L of spinosad, with component A accounting for approximately 84% and component D approximately 16%. The spinosad-producing strains obtained through mutagenesis and other methods, as reported in literature or patents, have a shake-flask fermentation yield of approximately 1-2 g / L (CN 113444659 B, a spinosad-producing polyspora spinosad).

[0004] *Paenibacillus polymyxa* was proposed as a bacterial species in 1994 by Ash et al. based on the molecular taxonomic characteristics of strain ATCC 842 (Ash C, Priest FG, Collins MD. Molecular identification of rRNA group 3bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus *Paenibacillus*. Antonie Van Leeuwenhoek 1993; 64:253-260.). *Paenibacillus polymyxa* is the type species of the genus *Paenibacillus*. It is a group of Gram-positive bacteria widely distributed in various natural environments, commonly found not only in soil and on plant surfaces, but also as an important component of plant endophytic bacteria. Currently, the main source of biocontrol polymyxin Bacillus is plant rhizosphere soil (Chen Xueli, Hao Zaibin, Wang Guanghua, et al. Isolation and purification of antibacterial protein of polymyxin BRF-1 [J]. Chinese Journal of Biological Control, 2007, 23(2):156-159; Liu Xunli, Sun Changpo, Ma Yingfei, et al. Isolation and identification of antagonistic bacteria of a silkworm pathogen [J]. Sericultural Science, 2004, 30(3):273-276; Zhao Deli, Zeng Zilin, Li Hui, et al. Preliminary study on antibacterial active substances and fermentation conditions of polymyxin Bacillus JW-725 [J]. Plant Protection, 2006, 32(1):47-50; Zhang Daojing, Gong Chunyan, Wei Honggang, et al. Chemical composition of polymyxin Bacillus HY96-2 [J]. Journal of East China University of Science and Technology: Natural Science Edition, 2008, 34(1):71-73.).

[0005] Polymyxins are among the few bacteria capable of producing antibiotics with clinical application value. Polymyxins B (PMB) and colistin produced by *Paenibacillus* are already used clinically to combat bacterial infections. Some strains within the *Paenibacillus* species are also important plant biocontrol bacteria and plant growth promoting rhizobacteria (PGPR), and have been widely used in agriculture (Dilfuza Egamberdiyeva. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different soils. Applied Soil Ecology, 2007, 36(2-3):184-189; Choong-Min Ryu, Jinwoo Kim, Okhee Choi, et al. Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame. Biological...). Control, 2006, 39(3):282-289; Shi Yingwu, Lou Kai, Li Chun, et al. Effects of endophytic polymyxa S-7 on photosynthesis, yield and quality of sugar beet [J]. Chinese Journal of Applied Ecology, 2009, 20(3):597-602; Zhang Qiuxia, Zhong Zengtao, Xu Yangchun, et al. Study on fluorescence in situ hybridization detection technology of polymyxa spores and its application in organic fertilizer fermentation [J]. Journal of Plant Nutrition and Fertilizers, 2010, 16(5):1276-1281.). Polymyxa and its metabolites also have applications in industrial and mining and wastewater treatment (Partha Patra, Natarajan KA. Surface chemical studies on selective separation of pyrite and galena in the presence of bacterial cells and metabolic products of Paenibacillus polymyxa. Journal of Colloid and Interface Science, 2006, 298(2):720-729).Based on the promising application prospects of Bacillus polymyxa in multiple fields, the U.S. Environmental Protection Agency (EPA) has listed Bacillus polymyxa as one of the microorganisms that can be commercially used, and my country's Ministry of Agriculture has also listed it as a first-class strain that is exempt from safety identification. Summary of the Invention

[0006] The purpose of this invention is to provide a co-culture agent of Polysporus spp. D2302 and Bacillus polymyxa D2303, its preparation method and application.

[0007] Firstly, this invention claims protection for a compound bacteria.

[0008] The composite bacteria claimed in this invention consists of Saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303.

[0009] The Saccharopolyspora spinosa D2302 strain has the accession number CGMCC No. 26388 from the China General Microbiological Culture Collection Center.

[0010] The accession number of the polymyxa (Paenibacillus polymyxa) D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0011] In the compound bacteria, the two bacteria are packaged separately.

[0012] Secondly, this invention claims protection for a complete set of products.

[0013] The complete product claimed by this invention is specifically composed of the compound bacteria and fermentation culture medium described in the first aspect above.

[0014] The solvent of the fermentation medium is water, and the solutes and their concentrations are as follows: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L; pH 7.2.

[0015] Furthermore, the complete product may also include seed culture media (such as the primary seed culture media and / or secondary seed culture media described below) for activating and culturing the strains.

[0016] Thirdly, the present invention claims protection for the use of the compound bacteria described in the first aspect above or the complete product described in the second aspect above in any of the following:

[0017] (A1) Production of spinosad;

[0018] (A2) Prepare products for the production of spinosad;

[0019] (A3) Simultaneously producing spinosad and indoleacetic acid;

[0020] (A4) Prepare a product for the simultaneous production of spinosad and indoleacetic acid;

[0021] (A5) Improve plant resistance to diseases and pests;

[0022] (A6) Prepare products to improve plant resistance to diseases and pests;

[0023] (A7) Improve plant resistance to diseases and pests while promoting plant growth;

[0024] (A8) Prepare products that can improve plant resistance to pests and diseases while promoting plant growth.

[0025] Fourthly, the present invention claims a method for producing spinosad.

[0026] The method for producing spinosad claimed in this invention may include the following steps: co-culturing Saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303 to obtain spinosad from the culture.

[0027] The Saccharopolyspora spinosa D2302 strain has the accession number CGMCC No. 26388 from the China General Microbiological Culture Collection Center.

[0028] The accession number of the polymyxa (Paenibacillus polymyxa) D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0029] Fifthly, the present invention claims a method for simultaneously producing spinosad and indoleacetic acid.

[0030] The present invention claims a method for simultaneously producing spinosad and indoleacetic acid, which may include the following steps: co-culturing Saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303 to simultaneously obtain spinosad and indoleacetic acid from the culture.

[0031] The Saccharopolyspora spinosa D2302 strain has the accession number CGMCC No. 26388 from the China General Microbiological Culture Collection Center.

[0032] The accession number of the polymyxa (Paenibacillus polymyxa) D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0033] Sixthly, the present invention claims protection for the use of Paenibacillus polymyxa D2303 in increasing the production of spinosad by Saccharopolyspora spinosa D2302.

[0034] The Saccharopolyspora spinosa D2302 strain has the accession number CGMCC No. 26388 from the China General Microbiological Culture Collection Center.

[0035] The accession number of the polymyxa (Paenibacillus polymyxa) D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0036] Seventhly, the present invention claims a method for increasing the yield of spinosad produced by Saccharopolyspora spinosa D2302.

[0037] The method for increasing the production of spinosad by *Saccharopolyspora spinosa* D2302, as claimed in this invention, may include the following steps: co-culturing *Saccharopolyspora spinosa* D2302 and *Paenibacillus polymyxa* D2303. The co-cultured culture exhibits a higher spinosad yield than a culture of *Saccharopolyspora spinosa* D2302 cultured alone under the same conditions.

[0038] The Saccharopolyspora spinosa D2302 strain has the accession number CGMCC No. 26388 from the China General Microbiological Culture Collection Center.

[0039] The accession number of the polymyxa (Paenibacillus polymyxa) D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0040] In the fourth, fifth, and seventh aspects mentioned above, the solvent of the fermentation medium used in the co-culture is water, and the solutes and their concentrations are: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L; pH 7.2.

[0041] In the fourth, fifth and seventh aspects above, the conditions for co-culturing are: rotation speed 220 rpm, temperature 28°C, and humidity 60%.

[0042] In the fourth, fifth and seventh aspects mentioned above, the co-culture time is 4 days.

[0043] In the methods described in the fourth, fifth, and seventh aspects above, before co-culturing the *Saccharopolyspora spinosa* D2302 and the *Paenibacillus polymyxa* D2303, the following steps may be included: The two bacteria (which can be obtained from slant agar—ISP2 medium) are sequentially activated in primary seed medium and secondary seed medium to obtain activated strains of the two bacteria. Then, the activated *Saccharopolyspora spinosa* D2302 is first inoculated into the fermentation medium for separate culture, and then the activated *Paenibacillus polymyxa* D230 is inoculated for co-culture.

[0044] Furthermore, the solvent for both the primary and secondary seed culture media is water, and the solutes and concentrations are as follows: glucose 10.0 g / L, soybean peptone 30.0 g / L, yeast extract 3.0 g / L, MgSO4 2.0 g / L; pH 7.2.

[0045] Furthermore, the conditions for the individual culture are: rotation speed 220 rpm, temperature 28°C, humidity 60%; the individual culture time is 2 days.

[0046] Eighthly, the present invention claims protection for any of the following strains or agents containing said strains:

[0047] (B1) Saccharopolyspora spinosa D2302, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No.26388;

[0048] (B2) Paenibacillus polymyxa D2303, whose accession number at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

[0049] Ninthly, the present invention claims protection for any of the following applications:

[0050] (C1) The use of Saccharopolyspora spinosa D2302 or an agent containing Saccharopolyspora spinosa D2302 in the production of spinosad as described in the eighth aspect above.

[0051] (C2) The use of Saccharopolyspora spinosa D2302 or an agent containing Saccharopolyspora spinosa D2302 as described in the eighth aspect above in the preparation of products for producing spinosad.

[0052] (C3) The use of Paenibacillus polymyxa D2303 or an agent containing Paenibacillus polymyxa D2303 as described in the eighth aspect above in the production of indoleacetic acid;

[0053] (C4) The use of Paenibacillus polymyxa D2303 or an agent containing Paenibacillus polymyxa D2303 as described in the eighth aspect above in the preparation of products for producing indoleacetic acid.

[0054] (C5) The application of Saccharopolyspora spinosa D2302 or an inoculum containing Saccharopolyspora spinosa D2302 as described in the eighth aspect above in improving plant resistance to diseases and pests.

[0055] (C6) The use of Saccharopolyspora spinosa D2302 or an inoculum containing Saccharopolyspora spinosa D2302 as described in the eighth aspect above in the preparation of products for improving plant resistance to pests and diseases.

[0056] (C7) The application of Paenibacillus polymyxa D2303 or an inoculum containing Paenibacillus polymyxa D2303 as described in the eighth aspect above in promoting plant growth;

[0057] (C8) The use of Paenibacillus polymyxa D2303 or an agent containing Paenibacillus polymyxa D2303 as described in the eighth aspect above in the preparation of products for promoting plant growth.

[0058] In the above aspects, the spinosad is spinosad A and / or spinosad D.

[0059] Experiments have shown that co-culturing *Saccharopolyspora spinosa* D2302 and *Paenibacillus polymyxa* D2303 significantly increases the former's production of spinosad without affecting the latter's production of indoleacetic acid. This invention is of great significance for improving crop resistance to diseases and pests, and promoting crop growth and development.

[0060] Preservation Instructions

[0061] Classification and nomenclature: Saccharopolyspora spinosa;

[0062] Biomaterial of ginseng: D2302;

[0063] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0064] The abbreviation for the depository institution is CGMCC.

[0065] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0066] Deposit date: January 6, 2023;

[0067] Registration number at the Preservation Center: CGMCC No. 26388.

[0068] Classification and nomenclature: Paenibacillus polymyxa;

[0069] Biological material of ginseng: D2303;

[0070] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0071] The abbreviation for the depository institution is CGMCC.

[0072] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0073] Deposit date: January 6, 2023;

[0074] Registered with the China National Collection Center (CGMCC) No. 26387. Attached Figure Description

[0075] Figure 1 Phylogenetic trees were constructed based on the 16S rRNA gene sequences of strain D2302 and related strains.

[0076] Figure 2 A phylogenetic tree was constructed based on the 16S rRNA gene sequences of strain D2303 and related strains.

[0077] Figure 3 The standard curve for IAA and the activity test graph of IAA production by strain D2303 are shown.

[0078] Figure 4 This paper presents the HPLC method for the determination of spinosad. A is the HPLC chromatogram of spinosad standard; B is the HPLC chromatogram of spinosad in the fermentation broth of strain D302; C is the HPLC chromatogram of spinosad in the co-culture fermentation broth of strains D302 and D2303. Detailed Implementation

[0079] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0081] Example 1: Isolation, screening, and identification of strains D2302 and D2303

[0082] I. Isolation of strains D2302 and D2303

[0083] The culture medium used for bacterial isolation was commercial medium R2A: yeast extract 0.5 g / L; peptone 0.5 g / L; casein hydrolysate 0.5 g / L; glucose 0.5 g / L; soluble starch 0.5 g / L; dipotassium hydrogen phosphate 0.3 g / L; anhydrous magnesium sulfate 0.024 g / L; sodium pyruvate 0.3 g / L; agar powder 20 g / L; pH 7.2.

[0084] Soil samples used for the isolation of strain D2302 were collected from the Taklamakan Desert in Xinjiang, while soil samples used for the isolation of strain D2303 were collected from the roots of Paris polyphylla plants in Dali Bai Autonomous Prefecture, Yunnan Province.

[0085] Microbial strain isolation procedure: Fresh soil samples were air-dried at room temperature for 2 weeks, then heat-dried at 120℃ for 15 min. 2g of the heat-dried soil was added to 18mL of sterile physiological saline and placed in a shaker at 28℃ for 40 min at 200 rpm to ensure complete suspension of soil particles. The mixture was then serially diluted to prepare 10... -4 Soil suspension with dilution.

[0086] Strain isolation and purification: Spread 0.2 mL of isolation medium onto an agar plate and incubate upside down at 28°C for 4 weeks. After 4 weeks, select different single colonies based on colony characteristics (shape, color, size, surface gloss, etc.) and transfer them to R2A agar plates for purification using the streak method. The obtained pure strains were preserved in liquid nitrogen and then frozen at -80°C using 20% ​​(v / v) glycerol as a cryoprotectant.

[0087] In the experiment, strains D2302 and D2303 were isolated and purified.

[0088] II. Screening and Identification of Strains D2302

[0089] 1. Morphological observation and physiological and biochemical characteristics detection of strain D2302

[0090] Strain D2302 was cultured at 28℃ on modified ISP2 solid medium (formulation: 10 g / L malt extract, 4 g / L yeast extract, 4 g / L glucose, 2 g / L calcium carbonate, 15 g / L agar; pH 7) for 14 days, and colony morphology and mycelial growth characteristics were observed on days 2, 4, 7, 10 and 14. The growth temperature was measured in the range of 4, 10, 15, 20, 25, 28, 30, 37, 40, 42, and 45 °C; the growth salt concentration (NaCl) was measured in 12 concentration gradients (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 15) between 0-10% and 15% (0-10 g / 100 mL and 15 g / 100 mL); and the growth pH was measured in 7 gradients (4, 5, 6, 7, 8, 9, and 10) between 4 and 10 (Xu P, Li WJ, Tang SK, Zhang YQ, Chen GZ, et al. Naxibacter alkalitoleransgen.nov., sp.nov., a novel member of the family Oxalobacteraceae isolated from China. Int J Syst Evol Microbiol 2005; 55:1149-1153). Physiological and biochemical functions of the strains were assessed using the API 50CH and API ZYM assay kits manufactured by bioMérieux, France, and the GEN III assay system manufactured by Biolog, USA. Other physiological characteristics of the strains, including Gram staining properties, motility, oxygen requirement, catalase activity, starch hydrolysis, gelatin liquefaction, indole production, H2S production, and cellulose hydrolysis activity, were primarily determined according to the *Handbook of Actinomycetes Systematics* (Xu L H. *Actinomycete systematics: principles, methods and practices* [M]. Beijing: Science Press, 2007, 93-108.).

[0091] Identification results showed that strain D2302 is a Gram-positive aerobic bacterium. On ISP2 medium, strain D2302 exhibited abundant substrate and aerial hyphae. The substrate hyphae were yellowish-brown to brown, with many fragments forming irregular rod-like shapes. The aerial hyphae were milky white to light brown, differentiating into loose, spiral chains of short spores. The spores had a spiny surface and were non-motile, producing a light yellow soluble pigment. The tolerance ranges for temperature, NaCl, and pH of strain D2302 were 20-40℃, 0-3% NaCl, and pH 6-8.0, respectively, with the optimal growth conditions being 28℃, 0% NaCl, and pH 7.0. The enzymes produced by oxidase, catalase, trypsin, esterase (C4), lipolipase (C8), lipolipase (C14), cystine aromatic aminoaminase, leucine aromatic aminoaminase, valine aromatic aminoaminase, β-galactosidase, α-glucosidase, β-glucosidase, alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase were positive; starch hydrolysis was positive; gelatin liquefaction, indole production, cellulose hydrolysis, and H2S production were negative. L-arabinose, D-galactose, melibiose, sucrose, and D-xylose can be used as the sole carbon and energy sources.

[0092] 2. Detection of cytochemical characteristics of strain D2302

[0093] The cytochemical components of strain D2302, including fatty acids, quinone types, polar lipid components, and cell wall amino acids, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC). (Sasser M. Identification of bacteria by gas ghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE:MIDI inc; 1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M et al. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984; 2:233–241; Lechevalier, MP & Lechevalier, HA (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, pp. 227–291. SIM Special Publication no. 6. Edited by A. Dietz & D.W. Thayer. Fairfax, VA: Society) for Industrial Microbiology.).

[0094] The results showed that the dominant fatty acid in strain D2302 cells was iso-C. 15:0 (19.0%), antesio-C 17:0 (15.3%), iso-C 16:0 (14.9%), iso-C 17:0 (13.0%), antesio-C 15:0 (6.3%), as well as small amounts of branched fatty acids and some trace amounts of saturated fatty acids; the main respiratory quinone in the respiratory chain is MK-9(H4), and small amounts of MK-9(H6) were also detected. Phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI) and phosphatidylcholine (PC) are the main polar lipid components, and the characteristic amino acid of the cell wall is meso-DAP.

[0095] 3. Determination of the phylogenetic position of strain D2302

[0096] Genomic DNA was extracted from strain D2302 and sequenced. The 16S rRNA gene sequence (SEQ ID No. 1) was then compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that strain D2302 of this invention is most closely related to species of the genus *Saccharopolysporum*. The 16S rRNA gene sequence of strain D2302 of this invention showed a 99.72% similarity to that of *Saccharopolyspora spinosa* NRRL 18395T in the database, significantly higher than the 98.65% threshold for distinguishing prokaryotic species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequences similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351.). This result preliminarily suggests that strain D2302 of this invention belongs to the *Saccharopolyspora* genus. A phylogenetic tree was constructed by retrieving the 16S rRNA gene sequences of model bacteria with high similarity to strain D2302 within the *Saccharopolyspora* genus and other related strains. Figure 1 The results show that strain D2302 falls within the genus *Saccharopolysporum*, and that strain D2302 and *Saccharopolysporum spinigerum* NRRL 18395T are on the same subclade, with an evolutionary distance of almost zero. This result further confirms that strain D2302 of this invention belongs to the *Saccharopolysporum spinigerum* species. Genomic sequence calculations show that the G+C content of strain D2302 is 75.9%.

[0097] Based on the culture characteristics, physiological and biochemical properties, cytochemical classification data, 16S rRNA gene sequence information, and phylogenetic analysis of strain D2302 of this invention, we confirm that strain D2302 of this invention is a Saccharopolyspora spinosa strain, labeled as Saccharopolyspora spinosa D2302.

[0098] Saccharopolyspora spinosa D2302 was deposited at the China General Microbiological Culture Collection Center on January 6, 2023, with accession number CGMCC No. 26388.

[0099] 4. Activity detection of spinosad produced by strain D2302

[0100] Fermentation of strain D2302: Well-grown cells from the ISP2 slant were transferred to 20 mL of primary seed culture medium and placed in a 250 mL Erlenmeyer flask. The flask was then incubated on a shaker at a constant temperature for 4 days. The incubation conditions were: shaker speed 220 rpm, temperature 28℃, humidity 60%. After 4 days, 2 mL of the primary seed culture was transferred to secondary seed culture medium. 20 mL of the secondary seed culture medium was then placed in a 250 mL Erlenmeyer flask and incubated on a shaker at a constant temperature for 4 days. The incubation conditions were: shaker speed 220 rpm, temperature 28℃, humidity 60%, incubation time 4 days.

[0101] Four days later, 10 mL of the secondary seed culture solution was transferred into the fermentation medium. 90 mL of the fermentation medium was placed in a 500 mL Erlenmeyer flask and incubated on a constant temperature shaker for 6 days. The incubation conditions were: shaker speed 220 rpm, temperature 28℃, and humidity 60%.

[0102] The primary and secondary seed culture media have the same formula, as follows: glucose 10.0 g / L, soybean peptone 30.0 g / L, yeast extract 3.0 g / L, MgSO4 2.0 g / L; pH 7.2.

[0103] Fermentation medium: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L; pH 7.2.

[0104] Determination of spinosad production activity of strain D2302: The fermentation broth and cells of strain D2302 were soaked in methanol at twice the volume (about 200 mL) for 2 h. After soaking in methanol, the mixture was centrifuged at 5000 rpm for 15 min. The supernatant was collected and the ability of strain D2302 to produce spinosad was determined by high performance liquid chromatography (HPLC) (Agilent 1200) (Zhang Yuan, Jin Zhihua, Lin Jianping, et al. Determination of spinosad by high performance liquid chromatography [J]. Pesticides, 2003, 42(10): 2.). The detection conditions were as follows: column C18 (15 cm × 3.2 mm); detection wavelength 250 nm; mobile phase methanol:hexane:water = 42.5:42.5:15 (volume ratio); flow rate 1 mL / min. The yield of spinosad produced by strain D2302 was determined by using pure spinosad (including spinosad A and spinosad D) as a control.

[0105] Based on the retention times of compounds in the HPLC chromatogram of strain D2302 fermentation broth and comparison with standards of spinosad A and spinosad D, it was determined that strain D2302 fermentation broth contains both spinosad A and spinosad D. Using peak area integration from the HPLC chromatogram, the spinosad yield produced by strain D2302 in this experiment was calculated to be 1.2 g / L according to the following formula.

[0106]

[0107] In the formula: A1 is the average peak area of ​​spinosad in the standard solution.

[0108] A2 represents the average peak area of ​​spinosad in the sample solution.

[0109] P represents the content of spinosad in the standard sample (mg / L).

[0110] III. Screening and Identification of Strains D2303

[0111] 1. Observation of cell morphology and detection of physiological and biochemical characteristics of strain D2303

[0112] After culturing strain D2303 at 30℃ on tryptone soybean agar medium (Solepro) for 48 h, cell morphology was observed using a transmission electron microscope (JEDL, JEM-1400, Japan). The growth temperature range for strain D2303 was 4, 10, 28, 30, 32, 37, 42, and 45℃; the growth salt concentration (NaCl) range was 0, 1, 3, 5, 7, and 10% (g / 100mL); and the growth pH range was eight gradients (4, 5, 6, 7, 8, 9, 10, and 11) between pH 4 and 11. The physiological and biochemical characteristics of the strain were determined using API 50CH, API ZYM, and BiOLOG GEN III carbon source assay kits. Other physiological characteristics of the strains, including Gram staining properties, oxygen requirements, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity, and cellulose hydrolysis activity, were mainly determined with reference to Bergey's Manual of Bacterial Identification (8th edition) and the Manual of Actinomycete Systematics (Xu LH (2007). Actinomycete systematics: principles, methods and practices. Beijing: Science Press, 93-108.).

[0113] Identification results showed that strain D2303 is a Gram-positive, aerobic, rod-shaped cell with peritrichous flagella, motile, with a cell size of (0.6-1.1) μm × (1.9-8.1) μm, and elliptical, centrally located spores. Strain D2303, when cultured at 30℃ on tryptone-soybean agar medium for 48 h, forms white to milky-white colonies with a slippery surface. The pH tolerance range of strain D2303 is 6.0-9.0, with an optimal growth pH of 7.0. It can tolerate 5% NaCl, and the optimal growth temperature is 28-32℃. Oxidase activity is mostly positive (occasionally negative), and catalase reaction is positive. Starch hydrolysis, gelatin hydrolysis, and urea hydrolysis tests are negative, while cellulose hydrolysis is positive, indicating nitrate reducing ability. It can assimilate various monosaccharides and oligosaccharides as an energy source.

[0114] 2. Detection of cytochemical characteristics of strain D2303

[0115] The cytochemical components of strain D2303, including fatty acids, quinone types, and polar lipid components, were detected by gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin-layer chromatography (TLC). (Sasser M. Identification of bacteria by gas ghromatography of cellular fatty acids, MIDI Technical Note 101. Newark, DE:MIDIinc; 1990. Minnikin DE, O'Donnell AG, Goodfellow M, Alderson G, Athalye M et al. An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Methods 1984; 2:233–241; Lechevalier, MP & Lechevalier, HA (1980). The chemotaxonomy of actinomycetes. In Actinomycete Taxonomy, pp. 227–291. SIM Special Publication no. 6. Edited by A. Dietz & D. W. Thayer. Fairfax, VA: Society for...) Industrial Microbiology.

[0116] The results showed that the main fatty acid of strain D2303 was antesio-C. 15:0 (41.3%), C 16:0 (17.1%), iso-C 16:0 (12.0%), antesio-C 17:0 (8.3%), iso-C 17:0 (7.7%) and other trace amounts of saturated fatty acids and branched fatty acids; in the cell membrane of strain D2303D, the respiratory quinone component is methylnaphthoquinone MK-7; the polar lipid components mainly contain diphosphatidylglycerol (DPG), polar lipid components include phosphatidylglycerol (PE), phosphatidylglycerol (PG) and some trace amounts of phospholipids (PLs) of unknown structure, as well as a small amount of amino polar lipids (ALs); the cell wall contains meso-DAP.

[0117] 3. Determination of the phylogenetic position of strain D2303

[0118] Genomic DNA was extracted from strain D2303 and sequenced. The 16S rRNA gene sequence (SEQ ID No. 2) was then compared online in an internationally authoritative bacterial taxonomy database (http: / / www.ezbiocloud.net / ) (Kim OS, Cho YJ, Lee K, et al. 2012, Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol, 62:716-721.). The results showed that strain D2303 of this invention is most closely related to species of the genus Bacillus. The 16S rRNA gene sequence of strain D2303 of this invention showed a 99.31% similarity to *Bacillus polymyxa* ATCC842T in the database, exceeding the 98.65% threshold for distinguishing prokaryotic species (Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014; 64:346–351.). This result preliminarily suggests that strain D2303 of this invention belongs to the *Bacillus polymyxa* species. A phylogenetic tree was constructed by retrieving the 16S rRNA gene sequences of model bacteria with high similarity to strain D2303 within the *Bacillus* genus, as well as other related strains. Figure 2 The results show that strain D2303 clusters within the genus *Bacillus*, and that strain D2303 and *Bacillus polymyxa* ATCC842T are on the same subbranch with an almost zero evolutionary distance. This result further confirms that strain D2303 of this invention belongs to the *Bacillus polymyxa* species. Genomic sequence calculations show that the G+C content of strain D2303 is 46.1%.

[0119] Based on the cellular morphological characteristics, physiological and biochemical properties, cytochemical classification data, 16S rRNA gene sequence information, and phylogenetic analysis of strain D2303 of this invention, we confirm that strain D2303 of this invention is a polymyxa bacillus, labeled as Paenibacillus polymyxa D2303.

[0120] Paenibacillus polymyxa D2303 was deposited at the China General Microbiological Culture Collection Center on January 6, 2023, with accession number CGMCC No. 26387.

[0121] 4. Detection of indoleacetic acid (IAA) production activity of strain D2303

[0122] The ability of strain D2303 to produce indoleacetic acid was determined by colorimetry (Bric et al., 1999). (Bric, JM, Bostock, RM, and Silverstone, SE (1991). Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl. Environ. Microbiol. 57, 535-538. doi:10.1128 / aem.57.2.535-538.1991). Standard curves were constructed using IAA concentrations of 0, 2, 4, 6, 8, 10, and 12 mg / L, and OD was measured. 540 nm absorbance value ( Figure 3 y = 0.0315x + 0.0236, r 2 =0.9901). Cells of the strain grown to the logarithmic growth phase were transferred to tryptone medium containing 3 mmol / L L-tryptophan (1% tryptophan content) and incubated at 30°C for 72 h. The OD of the culture medium was measured. 540 nm The absorbance value was measured. Based on the standard curve, the concentration of indoleacetic acid in the culture medium of strain D2303 was calculated to be 3.41 ± 0.09 mg / L. This confirms that strain D2303 has a strong ability to produce indoleacetic acid. This suggests that strain D2303 has the potential to promote plant growth.

[0123] Example 2: Co-culture of strains D2302 and D2303

[0124] Slant culture medium: ISP2 medium, with the following specific formula: 10 g / L malt extract, 4 g / L yeast extract, 4 g / L glucose, 2 g / L calcium carbonate, 15 g / L agar; pH 7.2.

[0125] The primary and secondary seed culture media used the following medium: glucose 10.0 g / L, soybean peptone 30.0 g / L, yeast extract 3.0 g / L, MgSO4 2.0 g / L; pH 7.2.

[0126] Fermentation medium: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L; pH 7.2.

[0127] Co-culture procedure of Polysporum sarcodactylum D2302 and Bacillus polymyxa D2303:

[0128] 1) Select cells that have grown well on ISP2 slant culture and transfer them to primary seed culture medium. 20 mL of primary seed culture medium is placed in a 250 mL Erlenmeyer flask and incubated on a constant-temperature shaker for 4 days. The incubation conditions are: shaker speed 220 rpm, temperature 28℃, humidity 60%. After 4 days, 2 mL of the primary seed culture is transferred to secondary seed culture medium. 20 mL of secondary seed culture medium is placed in a 250 mL Erlenmeyer flask and incubated on a constant-temperature shaker for 4 days. The incubation conditions are: shaker speed 220 rpm, temperature 28℃, humidity 60%, incubation time 4 days.

[0129] 2) Transfer 10 mL of the secondary seed culture of *Polysporium argentea* D2302 to the fermentation medium. Pour 80 mL of the fermentation medium into a 500 mL Erlenmeyer flask and incubate on a shaker for 2 days. The incubation conditions are: shaker speed 220 rpm, temperature 28℃, humidity 60%.

[0130] 3) Take 10 mL of the secondary seed culture of *Bacillus polymyxa* D2303 and transfer it to the above fermentation culture (culture broth of *Bacillus spp.* D2302 fermented for 2 days), and incubate on a constant temperature shaker for 4 days. The culture conditions are: shaker speed 220 rpm, temperature 28℃, humidity 60%.

[0131] 4) Detection of spinosad in the co-culture product of *Polysporium spp.* D2302 and *Bacillus polymyxa* D2303: High-performance liquid chromatography (HPLC) was used for detection, with spinosad standard as a control. Calculations based on the formula below showed that the spinosad yield in the co-culture fermentation of *Polysporium spp.* D2302 and *Bacillus polymyxa* D2303 was 2.41 g / L. Compared with the fermentation of *Polysporium spp.* D2302 alone (see step 2.4 in Example 1), the spinosad yield under the co-culture condition of *Polysporium spp.* D2302 and *Bacillus polymyxa* D2303 increased by 1.85 times. See details... Figure 4 .

[0132]

[0133] In the formula: A1 is the average peak area of ​​spinosad in the standard solution.

[0134] A2 represents the average peak area of ​​spinosad in the sample solution.

[0135] P represents the content of spinosad in the standard sample (mg / L).

[0136] 5) Detection of IAA in the co-culture products of *Polysporium spp.* D2302 and *Bacillus polymyxa* D2303: The IAA content in the co-culture fermentation broth was determined by colorimetry. D2302 and D2303 cells grown to the logarithmic growth phase were transferred to tryptone medium containing 3 mmol / L L-tryptophan (1% tryptophan content) and incubated at 30°C for 72 h. The OD of the culture medium was measured. 540nm The absorbance value. According to the standard curve (y = 0.0315x + 0.0236, r 2 =0.9901), the calculated concentration of indoleacetic acid (IAA) in the culture medium co-cultured with strains D2302 and 2303 was 3.291 ± 0.12 mg / L (which is basically consistent with the concentration of IAA in the culture medium of strain D2303 alone obtained in step 3.4 of Example 1). This suggests that the ability of strain D2303 to produce IAA is basically unaffected by strain D2302.

[0137] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Complex bacteria, composed of Polysporum spinosum (… Saccharopolyspora spinosa D2302 and Bacillus polymyxa ( Paenibacillus polymyxa Composed of D2303; said Microbispora sp. (Microbispora sp. Saccharopolyspora spinosa ) D2302 has a preservation number of CGMCC No. 26388 at the General Microbiological Center of China Microorganism Strain Preservation Management Committee; The Paenibacillus polymyxa (P. polymyxa) Paenibacillus polymyxa ) D2303 has a preservation number of CGMCC No. 26387 at the China General Microbiological Culture Collection Center.

2. A complete product, comprising the compound bacteria and fermentation culture medium as described in claim 1; The solvent of the fermentation medium is water, and the solutes and their concentrations are as follows: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, and FeSO4·7H2O 0.05 g / L.

3. The application of the compound bacteria of claim 1 or the complete product of claim 2 in any of the following: (A1) Production of spinosad; (A2) Prepare products for the production of spinosad; (A3) Simultaneously producing spinosad and indoleacetic acid; (A4) Prepare a product for the simultaneous production of spinosad and indoleacetic acid; (A5) Improve plant resistance to diseases and pests; (A6) Prepare products to improve plant resistance to diseases and pests; (A7) Improve plant resistance to diseases and pests while promoting plant growth; (A8) Prepare products that can improve plant resistance to pests and diseases while promoting plant growth.

4. A method for producing spinosad, comprising the following steps: mixing Polysporum spinosae (… Saccharopolyspora spinosa D2302 and Bacillus polymyxa ( Paenibacillus polymyxa D2303 was co-cultured to obtain spinosad from the culture; The polysporum spiculatum ( Saccharopolyspora spinosa D2302 has the accession number CGMCC No. 26388 at the China General Microbiological Culture Collection Center. The polymyxin Bacillus ( Paenibacillus polymyxa The accession number of D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

5. A method for simultaneously producing spinosad and indoleacetic acid, comprising the following steps: reacting Polysporum spinosae (… Saccharopolyspora spinosa D2302 and Bacillus polymyxa ( Paenibacillus polymyxa Co-culturing with D2303 yielded both spinosad and indoleacetic acid from the culture. The polysporum spiculatum ( Saccharopolyspora spinosa D2302 has the accession number CGMCC No. 26388 at the China General Microbiological Culture Collection Center. The polymyxin Bacillus ( Paenibacillus polymyxa The accession number of D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

6. Polymyxin Bacillus ( Paenibacillus polymyxa D2303 improves the efficacy of *Polysporium spp.* (…). Saccharopolyspora spinosa Application of D2302 in the production of spinosad; The polysporum spiculatum ( Saccharopolyspora spinosa D2302 has the accession number CGMCC No. 26388 at the China General Microbiological Culture Collection Center. The polymyxin Bacillus ( Paenibacillus polymyxa The accession number of D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

7. A method to enhance the growth of Polysporus spp. (Sacchariformis) Saccharopolyspora spinosa The method for producing spinosad by D2302 includes the following steps: ... (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) Saccharopolyspora spinosa D2302 and Bacillus polymyxa ( Paenibacillus polymyxa Co-culture with D2303; The polysporum spiculatum ( Saccharopolyspora spinosa D2302 has the accession number CGMCC No. 26388 at the China General Microbiological Culture Collection Center. The polymyxin Bacillus ( Paenibacillus polymyxa The accession number of D2303 at the China General Microbiological Culture Collection Center is CGMCC No. 26387.

8. The method according to claim 4, 5, or 7, characterized in that: The solvent for the fermentation medium used in the co-culture was water, and the solutes and their concentrations were as follows: glucose 50 g / L, plant protein hydrolysate 30 g / L, cottonseed meal 10 g / L, soybean oil 10 g / L, calcium carbonate 0.5 g / L, K2HPO4·3H2O 0.2 g / L, and FeSO4·7H2O 0.05 g / L.

9. The method according to claim 4, 5, or 7, characterized in that: The conditions for the co-culture were: rotation speed 220 rpm, temperature 28°C, and humidity 60%.

10. The method according to claim 4, 5, or 7, characterized in that: The co-culture period was 4 days.

11. The method according to claim 4, 5, or 7, characterized in that: Before co-culturing, the following steps are also included: activating the *Saccharomyces cerevisiae* (Saccharomyces cerevisiae) separately. Saccharopolyspora spinosa D2302 and the aforementioned Bacillus polymyxa ( Paenibacillus polymyxa D2303, then first the activated *Polysporium spp.* ( Saccharopolyspora spinosa D2302 was inoculated into the fermentation medium and cultured separately, and then inoculated with the activated Bacillus polymyxa (B2302). Paenibacillus polymyxa D230 was used for the co-culture.

12. The method according to claim 11, characterized in that: The conditions for the individual culture were: rotation speed 220 rpm, temperature 28°C, and humidity 60%.

13. The method according to claim 11, characterized in that: The individual culture period is 2 days.

Images