CN116121118A

CN116121118A - Salt-tolerant Xiaoyue and application thereof

Info

Publication number: CN116121118A
Application number: CN202211515558.4A
Authority: CN
Inventors: 赵显锋; 李丽丽; 刘凌风
Original assignee: Shandong Chunong Biotechnology Co ltd
Current assignee: Shandong Chunong Biotechnology Co ltd
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2023-05-16

Abstract

The invention discloses salt-tolerant aureobasidium parvulum and application thereof. The invention provides salt-tolerant Xiaoyue (Microlunatus halotolerans) L1102, which has a registration number of CGMCC No.25923 in the China general microbiological culture Collection center. The strain has the capabilities of dissolving phosphorus and producing indoleacetic acid (IAA), proves that the strain can play a role in promoting plant growth, and has wide application prospect in the aspects of eutrophic sewage treatment and saline-alkali soil improvement.

Description

Salt-tolerant Xiaoyue and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to salt-tolerant aureobasidium parvum and application thereof.

Background

The Microlunella (Microlunatus) strain has advantages in the aspect of treating the environmental pollution caused by phosphorus element, and has wide research and application development prospects. In 1995, nakamura et al established the genus Microsoft with Microsoft Phospholipidus (Microlunatus phosphovorus) as a model species. The genus Oenothera belongs to the family Propionibacterium (Propionibacterium), is a heterotrophic actinomycetes: aerobic and gram-positive staining of cells, the characteristic amino acid contained in the cell wall is LL-diaminopimelic acid, and the dominant respiratory quinone component is MK-9 (H) ₄ ) The phosphate lipids mainly include biphospholipid glycerol (DPG) and Phosphatidylglycerol (PG). The dominant fatty acids contained in the cells include antais-C _15:0 ,iso-C _15:0 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 genomic GC content ranges from 64.0 to 70.9mol%. Currently, this genus contains 23 species. Only 8 species were known before 2017. Nouioui et al, german scholars and China scholars Yang&Zhi system carding actinomycota in the research of systematic evolution of microorganisms, 9 species of Friedmanniella were reclassified into the genus aureobasidium (Nouioui I, carro L, garcI a-Lwave M, meier-Kolthoff JP, woyke T, kyrps NC, pukall R, klenk HP, goodfelt M,>

M(2018)Genome-Based Taxonomic Classification of the Phylum Actinobacteria.Front in Microbiol,9:2007；Yang L-L,Zhi X-Y.Reclassification of Friedmanniella endophytica,Lysinimicrobium sediminis and Lechevalieria rhizosphaerae as Microlunatus kandeliicorticis nom.nov.,Demequina sediminis comb.nov.and Lentzea rhizosphaeraecomb.nov.,rinjection J Syst Evol Microbiol2020; 70:3930-3931.). In recent years, new species have been reported successively, of which 3 species, although they have been reported with detailed taxonomic data, have so far been in an uneffective named state (https:// lpsn. Dsmz. De/genus/microrolunatus). Throughout the literature, members of the genus coleus are reported to be isolated from a variety of ecological environments: activated sludge, multiple types of soil environments, plant endophytic environments, caverns of different geological features, etc. (Xie YG, fang BZ, han MX, liu L, jiao JY, zhang XT, xiao M, li WJ. Microlunatus spincae sp. Nov., a novel actinobacterium isolated from a Karstic subterranean environment sample. Antonie Van Leeuwenhoek 2020; 113:117-125.).

The reported strains of the genus Micronothera are mostly mesophilic and less extensive actinomycetes with saline-alkali tolerance. Application studies on the genus Zostereum have only been seen with respect to biological phosphorus removal involving the strain of Zostereum (Santos MM, lemos PC, reis MA, santos H. Glucose metabolism and kinetics of phosphorus removal by the fermentative bacterium Microlunatus phosphorus. 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 bacteria, micro-organisms 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 phosphate remote 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,Fujita N.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), use of Polyhydroxyalkanoates (PHA) produced by Microbacterium (Chen GQ, wu Q, xi JZ. Micro production of biopoly esters-polyhydroxylkanoates, processein Nature Science,2000,10 (11): 843-850;Williams SF,Martin DP,Horowitz DM,Peoples OP.PHA applications:addressing the price performance issue:I.Tissue engineering.International Journal of Biological Macromolecules,1999,25 (1-3): 111-121;Chen GQ,Wu Q.Microbial production and applications of chiral 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 Chukchi Shelf marine sediments in the Arctic Ocean. Marinedrugs,2014,12 (3): 1281-1297.) and reports of participation in the decomposition of ginsenoside (AnDS, im WT, yoon MH. Microlunate panaciferae sp. Nov., abeta-glucosidase-producing bacterium isolated from soil in a ginseng field. International Journal of Systematic and Evolutionary Microbiology,2008,58 (12): 2734-2738.).

Disclosure of Invention

The object of the present invention is to provide a new species of the genus Oenothera which is tolerant to high salts.

In a first aspect, the invention claims a new species of the genus columbium.

The new species of the genus of the invention is specifically salt-tolerant Xiaoeria (Microlunatus halotolerans) L1102, which has a registration number of CGMCC No.25923 in the China general microbiological culture Collection center.

The salt-tolerant aureobasidium (Microlunatus halotolerans) L1102 is aerobic and has positive gram staining reaction. The cells are spherical, have no motility and do not produce endospores. On ISP 2 medium, milky-white to yellowish circular colony is formed, and the colony surface is dry and the edge is clean.

The salt-tolerant aureobasidium (Microlunatus halotolerans) L1102 can tolerate 8% (w/v) NaCl, has the optimal growth pH of 8.0, and expands the knowledge of the tolerance of the aureobasidium strain to the saline-alkali environment; strain L1102 not only shows a phosphorus-solubilizing effect on organic and inorganic phosphorus, but also is capable of producing the plant hormone indoleacetic acid which promotes plant growth.

In a second aspect, the invention claims a culture.

The culture claimed in the present invention is a culture of the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 described in the first aspect above in an actinomycete medium.

In the above culture, the substance includes metabolites of the salt-tolerant aureobasidium (Microlunatus halotolerans) L1102 (cell itself) and the salt-tolerant aureobasidium (Microlunatus halotolerans) L1102.

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

The term "culture" refers to a collective term for a liquid or solid medium in which a population of microorganisms has grown after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture. The term "culture" also includes subcultures obtained by passaging microorganisms, which may be a culture of a certain generation or a mixture of several generations.

The actinomycete culture medium is ISP (International Streptomyces study program) series culture medium. In a specific embodiment of the invention, the actinomycete medium is specifically ISP 2 medium.

In a third aspect, the invention claims a metabolite.

The claimed metabolite of the invention is a metabolite of the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 described in the first aspect hereinbefore.

The term "metabolite" refers to a primary metabolite and/or a secondary metabolite produced during metabolism of a microorganism. Primary metabolism refers to a process in which microorganisms absorb various nutrients from the outside and produce substances and energy that maintain vital activities through catabolism and anabolism. The primary metabolite is primary metabolite such as monosaccharide or monosaccharide derivative, nucleotide, vitamin, amino acid, fatty acid, and various macromolecular polymers composed of the same, such as protein, nucleic acid, polysaccharide, and lipid. Secondary metabolism refers to the process of synthesizing substances which have no definite function on the life activities of microorganisms by taking primary metabolites as precursors in a certain growth period of microorganisms. The secondary metabolite is the secondary metabolite, and most of the secondary metabolites are compounds with relatively complex molecular structures. Depending on their action, they can be classified into antibiotics, hormones, alkaloids, toxins, etc.

In a fourth aspect, the invention claims a microbial agent.

The claimed microbial agents contain a salt tolerant aureobasidium (Microlunatus halotolerans) L1102 as described in the first aspect hereinbefore, a culture as described in the second aspect hereinbefore and/or a metabolite as described in the third aspect hereinbefore.

The microbial inoculum is a microbial inoculum for dissolving phosphorus and/or producing indoleacetic acid.

In the microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the pesticide arts and which is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier can be mineral material, plant material or high molecular 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, soy 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.

Among the above-mentioned bacterial agents, the formulation of the bacterial agent can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.

In a fifth aspect, the invention claims the use of a salt tolerant aureobasidium (Microlunatus halotolerans) L1102 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore in any one of the following:

(A1) Dissolving phosphorus;

(A2) Preparing a product for dissolving phosphorus;

(A3) Producing indoleacetic acid;

(A4) Preparing a product for producing indoleacetic acid;

(A5) Degrading the organic phosphorus;

(A6) Preparing a product for degrading organic phosphorus;

(A7) A product having alkaline phosphatase activity is prepared.

Further, the application may be under normal conditions or under salt stress conditions.

Wherein, the salt stress can be 8% NaCl (80 g/L NaCl) at maximum.

In a sixth aspect, the invention claims the use of a salt tolerant aureobasidium (Microlunatus halotolerans) L1102 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a microbial inoculum as described in the fourth aspect hereinbefore in any one of the following:

(B1) Promoting plant growth;

(B2) Developing phosphate-dissolving bacterial manure;

(B3) Treating eutrophic sewage;

(B4) Improvement of saline-alkali soil;

Wherein, the salt stress can be 8% NaCl (80 g/L NaCl) at maximum.

In a seventh aspect, the invention claims a product for dissolving phosphorus.

The product for dissolving phosphorus claimed in the present invention has an active ingredient of the salt tolerant moon (Microlunatus halotolerans) L1102 described in the first aspect, the culture described in the second aspect, the metabolite described in the third aspect, or the microbial inoculum described in the fourth aspect.

Wherein, the phosphorus can be phosphorus under normal conditions or salt stress conditions.

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In an eighth aspect, the invention claims a product for degrading an organic phosphorus.

The product for degrading organic phosphorus claimed in the present invention has an active ingredient of the salt tolerant moonlight bacterium (Microlunatus halotolerans) L1102 described in the first aspect, the culture described in the second aspect, the metabolite described in the third aspect, or the microbial inoculum described in the fourth aspect.

Wherein, the degradation organic phosphorus can be the degradation organic phosphorus under normal condition or salt stress condition.

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In a ninth aspect, the invention claims a product having alkaline phosphatase activity.

The product with alkaline phosphatase activity as claimed in the present invention has an active ingredient of the salt tolerant moon (Microlunatus halotolerans) L1102 as described in the first aspect above or the culture as described in the second aspect above or the metabolite as described in the third aspect above or the microbial inoculum as described in the fourth aspect above.

Wherein the alkaline phosphatase activity may be that of normal conditions or under salt stress conditions.

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In a tenth aspect, the invention claims a product for use in the production of indoleacetic acid.

The product for producing indoleacetic acid claimed in the present invention has an active ingredient of the salt tolerant moon (Microlunatus halotolerans) L1102 described in the first aspect, the culture described in the second aspect, the metabolite described in the third aspect, or the microbial inoculum described in the fourth aspect.

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

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In an eleventh aspect, the invention claims a method of degrading an organic phosphorus.

The method for degrading the organic phosphorus, which is claimed by the invention, can comprise the following steps: the sample to be treated is treated with a salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 as described in the first aspect hereinbefore or a culture as described in the second aspect hereinbefore or a metabolite as described in the third aspect hereinbefore or a bacterial agent as described in the fourth aspect hereinbefore.

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In a twelfth aspect, the invention claims a method of promoting plant growth.

The method for promoting plant growth claimed by the invention can comprise the following steps: applying the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 as described in the first aspect above or the culture as described in the second aspect above or the metabolite as described in the third aspect above or the microbial inoculum as described in the fourth aspect above to a plant or plant growth substrate.

Wherein, the promotion of plant growth may be the promotion of plant growth under normal conditions or salt stress conditions.

Further, the salt stress may be 8% NaCl (80 g/L NaCl) at maximum.

In a thirteenth aspect, the invention claims the use of a salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 as described in the first aspect above for the preparation of a culture as described in the second aspect above or a metabolite as described in the third aspect above or a bacterial agent as described in the fourth aspect above.

Experiments prove that the strain L1102 has a part of obvious difference characteristics with the effective descriptive strain of the coleus which has been scientifically researched at present, including aspects of phenotype, physiology, biochemistry, cytochemistry and the like. Meanwhile, the phylogenetic analysis based on the gene level further shows that the strain L1102 can be distinguished from each effective species of the prior genus Microsoft, and fully proves that the strain L1102 represents a new species of the genus Microsoft and is named as salt-tolerant Microsoft (Microlunatus halotolerans). The strain has the capabilities of dissolving phosphorus and producing indoleacetic acid (IAA), proves that the strain can play a role in promoting plant growth, and has wide application prospect in the aspects of eutrophic sewage treatment and saline-alkali soil improvement. Can be used for sewage treatment, soil improvement, bacterial fertilizer preparation production and the like.

Preservation description

Classification naming: salt tolerant microyue (Microlunatus halotolerans);

biological materials according to: l1102;

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: 2022, 10, 17;

accession numbers of the preservation center: CGMCC No.25923.

Drawings

FIG. 1 is a photograph of a colony of strain L1102 grown for 7d at 28℃on ISP 2 medium.

FIG. 2 shows the results of detection of the polar lipid component of strain L1102.

FIG. 3 is a screen image of the phosphorus-dissolving capacity of strain L1102. Wherein the negative control is inactivated thallus L1102 residue, and the positive control is strain JXJ CY 19 ^T (Zhang B-H,Salam N,Cheng J,Li H-Q,Yang J-Y,Zha D-M,et al.(2016)Modestobacter lacusdianchii sp.nov.,aPhosphate-Solubilizing Actinobacterium with Ability to Promote Microcystis Growth.PLoS ONE 11(8):e0161069.doi:10.1371/journal.pone.0161069.)。

FIG. 4 is a graph of IAA standard curves and activity assays for IAA production by strain L1102.

FIG. 5 is a phylogenetic tree constructed based on the 16S rRNA gene sequence of strain L1102 and its related bacteria, showing the phylogenetic status of strain L1102.

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 and identification of salt tolerant Oenothera biennis (Microlunatus halotolerans) L1102

1. Isolation of Strain L1102

The strain L1102 is obtained by separating and purifying a plant rhizosphere soil sample collected from Lagenaria in Guide county of Hainan province.

The isolation medium for strain L1102 was ISP 2 medium. The ISP 2 medium was water as solvent and the solutes and concentrations were as follows: yeast extract 4.0g/L, malt extract 10.0g/L, glucose 4.0g/L, agar 15.0g/L, pH 7.5. Adding inhibitors and the concentration thereof into the culture medium are as follows: 30 mug/L potassium dichromate, 50 mug/L nystatin.

The strain separation, purification and preservation method and process are as follows: the collected soil sample is placed in a sterile ventilation place, and is air-dried at room temperature for 2 weeks and is dry-heated at 120 ℃ for 15min. 2g of the dry heat treated soil sample was taken and diluted with a sterile physiological saline gradient. From the preparation a dilution gradient of 10 ^-4 Is pipetted into 0.3mL, spread on a separation plate and incubated for 4 weeks at 28 ℃. Selecting single bacterial colonies with different forms and actinomycetes on the same flat plate on a prepared inclined plane ISP 2 culture medium; single colonies were isolated by four-way plate streaking. Purifying to obtain strain inclined plane, and preserving at 4deg.C for a short period; or placing in a glycerol pipe containing 20% (V/V), and preserving at-80deg.C for a long time.

A strain with the number L1102 is obtained in the experiment.

2. Identification of Strain L1102

1. Cell morphology observation and physiological and biochemical characteristic detection of strain L1102

The growth temperature detection range of strain L1102 was 4, 10, 15, 28, 30, 32, 37, 40, 42 and 45 ℃; 12 (0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15) concentration gradients with a growth salt concentration (NaCl) detection range of 0-15% (0-15 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, BIOLOG GEN III carbon source and the like. 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 the actinomycetes systems identification handbook (Xu L H. Actinomycete systems: principles, methods and practices [ M ] Beijing: science Press,2007,93-108.).

The identification result shows that the strain L1102 is heterotrophic aerobic bacteria and has positive gram staining reaction. The cells are spherical, have no motility and do not produce endospores. On ISP 2 medium, milky to pale yellow circular colonies were formed, the surfaces of the colonies were dried, the edges were clean, and the maximum colony diameter was 1.1mm (FIG. 1). Can grow in the temperature range of 10-42 ℃ and the concentration range of NaCl with pH of 6.0-10.0 and 0-8.0%; the most suitable growth conditions are pH8.0, naCl concentration 6-7% and 28 ℃. Can grow on ISP (International Streptomyces study program) series of culture media. Can ferment the following carbon sources to produce acid: glycerol, D-arabinose, L-arabinose, ribose, D-xylose, L-xylose, β -methyl-D-xyloside, glucose, sorbose, rhamnose, inositol, α -methyl-D-mannoside, α -methyl-D-glucoside, arbutin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, xylitol, gentiobiose, D-melibiose, D-lyxose D-fucose, L-fucose, D-arabitol, L-arabitol, gluconate, and 2-keto-gluconate. Has alkaline phosphatase, lipoid esterase, leucine arylamine enzyme, valine arylamine enzyme, cystine arylamine enzyme, trypsin, chymotrypsin, acid phosphatase, alpha-galactosidase, beta-galactosidase, alpha-glucosidase, beta-glucosidase, N-acetyl-glucosidase, beta-fucosidase, esterase, lipoidase activity, but does not have beta-uronic acid glycosidase, naphthol-phosphate hydrolase activity. Esculin hydrolysis, hydrogen sulfide production, starch hydrolysis, gelatin liquefaction, oxidase (API), VP experiments, methyl red experiments were negative, whereas nitrate reduction, catalase, urea hydrolysis reactions were positive.

2. Cell chemistry composition detection of Strain L1102

The cell chemical components such as fatty acids, quinone types, polar lipids, and 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, and 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 The method comprises the steps of carrying out a first treatment on the surface of the In strain L1102, the dominant menaquinone is MK-9 (H ₄ ) The method comprises the steps of carrying out a first treatment on the surface of the Polar lipid components include Phosphatidylglycerol (PG), dipesphatidylglycerol (dpp), phosphatidylinositol (PI), and some of the unknown Glycolipids (GLs), as shown in fig. 2; the cell wall contains LL-DAP and the whole cell hydrolysate contains glucose and arabinose.

Table 1 fatty acid composition of Strain L1102

Fatty acid component (%)	L1102
		anteiso-branched fatty acid
anteiso-C _15:0	40.1
		anteiso-C _17:0	1.3
iso-branched fatty acid
		iso-C _14:0	6.1
iso-C _15:0	10.1
		iso-C _16:0	34.5
iso-C _17:0	0.1
		Saturated fatty acids
C _16:0	0.6
		Unsaturated fatty acids
summed feature 5	0.7
		Hydroxy fatty acids
C _14:0 2-OH	0.8
		iso-C _14:0 3-OH	2.5

Taken together, the analysis results of the cell wall characteristic amino acids, sugar components, cell dominant fatty acid components, major respiratory quinones and polar lipid components of strain L1102 support the classification of strain L1102 into the genus coleus. Meanwhile, the specific composition of fatty acid of the strain L1102 and other strains of the genus Oenothera can be distinguished from each other. For example, the model strains of the strain of the genus Oenothera closely related species are identical in dominant fatty acid composition and all contain antesio-C _15:0 ，iso-C _16:0 And iso-C _15:0 Etc. as main components. However, there was a significant difference in the fatty acid fraction composition between strain L1102 and the model strain of the kindred species of the genus Oenothera, e.g., part of the strain did 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, microilutus sol sp.nov., isolated from soil International Journal of Systematic and Evolutionary Microbiology, pp 824-827 (2010)), while strain L1102 contained Hydroxy fatty acids (C) _14:0 2-OH，iso-C _14:0 3-OH) (Table 1). These results all support that strain L1102 represents a new species of the genus Oenothera.

3. Alkaline phosphatase Activity assay of Strain L1102

Plate screening strains for alkaline phosphatase activity was performed using lecithin as the sole phosphorus source in the plates. The method comprises the following specific steps: preparing organic phosphorus solid screening culture medium (glucose 3 g.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 0.03 g.L ^-1 5 g.L of calcium carbonate ^-1 Lecithin 1 g.L ^-1 Agar 15 g.L ^-1 The method comprises the steps of carrying out a first treatment on the surface of the pH 7), and autoclaving at 115℃for 30min. Then, bacterial strain L1102 in the logarithmic growth phase was inoculated onto an organic phosphorus solid screening medium by picking single colonies, and after culturing at 28℃for 4 days, the bacterial colony diameter (D) and the diameter of the phosphate solubilizing ring (D) were measured, respectively, to calculate the solubility index (D/D).

The experimental results show that the strain L1102 can generate a phosphate solubilizing ring on the organic phosphorus solid screening medium (figure 3), and the solubility index is 3.92 (the average value of the results of three independent experiments is taken).

Meanwhile, strain L1102 was subjected to liquid screening for alkaline phosphatase activity. Inoculating strain L1102 in logarithmic growth phase into 20mL of organic phosphorus liquid culture medium, culturing at 28deg.C for 14 days with blank culture medium without inoculating bacteria, centrifuging to obtain supernatant, measuring organic phosphorus concentration in culture solution, and calculating PO generated by degrading organic phosphorus with blank culture medium as control ₄ ³⁺ Concentration variation (in terms of DeltaPO) ₄ ³⁺ Representation). Preparing a standard curve according to phosphate standard solution, wherein M0 is PO detected in a blank control group ₄ ³⁺ Concentration, M1 is PO detected in the experimental group ₄ ³⁺ Concentration.

ΔPO ₄ ³⁺ concentration=m1-M0

The strain L1102 of the invention has alkaline phosphatase activity and can degrade delta PO generated by organic phosphorus ₄ ³⁺ The concentration value was 0.59mg/L (average of the results of three independent experiments).

In addition, the genome data of strain L1102 was compared with the egNOG numbers, respectively, using diamond softwareThe database (http:// egnogog. Embl. De /) is compared with the KEGG database (http:// kobas. Cbi. Pku. Edu. Cn/home. Do) and the E value is set to be lower than E ^-5 . We searched for the alkaline phosphatase A gene (phoA) and alkaline phosphatase E (phoE) genes from the genome of this strain.

Genomic analysis and phenotypic experimental results fully confirm that strain L1102 has phosphorus-dissolving potential.

4. Detection of indoleacetic acid (3-Indoleacetic acid, IAA) production activity of Strain L1102

The capacity of strain L1102 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 0,0.675,1.25,2.5,5,10,20,40,50,60,80,100mg/L, and OD was measured ₅₄₀ _nm Absorbance values of (fig. 4, y=0.0244 x+0.0482, r) ² =0.9998). Cells of the strain grown to the logarithmic phase were transferred to a medium containing 3mmol/L of L-tryptophan and containing tryptone (1%) (w/v), and incubated at 30℃for 3 days. 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 L1102 is calculated to be 8.27+/-0.19 mg/L. The strain L1102 is proved to have stronger capacity of producing indoleacetic acid.

5. Determination of the phylogenetic status of strains

Genomic DNA of strain L1102 was extracted and sequenced and the 16S rRNA gene sequence therein (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 similarity values between the 16SrRNA gene sequence (1567 bp) of the strain L1102 of the present invention and known strains are shown in Table 2. The results show that: the strain L1102 has the highest similarity with the strain of the genus Microsoft of the family Propionibacteriaceae, and the strain similarity of the strain L1102 with the strain of the genus Microsoft ranges from 93.5% to 98.0%. These values are all below 98.65% of the threshold for differentiating procaryotic microorganisms (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.). The 16S rRNA gene sequences of all the effective species strains of the genus Microsoft were selected and phylogenetic tree was constructed from the partial species strains of the genus Microsoft in the family Propionibacteriaceae (FIG. 5). Strain L1102 was shown to be included in the genus Oenothera and effectively described the species to be collected on a stable evolutionary branch, strain L1102 occupied the position of one species.

Genomic sequence analysis of strain L1102 gave a G+C content of 65.9mol% for the genome of strain L1102.

Table 2, 16S rRNA Gene alignment results of Strain L1102 and kindred bacteria

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In summary, the analysis of the multiphase taxonomy study data shows that the strain L1102 of the invention has a part of obvious difference characteristics from the effective descriptive strain of the Zostereum which is scientifically studied at present, including aspects of phenotype, physiological biochemistry, cytochemistry components and the like. Meanwhile, the phylogenetic analysis based on the gene level further shows that the strain L1102 can be distinguished from each effective species of the prior genus Microsoft, and fully proves that the strain L1102 represents a new species of the genus Microsoft and is named as salt-tolerant Microsoft (Microlunatus halotolerans). Meanwhile, the bacterial strain L1102 has the capabilities of dissolving phosphorus and producing indoleacetic acid (IAA), and the bacterial strain L1102 has the capability of promoting plant growth, and has wide application prospects in the aspects of eutrophic sewage treatment and saline-alkali soil improvement. Can be used for sewage treatment, soil improvement, bacterial fertilizer preparation production and the like.

The salt-tolerant small month fungus (Microlunatus halotolerans) L1102 is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 25923 at the 10 th month and 17 th year of 2022.

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

1. The registration number of the salt-tolerant Xiaoyue (Microlunatus halotolerans) L1102 in the China general microbiological culture Collection center is CGMCC No.25923.

2. The culture of the salt-tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 of claim 1, which is obtained by culturing the salt-tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 of claim 1 in an actinomycete medium.

3. A metabolite of the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 of claim 1.

4. The microbial inoculum is characterized in that: the microbial inoculum comprises the salt-tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1, the culture of claim 2 and/or the metabolite of claim 3.

5. The microbial agent of claim 4, wherein: the microbial inoculum is a microbial inoculum for dissolving phosphorus and/or producing indoleacetic acid.

6. Use of the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5 in any one of the following:

(A1) Dissolving phosphorus;

(A2) Preparing a product for dissolving phosphorus;

(A3) Producing indoleacetic acid;

(A4) Preparing a product for producing indoleacetic acid;

(A5) Degrading the organic phosphorus;

(A6) Preparing a product for degrading organic phosphorus;

(A7) Preparing a product having alkaline phosphatase activity;

further, the application is under normal conditions or under salt stress conditions.

7. Use of the salt tolerant aureobasidium pullulans (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5 in any one of the following:

(B1) Promoting plant growth;

(B2) Developing phosphate-dissolving bacterial manure;

(B3) Treating eutrophic sewage;

(B4) Improvement of saline-alkali soil;

8. A product for dissolving phosphorus, the active ingredient of which is the salt-tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5;

or (b)

A product for degrading organic phosphorus, the active ingredient of which is the salt-tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5;

or (b)

A product having alkaline phosphatase activity, the active ingredient of which is the salt-tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5;

or (b)

A product for the production of indoleacetic acid, the active ingredient of which is the salt-tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5.

9. A method of degrading an organophosphorus comprising the steps of: treating a sample to be treated with the salt tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5;

or (b)

A method of promoting plant growth comprising the steps of: the application of the salt tolerant aureobasidium (Microlunatus halotolerans) L1102 of claim 1 or the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5 to a plant or plant growth substrate.

10. Use of the salt tolerant aureobasidium parvulum (Microlunatus halotolerans) L1102 of claim 1 for the preparation of the culture of claim 2 or the metabolite of claim 3 or the microbial inoculum of claim 4 or 5.