Salt-tolerant Scutellaria baicalensis and its application

Abstract

The present invention discloses salt-tolerant Microlunatus halotolerans and its application. The present invention provides salt-tolerant Microlunatus halotolerans L1102, which has a registration number of CGMCC No. 25923 in the General Microbiological Center of China Microbiological Culture Collection Administration. The strain has the ability to dissolve phosphorus and produce indoleacetic acid (IAA), which proves that the strain of the present invention can promote plant growth and has broad application prospects in eutrophic sewage treatment and saline-alkali land improvement.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to salt-tolerant Sphenanthellus sphaeroides and applications thereof. Background Art

[0002] Microlunatus strains have advantages in treating environmental pollution caused by phosphorus, and have broad prospects for research and application development. In 1995, Nakamura et al. established the Microlunatus genus using Microlunatus phosphovorus as the model species. Microlunatus belongs to the Propionibacteriaceae family and is a type of chemoheterotrophic actinomycetes: aerobic, Gram-positive cells, the characteristic amino acid contained in the cell wall is LL-diaminopimelic acid, the dominant respiratory quinone component is MK-9 (H4), and the phospholipids mainly include diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). The dominant fatty acids contained in the cells include anteiso-C 15:0 ,iso-C 15:0 and iso-C 16:0 (Hanada S, Nakamura K. Genus Microlunatus. Nakamura, Hiraishi, Yoshimi, Kawaharasaki, Masuda and Kamagata 1995,21 VP In:Goodfellow M, P, Busse HP, Trujillo ME, Suzuki K, Ludwig W, Whitman WB (editors). Bergey's Manual of Systematic Bacteriology: The Actinobacteria, Part B, 2nd edn, vol. 5. New York: Springer; 2012. pp. 1168–1172.). The GC content of the genome ranges from 64.0 to 70.9 mol%. Currently, the genus contains 23 species. Before 2017, only 8 species were known. German scholars Nouioui et al. and Chinese scholars Yang & Zhi reclassified 9 species of Friedmanniella into the genus Lupinella in their systematic evolutionary study of microorganisms in the Actinobacteria (Nouioui I, Carro L, García-López M, Meier-Kolthoff JP, Woyke T, Kyrpides NC, Pukall R, Klenk HP, Goodfellow M, M (2018) Genome-Based Taxonomic Classification of the Phylum Actinobacteria. Front in Microbiol, 9: 2007; Yang LL, Zhi XY. Reclassification of Friedmanniella endophytica, Lysinimicrobium sediminis and Lechevalieria rhizosphaerae as Microlunatuskandeliicorticis nom.nov., Demequina sediminis comb.nov. and Lentzearhizosphaeraecomb.nov., respectively. Int J Syst Evol Microbiol 2020; 70: 3930–3931.). In recent years, new species have been reported one after another. Among them, although detailed taxonomic data of three species have been reported, they are still in the state of not being named effectively (https: / / lpsn.dsmz.de / genus / microlunatus). According to relevant literature reports, members of the genus Microlunatus were isolated from a variety of ecological environments: activated sludge, multiple types of soil environments, plant endophytic environments, and caves with different geological characteristics (Xie YG, Fang BZ, Han MX, Liu L, Jiao JY, Zhang XT, Xiao M, Li WJ. Microlunatus speluncae sp. nov., a novel actinobacterium isolated from a Karstic subterranean environment sample. Antonie Van Leeuwenhoek 2020; 113: 117-125.).

[0003] Most of the reported strains of the genus Lumpina are mesophilic actinomycetes with a narrow range of salt and alkali tolerance. Regarding the application research of Microlunatus, only the biological phosphorus removal involving Microlunatus strains has been found (Santos MM, Lemos PC, Reis MA, Santos H. Glucose metabolism and kinetics of phosphorus removal by the fermentative bacterium Microlunatus phosphovorus. Applied and Environmental Microbiology, 1999, 65(9): 3920-3928; Tanaka S, Lee SO, Hamaoka K, Kato J, Takiguchi N, Nakamura K, Ohtake H, Kuroda A. Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium. Microlunatus phosphovorus. Journal of Bacteriology, 2003, 185(18): 5654-5656; Begum SA, Batista JR. Microbial selection of polyphosphate-accumulating bacteria in activated sludge wastewater treatment processes for enhanced biological phosphateremoval.World Journal of Microbiology&Biotechnology,2012,65(3):341-348;awakoshi A,Nakazawa H,Fukada J,Sasagawa M,Katano Y,Nakamura S,Hosoyama A,Sasaki H,Ichikawa N,Hanada S,Kamagata Y,Nakamura K,Yamazaki S,FujitaN.Deciphering the genome of polyphosphate accumulating actinobacterium Microlunatus phosphovorus.DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes, 2012, 19(5): 383-394.), Application of polyhydroxyalkanoates (PHA) produced by Microbial fungus (Chen GQ, Wu Q, Xi JZ. Microbial production of biopolyesters-polyhydroxyalkanoates. Progressin Natural Science, 2000, 10 (11): 843-850; Williams SF, Martin DP, Horowitz DM, Peoples OP. hydroxyalkanoates.Applied Microbiology&Biotechnology,2005,67(5):17; Yuan M,Yu Y,Li HR,Dong N,Zhang XH. Phylogenetic diversity and biological activity of actinobacteria isolated from the ChukchiShelf marine sediments in the Arctic Ocean. Marine Drugs, 2014, 12 (3): 1281-1297.) and reports of involvement in the decomposition of ginsenosides (An DS, Im WT, Yoon MH. Microlunatus panaciterraesp.nov., abeta-glucosidase-producing bacterium isolated from soil in a ginsengfield.International Journal of Systematic and Evolutionary Microbiology,2008,58(12):2734-2738.). . Summary of the invention

[0004] The purpose of the present invention is to provide a new species of the genus Lumpina that can tolerate high salt.

[0005] In a first aspect, the present invention claims protection for a new species of the genus Lumpina.

[0006] The new species of the genus Microlunatus that is claimed to be protected by the present invention is specifically the salt-tolerant Microlunatus (Microlunatushalotolerans) L1102, and its registration number in the General Microbiological Center of China National Microbiological Culture Collection Administration is CGMCC No.25923.

[0007] The salt-tolerant Microlunatus halotolerans L1102 is an aerobic bacterium with a positive Gram staining reaction. The cells are spherical, non-motile, and do not produce endospores. On the ISP 2 medium, a milky white to light yellow circular colony is formed, and the colony surface is dry and the edges are neat.

[0008] The salt-tolerant Microlunatus halotolerans L1102 can tolerate 8% (w / v) NaCl, with an optimal growth pH of 8.0, which expands the understanding of the tolerance of Microlunatus halotolerans strains to saline-alkali environments; the strain L1102 not only shows a phosphorus-dissolving effect on organic phosphorus and inorganic phosphorus, but also can produce the plant hormone indoleacetic acid that promotes plant growth.

[0009] In a second aspect, the invention claims a culture.

[0010] The culture claimed in the present invention is a substance obtained by culturing the Microlunatus halotolerans L1102 described in the first aspect above in an actinomycete culture medium.

[0011] In the above culture, the substances include the Microlunatus halotolerans L1102 (the bacteria itself) and metabolites of the Microlunatus halotolerans L1102.

[0012] In the above culture, the actinomycete culture medium may be a solid culture medium or a liquid culture medium.

[0013] The term "culture" refers to a general term for liquid or solid culture media that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or it can contain a certain amount of culture medium, metabolites or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing microorganisms, which can be a culture of a certain generation or a mixture of several generations.

[0014] The actinomycete culture medium is an ISP (International Streptomyces Research Program) series culture medium. In a specific embodiment of the present invention, the actinomycete culture medium is specifically an ISP 2 culture medium.

[0015] In a third aspect, the present invention claims a metabolite.

[0016] The metabolite claimed in the present invention is the metabolite of Microlunatushalotolerans L1102 described in the first aspect above.

[0017] The term "metabolite" refers to the primary metabolites and / or secondary metabolites produced during the metabolism of microorganisms. Primary metabolism refers to the process in which microorganisms absorb various nutrients from the outside world and generate substances and energy to maintain life activities through catabolism and anabolism. The products of primary metabolism are primary metabolites, such as monosaccharides or monosaccharide derivatives, nucleotides, vitamins, amino acids, fatty acids and other monomers, as well as various macromolecular polymers composed of them, such as proteins, nucleic acids, polysaccharides, lipids, etc. Secondary metabolism refers to the process in which microorganisms use primary metabolites as precursors to synthesize some substances that have no clear function in the life activities of microorganisms during a certain growth period. The products of secondary metabolism are secondary metabolites, most of which are compounds with relatively complex molecular structures. According to their functions, they can be divided into types such as antibiotics, hormones, alkaloids, toxins, etc.

[0018] In a fourth aspect, the present invention claims protection for a bacterial agent.

[0019] The bacterial agent claimed in the present invention contains the salt-tolerant Microlunatushalotolerans L1102 described in the first aspect above, the culture described in the second aspect above, and / or the metabolites described in the third aspect above.

[0020] The bacterial agent is a bacterial agent for dissolving phosphate and / or producing indoleacetic acid.

[0021] In the above-mentioned microbial agent, in addition to the active ingredient, the microbial agent also contains a carrier. The carrier may be a carrier commonly used in the field of pesticides and biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier may be a mineral material, a plant material or a polymer compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, soybean flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol; the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0022] The above-mentioned bacterial agents may be in various dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0023] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0024] In a fifth aspect, the present invention claims the use of the salt-tolerant Microlunatushalotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above in any of the following:

[0025] (A1) Phosphate dissolution;

[0026] (A2) preparing a product for dissolving phosphate;

[0027] (A3) producing indoleacetic acid;

[0028] (A4) preparing a product for producing indoleacetic acid;

[0029] (A5) degradation of organic phosphorus;

[0030] (A6) preparing products for degrading organic phosphorus;

[0031] (A7) Preparing a product having alkaline phosphatase activity.

[0032] Furthermore, the application may be under normal conditions or under salt stress conditions.

[0033] The strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0034] In a sixth aspect, the present invention claims the use of the salt-tolerant Microlunatushalotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above in any of the following:

[0035] (B1) Promote plant growth;

[0036] (B2) Develop phosphate-dissolving bacterial fertilizers;

[0037] (B3) Eutrophication wastewater treatment;

[0038] (B4) Improvement of saline-alkali land;

[0039] Furthermore, the application may be under normal conditions or under salt stress conditions.

[0040] The strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0041] In a seventh aspect, the present invention claims a product for dissolving phosphorus.

[0042] The active ingredient of the product for dissolving phosphate claimed in the present invention is the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0043] The dissolved phosphorus may be dissolved phosphorus under normal conditions or under salt stress conditions.

[0044] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0045] In an eighth aspect, the present invention claims a product for degrading organic phosphorus.

[0046] The product for degrading organic phosphorus claimed in the present invention has the active ingredient of the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0047] The degraded organic phosphorus may be degraded organic phosphorus under normal conditions or under salt stress conditions.

[0048] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0049] In a ninth aspect, the present invention claims a product having alkaline phosphatase activity.

[0050] The product with alkaline phosphatase activity claimed in the present invention has the active ingredient of the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0051] The alkaline phosphatase activity may be alkaline phosphatase activity under normal conditions or under salt stress conditions.

[0052] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0053] In a tenth aspect, the present invention claims a product for producing indoleacetic acid.

[0054] The product for producing indoleacetic acid claimed in the present invention has the active ingredient of the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0055] Wherein, the production of indoleacetic acid can be the production of indoleacetic acid under normal conditions or under salt stress conditions.

[0056] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0057] In an eleventh aspect, the present invention claims a method for degrading organic phosphorus.

[0058] The method for degrading organic phosphorus claimed in the present invention may include the following steps: treating the sample to be treated with the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above.

[0059] The degraded organic phosphorus may be degraded organic phosphorus under normal conditions or under salt stress conditions.

[0060] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0061] In a twelfth aspect, the present invention claims a method for promoting plant growth.

[0062] The method for promoting plant growth claimed in the present invention may include the following steps: applying the salt-tolerant Microlunatus halotolerans L1102 described in the first aspect above, or the culture described in the second aspect above, or the metabolite described in the third aspect above, or the bacterial agent described in the fourth aspect above to plants or plant growth substrates.

[0063] Wherein, the promoting plant growth may be promoting plant growth under normal conditions or under salt stress conditions.

[0064] Furthermore, the strongest salt stress may be 8% NaCl (80 g / L NaCl).

[0065] In the thirteenth aspect, the present invention claims protection for the use of the salt-tolerant Microlunatushalotolerans L1102 described in the first aspect above in the preparation of the culture described in the second aspect above or the metabolite described in the third aspect above or the bacterial agent described in the fourth aspect above.

[0066] Experiments have shown that the strain L1102 of the present invention has some obvious differences from the effectively described strains of the genus Microlunatus that have been scientifically studied, including phenotype, physiological and biochemical and cytochemical components. At the same time, the systematic evolutionary analysis based on the gene level further illustrates that the strain L1102 can be distinguished from the existing effective species of the genus Microlunatus, which fully proves that the strain L1102 of the present invention represents a new species of the genus Microlunatus, named Microlunatus alotolerans. The strain has the ability to dissolve phosphorus and produce indoleacetic acid (IAA), which proves that the strain of the present invention can promote plant growth and has broad application prospects in eutrophic sewage treatment and saline-alkali land improvement. It can be used for sewage treatment, soil improvement, and production of bacterial fertilizer preparations.

[0067] Collection Instructions

[0068] Classification and nomenclature: Microlunatus halotolerans;

[0069] Reference biological materials: L1102;

[0070] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0071] Abbreviation of depository institution: CGMCC;

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

[0073] Deposit date: October 17, 2022;

[0074] The registration number of the Collection Center is: CGMCC No.25923. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is a colony photograph of strain L1102 grown on ISP 2 medium at 28°C for 7 days.

[0076] Figure 2 This is the detection result of polar lipid components of strain L1102.

[0077] Figure 3 This is a picture of the screening of the phosphate solubilization ability of strain L1102. The negative control is the residue of inactivated bacteria L1102, and the positive control is strain JXJ CY 19 T (Zhang BH,Salam N,Cheng J,Li HQ,Yang JY,Zha DM,etal.(2016)Modestobacter lacusdianchii sp.nov.,aPhosphate-SolubilizingActinobacterium with Ability to Promote Microcystis Growth.PLoS ONE 11(8):e0161069.doi:10.1371 / journal.pone.0161069.).

[0078] Figure 4 This is the IAA standard curve and the activity test chart of IAA produced by strain L1102.

[0079] Figure 5 This is a phylogenetic tree constructed based on the 16S rRNA gene sequences of strain L1102 and its closely related bacteria, showing the phylogenetic status of strain L1102. DETAILED DESCRIPTION

[0080] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0081] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0082] Example 1. Isolation and identification of salt-tolerant Microlunatus halotolerans L1102

[0083] 1. Isolation of strain L1102

[0084] The strain L1102 of the present invention is separated and purified from a plant rhizosphere soil sample collected from Laji Mountain, Guide County, Hainan Prefecture, Qinghai Province.

[0085] The isolation medium of strain L1102 is ISP 2 medium. The solvent of ISP 2 medium is water, and the solutes and concentrations are as follows: yeast extract 4.0 g / L, malt extract 10.0 g / L, glucose 4.0 g / L, agar 15.0 g / L, pH 7.5. Inhibitors are added to the medium and the concentrations are: potassium dichromate 30 μg / L, nystatin 50 μg / L.

[0086] Method and process for strain isolation, purification and preservation: Place the collected soil samples in a sterile and ventilated place, air-dry at room temperature for 2 weeks, and dry-heat at 120°C for 15 minutes. Take 2g of the dry-heat-treated soil sample and dilute it with sterile physiological saline. -4 0.3 mL of soil suspension was drawn from the plate, spread on the separation plate, and placed at 28°C for 4 weeks. Single colonies of bacteria and actinomycetes with different morphologies on the same plate were picked and placed on the ISP 2 medium prepared in advance; single colonies were separated by streaking on the quartered plate. The purified strain slant was placed at 4°C for short-term storage; or placed in a tube containing 20% ​​(V / V) glycerol and stored at -80°C for long-term storage.

[0087] A strain numbered L1102 was obtained in this experiment.

[0088] 2. Identification of strain L1102

[0089] 1. Cell morphology observation and physiological and biochemical characteristics detection of strain L1102

[0090] The growth temperature detection range of strain L1102 is 4, 10, 15, 28, 30, 32, 37, 40, 42 and 45°C; the growth salt concentration (NaCl) detection range is 12 (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15) concentration gradients of 0-15% (0-15g / 100ml); the growth pH detection range is 8 (4, 5, 6, 7, 8, 9, 10, 11) gradients between pH4-11. The physiological and biochemical characteristics of the strain were detected using detection kits such as API 50CH, API ZYM, and BIOLOG GEN III carbon source. Other physiological characteristics of the strains, including Gram staining properties, oxygen requirement, catalase activity, oxidase activity, gelatin hydrolysis activity, starch hydrolysis activity and cellulose hydrolysis activity, were mainly determined with reference to the Manual of Actinomycete Systematics (Xu L H. Actinomycete systematics: principles, methods and practices [M]. Beijing: Science Press, 2007, 93-108.).

[0091] The identification results showed that strain L1102 was a chemoheterotrophic aerobic bacterium with a positive Gram staining reaction. The cells were spherical, non-motile, and did not produce endospores. On ISP 2 medium, it formed milky white to light yellow circular colonies with dry surface and neat edges. The maximum colony diameter was 1.1 mm ( Figure 1). Able to grow in pH 6.0-10.0, NaCl concentration range of 0-8.0%, temperature range of 10-42°C; the optimal growth conditions are pH 8.0, NaCl concentration of 6-7%, 28°C. Able to grow on ISP (International Streptomyces Research Program) series of media. Able to ferment the following carbon sources to produce acid: glycerol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, β-methyl-D-xylose, glucose, sorbitol, rhamnose, inositol, α-methyl-D-mannosyl, α-methyl-D-glucoside, arbutin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, xylitol, gentiobiose, D-turanose, D-lyxose, D-rockose, L-rockose, D-arabitol, L-arabitol, gluconate and 2-keto-gluconate. It has alkaline phosphatase, lipid esterase, leucine arylamine, valine arylamine, cystine arylamine, trypsin, chymotrypsin, acid phosphatase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, N-acetyl-glucosaminidase, β-fucosidase, esterase, lipase activity, but does not have β-uronidase, naphthol-phosphohydrolase activity. The results of esculin hydrolysis, hydrogen sulfide production, starch hydrolysis, gelatin liquefaction, oxidase (API), VP test, and methyl red test are negative, while the nitrate reduction, catalase, and urea hydrolysis reactions are positive.

[0092] 2. Detection of cytochemical composition of strain L1102

[0093] The cell chemical components such as fatty acids, quinone types, polar lipids, characteristic amino acids and sugar components of strain L1102 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.). The fatty acid composition of strain L11023 is shown in Table 1. The results show that the main fatty acid of strain L1102 is antesio-C 15:0,iso-C 16:0 and iso-C 15:0 ; In strain L1102, the dominant menaquinone is MK-9(H4); polar lipid components include phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI) and some unknown glycolipids (GLs), such as Figure 2 As shown; the cell wall contains LL-DAP, and the whole cell hydrolysate contains glucose and arabinose.

[0094] Table 1. Fatty acid composition of strain L1102

[0095] Fatty acid composition (%) L1102 anteiso-branched fatty acid <![CDATA[anteiso-C 15:0 ]]> 40.1 <![CDATA[anteiso-C 17:0 ]]> 1.3 iso-branched fatty acid <![CDATA[iso-C 14:0 ]]> 6.1 <![CDATA[iso-C 15:0 ]]> 10.1 <![CDATA[iso-C 16:0 ]]> 34.5 <![CDATA[iso-C 17:0 ]]> 0.1 Saturated fatty acids <![CDATA[C 16:0 ]]> 0.6 Unsaturated fatty acids summed feature 5 0.7 Hydroxy fatty acids <![CDATA[C 14:0 2-OH]]> 0.8 <![CDATA[iso-C 14:0 3-OH]]> 2.5

[0096] In summary, the analysis results of the cell wall characteristic amino acids, sugar components, cell dominant fatty acid components, main respiratory quinones and polar lipid components of strain L1102 support the classification of strain L1102 into the genus Luna. At the same time, the specific composition of fatty acids of strain L1102 can be distinguished from other species of the genus Luna. For example, the model strains of closely related species of the genus Luna are consistent in the dominant fatty acid composition, and both contain antesio-C 15:0 ,iso-C 16:0 and iso-C 15:0 However, there are obvious differences in the composition of fatty acid groups between strain L1102 and the model strains of closely related species of the genus Lumpina. For example, some strains do not contain Hydroxy fatty acids (C 14:0 2-OH, iso-C 14:0 3-OH)(P. Chiu-Chung Young, H.-J. Busse, et al. Microlunatus soli sp. nov., isolated from soil. International Journal of Systematic and Evolutionary Microbiology 60, pp824-827 (2010)), while strain L1102 contained Hydroxy fatty acids (C 14:0 2-OH, iso-C 14:0 3-OH) (Table 1). These results support that strain L1102 represents a new species of the genus Luna.

[0097] 3. Determination of alkaline phosphatase activity of strain L1102

[0098] Using lecithin as the only phosphorus source in the plate, the alkaline phosphatase activity of the strain was screened on the plate. The specific steps are as follows: prepare an organophosphorus solid screening medium (glucose 3g·L -1 , ammonium sulfate 0.5 g·L -1 , sodium chloride 0.3 g·L -1 , potassium chloride 0.3 g·L -1 , ferrous sulfate heptahydrate 0.03 g·L -1 , manganese sulfate heptahydrate 0.03g·L -1 , calcium carbonate 5g·L -1 , lecithin 1g·L -1 , agar 15 g·L -1 ; pH 7), sterilized at 115°C for 30 min. Then, single colonies of strain L1102 in the logarithmic growth phase were picked and inoculated on the organophosphorus solid screening medium. After culturing at 28°C for 4 days, the diameter of the bacterial colony (d) and the diameter of the phosphate solubility zone (D) were measured, and the solubility index (D / d) was calculated.

[0099] The experimental results show that the strain L1102 of the present invention can produce a phosphate-dissolving circle ( Figure 3 ), the solubility index was 3.92 (the average of three independent experimental results).

[0100] At the same time, liquid screening of alkaline phosphatase activity was performed on strain L1102. Strain L1102 in the logarithmic growth phase was inoculated into 20 mL of liquid organophosphorus culture medium, and a blank culture medium without bacteria was used as a control group. After culturing at 28°C for 14 days, the supernatant was centrifuged to determine the organophosphorus concentration in the culture medium, and the blank group was used as a control to calculate the PO4 produced by the strain in degrading organophosphorus. 3+ (mg / L) concentration change (using △PO4 3+ The standard curve was prepared according to the phosphate standard solution, and M0 was the PO4 detected in the blank control group. 3+ concentration, M1 is the PO4 detected in the experimental group 3+ concentration.

[0101] ΔPO4 3+ Concentration = M1-M0

[0102] The strain L1102 of the present invention has alkaline phosphatase activity and can degrade △PO4 produced by organic phosphorus. 3+ The concentration value was 0.59 mg / L (the average value of three independent experimental results).

[0103] In addition, the genome data of strain L1102 were compared with the eggNOG database (http: / / eggnog.embl.de / ) and the KEGG database (http: / / kobas.cbi.pku.edu.cn / home.do) using the diamond software, and the E value was set lower than e -5 We searched for alkaline phosphatase A gene (pho A) and alkaline phosphatase E (pho E) genes in the genome of this strain.

[0104] The results of genomic analysis and phenotypic experiments fully confirmed that strain L1102 has the potential to solubilize phosphate.

[0105] 4. Detection of 3-Indoleacetic acid (IAA) activity produced by strain L1102

[0106] The ability of strain L1102 to produce indoleacetic acid was determined by colorimetry (Bric et al., 199Bric, 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). The concentrations of IAA were 0, 0.675, 1.25, 2.5, 5, 10, 20, 40, 50, 60, 80, 100 mg / L, and a standard curve was prepared to determine the OD 540 nm The absorbance value ( Figure 4 ,y=0.0244x+0.0482,r 2 =0.9998). The cells of the strain growing to the logarithmic phase were transferred to a culture medium containing 3 mmol / L L-tryptophan and tryptone (1%) (w / v) and incubated at 30°C for 3 days. The OD of the culture medium was measured. 540 nm According to the standard curve, the concentration of indoleacetic acid in the culture medium of strain L1102 was calculated to be 8.27±0.19 mg / L. This confirmed that strain L1102 has a strong ability to produce indoleacetic acid.

[0107] 5. Determination of the phylogenetic status of strains

[0108] The genomic DNA of strain L1102 was extracted for sequencing, and the 16S rRNA gene sequence (SEQ ID No. 1) thereof was compared online in the international authoritative bacterial taxonomy analysis 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 similarity values ​​of the 16S rRNA gene sequence (1567 bp) of the strain L1102 of the present invention and the known species are listed in Table 2. The results showed that the strain L1102 of the present invention had the highest similarity with the species of the genus Lunar spp. of the Propionibacteriaceae family, and the similarity between the strain L1102 and the species of the genus Lunar spp. was in the range of 93.5-98.0%. These values ​​are all lower than the threshold value of 98.65% for distinguishing prokaryotic microorganisms (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.). The 16S rRNA gene sequences of all valid strains of the genus Luna and some strains of the genera adjacent to the genus Luna in the Propionibacteriaceae were selected to construct a phylogenetic tree ( Figure 5 ). It showed that strain L1102 was included in the genus Lunar Newton, and the effectively described species in the genus Lunar Newton were clustered on a stable evolutionary branch, and strain L1102 occupied the position of a species.

[0109] The genome sequence analysis of strain L1102 showed that the G+C content of the genome of strain L1102 was 65.9 mol%.

[0110] Table 2. Comparison results of 16S rRNA genes of strain L1102 and closely related bacteria

[0111]

[0112]

[0113] In summary, the data analysis of the multiphase taxonomic research shows that the strain L1102 of the present invention has some obvious differences from the effectively described strains of the genus Microlunatus that have been scientifically studied, including phenotype, physiological and biochemical and cytochemical components. At the same time, the systematic evolutionary analysis based on the gene level further illustrates that the strain L1102 can be distinguished from the existing effective species of the genus Microlunatus, which fully proves that the strain L1102 of the present invention represents a new species of the genus Microlunatus, named as salt-tolerant Microlunatus (Microlunatus halotolerans). At the same time, it was found that the strain L1102 has the ability to dissolve phosphorus and produce indoleacetic acid (IAA), which proves that the strain L1102 of the present invention can promote plant growth and has broad application prospects in the treatment of eutrophic sewage and the improvement of saline-alkali land. It can be used for sewage treatment, soil improvement, and the production of bacterial fertilizer preparations.

[0114] Salt-tolerant Microlunatus halotolerans L1102 was deposited in the China General Microbiological Culture Collection Center on October 17, 2022, and its deposit number is CGMCC No.25923.

[0115] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Salt-tolerant Scutellaria spp. Microlunatus halotoleran s) L1102, whose registration number in the General Microbiological Center of China Culture Collection Administration is CGMCC No.25923.

2. The salt-tolerant Scutellaria baicalensis of claim 1 ( Microlunatus halotoleran s) The culture of L1102 is the salt-tolerant Scutellaria baicalensis ( Microlunatus halotoleran s) Biologically pure culture of L1102.

3. A microbial agent, characterized in that: The bacterial agent contains the salt-tolerant Scutellaria baicalensis according to claim 1 ( Microlunatus halotoleran s) L1102 and / or the culture described in claim 2.

4. The bacterial agent according to claim 3, characterized in that: The bacterial agent is a bacterial agent for dissolving phosphate and / or producing indoleacetic acid.

5. The salt-tolerant Scutellaria baicalensis of claim 1 ( Microlunatus halotoleran s) Use of L1102 or the culture of claim 2 or the bacterial agent of claim 3 or 4 in any of the following: (A1) Phosphate dissolution; (A2) preparing products for phosphate dissolution; (A3) Production of indoleacetic acid; (A4) preparing a product for producing indoleacetic acid; (A5) Degradation of organic phosphorus; (A6) preparing products for the degradation of organic phosphorus; (A7) Preparing a product having alkaline phosphatase activity.

6. The use according to claim 5, characterized in that: The application is application under normal conditions or under salt stress conditions.

7. The salt-tolerant Scutellaria baicalensis of claim 1 ( Microlunatus halotoleran s) Use of L1102 or the culture of claim 2 or the bacterial agent of claim 3 or 4 in any of the following: (B1) Promote plant growth; (B2) Develop phosphate-dissolving bacterial fertilizers; (B3) Eutrophication wastewater treatment.

8. The use according to claim 7, characterized in that: The application is application under normal conditions or under salt stress conditions.

9. A product for dissolving phosphate, the active ingredient of which is the salt-tolerant Scutellaria baicalensis ( Microlunatus halotoleran s) L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

10. A product for degrading organic phosphorus, the active ingredient of which is the salt-tolerant Scutellaria baicalensis ( Microlunatus halotoleran s) L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

11. A product having alkaline phosphatase activity, wherein the active ingredient is the salt-tolerant Scutellaria baicalensis according to claim 1 ( Microlunatus halotoleran s) L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

12. A product for producing indoleacetic acid, the active ingredient of which is the salt-tolerant Scutellaria baicalensis ( Microlunatus halotoleran s) L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

13. A method for degrading organic phosphorus, comprising the following steps: using the salt-tolerant Scutellaria baicalensis ( Microlunatus halotoleran s) The sample to be treated is treated with L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4.

14. A method for promoting plant growth, comprising the following steps: Microlunatus halotoleran s) L1102 or the culture described in claim 2 or the bacterial agent described in claim 3 or 4 is applied to plants or plant growth substrates.

15. The salt-tolerant Scutellaria baicalensis of claim 1 ( Microlunatus halotoleran s) Use of L1102 in the preparation of the culture according to claim 2 or the bacterial agent according to claim 3 or 4.

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