CN116376748A - Co-culture microbial inoculum of saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303 as well as preparation method and application thereof - Google Patents
Co-culture microbial inoculum of saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303 as well as preparation method and application thereof Download PDFInfo
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- CN116376748A CN116376748A CN202310070389.6A CN202310070389A CN116376748A CN 116376748 A CN116376748 A CN 116376748A CN 202310070389 A CN202310070389 A CN 202310070389A CN 116376748 A CN116376748 A CN 116376748A
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- paenibacillus polymyxa
- cgmcc
- saccharopolyspora spinosa
- spinosyn
- spinosad
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- C12N1/20—Bacteria; Culture media therefor
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303 co-culture microbial inoculum, a preparation method and application thereof. The invention provides a compound bacterium consisting of saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 (CGMCC No. 26388) and paenibacillus polymyxa (Paenibacillus polymyxa) D2303 (CGMCC No. 26387). Co-culturing the saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and the paenibacillus polymyxa (Paenibacillus polymyxa) D2303 can remarkably improve the yield of spinosad and does not influence the yield of indoleacetic acid. The invention has important significance for improving stress resistance of crops such as disease and insect resistance and the like and promoting the growth and development of crops.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a co-culture microbial inoculum of saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303, and a preparation method and application thereof.
Background
Saccharopolyspora spinosa (Saccharopolyspora spinosa) is a widely studied actinomycete species within the genus Saccharopolyspora. The spinosad was initially isolated from a waste brewery by a chemist (Mertz FP, yao RC. Saccharomyces spinosa sp. Nov. Isolated from soil collected in a sugar mill rum still. Int J System bacteriol.1990;40 (1): 34-39.Doi: 10.1099/00207713-40-1-34.) and later studied to find a broad distribution of spinosad in various types of soil environments. Part of the spinosyn strains are fermented under aerobic conditions to produce spinosyns. Spinosad belongs to a class of macrolide biopesticides (Baker D R, fenies J G, steffens J J, et al Synthesis and Chemistry of Agrochemicals III [ M ]. Washington D C: american Chemical Society,1992.214-225;Kirst H,Michel K H,Martin J W,et al.A83543A-D, unique fermentation-derived tetracyclic acrolides [ J ]. Tetrahedron Lett,1991,32 (37): 4839-4842;Boeck LD,Chio H,Eaton T E,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; the spinosad is mainly used as a high-efficiency biological pesticide, is mainly used for preventing and controlling agriculture and forestry insect pests, grain storage insect pests, sanitary insect pests, livestock parasitic insect pests, and is mainly used for crops such as cotton, fruits and vegetables, tea, tobacco, chinese herbal medicines, grains and the like (Salgado V L, sheers J J, watson G B, 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), has the advantages of high specificity, high activity, environmental safety and the like, is widely used in a plurality of countries, has the advantages of broad insecticidal spectrum, high biological activity, low toxicity, low residue, extremely high safety limits on mammals, fishes, birds and most beneficial insects, and the like, obtains a general green chemical challenge prize in the United states, and uses the spinosad as a grain storage protective agent (Thompson GD, dutton R, spark TC. Spinosad-a case study an example from a natural products discovery programme [ J ]. Pest Manag Sci 2000,56 (8): 696-702; wu Xia spinosad—an example of the development of new pesticides from natural products [ J ]. World pesticides, 2004,1:24-28; xu Zhigong, jiang Zhisheng. Toxic symptoms and mechanism of action of biopesticide spinosad [ J ]. Pesticide science and management, 2004, 25 (2): 25-28). Today, where synthetic biology is widely used, the actual production of the biopesticide spinosad still depends on the fermentative production of spinosad. Therefore, the wild spinosyn resource with excellent quality is mined, the efficiency of spinosyn production by spinosyns is improved, and the research significance is great.
The secondary metabolite spinosyns produced by fermentation of spinosyns contain a mixture of spinosyns A (Spinosyns A) and spinosyns D (Spinosyns D), which are structurally similar compounds (Gary D, thomas c.return advance in the chemistry of spinosyns [ J ]. Pest Manag Sci,2001,57 (2): 177-185;Thompson G D,Dutton R,Sparks T C.Spinosad-a case student: an example from a natural products discovery programme [ J ]. Pest Manage Sci,2000,56 (8): 696-702). Currently, the wild-type strain NRRL18395 of spinosyns, which was investigated as a model, produced a yield of spinosad of 78.7mg/L, with a component A of about 84% and a component D of about 16%. The shaking flask fermentation yield of the spinosad high-yield strain obtained by mutagenesis and other methods reported in the literature or patent is about 1-2g/L (CN 113444659B, a spinosyn high-yield spinosad).
Paenibacillus polymyxa (Paenibacillus polymyxa) was proposed to establish a bacterial species (Ash C, priest FG, collins MD.molecular identification of rRNA group bacterial (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.) based on the results of molecular classification characterization studies of strain ATCC 842 in 1994, and Paenibacillus polymyxa was identified as a model species of Paenibacillus. Paenibacillus polymyxa is a gram-positive bacterium widely distributed in various natural environments, and is not only commonly found on soil and plant surfaces, but also an important component of plant endophytic bacteria. At present, the main sources of the biocontrol Paenibacillus polymyxa are plant rhizosphere soil (Chen Xueli, hao Zaibin, wang Guanghua, etc.. The isolation and purification of Paenibacillus polymyxa BRF-1 antimicrobial protein [ J ]. Chinese biocontrol, 2007, 23 (2) [ J ]. 156-159; liu Xunli, sun Changpo, makeup, etc.. The isolation and identification of a silkworm pathogen antagonistic bacterium [ J ]. Silkworm science, 2004, 30 (3) [ 273-276 ], zhao Deli, zeng Zilin, li Hui, etc.. The antibacterial active substance of Paenibacillus polymyxa JW-725 and the preliminary study of fermentation conditions thereof [ J ]. Plant protection, 2006, 32 (1) [ 47-50 ], zhangdaojing, chunyan, wei Honggang, etc.. The chemical composition of Paenibacillus polymyxa HY96-2 [ J ]. University of Huadong university: nature science edition, 2008, 34 (1) [ 71-73 ].
Paenibacillus polymyxa is a small group of bacteria that can produce antibiotics of clinical utility. Polymyxin B (PMB) and polymyxin E (colistin) produced by paenibacillus have been used clinically against bacterial infections. Part of the strains within Paenibacillus polymyxa species are also important plant biocontrol bacteria and plant rhizosphere growth promoters (plant growth promoting rhizobacteria, PGPR), and have been widely used in the agricultural field (Dilfuza Egamberdiyva. The effect of plant growth promoting bacteria on growth and nutrient uptake of maize in two different ecological sciences, 2007,36 (2-3): 184-189; chooong-Min Ryu, jinwao Kim, okhee Choi, et al.improved of biological Control capacity of Paenibacillus polymyxa E681 by seed pelleting on se same. Biological Control,2006,39 (3): 282-289; shi Yingwu, lo , li Chun, etc.. The influence of endophytic Paenibacillus polymyxa S-7 on beet photosynthesis and yield and quality [ J ]. Applied ecological sciences, 2009, 20 (3): 597-602; zhang Qiuxia, zhong Zengtao, xu Yangchun, etc.. Bacillus polymyxa fluorescence in situ hybridization detection techniques and their use in organic fertilizer fermentation [ J ]. Applied fertilizer research [ J ]. Is provided herein and fertilizer (1286.: 6). Paenibacillus polymyxa and its metabolites have also found application in industry and waste water 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 fact that Paenibacillus polymyxa has good application prospects in various fields, the U.S. Environmental Protection Agency (EPA) lists Paenibacillus polymyxa as one of commercially applicable microorganisms, and the agricultural department of China also lists Paenibacillus polymyxa as a first-class strain free of safety identification.
Disclosure of Invention
The invention aims to provide a co-culture microbial inoculum of saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303, and a preparation method and application thereof.
In a first aspect, the invention claims a composite bacterium.
The invention discloses a composite bacterium which consists of saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and paenibacillus polymyxa (Paenibacillus polymyxa) D2303.
The preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388.
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
In the composite bacteria, the two bacteria are respectively and independently packaged.
In a second aspect, the invention claims a kit.
The kit of parts claimed in the present invention consists in particular of the complex bacteria described in the first aspect above and the fermentation medium.
The solvent of the fermentation medium is water, and the solute and the concentration are as follows: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O0.05g/L；pH 7.2。
Still further, a seed medium (primary seed medium and/or secondary seed medium as described below) for activating the seed species may be included in the kit.
In a third aspect, the invention claims the use of a complex bacterium as described in the first aspect hereinbefore or a kit as described in the second aspect hereinbefore in any of the following:
(A1) Producing spinosad;
(A2) Preparing a product for producing spinosad;
(A3) Simultaneously producing spinosad and indoleacetic acid;
(A4) Preparing a product for simultaneously producing spinosad and indoleacetic acid;
(A5) The resistance of plants to plant diseases and insect pests is improved;
(A6) Preparing a product for increasing the resistance of a plant to a pest;
(A7) The plant growth is promoted while the plant resistance to plant diseases and insect pests is improved;
(A8) A product for promoting plant growth while increasing plant resistance to pests is prepared.
In a fourth aspect, the invention claims a method of producing spinosad.
The method for producing spinosad claimed in the present invention may comprise the steps of: the spinosyns (Saccharopolyspora spinosa) D2302 and Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 were co-cultured to obtain spinosad from the culture.
The preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388.
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
In a fifth aspect, the invention claims a method for simultaneously producing spinosad and indoleacetic acid.
The method for simultaneously producing spinosad and indoleacetic acid, which is claimed by the invention, can comprise the following steps: the spinosyns (Saccharopolyspora spinosa) D2302 and the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 were co-cultured to obtain spinosad and indoleacetic acid simultaneously from the culture.
The preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388.
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
In a sixth aspect, the invention claims the use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 to increase the yield of spinosyns produced by spinosyns (Saccharopolyspora spinosa) D2302.
The preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388.
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
In a seventh aspect, the invention claims a method of increasing the yield of spinosyns produced by spinosyns (Saccharopolyspora spinosa) D2302.
The method for improving the yield of spinosyn produced by spinosyn (Saccharopolyspora spinosa) D2302, which is claimed by the invention, can comprise the following steps: co-culturing of Saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and Paenibacillus polymyxa (Paenibacillus polymyxa) D2303. The co-cultivated cultures produced higher yields of spinosad than cultures in which the spinosyns (Saccharopolyspora spinosa) D2302 were cultivated alone under the same conditions.
The preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388.
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
In the fourth, fifth and seventh aspects, the fermentation medium used in performing the co-cultivation is water as a solvent, and the solute and concentration are as follows: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O 0.05g/L；pH 7.2。
In the fourth, fifth and seventh aspects above, the co-culturing is performed under the following conditions: the rotation speed is 220rpm, the temperature is 28 ℃, and the humidity is 60%.
In the fourth, fifth and seventh aspects described above, the co-cultivation is performed for a period of 4 days.
In the method according to the fourth, fifth and seventh aspects, the method further comprises the step of, before co-culturing the polysaccharide sulfate (Saccharopolyspora spinosa) D2302 and the paenibacillus polymyxa (Paenibacillus polymyxa) D2303: the two bacteria (which can be taken from a slant culture medium-ISP 2 culture medium) are respectively and sequentially subjected to activation culture in a first seed culture medium and a second seed culture medium to obtain activated strains of the two bacteria. Then, the activated polysaccharide gum spore bacteria (Saccharopolyspora spinosa) D2302 is inoculated into the fermentation medium for separate culture, and then the activated polysaccharide gum spore bacteria (Paenibacillus polymyxa) D230 is inoculated for the co-culture.
Further, the primary seed culture medium and the secondary seed culture medium are both water as solvents, and the solutes and the concentrations are as follows: glucose 10.0g/L, soytone 30.0g/L, yeast extract 3.0g/L, mgSO 4 2.0g/L；pH 7.2。
Further, the conditions under which the individual culture is performed are: the rotation speed is 220rpm, the temperature is 28 ℃, and the humidity is 60%; the incubation alone was performed for a period of 2 days.
In an eighth aspect, the invention claims any of the following strains or inoculants comprising said strains:
(B1) Saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 with a collection number of CGMCC No.26388 in China general microbiological culture Collection center;
(B2) Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 has a collection number of CGMCC No.26387 in China general microbiological culture Collection center.
In a ninth aspect, the invention claims any of the following applications:
(C1) Use of a spinosyn (Saccharopolyspora spinosa) D2302 as described in the eighth aspect hereinbefore or a bacterial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 for the production of spinosad;
(C2) Use of a spinosyn (Saccharopolyspora spinosa) D2302 or a bacterial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 as described in the eighth aspect hereinbefore for the manufacture of a product for the production of spinosad;
(C3) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a bacterial agent comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 as described in the eighth aspect hereinbefore to produce indoleacetic acid;
(C4) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a bacterial agent comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 as described in the eighth aspect hereinbefore in the manufacture of a product for the production of indoleacetic acid;
(C5) Use of a spinosyn (Saccharopolyspora spinosa) D2302 as described in the eighth aspect hereinbefore or a microbial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 to increase plant resistance to insect disease;
(C6) Use of a spinosyn (Saccharopolyspora spinosa) D2302 or a microbial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 as described in the eighth aspect hereinbefore for the manufacture of a product for increasing plant resistance to pests;
(C7) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a microbial inoculant comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 as described in the eighth aspect hereinbefore to promote plant growth;
(C8) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a microbial inoculant comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 as described in the eighth aspect hereinbefore in the manufacture of a product for promoting plant growth.
In the above aspects, the spinosad is spinosad a and/or spinosad D.
Experiments prove that the co-culture of the saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and the paenibacillus polymyxa (Paenibacillus polymyxa) D2303 can remarkably improve the yield of spinosad and does not influence the yield of indoleacetic acid. The invention has important significance for improving stress resistance of crops such as disease and insect resistance and the like and promoting the growth and development of crops.
Preservation description
Classification naming: saccharopolyspora spinosa (Saccharopolyspora spinosa);
biological materials according to: d2302;
preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);
the preservation organization is abbreviated as: CGMCC;
address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;
preservation date: 2023, 1, 6;
accession numbers of the preservation center: CGMCC No.26388.
Classification naming: paenibacillus polymyxa (Paenibacillus polymyxa);
biological materials according to: d2303;
preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection);
the preservation organization is abbreviated as: CGMCC;
address: beijing, chaoyang area, north Chenxi Lu No. 1, 3;
Preservation date: 2023, 1, 6;
accession numbers of the preservation center: CGMCC No.26387.
Drawings
FIG. 1 is a phylogenetic tree constructed based on the 16S rRNA gene sequences of strain D2302 and related strains.
FIG. 2 is a phylogenetic tree constructed based on the 16S rRNA gene sequences of strain D2303 and related strains.
FIG. 3 is a graph of IAA standard curve and activity test for IAA production by strain D2303.
FIG. 4 shows the detection of spinosad by HPLC. A is HPLC of spinosad standard; b is an HPLC (high Performance liquid chromatography) spectrum of spinosad in single-strain fermentation broth of the strain D302; c is the HPLC spectrum of spinosad in the co-culture fermentation broth of the strains D302 and D2303.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 isolation screening and identification of Strain D2302 and Strain D2303
1. Isolation of Strain D2302 and Strain D2303
The strain isolation medium used was commercial medium R2A: 0.5g/L yeast extract powder; peptone 0.5g/L; 0.5g/L casein hydrolysate; glucose 0.5g/L; soluble starch 0.5g/L; dipotassium hydrogen phosphate 0.3g/L; anhydrous magnesium sulfate 0.024g/L; sodium pyruvate 0.3g g/L; 20g/L of agar powder; pH7.2.
The soil sample for the separation of the strain D2302 was collected from the dry desert of Takara-ma in Xinjiang, and the soil sample for the separation of the strain D2303 was collected from the root of Paris polyphylla plant in white autonomous state in Yunnan province.
The strain separation flow comprises the following steps: after the fresh soil sample was air-dried at room temperature for 2 weeks, it was dry-heated at 120℃for 15min. Taking 2g of dry and hot soil, adding into 18mL of sterile physiological saline, placing into a shaking table at 28 ℃ for 40min at a rotating speed of 200r/min to fully suspend soil particles, and performing gradient dilution to prepare 10 -4 Dilutions of soil sample suspension.
Separating and purifying strains: 0.2mL of the culture medium was spread on a plate of the isolation medium, and the culture was inverted at 28℃for 4 weeks. After 4 weeks, different single colonies were picked on R2A medium plates according to colony characteristics (shape, color, size, surface gloss, etc.), and cultures were purified by the four-fold streaking method. The obtained pure strain is preserved in liquid nitrogen and frozen at-80 ℃ by taking 20% (v/v) glycerol as a protective agent.
In the experiment, the strain D2302 and the strain D2303 are obtained through separation and purification.
2. Screening and identification of Strain D2302
1. Morphological observation and physiological and biochemical feature detection of strain D2302
Strain D2302 was cultured on modified ISP2 solid medium (formulation: malt extract 10g/L, yeast extract 4g/L, glucose 4g/L, calcium carbonate 2g/L, agar 15g/L; pH 7) at 28℃for 14 days, and the colony morphology and the growth characteristics of mycelia were observed on days 2, 4, 7, 10, and 14, respectively. The growth temperature detection ranges are 4, 10, 15, 20, 25, 28, 30, 37, 40, 42 and 45 ℃; 12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 15) concentration gradients ranging from 0-10% and 15% (0-10 g/100mL and 15g/100 mL) of growth salt concentration (NaCl); the growth pH detection range was 7 (4, 5, 6, 7, 8, 9, 10) gradients (Xu P, li WJ, tang SK, zhang YQ, chen GZ, et al Naxibacter akalitolians gen.nov., sp.nov., a novel member of the family Oxalobacteraceae isolated from) between 4 and 10China, int J Syst Evol Microbiol 2005; 55:1149-1153). The physiological and biochemical functions of the strain were performed using the detection kit API 50CH, API ZYM, manufactured by Meriera, france, and the detection system GEN III, manufactured by BiOLOG, USA. Other strain physiological characteristics including gram stain properties, motility, oxygen demand, thixotropic activity, starch hydrolysis, gelatin liquefaction, indole production, H 2 S production and cellulolytic Activity are mainly carried out by reference to the "actinomycetes System identification Manual" (Xu L H. Actinomyces systems: principles, methods and practices [ M)].Beijing:Science Press,2007,93-108.)。
The identification result shows that: strain D2302 is a gram-positive aerobic bacterium. On ISP2 culture medium, the mycelium in the basal hypha and aerial hypha of the strain D2302 are rich, the mycelium in the basal hypha is yellow to brown, the cephalosporin in the basal hypha is broken into irregular rods, the aerial hypha is milky white to light brown, the aerial hypha is differentiated to form loose spiral short spore chains, the surface of the spores is thorny, and the spores do not move. A pale yellow soluble pigment is produced. Strain D2302 has tolerance ranges of 20-40deg.C, 0-3% NaCl, and pH 6-8.0 for temperature, naCl and pH, and the optimal growth conditions are 28deg.C, 0% NaCl, and pH 7.0. Positive production of oxidase, thixose, trypsin, esterase (C4), lipoid esterase (C8), lipoid enzyme (C14), cystine arylamidase, leucine arylaminase, valine arylaminase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, alkaline phosphatase, acid phosphatase, and naphthol-AS-BI-phosphate hydrolase; starch hydrolysis is positive; gelatin liquefaction, indole production, cellulose hydrolysis and H 2 The S-production test was negative. L-arabinose, D-galactose, melibiose, sucrose, D-xylose can be utilized as the sole carbon source and energy source.
2. Cytochemical characterization of strain D2302
The fatty acid, quinone type, polar lipid component, cell wall amino acid and other cytochemical components of the strain D2302 were detected by GC gas chromatography, HPLC liquid chromatography and TLC thin layer chromatography (Sasser M.identification of bacteria by gas ghromatography of cellular fatty acids, MIDI Technical Note101.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, M.P. & Lechevalier, H.A. (1980). The chemotaxonomy of actinomies.In Actinomycete Taxonomy, pp.227-291.SIM Special Publication no.6.Edited by A.Dietz&D.W.Thayer.Fairfax,VA:Society for Industrial Microbiology.).
The results show that: in the strain D2302 cells, the dominant fatty acid is 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 fatty acids with branched structures and some traces of saturated fatty acids; the major respiratory quinone in the respiratory chain is MK-9 (H 4 ) A small amount of MK-9 (H) 6 ) Phosphatidylglycerol (PG), biphospholipid glycerol (DPG), phosphatidylinositol (PI) and Phosphatidylcholine (PC) are the major polar lipid components, and the cell wall characteristic amino acid is meso-DAP.
3. Determination of the phylogenetic status of Strain D2302
Genomic DNA of strain D2302 was extracted and sequenced, and the 16S rRNA gene sequence (SEQ ID No. 1) was aligned on-line in the International authoritative bacterial taxonomic analysis database (http:// www.ezbiocloud.net /) (KimOS, cho YJ, lee K, et al 2012, introducing EzTaxon-e: a prokaryotic16S rRNA gene sequence database with phylotypes that represent uncultured patterns.int J Syst Evol Microbiol, 62:716-721.). The results showed that the near-edge relationship between the strain D2302 of the present invention and the species of Saccharopolyspora was recent. The similarity between the 16S rRNA gene sequence of the strain D2302 and the Saccharopolyspora spinosa (Saccharopolyspora spinosa) NRRL 18395T in the database is up to 99.72%, which is obviously higher than the limit of 98.65% for distinguishing the species of the prokaryotic microorganism (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 primarily suggests that the strain D2302 of the present invention is a member of the Saccharopolyspora species. A phylogenetic tree was constructed by taking the 16S rRNA gene sequences of model bacteria having high similarity to the 16S rRNA gene sequences of strain D2302 and other related strains within the genus Saccharopolyspora (FIG. 1). Strain D2302 was shown to fall within the genus saccharicterium, with strain D2302 and saccharicterium NRRL 18395T on the same sub-branch, with an evolutionary distance of almost zero. This result further demonstrates that the strain D2302 of the present invention belongs to a member of the Saccharopolyspora spinosa species. As a result of calculation of the genomic sequence, the G+C content of the strain D2302 was 75.9%.
By combining the culture characteristics, physiological and biochemical characteristics, cytochemical classification data and 16S rRNA gene sequence information and systematic evolution analysis of the strain D2302, we confirm that the strain D2302 is a strain of Saccharopolyspora spinosa, and the strain is marked as Saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302.
Saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 is preserved in China general microbiological culture collection center (CGMCC) No.26388 at 1 and 6 of 2023.
4. Activity detection of spinosyn produced by Strain D2302
Fermentation of strain D2302: selecting good thallus growing on ISP2 inclined plane, transferring to primary seed culture medium 20mL, placing in a 250mL triangular flask, and culturing on a constant temperature shaking table for 4 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity. After 4 days, 2mL was aspirated from the primary seed culture and transferred to secondary seed culture. 20mL of the secondary seed culture medium is placed in a triangular flask with the capacity of 250mL, and the secondary seed culture medium is placed on a constant temperature shaking table for 4 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity for 4 days.
After 4 days, 10mL of the secondary seed culture solution was transferred to the fermentation medium. 90mL of fermentation medium is placed in a 500mL triangular flask, and placed on a constant temperature shaking table for 6 days under the culture conditions that the rotation speed of the shaking table is 220rpm, the temperature is 28 ℃ and the humidity is 60%.
The formula of the primary seed culture medium and the secondary seed culture medium is the same, and the specific steps are as follows: glucose 10.0g/L, soybean eggBai 30.0.0 g/L, yeast extract 3.0g/L, mgSO 4 2.0g/L；pH 7.2。
Fermentation medium: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O 0.05g/L；pH 7.2。
Activity determination of spinosyn produced by Strain D2302: the fermentation broth and the cells of strain D2302 were soaked with 2 volumes (about 200 mL) of methanol, after 2h of methanol soaking, centrifuged at 5000rpm for 15min, the supernatant was taken and the ability of strain D2302 to produce spinosad was measured by High Performance Liquid Chromatography (HPLC) (Agilent 1200) (Zhang Yuan, jin Zhihua, lin Jianping, et al. High performance liquid chromatography of spinosad [ J ]. Pesticide, 2003, 42 (10): 2.). The detection conditions are as follows: column C18 (15 cm. Times.3.2 mm); the detection wavelength is 250nm; the mobile phase adopts methanol, capronitrile and water=42.5:42.5:15 (volume ratio); the flow rate was 1mL/min. The yield of spinosad produced by strain D2302 was determined using spinosad pure products (including spinosad a and spinosad) as controls.
And (3) according to the comparison and judgment of the compound retention time in the HPLC (high performance liquid chromatography) spectrum of the strain D2302 fermentation liquor and the spinosad A and spinosad D standard substances, the strain D2302 fermentation liquor contains spinosad A and spinosad. Using the integration of the peak areas of the HPLC profile, the yield of spinosyn produced by strain D2302 in this experiment was calculated to be 1.2g/L according to the following formula.
Wherein: a is that 1 As peak area average value of spinosad in standard solution
A 2 Mean value of peak area of spinosad in sample solution
P is the content (mg/L) of spinosad in the standard sample
3. Screening and identification of Strain D2303
1. Cell morphology observation and physiological and biochemical characteristic detection of strain D2303
After culturing strain D2303 on tryptone soybean agar medium (Soy Corp.) at 30℃for 48 hours, cell morphology was observed using a transmission electron microscope (Japanese electron JEDL, JEM-1400). The growth temperature detection range of strain D2303 was 4, 10, 28, 30, 32, 37, 42 and 45 ℃; the detection range of the growth salt concentration (NaCl) is 0, 1, 3, 5, 7 and 10 percent (g/100 mL); the growth pH detection range is 8 (4, 5, 6, 7, 8, 9, 10, 11) gradients between pH 4-11. The physiological and biochemical characteristics of the strain are detected by using detection kits such as API 50CH, API ZYM, and BiOLOG GEN III carbon source. Other strain physiological characteristics, including gram staining attributes, oxygen demand, contact enzyme activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, are mainly described in Berger's bacteria identification handbook (8 th edition) and actinomycete systems identification handbook (Xu L H (2007). Actinomycete systematics: principles, methods and acts.Beijing: science Press, 93-108.).
The identification result shows that: strain D2303 is a gram positive bacterium, aerobic, long rod-shaped cell, periphyton, mobile, cell size (0.6-1.1) μm× (1.9-8.1) μm, and in oval spore. Strain D303 was cultured on tryptone soy agar medium at 30℃for 48 hours to form white to milky colonies, which were wet and slippery on the surface. The pH tolerance range of strain D2303 was 6.0-9.0, and the optimum pH for growth was 7.0. Can tolerate 5% NaCl, and the optimum growth temperature is 28-32 ℃. Oxidase is mostly positive (even negative), and catalase reaction is positive. Starch hydrolysis, gelatin hydrolysis and urea hydrolysis experiments are negative, cellulose hydrolysis is positive, and nitrate reduction capability is achieved. Can assimilate a variety of mono-and oligosaccharides as an energy source.
2. Cytochemical characterization of strain D2303
The cell chemical components such as fatty acid, quinone type, polar lipid component, etc. of the strain D2303 were detected by GC gas chromatography, HPLC liquid chromatography and TLC thin layer chromatography (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, M.P. & Lechevalier, H.A. (1980) The chemotaxonomy of actinomycete.In Actinomycete Taxonomy, pp.227-291.SIM Special Publication no.6.Edited by A.Dietz&D.W.Thayer.Fairfax,VA:Society for Industrial Microbiology.).
The results show that: the main fatty acid of strain D2303 is 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 some other trace amounts of saturated fatty acids and fatty acids with branched structures; in the cell membrane of strain D2303D, the respiratory quinone component is menaquinone MK-7; the polar lipid component mainly contains di-phosphatidyl glycerol (DPG), the polar lipid component comprises phosphatidyl glycerol (PE), phosphatidyl Glycerol (PG) and some tiny amount of Phospholipids (PLs) with unknown structures, and a small amount of amino polar lipids (ALs); the cell wall contains meso-DAP.
3. Determination of the phylogenetic status of Strain D2303
Genomic DNA of strain D2303 was extracted and sequenced, and the 16S rRNA gene sequence (SEQ ID No. 2) was aligned on-line in the International authoritative bacterial taxonomic analysis database (http:// www.ezbiocloud.net /) (KimOS, cho YJ, lee K, et al 2012, introducing EzTaxon-e: a prokaryotic16S rRNA gene sequence database with phylotypes that represent uncultured patterns.int J Syst Evol Microbiol, 62:716-721.). The results showed that the near-edge relationship between the strain D2303 of the present invention and the species of Paenibacillus was recent. The similarity between the 16S rRNA gene sequence of the strain D2303 of the invention and Paenibacillus polymyxa ATCC842T in the database is up to 99.31%, which is higher than 98.65% of the limit for distinguishing the species of the prokaryotic microorganism (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 Microbiol2014;64:346-351.). This result primarily suggests that the strain D2303 of the present invention is a member of Paenibacillus polymyxa species. A phylogenetic tree was constructed by calling the 16S rRNA gene sequences of model bacteria having high similarity to the 16S rRNA gene sequences of strain D2303 and other related strains within Paenibacillus genus (FIG. 2). Strain D2303 was shown to be concentrated in one paenibacillus, with strain D2303 and paenibacillus polymyxa ATCC842T on the same subfraction, with an evolutionary distance of almost zero. This result further demonstrates that the strain D2303 of the present invention belongs to a member of the Paenibacillus polymyxa species. As a result of calculation of the genomic sequence, the G+C content of the strain D2303 was 46.1%.
By combining the morphological characteristics, physiological and biochemical characteristics, cytochemical classification data and 16S rRNA gene sequence information of the strain D2303 and systematic evolution analysis, we confirm that the strain D2303 is a Paenibacillus polymyxa and marked as Paenibacillus polymyxa (Paenibacillus polymyxa) D2303.
Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 is preserved in China general microbiological culture collection center (CGMCC) No.26387 at 2023, 1 and 6.
4. Detection of indoleacetic acid (3-Indoleacetic acid, IAA) production activity of Strain D2303
The capacity of strain D2303 to produce indoleacetic acid was determined colorimetrically (Bric et al, 199Bric, J.M., bostock, R.M., and Silverstone, S.E. (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). A standard curve was prepared with IAA-containing concentrations of 0,2,4,6,8,10,12mg/L, respectively, and OD was measured 540 nm Is (fig. 3, y=0.0315x+0.0236, r) 2 = 0.9901). Cells of the strain grown to the logarithmic phase were transferred to a tryptone culture medium containing 3mmol/L of L-tryptophan (tryptone content in the medium: 1%) and incubated at 30℃for 72 hours. Determination of OD of culture solution 540 nm Is used for the light absorption value of (a). According to the standard curve, the concentration of indoleacetic acid in the culture solution of the strain D2303 is calculated to be 3.41+/-0.09 mg/L. The strain D2303 is proved to have stronger capacity of producing indoleacetic acid. Suggesting that strain D2303 has the potential to promote plant growth.
Example 2 Co-cultivation of Strain D2302 and Strain D2303
Slant culture medium: ISP2 medium, the specific formulation is as follows: malt extract 10g/L, yeast extract 4g/L, glucose 4g/L, calcium carbonate 2g/L, and agar 15g/L; pH 7.2.
The primary and secondary seed culture mediums are selected from the following culture mediums: glucose 10.0g/L, soytone 30.0g/L, yeast extract 3.0g/L, mgSO 4 2.0g/L；pH 7.2。
Fermentation medium: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O 0.05g/L；pH 7.2。
Co-cultivation procedure of polysaccharide-bearing fungus D2302 and Paenibacillus polymyxa D2303:
1) Selecting good thalli growing on an ISP2 inclined plane, and transferring the thalli into a first-stage seed culture medium. 20mL of the primary seed culture medium is placed in a triangular flask with the capacity of 250mL and placed on a constant temperature shaking table for 4 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity. After 4 days, 2mL was aspirated from the primary seed culture and transferred to secondary seed culture. 20mL of the secondary seed culture medium is placed in a triangular flask with the capacity of 250mL, and the secondary seed culture medium is placed on a constant temperature shaking table for 4 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity for 4 days.
2) 10mL of the secondary seed culture solution of the spinosyn D2302 was transferred into a fermentation medium. 80mL of fermentation medium is placed in a 500mL triangular flask, and placed on a constant temperature shaking table for 2 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity.
3) 10mL of the second seed culture solution of Paenibacillus polymyxa D2303 was transferred into the fermentation culture solution (the culture solution obtained by fermenting Paenibacillus spinosus D2302 for 2 days), and the culture was carried out on a constant temperature shaking table for 4 days. The culture conditions were 220rpm on a shaker at 28℃and 60% humidity.
4) Detection of spinosad in the co-culture product of spinosad D2302 and Paenibacillus polymyxa D2303: high Performance Liquid Chromatography (HPLC) is used for detection, and spinosad standard is used as a control. According to the calculation of the following formula, the co-culture fermentation of the spinosyns D2302 and the Paenibacillus polymyxa D2303 is obtained, the yield of spinosyns is 2.41g/L, and compared with the single culture fermentation of the spinosyns D2302 (see the step two 4 of the example 1), the yield of spinosyns is improved by 1.85 times under the co-culture condition of the spinosyns D2302 and the Paenibacillus polymyxa D2303. See in particular fig. 4.
Wherein: a is that 1 As peak area average value of spinosad in standard solution
A 2 Mean value of peak area of spinosad in sample solution
P is the content (mg/L) of spinosad in the standard sample
5) Detection of IAA in the Co-culture product of the Saccharopolyspora spinosa D2302 and Paenibacillus polymyxa D2303: and determining the IAA content in the double-bacterium co-culture fermentation broth by adopting a colorimetric method. D2302 and D2303 cells grown to logarithmic phase were transferred to tryptone culture medium containing 3mmol/L of L-tryptophan (tryptone content of 1% in this medium) and incubated at 30℃for 72 hours. Determination of OD of culture solution 540nm Is used for the light absorption value of (a). According to the standard curve (y=0.0315x+0.0236, r 2 = 0.9901), the concentration of indoleacetic acid in the culture broth of the co-culture of the strains D2302 and 2303 was calculated to be 3.291 ±0.12mg/L (substantially identical to the concentration of indoleacetic acid in the individual culture broth of the strain D2303 obtained in step three 4 of example 1, 3.41±0.09 mg/L). The ability of strain D2303 to produce indoleacetic acid was suggested to be substantially unaffected by strain D2302.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. The composite bacteria consist of saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and paenibacillus polymyxa (Paenibacillus polymyxa) D2303;
the preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388;
the collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
2. A kit of parts consisting of the complex bacterium of claim 1 and a fermentation medium;
the solvent of the fermentation medium is water, and the solute and the concentration are as follows: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O0.05g/L。
3. Use of a complex bacterium according to claim 1 or a kit according to claim 2 in any of the following:
(A1) Producing spinosad;
(A2) Preparing a product for producing spinosad;
(A3) Simultaneously producing spinosad and indoleacetic acid;
(A4) Preparing a product for simultaneously producing spinosad and indoleacetic acid;
(A5) The resistance of plants to plant diseases and insect pests is improved;
(A6) Preparing a product for increasing the resistance of a plant to a pest;
(A7) The plant growth is promoted while the plant resistance to plant diseases and insect pests is improved;
(A8) A product for promoting plant growth while increasing plant resistance to pests is prepared.
4. A method of producing spinosad comprising the steps of: co-culturing spinosyn (Saccharopolyspora spinosa) D2302 and Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 to obtain spinosad from the culture;
the preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388;
the collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
5. A method for simultaneously producing spinosad and indoleacetic acid, comprising the steps of: co-culturing spinosyns (Saccharopolyspora spinosa) D2302 and Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 to obtain spinosad and indoleacetic acid simultaneously from the culture;
the preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388;
The collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
6. Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 to increase the yield of spinosyns produced by spinosyn (Saccharopolyspora spinosa) D2302;
the preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388;
the collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
7. A method of increasing the yield of spinosyns produced by spinosyns (Saccharopolyspora spinosa) D2302, comprising the steps of: co-culturing the spinosyns (Saccharopolyspora spinosa) D2302 and Paenibacillus polymyxa (Paenibacillus polymyxa) D2303;
the preservation number of the spinosyn (Saccharopolyspora spinosa) D2302 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26388;
the collection number of the Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 in the China general microbiological culture Collection center (CGMCC) is CGMCC No.26387.
8. The method according to claim 4 or 5 or 7, characterized in that: the solvent of the fermentation medium adopted in the co-cultivation is water, and the solute and the concentration are as follows: glucose 50g/L, vegetable protein hydrolysate 30g/L, cotton seed powder 10g/L, soybean oil 10g/L, calcium carbonate 0.5g/L, K 2 HPO 4 ·3H 2 O 0.2g/L，FeSO 4 ·7H 2 O0.05g/L; and/or
The conditions under which the co-cultivation was performed were: the rotation speed is 220rpm, the temperature is 28 ℃, and the humidity is 60%; and/or
The co-cultivation was performed for 4 days;
and/or
The method further comprises the following steps before the co-culture: activating the saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 and the paenibacillus polymyxa (Paenibacillus polymyxa) D2303 respectively, inoculating the activated saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 into the fermentation medium for independent culture, and inoculating the activated paenibacillus polymyxa (Paenibacillus polymyxa) D230 for co-culture;
further, the conditions under which the individual culture is performed are: the rotation speed is 220rpm, the temperature is 28 ℃, and the humidity is 60%; and/or
Further, the separate culture was performed for 2 days.
9. Any one of the following strains or a microbial agent containing the same:
(B1) Saccharopolyspora spinosa (Saccharopolyspora spinosa) D2302 with a collection number of CGMCC No.26388 in China general microbiological culture Collection center;
(B2) Paenibacillus polymyxa (Paenibacillus polymyxa) D2303 has a collection number of CGMCC No.26387 in China general microbiological culture Collection center.
10. Any of the following applications:
(C1) Use of spinosyn (Saccharopolyspora spinosa) D2302 as claimed in claim 9 or a microbial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 for the production of spinosyn;
(C2) Use of spinosyn (Saccharopolyspora spinosa) D2302 or a bacterial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 as described in claim 9 in the manufacture of a product for the production of spinosad;
(C3) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a bacterial agent comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 according to claim 9 for the production of indoleacetic acid;
(C4) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a bacterial agent comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 as described in claim 9 in the manufacture of a product for the production of indoleacetic acid;
(C5) Use of the spinosyn (Saccharopolyspora spinosa) D2302 or a microbial agent comprising said spinosyn (Saccharopolyspora spinosa) D2302 as claimed in claim 9 to increase plant resistance to insect pests;
(C6) Use of a spinosyn (Saccharopolyspora spinosa) D2302 as claimed in claim 9 or a microbial inoculum containing said spinosyn (Saccharopolyspora spinosa) D2302 for the manufacture of a product for increasing plant resistance to pests;
(C7) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a microbial inoculum comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 according to claim 9 for promoting plant growth;
(C8) Use of paenibacillus polymyxa (Paenibacillus polymyxa) D2303 or a microbial inoculant comprising said paenibacillus polymyxa (Paenibacillus polymyxa) D2303 according to claim 9, for the preparation of a product for promoting plant growth.
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| CN202310070389.6A CN116376748A (en) | 2023-02-07 | 2023-02-07 | Co-culture microbial inoculum of saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303 as well as preparation method and application thereof |
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| CN202310070389.6A CN116376748A (en) | 2023-02-07 | 2023-02-07 | Co-culture microbial inoculum of saccharopolyspora spinosa D2302 and paenibacillus polymyxa D2303 as well as preparation method and application thereof |
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