CN113355258A

CN113355258A - Sphingobacterium HP10 and application thereof

Info

Publication number: CN113355258A
Application number: CN202110287417.0A
Authority: CN
Inventors: 郭琪; 彭轶楠; 张军; 王沛雅; 杨晖; 王治业
Original assignee: Institute of Biology of Gansu Academy of Sciences
Current assignee: Institute of Biology of Gansu Academy of Sciences
Priority date: 2021-03-17
Filing date: 2021-03-17
Publication date: 2021-09-07
Anticipated expiration: 2041-03-17
Also published as: CN113355258B

Abstract

The invention relates to the technical field of microorganisms, and particularly relates to a sphingosine bacillus HP10 and application thereof, wherein the sphingosine bacillus HP10 is preserved in China general microbiological culture collection management center at 1 month and 13 months in 2021, and the preservation number is CGMCC No. 21618. The strain has high azotase activity, can degrade spirodiclofen, can inhibit bacteria and secrete indoleacetic acid. Can be used for microbial nitrogen fertilizer and can also be used as a soil residual pesticide degradation agent; the sphingosine bacillus HP10 has great significance for planting hybrid broussonetia papyrifera in northwest region.

Description

Sphingobacterium HP10 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to a sphingosine bacillus HP10 and application thereof.

Background

The broussonetia papyrifera of the family of Moraceae is perennial fallen leaf arbor, is widely distributed in China, has long application history, and has very good development and utilization prospects. Currently, its main economic value is concentrated in the following 3 aspects: firstly, the leaves are used as the feed to replace grain crops to feed livestock, and the tree is used for replacing grain to become a new way for relieving the crisis of feed raw materials and ensuring the food safety; secondly, developing barks and branches as high-grade papermaking raw materials; thirdly, felling plants as medicinal materials. The hybrid broussonetia papyrifera is a new species with excellent properties, which is screened out by the Chinese academy of sciences plant research institute through wild broussonetia papyrifera resource collection, the core germplasm is taken as a male parent, the broussonetia papyrifera which belongs to the same shrub is taken as a female parent, the modern breeding technology and the conventional technology are combined for interspecific hybridization, and space carrying mutagenesis is carried out. Compared with wild broussonetia papyrifera, the broussonetia papyrifera hybrid has obvious heterosis, has the characteristics of strong adaptability, drought resistance, saline-alkali resistance, barren resistance, pollution resistance, plant diseases and insect pests resistance and the like, and has the advantages of high growth speed, high yield, high nutritional value and the like. When the feed is used as protein feed, the fresh weight yield per hectare in the high-yield period can be up to 150t at most, the content of crude protein in stems and leaves is 19.2 percent, the feed is superior to alfalfa, and the contents of trace elements such as calcium, phosphorus, zinc, iron, manganese, copper and the like, and functional active substances such as amino acid, flavonoid and the like are rich, so the feed is a high-quality woody crude feed source for livestock and poultry. In 2016 to 2020, hybrid paper mulberry trees have been widely planted in more than 20 provinces (cities) such as inner Mongolia, Henan, Guizhou, Shandong, Shanxi, etc. However, in northwest arid and semi-arid regions such as Gansu province and the like, the problems of low yield, low green return rate in the next year and the like exist in the growth of the hybrid paper mulberry, the development of large-scale production of tissue culture of the hybrid paper mulberry is restricted, and the large-area popularization of the hybrid paper mulberry in the northwest arid and semi-arid regions is hindered.

Meanwhile, mites are main insect pests after the tissue culture seedlings of the hybrid paper mulberry are transplanted to a field, spirodiclofen has a strong mite killing effect and is widely used for preventing and controlling red spiders and tetranychus urticae koch of the hybrid paper mulberry, but the residual period of the spirodiclofen is long, so that pollution to the soil environment and the water body is caused, potential harm is caused to the growth, quality safety and livestock and poultry health of the hybrid paper mulberry, and the development of livestock and poultry breeding industry needing green organic feed is seriously hindered.

Sphingobacteriaceae belong to the kingdom bacteriaceae (Bacteria), Bacteroidetes (bacteroides), Sphingobacteriaceae (sphingobacteriia), Sphingobacteriales (Sphingobacteriales), Sphingobacteriaceae (Sphingobacteriaceae), a class of gram-negative non-fermenting rod-shaped Bacteria, rarely cause human infection, and are mainly characterized by containing a large amount of cell membrane sphingomyelin. The current research shows that the Sphingobacterium has the function of biological desulfurization (see patent CN106754462B), the function of catalyzing the synthesis of L (+) -tartaric acid or salt thereof (see patent CN110591954B), or the function of degrading cembratriene-4, 6-diol (see patent CN107058162B) and aniline (see patent CN 106635904B). However, there is no disclosure in any study that sphingosine bacteria have a nitrogen-fixing effect, nor is there any disclosure that sphingosine bacteria have a spirodiclofen-degrading effect.

Aiming at the technical problems, the inventor unexpectedly separates a new sphingobacterium from rhizosphere soil of a hybrid broussonetia papyrifera, and the sphingobacterium has the advantages of fixing nitrogen, inhibiting bacteria in the rhizosphere soil, degrading spirodiclofen and the like.

Disclosure of Invention

The first purpose of the invention is to provide a sphingobacterium (Sphingobium aborkinnse) HP10 separated from rhizosphere soil of a broussonetia papyrifera, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.21618, and the address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, and the preservation time is 2021 year, 1 month and 13 days.

The second purpose of the invention is to provide the application of the Sphingobacterium aborkinse HP10 in preparing the biological fermentation organic fertilizer.

The third purpose of the invention is to provide the application of the Sphingobacterium aborkinse HP10 as a soil residual pesticide degrading agent.

Preferably, the soil residual pesticide is spirodiclofen.

The fourth purpose of the invention is to provide the application of the Sphingobacterium aborkinnse HP10 in preparing a microbial bacteriostatic agent for controlling bacterial pathogenic bacteria and fungal pathogenic bacteria.

Preferably, the bacterial pathogenic bacteria are one or more of fusarium solani and fusarium oxysporum, and the fungal pathogenic bacteria are alternaria solani.

The fifth purpose of the invention is to provide the application of the Sphingobacterium aborkinnse HP10 in promoting plant growth.

Preferably, the plant is a hybrid paper mulberry.

The sixth purpose of the invention is to provide the application of the Sphingobacterium aborkinse HP10 in the preparation of soil conditioners.

The seventh purpose of the invention is to provide a microbial agent, which contains the sphingosine (Sphingobium abikonense) HP 10.

The invention has the beneficial effects that: firstly, the invention separates a sphingosine bacillus HP10 from the rhizosphere soil of the hybrid broussonetia papyrifera, which is preserved in China general microbiological culture Collection center (CGMCC),the preservation number is CGMCC No. 21618; ② the Sphingobacterium HP10 has higher nitrogen-fixing enzyme activity, the nitrogen-fixing enzyme activity is 177.5nmol (C)₂H₄)·h^-1·mL^-1Can be used as a microbial nitrogen fertilizer for planting the tissue culture seedlings of the hybrid broussonetia papyrifera; thirdly, the sphingosine bacillus HP10 can degrade the spirodiclofen, and is used as a soil residual pesticide degradation agent to repair soil polluted by spirodiclofen, improve the quality safety of the hybrid broussonetia papyrifera and protect the health of livestock and poultry; the sphingosine bacillus HP10 has the capability of inhibiting fusarium solani, alternaria solani and fusarium oxysporum, the inhibition rates are respectively 44.44%, 45.83% and 26.82%, and the sphingosine bacillus HP10 can be used as a microbial antibacterial agent for preventing and treating plant pathogenic bacteria; the sphingosine bacillus HP10 can secrete indoleacetic acid with the secretion amount of 2.26 mug. mL^-1Can promote the growth of plants. In conclusion, the sphingosine bacillus HP10 has great significance for planting hybrid broussonetia papyrifera in northwest China.

Drawings

FIG. 1 photograph of sphingosine bacillus HP10 colony

FIG. 2 Effect of Sphingobacterium HP10 on antagonizing pathogenic microorganisms

Detailed Description

The present invention is described in detail below with reference to specific embodiments, which will assist those skilled in the art in further understanding the present invention, but do not limit the present invention in any way.

The experimental procedures used in the following examples are conventional unless otherwise specified.

The Ashby medium, LB broth, and MSM broth used in the following examples were conventional products.

The spirodiclofen standards used in the following examples were provided by Beijing Zhongke quality inspection Biotechnology Ltd.

Fusarium solani GSICC60605 and Azotobacter chroococcum GSICC30117 used in the following examples were obtained from the Ministry of Gansu of China center for culture Collection of Industrial microorganisms to repeat the experiments of the present application.

Alternaria solani ACCC37717 and Fusarium oxysporum ACCC30373, which are used in the following examples, were publicly available from the China general culture Collection for microorganisms to repeat the experiments of the present application.

The "salt tolerance test" described in the following examples is a test for detecting the salt tolerance of microorganisms using media containing sodium chloride at various concentrations.

The "sugar fermentation test" described in the following examples was conducted to examine the ability of microorganisms to utilize various carbon sources differently, on the principle that different microorganisms have different enzyme systems. Most bacteria can use sugars as a carbon source, but they have a large difference in their ability to decompose sugar substances. Some bacteria can decompose certain sugar to generate organic acid (such as lactic acid, acetic acid, propionic acid and the like) and gas (such as hydrogen, methane, carbon dioxide and the like), and some bacteria only generate acid and do not generate gas.

The "starch hydrolysis test" described in the following examples is a test in which starch, a polysaccharide, is hydrolyzed to monosaccharides. The microorganisms cannot directly utilize macromolecular starch, and the macromolecular substances can only be absorbed and utilized by the microorganisms by decomposing the macromolecular substances by the produced extracellular enzymes. Extracellular enzymes are primarily hydrolases which act to degrade substances with a high molecular weight into smaller compounds which can be transported into the cell. For example, amylases hydrolyze starch to small dextrins, disaccharides and monosaccharides. The starch turns blue when encountering iodine liquid.

The citrate utilization test described in the following examples is a test in which some bacteria such as Aerobacter aerogenes can utilize sodium citrate as a carbon source, and thus can grow on a citrate medium, and decompose citrate to produce carbonate, thereby making the medium alkaline. The bromothymol blue indicator in the medium changed from green to dark blue at this time. Bacteria that cannot utilize citrate as a carbon source do not grow on the medium and the medium does not change color.

"catalase reaction" as described in the following examples means that the number of bacteria is estimated from the floating time in a test tube containing H2O2 by counting the number of catalase-containing disks. The catalase reacts with H2O2 to release oxygen, the higher the content of catalase-positive bacteria in the sample is, the more oxygen is released, and the shorter the floating time of the paper disc is; conversely, the longer the paper tray floats. Most of spoilage microorganisms are psychrophilic bacteria, and most of them are positive for exposure to enzymes, so they can be assayed for psychrophilic bacteria in food by exposure to enzyme reaction.

The "acetylene reduction method" described in the following examples refers to a method of measuring the amount of acetylene reduced to ethylene by a gas-fixed enzyme using a gas chromatograph to estimate the activity of the nitrogenase or the amount of nitrogen. The nitrogen-fixing enzyme can not only catalyze N₂Reduction to NH₃And can also catalyze the reduction of acetylene to ethylene. Ethine and N₂The affinities to the azotase are similar, and the reduction processes of the azotase and the azotase are related in parallel. Acetylene and ethylene can be separated by gas chromatography, and the quantitative determination can be carried out sensitively and rapidly, thereby indirectly estimating the nitrogen-fixing enzyme activity of the organism. Reaction formula N₂+6H ⁺+6e^-→2NH₃，3C₂H₂+6H⁺+6e^-→3C₂H₄It is shown that reducing 3 g of acetylene is equivalent to reducing 1g of nitrogen, so the amount of nitrogen fixation can be estimated from this. In 1968, the activity of biological nitrogen fixation enzyme is measured by using an acetylene reduction method, such as hayashi (R.W.F.Harday), and the biological nitrogen fixation amount in laboratories and fields is calculated according to the activity. The quantitative calculation method of the azotase activity comprises an external standard method, an area normalization method, an internal standard method, an external pure sample method and the like. The method is divided into the following steps according to the measurement of the azotobacter activity of different azotobacter systems: the whole plant measuring method includes culturing plant in sealed apparatus for 1 day, injecting acetylene, sampling at certain time and measuring the amount of ethylene reduced. The method can be used for measuring the nitrogenase activity of leaves and roots. ② in vitro root system or root nodule determination method, taking root system segment or root nodule from soil, in vitro culturing for a certain time, injecting acetylene to determine its azotobacter activity. Thirdly, in-situ measurement, inserting a metal cylinder into soil around the plant, sealing the whole plant by using a plastic bag, injecting acetylene, and periodically measuring the ethylene production. This method is commonly used to determine the activity of the rhizosphere-associated nitrogenase in rice.

EXAMPLE 1 Strain isolation

Weighing 10g of soil sample (collected from Lanzhou city, Gansu province), filling into a triangular flask, adding 100mL of sterile water, standing for 20min, rotating at 28 ℃ for 200 r.min^-1And shaking for 30min to form a suspension. 5mL of the suspension is sucked and added into 100mLAshby liquid culture medium, the temperature is 28 ℃, and the rotating speed is 180 r.min^-1After enrichment culture for 72h, the product is prepared into 10 by adopting a conventional gradient dilution method^-4、10^-5、 10^-6、10^-7After the gradient bacterial suspension is diluted, 0.1mL of bacterial suspension is sucked and coated on an Ashby culture medium plate, each gradient is repeated for 3 times, and the culture is carried out for 5 d-7 d at the constant temperature of 28 ℃. The above separation operation was repeated 3 times. After the isolated colonies grew out, the strain was further purified on an Ashby medium plate by streaking. The bacterial strain with the diameter of 0.5-1 mm, the color of the bacterial colony being milky white, the bacterial colony being opaque, the surface being wrinkled, the edge being irregular and the later stage being brown is obtained, the specific morphology is shown in figure 1, the bacterial strain is named as HP10, and the bacterial strain is identified by separation treatment.

Example 2 strain identification.

The isolated and purified strains were identified from the following three aspects.

1. Morphological identification

The HP10 isolated in example 1 was subjected to single colony culture and colonies were observed, mainly including the size, color, transparency, surface state (whether flat, convex, wrinkled or concave, etc.), edge state (whether neat, irregular or radial, etc.), and late growth state of the colonies. And performing microscopic examination after staining by a biological stain.

The results show that the strain HP10 obtained after isolation and purification had the colony: the diameter of the bacterial colony is 0.5-1 mm, the color of the bacterial colony is milky white, the bacterial colony is opaque, the surface is wrinkled, the edge is irregular, and the later stage is brownish. The microscopic examination result is gram negative, short rod shape and no spore.

2.16s rDNA sequence homology isolation

Culturing the strain HP10 separated and purified by the steps by a conventional method, extracting single colony genomic DNA by using a bacterial genomic DNA extraction kit (Beijing Soilebao science and technology Co., Ltd.), sending the single colony genomic DNA to 16S rDNA of bioengineering (Shanghai) GmbH for sequencing, judging the type of bacteria after homology comparison between the sequencing result and a known sequence in NCBI GenBank, and dividing the bacteria into genera or species.

The 16s rDNA gene fragment 1401bp of the strain HP10 has the following sequence:

CCGGTGCGGGCATGCCTATACATGCAGTCGAACGAGATCTTCGGATCTAGTGGCGCACG GGTGCGTAACGCGTGGGAATCTGCCCTTGGGTTCGGAATAACAGTTGGAAACGACTGCT AATACCGGATGATGACGAAAGTCCAAAGATTTATCGCCCAAGGATRARSSMGYKKAGG WYYRSSTAGKWKKWGSKRKRWWGKSSKWYMARSSYRWCRRYSMYKAKCTRGYYKKM GAGRRKRWKMWSMSMMMMYKGGACTGAGACACGGYCCAGACTCCTACGGGAGGCA GCAGTAGGGAATATTGGACAATGGGGGCAMCCYKRWYCCAGCAATGCCGCGTGAGTGA TGAAGGCSTWRGGGTTGTAAAGCTCYTTTACCCGGGATGATAATGACAGTACCGGGAGA ATAAGCCCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGGGCTAGCGTTGT TCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGCGATTTAAGTCAGAGGTGAAAGCC CGGGGCTCAACCCCGGAACTGCCTTTGAGACTGGATTGCTTGAATCCGGGAGAGGTGA GTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAA GGCGGCTCACTGGACCGGCATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGG ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGCCGGGGCACATGGT GTTTCGGTGGCGCAGCTAACGCATTAAGTTATCCGCCTGGGGAGTACGGTCGCAAGATT AAAACTCAAAGGAATTGACGGGGGCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCG AAGCAACGCGCAGAACCTTACCAACGTTTGACATCCTCATCGCGATTTCCAGAGATGGA TTTCTTCAGTTCGGCTGGATGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCG TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTGCCAGCATTT AGTTGGGTACTCTAAAGGAACCGCCGGTGATAAGCCGGAGGAAGGTGGGGRWKRMSK YMAGTCYYMWKRKCCCTTACGCGTKGSTACAACGTGCTACAYRCGWSYWMMRWKGS MGMSWMYMGYGRGSRGMGMYMTCSYSMRRRRRWSCKWMTCTCMRRWWSTCGTCTC AGTTCGGATCGTTCTCTGCAACTCGAGAGCGTGAAGGCGGAATCGCTAGTAATCGCGGA TCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTCACACCATGG GAGTTGGATTCACTCGAAGGCGTTGAGCTAACCCGCAAGGGAGGCAGGCGACCACAGT GGTTGCTC

after the sequence determination, the homology of HP10 with Sphingobacterium (Sphingobium abikonense) is the highest and reaches 98.70 percent by online homology comparison with the published 16s rDNA sequence in NCBI database.

3. Physiological and biochemical characteristic analysis

The determination result of the physiological and biochemical characteristics of the sphingosine bacillus HP10 is as follows:

growth temperature: no growth at 4 ℃, growth at 28 ℃ and growth at 37 ℃;

salt tolerance test: 0.5% NaCl grows, 3% NaCl grows, and 5-10% NaCl does not grow;

sugar fermentation experiment: glucose positive, D-xylose positive, mannitol negative, lactose positive, cellulose positive and L-arabose positive;

hydrolysis starch test: negative;

using citrate: positive;

hydrolyzing casein: positive;

and (3) carrying out a catalase reaction: positive;

lipase reaction: and (4) negativity.

In conclusion, HP10 can grow on a culture medium containing 0.5% -3% NaCl, has salt tolerance, and can utilize glucose, D-xylose, lactose, cellulose and L-arabinose; the ability to utilize citrate; is capable of hydrolyzing casein; and produce a peroxidase.

In view of the above morphology, 16s rDNA sequence homology analysis and physiological and biochemical characteristic analysis results, the strain isolated and purified in example 1 was determined to be Sphingobacterium in the bacterial domain, named Sphingobacterium aborkinse (HP 10), and deposited in the China general microbiological culture Collection center (CGMCC) at 1/13/2021, with the preservation number of CGMCC No.21618, address: the contact telephone number of Xilu No. 1 Hospital No. 3, Beijing, Chaoyang, the district of rising Yang: 010-64807288.

In the following examples, sphingobacterium (Sphingobium abikonense) HP10 is abbreviated as sphingobacterium HP10 to more clearly show the technical solution of the present invention.

Example 3 Nitrosclerobacillus HP10 Activity assay for Nitrogen-fixing enzymes

The activity of the nitrogen-fixing enzyme of the sphingosine bacillus HP10 separated and purified in the example 1 is tested by adopting an acetylene reduction method, and the specific method is as follows:

one ring of sphingosine bacillus HP10 was inoculated into a serum bottle containing 5mL of semi-solid Ashby medium and cultured at 28 ℃ for 48 h. The serum bottle cap was replaced with a rubber stopper in a sterile bench, 1mL of gas was evacuated with a sterile syringe, then 1mL of acetylene was injected, sealed, and cultured at 28 ℃ for 36 h. 0.2ml of the mixed gas was extracted from the serum bottle by a sterile syringe and injected into a gas chromatograph, and the amount of ethylene produced was measured to calculate the nitrogenase activity according to the following formula.

N ═ hxCV/(24.9 hst). Wherein N is the concentration of produced ethylene nmol (C)₂H₄)·h^-1·mL^-1Namely, the azotase activity, hx is the area of the ethylene peak of the sample, and C is the standard ethylene concentration (nmol. ml)^-1) V is the culture vessel volume (mL), 24.9 is a constant, hs is the standard ethylene peak area value, and t is the sample incubation time (h). Control azotobacter chroococcum is cultured in nitrogen-fixing medium, and its azotase activity is determined by the same method as reference. The above experiments were repeated in three groups.

The results showed that the isolated and purified Sphingobacterium HP10 CGMCC No.21618 of example 1 has a nitrogenase activity of 177.5nmol (C)₂H₄)·h^-1·mL^-1The azotobacter chroococcum has the azotobacter activity of only 124.60nmol (C)₂H₄)·h^-1·mL^-1. The result shows that the sphingosine bacillus HP10 has higher nitrogen fixation capacity than the strain azosine toruloides used in the traditional biological nitrogen fixation fertilizer, and can be used for preparing nitrogen fixation microbial agents and producing biological organic fertilizers.

Example 4 Spiromesifen degradability of Sphingobacterium HP10

Inoculating Sphingobacterium HP10 into LB liquid medium, and culturing at 28 deg.C for 180r min^-1Shaking culture at 4 deg.C and 10000 r.min^-1Centrifuging the bacterial liquid for 5min, washing with MSM culture medium for 1 time, suspending sufficiently to logarithmic phase bacterial count, inoculating with inoculum size of 5% containing 50, 100, 150 mg.L^-1MSM culture medium of spirodiclofen standard sample at 28 deg.c for 180r min^-1Shaking table culture, adding 2mL of culture solution into a test tube with a plug every 24h, adding 1g of sodium chloride and 2mL of ethyl acetate, shaking vigorously for 5min, and standing. Taking 1mL of the upper layer solvent to volatilize, then adding acetonitrile to a constant volume of 5mL, and then adding 0.45Mu.m filter membrane filtration, using HPLC (chromatograph model: Agilent model 1260; chromatographic column: pre-separation column + Eclipse plus C18, 4.6 mm. times.100 mm, 3.5 μm; mobile phase: acetonitrile, flow rate 1 mL. min.)^-1Detecting the content of the spirodiclofen at the wavelength of 250nm, the column temperature of 30 ℃ and the sample injection amount of 10 mu L) according to a formula: degradation rate (1-C)_{Spirodiclofen concentration at sampling}/C_{Initial spirodiclofen concentration}) X 100%, calculating the degradation rate of spirodiclofen in the culture solution, and the experimental results are shown in table 1:

TABLE 1 degradation rate of spirodiclofen in the culture broth

The results show that the Sphingobacterium HP10 has obvious degradation effect on the spirodiclofen, and when the concentration of the spirodiclofen is 100 mg.L^-1When the bacillus sphingosine HP10 is used, the degradation rate of the spirodiclofen is 56.38% at 72 hours, and the degradation efficiency is highest. In conclusion, the sphingosine bacillus HP10 can be used as a soil conditioner to degrade residual spirodiclofen in soil.

Example 5 inhibition of Sphingobacterium HP10 against Alternaria solani (Alternaria solani), Fusarium solani (Fusarium solani), Fusarium oxysporum (Fusarium oxysporum) test

The inhibition of the bacterium Alternaria solani (Alternaria solani), Fusarium solani (Fusarium solani) and Fusarium oxysporum (Fusarium oxysporum) by the Sphingobacterium HP10 was determined by the paper strip method. Culturing Sphingobacterium HP10 in LB liquid culture medium until the concentration of the bacterial liquid reaches OD₆₀₀When the strain is equal to 1, taking the bacterial liquid for later use. A7 mm diameter piece of mycelia of Alternaria solani (Alternaria solani), Fusarium solani (Fusarium solani), and Fusarium oxysporum (Fusarium oxysporum) was inoculated in the center of a PDA medium plate, respectively, and the following three treatments were performed:

a comparison: no treatment is carried out;

b sterile water filter paper strip control: placing filter paper strips soaked with sterile water at a distance of 1.5cm from both sides of the hypha block;

c antagonism test: and placing filter paper strips soaked with the azotobacter to-be-detected bacteria liquid at the positions 1.5cm away from the two sides of the hypha block, culturing at 28 ℃ for 7d, and observing the bacteriostatic effect.

When the sterile water filter paper strip contrast is paved on the whole flat plate, the contrast and the diameter of the treated bacterial colony are measured in the direction vertical to the filter paper strip, and the inhibition rate is calculated.

Inhibition ═ inhibition (sterile water filter strip control colony diameter-treated c colony diameter)/sterile water filter strip control colony diameter × 100%.

As shown in FIG. 2, the inhibition ratios of Sphingobacterium HP10 against Alternaria solani (Alternaria solani), Fusarium solani (Fusarium solani) and Fusarium oxysporum (Fusarium oxysporum) were 44.44%, 45.83% and 26.82%, and they were used as microbial bacteriostatic agents.

Example 6 determination of the ability of Sphingobacterium HP10 to secrete Indolylacetic acid

Inoculating isolated and purified Sphingobacterium HP10 into LB liquid medium containing L-tryptophan (100mg/L), and culturing at 28 deg.C for 180 r.min^-1OD600 of suspension of bacteria cultured on shaking table_nmWhen the temperature is 1 ℃, 4 ℃, 10000 r.min^-1Centrifuging for 10min, collecting supernatant, adding Salkowski (50mL 35% HClO)₄+1mL0.5M FeCl₃) Standing the colorimetric solution in dark place for 30min, and determining its OD_530nmThe value of (c). Calculating the bacterial concentration OD_600nmAt a value of 1, the content of indoleacetic acid per unit volume of fermentation broth. The standard curve was prepared by gradient dilution of analytically pure indoleacetic acid.

As a result, the amount of IAA secreted from the suspension of Sphingobacterium HP10 was 2.26. mu.g/mL^-1Has the capability of promoting the growth of plants.

In conclusion, the invention separates a sphingosine bacillus HP10 from the rhizosphere soil of the hybrid broussonetia papyrifera, which is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of CGMCC No. 21618; the Sphingobacterium HP10 has high nitrogenase activity, and the nitrogenase activity is 177.5nmol (C)₂H₄)·h^-1·mL^-1Can be used as a microbial nitrogen fertilizer for planting the tissue culture seedlings of the hybrid broussonetia papyrifera; the Sphingobacterium HP10 can degrade spirodiclofen serving as soil residueThe pesticide degradation agent is remained to repair the soil polluted by the spirodiclofen, improve the quality safety of the hybrid broussonetia papyrifera and protect the health of the livestock and poultry; the sphingosine bacillus HP10 has the capability of inhibiting fusarium solani, alternaria solani and fusarium oxysporum, the bacteriostasis rates are respectively 44.44%, 45.83% and 26.82%, and the sphingosine bacillus HP10 can be used as a microbial bacteriostat for preventing and treating bacterial pathogenic bacteria; the sphingosine bacillus HP10 can secrete indoleacetic acid with the secretion amount of 2.26 mu g/mL^-1The sphingosine bacillus HP10 has great significance for planting hybrid broussonetia papyrifera in northwest region.

Claims

1. A sphingosine bacillus (Sphingobium abikonense) HP10 is characterized in that the Sphingobium bacillus (Sphingobium abikonense) HP10 is preserved in China general microbiological culture collection center at 1 month and 13 months of 2021, and the preservation number is CGMCC No. 21618.

2. Use of a sphingobacterium (Sphingobium abikonense) HP10 according to claim 1 for the preparation of a biofermentation organic fertilizer.

3. Use of a sphingobacterium (Sphingobium abikonense) HP10 according to claim 1 as a soil residual pesticide degrading agent.

4. The use according to claim 3, wherein the soil pesticide residue is spirodiclofen.

5. Use of a sphingobacterium (Sphingobium abikonense) HP10 according to claim 1 for the preparation of a microbial bacteriostatic for the control of bacterial pathogens.

6. The use according to claim 5, wherein the bacterial pathogen is one or more of Fusarium solani (Fusarium solani) and Fusarium oxysporum (Fusarium oxysporum), and the fungal pathogen is Alternaria solani (Alternaria solani).

7. Use of a bacterium sphingosine (Sphingobium abikonense) HP10 according to claim 1 for promoting plant growth.

8. The use of claim 7, wherein the plant is a Broussonetia papyrifera.

9. Use of a sphingobacterium (Sphingobium abikonense) HP10 according to claim 1 for the preparation of a soil amendment.

10. A microbial agent comprising the bacterium Sphingobacterium (Sphingobium abikonense) HP10 according to claim 1.