CN111518726B - Pseudomonas aeruginosa and screening method and application thereof - Google Patents

Pseudomonas aeruginosa and screening method and application thereof Download PDF

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CN111518726B
CN111518726B CN202010388306.4A CN202010388306A CN111518726B CN 111518726 B CN111518726 B CN 111518726B CN 202010388306 A CN202010388306 A CN 202010388306A CN 111518726 B CN111518726 B CN 111518726B
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pseudomonas aeruginosa
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马江山
刘高强
李培旺
黎继烈
刘汝宽
李昌珠
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Central South University of Forestry and Technology
Hunan Academy of Forestry
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Abstract

Pseudomonas aeruginosa and screening method and application thereof, wherein pseudomonas aeruginosa (pseudomonas aeruginosa)Pseudomonas aeruginosa) The strain number of the strain is LYT-4, and the strain is preserved in the China general microbiological culture Collection center on 26 months in 2018, wherein the preservation number is CGMCC No. 15519; the screening method comprises the following steps: (1) adding water into the naturally piled and fermented plant meal, uniformly stirring, and standing; (2) gradient dilution, coating inoculation and constant temperature culture; (3) and (4) picking out the grown colonies, repeatedly carrying out streak inoculation, screening the strains with high content of amino nitrogen, and identifying the 16SrDNA as the pseudomonas aeruginosa. The pseudomonas aeruginosa is applied to the protein fermentation of plant meal or antagonism of plant pathogenic fungi. The method is convenient and quick, and the purity of the screened strains is high. The invention has simple application and operation and low cost, and is suitable for agricultural production.

Description

Pseudomonas aeruginosa and screening method and application thereof
Technical Field
The invention particularly relates to pseudomonas aeruginosa and a screening method and application thereof.
Background
The cake is a plant residue left after the extraction of the plant oil, and the protein content of the cake is high, but the cake is difficult to use and is mostly discarded. For example, the tung oil cake (tung meal) is a main byproduct left after tung oil is extracted from tung seeds, the protein content of the tung oil cake can reach 36-45%, the amino acid content and the essential amino acid content extracted from the tung oil cake can respectively reach 93.8% and 45.4%, and the tung oil cake is an excellent plant protein resource for development and utilization. Besides high-content protein, the tung meal also contains 1-3% of phosphorus and 1-2% of potassium and other trace elements required by plants, and is an ideal plant fertilizer matrix capable of being developed and utilized. Although China has the first tung oil yield in the world and the annual tung meal yield reaches 50 ten thousand tons, the utilization rate of the tung meal resources is less than 20 percent at present, most of the tung meal resources are discarded, the application value of the resources is not exerted, and the tung meal resources can also become a new pollution source. With the rise of the sustainable development trend of green agriculture and forestry in the world, the method for fermenting the tung seed meal into the amino acid organic fertilizer with high added value by using related functional microorganisms is a tung seed meal utilization mode with great prospect at present based on the strategic demand of zero increase of chemical fertilizer and pesticide in China. There are few reports on the protein fermentation of pseudomonas aeruginosa, particularly the crude protein in tung meal.
Antagonistic plant pathogenic fungi are key factors for ensuring high yield of crops. Since the abuse of pesticides does not meet the general pursuit of green foods, microbial fungicides are a natural choice for the inhibition of plant pests.
CN109439561A discloses a Pseudomonas aeruginosa strain and application thereof, wherein the Pseudomonas aeruginosa strain comprises cabbage black spot pathogen, sugarcane pineapple pathogenic bacteria, kumquat husky bark pathogen and rice flax leafThe alternaria alternata and the rhizoctonia solani and the like show stronger antagonistic performance, but the anthracnose of flax (A), (B) and (C)Colletotrichum lini) And Fusarium oxysporum (F.), (Fusarium oxysporum) And the antagonistic performance of the strain is tested, and the strain is not researched to be applied to the fermentation of the plant meal.
CN 11061616179A discloses a pseudomonas aeruginosa DGNK-JL2 and application thereof, the pseudomonas aeruginosa shows a good phosphate solubilizing effect, but the capability of fermenting plant meal is not researched; the pseudomonas aeruginosa has a certain antagonistic effect on pathogenic fungi such as pepper anthracnose and bitter melon fusarium wilt, but does not mention the bacteriostasis rate, and does not have the effect on flax anthracnose bacteria: (Colletotrichum lini) Flax anthracnose bacterium (A)Colletotrichum lini) And Fusarium oxysporum (F.), (Fusarium oxysporum) Antagonistic performance studies were performed.
At present, microbial resources with functions of tung meal crude protein fermentation and plant pathogenic fungus antagonism are reported, and screening and development of the strain resources with double functions are beneficial to high-value utilization of high-protein plant meal resources and improvement of economic activity of plant oil processing industry, and development of the strain resources into cake meal-based organic fertilizer with functions of fertilizer supply and prevention can avoid dependence of crops on chemical fertilizers and pesticides to a certain extent.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides pseudomonas aeruginosa with functions of plant meal protein fermentation and plant pathogenic fungi antagonism.
The invention further aims to solve the technical problem of overcoming the defects in the prior art and providing a screening method for screening pseudomonas aeruginosa with high strain purity, which is convenient and quick.
The invention further aims to solve the technical problem of overcoming the defects in the prior art and provide the application of the pseudomonas aeruginosa for the agricultural production, wherein the amino acid conversion rate of the protein fermentation of the plant meal is high, the effect of resisting plant pathogenic fungi is obvious, the operation is simple and convenient, the cost is low, and the application is suitable.
The technical scheme adopted by the invention for solving the technical problems is as follows: pseudomonas aeruginosa, the pseudomonas aeruginosaPseudomonas aeruginosa) The strain number of the strain is LYT-4, and the strain is preserved in the China general microbiological culture Collection center on 26 months in 2018, and the preservation number is CGMCC number 15519. Said Pseudomonas aeruginosaPseudomonas aeruginosa) The sequence of 16S rDNA (GenBank: MF 967440.1) of LYT-4 is shown in SEQ ID NO: 1.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) LYT-4 has rod-like thallus size of 0.5 μm × 1.5 μm-1 μm × 4 μm, gram-negative property, flagellum, and no fluorescence; colonies on the NA medium are yellowish green moist, flat and moist, irregular in edges and smooth in surface.
The technical scheme adopted by the invention for further solving the technical problems is as follows: a screening method of pseudomonas aeruginosa comprises the following steps:
(1) adding the naturally piled and fermented plant meal into sterile water, stirring uniformly, and standing to obtain a strain stock solution;
(2) after being diluted in a gradient manner, the strain stock solution obtained in the step (1) is coated and inoculated on an NA culture medium and cultured at a constant temperature;
(3) and (3) picking out colonies growing on the NA culture medium in the step (2), repeatedly streaking and inoculating the colonies to a new NA culture medium, screening a strain with high content of amino nitrogen, and identifying the 16SrDNA as pseudomonas aeruginosa.
Preferably, in the step (1), the fermentation conditions of the naturally piled and fermented plant meal are outdoor natural piling.
Preferably, in the step (1), the protein content in the plant meal is 36-45%.
Preferably, in the step (1), the plant meal is tung meal or the like.
Preferably, in the step (1), the mass-to-volume ratio (g/mL) of the plant meal to the sterile water is 1: 8-10.
Preferably, in the step (1), the stirring time is 10-30 min.
Preferably, in the step (1), the standing time is 25-35 min.
Preferably, in step (2), the dilution gradient is 10 times-8～10-9。
Preferably, in the step (2), the temperature of the constant-temperature culture is 25-35 ℃ and the time is 30-40 h.
Preferably, in steps (2) and (3), the formula of the NA culture medium is as follows: 10.0 g/L of peptone, 3.0 g/L of beef extract, 5.0 g/L of NaCl, 15.0 g/L of agar and 7.0-7.2 of pH.
Preferably, in the step (3), the number of times of the repeated streaking is 3-4.
In the step (3), the bacterial strain with high content of amino nitrogen is identified and screened as pseudomonas aeruginosa by combining the colony morphological characteristics, physiological and biochemical characteristics and the system analysis result based on the 16S rDNA gene sequencePseudomonas aeruginosa。
The technical scheme adopted by the invention for further solving the technical problems is as follows: the application of the pseudomonas aeruginosa in the plant meal protein fermentation or the antagonism of plant pathogenic fungi is disclosed. The pseudomonas aeruginosa can secrete high-activity protease to decompose crude protein in plant meal into amino acid, and the content change of the produced amino acid is measured by detecting the content of amino nitrogen, wherein the produced amino acid is from the degradation of the crude protein; in addition, pseudomonas aeruginosa can also secrete a plurality of secondary metabolites with bacteriostatic effects such as siderophins, phenazines, pyoluteorins and the like, and the pathogenic fungi are antagonized through the secondary metabolites.
Preferably, the protein content in the plant meal is 36-45%.
Preferably, the plant meal is tung meal or the like.
Preferably, the specific operation of the plant meal protein fermentation is as follows: inoculating the seed liquid of the pseudomonas aeruginosa to a plant meal solid fermentation culture medium, uniformly mixing, and fermenting to obtain a fermentation product. In the fermentation process, the pseudomonas aeruginosa strain grows by using plant meal as a carbon source and an energy source substance, and crude protein in the plant meal can be degraded by protease secreted by the pseudomonas aeruginosa strain to generate amino acid micromolecules.
Preferably, the volume-to-mass ratio (mL/g) of the seed liquid to the plant meal is 8-12: 100. Too small or too large a vaccination volume may affect the crude protein degradation effect.
Preferably, the solid fermentation medium consists of plant meal and an inorganic salt solution in a mass-to-volume ratio (g/mL) of 2.5-3.5: 1. The fermentation is solid state fermentation, the plant meal is simultaneously used as a carbon source, and the inorganic salt solution enables the water content of the plant meal to reach more than 50%.
Preferably, the formulation of the inorganic salt solution is: (NH)4)2SO4 1.5～2.5g/L，K2HPO4 1.5～2.5g/L，KH2PO4 1.5～2.5g/L，MgSO4 0.15～0.25g/L，CaCl2 0.05～0.15g/L，MnSO4 0.015～0.025g/L，FeSO40.045-0.055 g/L and pH 7.0-7.2. The inorganic salt ions in the formula can provide trace elements required by the growth of the strain.
Preferably, the fermentation temperature is 25-35 ℃, the relative humidity is 70-80%, and the fermentation time is 5-6 days. The fermentation conditions are more favorable for the growth of the strain, the production of protease and the degradation of crude protein in the plant meal.
Preferably, the phytopathogenic fungus is Colletotrichum flaccidum (B.) (Colletotrichum lini) Linum Linearioides (C. Linearioides) ((C. Linearioides))Rhizoctonia solani) Or Fusarium oxysporum (F.), (Fusarium oxysporum) And the like. The pseudomonas aeruginosa can secrete a plurality of secondary metabolites with bacteriostatic effects such as siderophins, phenazines, pyoluteorins and the like, and the pathogenic fungi are antagonized by the secondary metabolites.
Preferably, said antagonistic phytopathogenic fungi are specifically operated by: and (3) inoculating the seed solution of the pseudomonas aeruginosa to a PDA culture medium on which the plant pathogenic fungi grow, and carrying out inverted culture to obtain the pseudomonas aeruginosa seed solution.
Preferably, the inoculation amount of the seed solution of the pseudomonas aeruginosa is 80-120 mu L.
Preferably, the inoculation position is 2-3 cm away from the cross point in the center of the flat plate. Preferably, the inoculation positions are equidistant from each other.
Preferably, the temperature of the inverted culture is 20-30 ℃, and the time is 4-6 days.
Preferably, the preparation method of the PDA culture medium for growing the plant pathogenic fungi comprises the following steps: inoculating the plant pathogenic fungi to the center of a PDA culture medium, and performing inverted culture at the temperature of 20-30 ℃ for 20-30 hours until hyphae grow out.
Preferably, the formula of the PDA culture medium is as follows: 200g/L of potatoes, 20g/L of glucose and 15-20 g/L of agar.
Preferably, the concentration of the seed liquid is 3.0 × 106～3.0×108cfu/mL。
Preferably, the seed solution is obtained by inoculating the pseudomonas aeruginosa into an NB liquid culture medium and culturing the pseudomonas aeruginosa to a logarithmic phase.
Preferably, the formula of the NB liquid medium is as follows: 10.0 g/L of peptone, 3.0 g/L of beef extract, 5.0 g/L of sodium chloride and 7.2-7.5 of pH.
The invention has the following beneficial effects:
(1) the invention relates to pseudomonas aeruginosaPseudomonas aeruginosa) The protease activity after LYT-4 fermentation of the tung meal can reach 2.18U/mL, which is equivalent to 1.7 times of the maximum enzyme activity value of natural fermentation, the content of amino nitrogen released after 5 days of fermentation can reach 37.3 mg/mL, which is equivalent to 1.4 times of natural fermentation, and the content of amino acid in the tung meal after 6 days of fermentation is obviously improved and is higher than that of natural fermentation; and the pseudomonas aeruginosa LYT-4 can effectively antagonize colletotrichum lini, (B) fColletotrichum lini) Linum Linearioides (C. Linearioides) ((C. Linearioides))Rhizoctonia solani) And Fusarium oxysporum (F.), (Fusarium oxysporum) And other plant pathogenic fungi; the screened pseudomonas aeruginosa LYT-4 has the functions of plant meal protein fermentation and plant pathogenic fungi antagonism, belongs to excellent microbial resources, and has the potential of being developed as biocontrol bacteria;
(2) the screening method is convenient and quick, and the purity of the screened strains is high;
(3) the pseudomonas aeruginosa LYT-4 of the invention has wide application, high amino acid conversion rate of protein fermentation, obvious effect of antagonizing plant pathogenic fungi, simple operation and low cost, can convert plant dregs into amino acid organic fertilizer with high added value and biocontrol function, and is suitable for agricultural production.
Biological material deposit information description: classification nomenclature of biological materials: pseudomonas aeruginosa; latin literature name of biomaterial:Pseudomonas aeruginosa(ii) a The preservation unit is called as follows: china general microbiological culture Collection center; the preservation unit is abbreviated as: CGMCC; address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 26 months 3 in 2018; the preservation number is as follows: CGMCC number 15519.
Drawings
FIG. 1 is a colony morphology of the invention Pseudomonas aeruginosa LYT-4 on NA medium;
FIG. 2 is a phylogenetic tree diagram of the invention, Pseudomonas aeruginosa LYT-4, based on the 16S rDNA sequence;
FIG. 3 is a graph comparing the change in protease activity during LYT-4 fermentation of Pseudomonas aeruginosa according to the invention with naturally fermented tung meal;
FIG. 4 is a graph comparing the variation of amino nitrogen content during fermentation of Pseudomonas aeruginosa LYT-4 of the present invention with naturally fermented tung meal;
FIG. 5 is a graph comparing the amino acid content of naturally fermented and unfermented tung meal after LYT-4 fermentation of Pseudomonas aeruginosa according to the invention;
FIG. 6 shows the combination of Pseudomonas aeruginosa LYT-4 and Colletotrichum flaccidum of the present invention: (A)Colletotrichum lini) Linum Linearum Raeumannomyces (L.) KuntzeRhizoctonia solani) And Fusarium oxysporum (F.), (Fusarium oxysporum) The flat plate of (2) is faced to each other.
Detailed Description
The invention is further illustrated by the following examples and figures.
The tung meal used in the embodiment of the invention is purchased from Chenzhou, Hunan, China, Bio-energy resources, Inc., and has a protein content of 41 percent; natural heap for useThe fermentation condition of the fermented tung meal is outdoor natural accumulation; the formula of the NA culture medium is as follows: 10.0 g/L of peptone, 3.0 g/L of beef extract, 5.0 g/L of NaCl, 15.0 g/L of agar and pH 7.2; the formula of the NB liquid medium used was: 10.0 g/L of peptone, 3.0 g/L of beef extract, 5.0 g/L of NaCl and 7.2 of pH; the colletotrichum flaxseed (B) usedColletotrichum lini) Linum Linearioides (C. Linearioides) ((C. Linearioides))Rhizoctonia solani) And Fusarium oxysporum (F.), (Fusarium oxysporum) Purchased from the national institute of agricultural science, hemp research; the formula of the PDA culture medium is as follows: 200g/L of potato, 20g/L of glucose and 15g/L of agar; the raw materials, bacterial species and chemical reagents used in the examples of the present invention were obtained by conventional commercial routes unless otherwise specified.
Pseudomonas aeruginosa and screening method embodiment thereof
A screening method of pseudomonas aeruginosa comprises the following steps:
(1) adding 10g of naturally piled and fermented tung meal into 90mL of sterile water, stirring for 20min until the mixture is uniform, and standing for 30 min to obtain a strain stock solution;
(2) passing the strain stock solution obtained in the step (1) through a filter 10-8After dilution in a gradient manner, coating and inoculating the diluted solution on an NA culture medium, and culturing for 36 hours at a constant temperature of 30 ℃;
(3) picking out colonies growing on the NA culture medium in the step (2), repeatedly carrying out streak inoculation for 4 times to a new NA culture medium, screening a strain with high content of amino nitrogen, and identifying 16SrDNA as pseudomonas aeruginosa.
And (3) performing colony morphological characteristic analysis, physiological and biochemical experiments and 16SrDNA sequence analysis on the obtained pseudomonas aeruginosa.
a. And (3) colony morphological characteristic analysis:
and (3) streaking and inoculating the single colony of the pseudomonas aeruginosa strain of the embodiment of the invention to an NA culture medium, then inverting the NA culture medium into a constant temperature incubator at 30 ℃, culturing for 18h, and observing the morphology of the grown colony.
Through detection, the pseudomonas aeruginosa thallus in the embodiment of the invention is rod-shaped, the average size of the thallus is 0.5 Mum multiplied by 1.5 Mum multiplied by 4 Mum, gram staining is negative, and the pseudomonas aeruginosa thallus has terminal flagellum and no fluorescence characteristic; as shown in FIG. 1, the colonies of Pseudomonas aeruginosa in the example of the present invention on the NA medium are yellowish green, moist, flat, irregular in edges, and smooth in surface.
b. Physiological and biochemical experimental analysis of the strain:
the pseudomonas aeruginosa strain of the embodiment of the invention is respectively subjected to physiological and biochemical test detection of citric acid utilization, nitrate reduction, hydrogen sulfide production test, V-P test, methyl red test, indole production test, oxidase test, gelatin hydrolysis test and phosphorus dissolving test, and the results are shown in table 1.
TABLE 1 Table of results of physiological and biochemical tests on Pseudomonas aeruginosa strains according to the present invention
Figure 402264DEST_PATH_IMAGE001
Note: in the table, "+" is positive and "-" is negative.
As can be seen from Table 1, the Pseudomonas aeruginosa strains according to the embodiments of the present invention can utilize citrate, nitrate reduction, hydrogen sulfide production, oxidase production, gelatin hydrolysis and phosphate solubilization, no indole production, and negative in V-P test and methyl red test, and these physiological and biochemical characteristics and methyl red test are negativePseudomonas aeruginosaThe genera of the bacteria are basically consistent.
c. 16S rDNA analysis of the strains:
extracting the genome DNA of the pseudomonas aeruginosa strain in the embodiment of the invention by using a bacterial genome DNA extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.), taking the genome DNA as a template, amplifying a 16S rDNA sequence fragment of the pseudomonas aeruginosa strain in the embodiment of the invention by using a bacterial 16S rDNA universal primer 27F/1492R through PCR reaction, and sequencing the amplified fragment by Shanghai biological engineering Co., Ltd (the 16S rDNA sequence is shown as SEQ ID NO:1, GenBank: MF 967440.1); BLAST comparison analysis is carried out on the 16S rDNA sequence of the pseudomonas aeruginosa strain obtained by sequencing and a nucleic acid database of NCBI, and a phylogenetic tree (shown in figure 2) based on the 16S rDNA sequence is constructed by adopting MEGA 5.1 software based on a proximity method, so as to obtain: what is needed isPseudomonas aeruginosaPseudomonas aeruginosa) Having 16S rDNA (GenBank: MF 967440.1) shown in SEQ ID NO:1, andPseudomonas aeruginosa the homology of S3 is the highest and is 99.79 percent, which shows that the strain has the highest similarity with the strain, namely the strain belongs to pseudomonas aeruginosa (Pseudomonas aeruginosa) (Pseudomonas aeruginosa)Pseudomonas aeruginosa) Belongs to the genus of bacteria.
In conclusion, the pseudomonas aeruginosa of the embodiment is identified as the pseudomonas aeruginosa based on the colony morphology characteristics, physiological and biochemical characteristics and the system analysis result based on the 16S rDNA gene sequencePseudomonas aeruginosaThe strain number of the strain belongs to LYT-4, and the strain is preserved in the China general microbiological culture Collection center on 26 months in 2018, and the preservation number is CGMCC number 15519.
Application embodiment of pseudomonas aeruginosa
Inoculating said Pseudomonas aeruginosa LYT-4 to NB liquid medium, culturing to log phase to obtain 3.9 × 107cfu/mL of seed solution of Pseudomonas aeruginosa LYT-4.
(1) The plant meal protein fermentation application comprises: inoculating 5mL of seed liquid of the Pseudomonas aeruginosa LYT-4 into a plant meal solid fermentation culture medium (50 g of tung meal and 17mL of inorganic salt solution), uniformly mixing, and fermenting for 5 days at 30 ℃ and under the condition that the relative humidity is 75% to obtain a LYT-4 fermentation product; the formula of the inorganic salt solution is as follows: (NH)4)2SO4 2.0g/L，K2HPO4 2.0g/L，KH2PO4 2.0g/L，MgSO4 0.20g/L，CaCl2 0.10g/L，MnSO4 0.020g/L，FeSO4 0.050g/L，pH 7.2。
Comparative example of natural fermentation: under the same conditions as all the conditions, only the seed solution of the pseudomonas aeruginosa LYT-4 in the plant meal protein fermentation application is replaced by sterile water, and the fermentation product is obtained.
In order to research the optimal fermentation days of the tung meal, in the process of fermenting the tung meal for 6 days, a pseudomonas aeruginosa LYT-4 fermentation product and a natural fermentation product are respectively taken every 24 hours, the fermentation product and deionized water are mixed according to the mass-to-volume ratio (g/L) of 1:10 and are placed on a shaking table, after shaking at 170rpm for 24 hours, the mixture is centrifuged at 12000 rpm and 4 ℃ for 10min, supernatant is taken to carry out detection and analysis on protease activity and amino nitrogen content, and the amino acid content in the final fermentation product is respectively detected after fermenting for 6 days.
a. Protease activity change assay:
the protease activity detection adopts casein as a substrate, and the detection method comprises the following steps: adding 1 mL of the centrifuged fermentation liquid supernatant diluted to 100 times into a centrifuge tube, placing the centrifuge tube in a water bath at 40 ℃ for preheating for 10min, adding 1 mL of casein solution preheated to 40 ℃ and having a mass concentration of 2%, and preparing for timely heat preservation for 10 min; then adding 2 mL of trichloroacetic acid solution and 0.4 mol/L of trichloroacetic acid solution, uniformly mixing, standing for 15 min, centrifuging, taking 1 mL of supernatant, adding 5mL of sodium carbonate solution and 0.4 mol/L of sodium carbonate solution, finally adding 1 mL of Folin-phenol reagent, uniformly mixing, placing in a water bath at 40 ℃ for 20min, measuring absorbance at the wavelength of 680 nm, and taking an inactivated enzyme sample as a blank control; the results of the change in protease activity during fermentation are shown in FIG. 3.
As can be seen from FIG. 3, the protease activities produced by both the natural fermentation and the LYT-4 fermentation remained on the rising trend during the first 3 days of the fermentation process; after the protease activity value in the natural fermentation process is slightly reduced at day 4, the protease activity value is increased at day 5 and reaches the maximum value of 1.29U/mL; the protease activity value in the LYT-4 fermentation process is increased until the maximum value is reached at day 5, the value is 2.18U/mL, which is equivalent to 1.7 times of the maximum enzyme activity value in natural fermentation, which shows that the LYT-4 can generate protease with higher activity in the fermentation process, and the time for reaching the maximum protease activity is shortened.
b. Analyzing the content change of the amino nitrogen:
the content of amino nitrogen is detected by an Amino Acid (AA) detection kit (ninhydrin colorimetry) (purchased from Beijing Lei Gen Biotech Co., Ltd.), and the detection steps are carried out according to the instruction; the results of the change in the content of amino nitrogen during the fermentation are shown in FIG. 4.
As can be seen from FIG. 4, in the fermentation process, the content of amino nitrogen generated by natural fermentation and LYT-4 fermentation is increased until 5 days of fermentation, the content of amino nitrogen released in the LYT-4 fermentation process is significantly higher than that in natural fermentation, and the content of amino nitrogen released in the LYT-4 fermentation process can reach 37.3 mg/mL, which is 1.4 times of that in natural fermentation (25.8 mg/mL), which indicates that the content of amino acid released by protease degradation of crude protein in tung meal in the LYT-4 fermentation process is higher than that in natural fermentation, i.e. LYT-4 has stronger crude protein degradation capability.
c. Comparative analysis of amino acid content:
the pretreatment method of the amino acid content detection sample comprises the following steps: adding 2 mL of hydrochloric acid of 6mol/L into 1 mL of fermentation product solution, adding phenol as a protective agent, hydrolyzing at 110 ℃ for 23 h, removing acid from hydrolysate for 3 times, dissolving in 0.1mol/L dilute hydrochloric acid, and passing through a 0.45 mu m microporous filter membrane for subsequent amino acid detection and analysis.
The amino acid detection adopts a high performance liquid chromatography: agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); symmetry C18 column; the column temperature is 37 ℃; the flow rate is 2.0 mL/min; excitation wavelength of 250 nm and emission wavelength of 395 nm; the sample volume is 10 mu L; mobile phase: a is phosphate buffer solution, B is pure acetonitrile, C is ultrapure water, and the gradient elution procedure is as follows: 0min, 100% A; 0.5 min, 98% A +2.0% B; 0.5-9.0 min, 96.5% of A +3.5% of B; 9.0-9.5 min, 95.0% A +5.0% B; 9.5-11.5 min, 91.5% of A and 8.5% of B; 11.5-13.0 min, 83.0% A +17.0% B; 13.0-19.0 min, 60.0% B +40% C; 19.0-23.0 min, 100% A; qualitative and quantitative analysis of the amino acids in the samples were performed according to the retention time of the amino acid standards and the standard curve equation, and the results are shown in fig. 5.
From fig. 5, it can be seen that the contents of the 17 amino acids generated after natural fermentation and LYT-4 fermentation are increased compared with the un-fermented tung meal, and particularly, the percentage of the amino acid content generated by LYT-4 fermentation reaches 55.5%, which is higher than 46.5% of the amino acid content generated by natural fermentation, which is consistent with the previous results of protease activity and amino nitrogen content, further confirming the ability of LYT-4 to degrade crude tung meal proteins into amino acids.
(2) The application of antagonistic plant pathogenic fungi comprises the following steps: respectively inoculating 100 μ L of LYT-4 seed solution of Pseudomonas aeruginosa to the cross point 2.5cm away from the center of the flat plate, and inoculating to the strain grown with colletotrichum flaxseed ((L.) (Colletotrichum lini）、Linum Linearioides (C. Linearioides) ((C. Linearioides))Rhizoctonia solani) And Fusarium oxysporum (F.), (Fusarium oxysporum) Inoculating on PDA culture medium, culturing at 25 deg.C for 5 days; the preparation method of the PDA culture medium for growing the plant pathogenic fungi comprises the following steps: inoculating the plant pathogenic fungi into the center of PDA culture medium with a punch with diameter of 0.5cm, and culturing at 25 deg.C under inverted state for 24 hr until hyphae grow out.
Blank control: under the same conditions as all the conditions described above, only the seed solution of the P.aeruginosa LYT-4 in the application against phytopathogenic fungi was replaced with sterile water.
As shown in FIG. 6, P.aeruginosa LYT-4 is responsible for anthrax Linnaeus bacteria: (A), (B)Colletotrichum lini) Linum Linearioides (C. Linearioides) ((C. Linearioides))Rhizoctonia solani) And Fusarium oxysporum (F.), (Fusarium oxysporum) The inhibition rates of the compounds reach 40.1%, 31.3% and 35.5% respectively, and the compounds show better antagonistic performance; the genome of the pseudomonas aeruginosa consists of a highly conserved core genome and different additional genomes, and because additional genome elements are derived from the transverse transfer of genetic materials of different species or genera of bacteria, the genome endows different pseudomonas aeruginosa with the capability of producing different secondary metabolites, and the better antagonistic performance shown by the LYT-4 of the pseudomonas aeruginosa indicates that the LYT-4 of the pseudomonas aeruginosa possibly secretes secondary metabolites with better antibacterial performance, which indicates that the LYT-4 of the pseudomonas aeruginosa has the potential of being developed as a biocontrol bacterium.
Sequence listing
<110> technical university of the middle and south forestry
Hunan Academy of Forestry Sciences
<120> pseudomonas aeruginosa strain and screening method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1444
<212> DNA
<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)
<400> 1
ggcctgtgcg ggcaggccta cacatgcagt cgagcggatg aagggagctt gctcctggat 60
tcagcggcgg acgggtgagt aatgcctagg aatctgcctg gtagtggggg ataacgtccg 120
gaaacgggcg ctaataccgc atacgtcctg agggagaaag tgggggatct tcggacctca 180
cgctatcaga tgagcctagg tcggattagc tagttggtgg ggtaaaggcc taccaaggcg 240
acgatccgta actggtctga gaggatgatc agtcacactg gaactgagac acggtccaga 300
ctcctacggg aggcagcagt ggggaatatt ggacaatggg cgaaagcctg atccagccat 360
gccgcgtgtg tgaagaaggt cttcggattg taaagcactt taagttggga ggaagggcag 420
taagttaata ccttgctgtt ttgacgttac caacagaata agcaccggct aacttcgtgc 480
cagcagccgc ggtaatacga agggtgcaag cgttaatcgg aattactggg cgtaaagcgc 540
gcgtaggtgg ttcagcaagt tggatgtgaa atccccgggc tcaacctggg aactgcatcc 600
aaaactactg agctagagta cggtagaggg tggtggaatt tcctgtgtag cggtgaaatg 660
cgtagatata ggaaggaaca ccagtggcga aggcgaccac ctggactgat actgacactg 720
aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gccgtaaacg 780
atgtcgacta gccgttggga tccttgagat cttagtggcg cagctaacgc gataagtcga 840
ccgcctgggg agtacggccg caaggttaaa actcaaatga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttacctgg ccttgacatg 960
ctgagaactt tccagagatg gattggtgcc ttcgggaact cagacacagg tgctgcatgg 1020
ctgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gtaacgagcg caacccttgt 1080
ccttagttac cagcacctcg ggtgggcact ctaaggagac tgccggtgac aaaccggagg 1140
aaggtgggga tgacgtcaag tcatcatggc ccttacggcc agggctacac acgtgctaca 1200
atggtcggta caaagggttg ccaagccgcg aggtggagct aatcccataa aaccgatcgt 1260
agtccggatc gcagtctgca actcgactgc gtgaagtcgg aatcgctagt aatcgtgaat 1320
cagaatgtca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga 1380
gtgggttgct ccagaagtag ctagtctaac cgcaaggggg acggttacca cgagtattcc 1440
tttt 1444

Claims (6)

1. Pseudomonas aeruginosaPseudomonas aeruginosa) LYT-4, characterized by: the pseudomonas aeruginosa LYT-4 is preserved in China general microbiological culture Collection center in 2018, 3 and 26 months, and the preservation number is CGMCC number 15519.
2. The Pseudomonas aeruginosa of claim 1 (M.aeruginosa)Pseudomonas aeruginosa) Use of LYT-4, characterized by: the pseudomonas aeruginosa (a)Pseudomonas aeruginosa) LYT-4 is applied to plant meal protein fermentation or antagonistic plant pathogenic fungi; the plant pathogenic fungus is colletotrichum flaxseed (B)Colletotrichum lini) Linum Linearum Raeumannomyces (L.) KuntzeRhizoctonia solani) Or Fusarium oxysporum (F.), (Fusarium oxysporum) One or more of them.
3. The Pseudomonas aeruginosa according to claim 2 (M.aeruginosa)Pseudomonas aeruginosa) Use of LYT-4, characterized by: the protein content in the plant meal is 36-45%; the plant meal is tung meal; the specific operation of the plant meal protein fermentation is as follows: the pseudomonas aeruginosa (a)Pseudomonas aeruginosa) Inoculating the seed solution of LYT-4 into the solid fermentation culture medium of plant meal, mixing, and fermenting to obtain the fermentation product.
4. The Pseudomonas aeruginosa according to claim 3 (M.aeruginosa)Pseudomonas aeruginosa) Use of LYT-4, characterized in that: the volume-mass ratio of the seed liquid to the plant meal is 8-12: 100; the fixing partThe bulk fermentation culture medium consists of plant meal and an inorganic salt solution in a mass-volume ratio of 2.5-3.5: 1; the formula of the inorganic salt solution is as follows: (NH)4)2SO4 1.5～2.5g/L，K2HPO4 1.5～2.5g/L，KH2PO4 1.5～2.5g/L，MgSO4 0.15～0.25g/L，CaCl2 0.05～0.15g/L，MnSO4 0.015～0.025g/L，FeSO40.045-0.055 g/L, pH 7.0-7.2; the fermentation temperature is 25-35 ℃, the relative humidity is 70-80%, and the fermentation time is 5-6 days.
5. The Pseudomonas aeruginosa according to claim 2 (M.aeruginosa)Pseudomonas aeruginosa) Use of LYT-4, characterized by: the specific operation of the antagonistic plant pathogenic fungi is as follows: the pseudomonas aeruginosa (a)Pseudomonas aeruginosa) Inoculating the seed solution of LYT-4 to PDA culture medium with the plant pathogenic fungi, and performing inverted culture; the pseudomonas aeruginosa (Pseudomonas aeruginosa) The inoculation amount of the LYT-4 seed solution is 80-120 mu L; the inoculation position is 2-3 cm away from the central cross point of the flat plate; the temperature of the inverted culture is 20-30 ℃, and the time is 4-6 days; the preparation method of the PDA culture medium for growing the plant pathogenic fungi comprises the following steps: inoculating the plant pathogenic fungi to the center of a PDA culture medium, and performing inverted culture at the temperature of 20-30 ℃ for 20-30 hours until hyphae grow out; the PDA culture medium comprises the following components in percentage by weight: 200g/L of potato, 20g/L of glucose and 15-20 g/L of agar.
6. Pseudomonas aeruginosa according to any one of claims 3 to 5Pseudomonas aeruginosa) Use of LYT-4, characterized by: the concentration of the seed liquid is 3.0 x 106～3.0×108cfu/mL; the seed liquid is prepared by mixing the pseudomonas aeruginosa (Pseudomonas aeruginosa)Pseudomonas aeruginosa) Inoculating LYT-4 into NB liquid culture medium, and culturing to logarithmic phase; the NB liquid culture medium comprises the following formula: 10.0 g/L of peptone, 3.0 g/L of beef extract, 5.0 g/L of sodium chloride and 7.2-7.5 of pH.
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