CN117305388B

CN117305388B - Application of bacillus bailii SL-K2 in preparation of guanine extract, preparation method of guanine extract and application

Info

Publication number: CN117305388B
Application number: CN202311245655.0A
Authority: CN
Inventors: 毛丽强
Original assignee: Qingdao Rishengyuan Crop Nutrition Co ltd
Current assignee: Qingdao Rishengyuan Crop Nutrition Co ltd
Priority date: 2023-09-25
Filing date: 2023-09-25
Publication date: 2024-04-26
Anticipated expiration: 2043-09-25
Also published as: CN117305388A

Abstract

The invention discloses an application of bacillus beleiensis SL-K2 in preparation of guanine extract, a preparation method of the guanine extract and an application thereof, wherein the bacillus beleiensis can be used for preparing the guanine extract and has a preservation number of CGMCC 28155. The preparation method of guanine extract comprises: s1, centrifuging fermentation liquor of bacillus belicus SL-K2, and reserving a fermentation liquor supernatant; s2, drying and grinding the supernatant of the fermentation broth into dry powder; s3, carrying out alcohol precipitation on the dry powder by 8-12 times of ethanol, keeping for 20-30 hours, and filtering and precipitating to obtain a primary extract; s4, removing ethanol in the precipitate, preparing dry powder, dissolving the dry powder, performing dialysis treatment, and removing macromolecules above 500Da to obtain a secondary extract; s5, performing HPLC chromatographic purification on the secondary extract, and separating to obtain guanine extract. The guanine extract is used as bacterial fermentation broth extract, has good Fusarium resistance and plant growth promotion effects, and has the advantages of extremely low cost, strong process adaptability, green and safe.

Description

Application of bacillus bailii SL-K2 in preparation of guanine extract, preparation method of guanine extract and application

Technical Field

The invention relates to the technical field of microbial extracts, in particular to application of bacillus beijerinus SL-K2 in preparation of guanine extracts, and a preparation method and application of the guanine extracts.

Background

Potatoes are annual herbs of the genus solanum of the family solanaceae, which are a non-cereal food crop for human consumption, the first one in the potato rank is the fourth largest crop next to rice, corn and wheat, playing a significant role in food safety in developing countries. The mountain land of the tropical america of the original potato is transferred to india in the 16 th century and then to china, and is now widely planted in the temperate region of the world. About 13 million people in china and india use fresh potatoes as staple food.

However, in potato planting, fungal diseases have been an important cause of the influence on the yield. Among the various soil-borne diseases, fusarium is the most common pathogen responsible for yield and commercial losses in potato production. At present, the control method of Fusarium potato diseases is mainly focused on chemical bactericides, rotation and breeding of resistant varieties. However, fusarium diseases are difficult to control due to rapid differentiation of pathogenicity of pathogenic bacteria, increase of drug-resistant pathogenic bacteria, and lack of high-resistance fine varieties.

At present, the main pathogenic bacteria causing fusarium diseases of potatoes in Shandong and other areas are fusarium wilt of crabapple (the scholaris name: fusarium foetens), the report of the novel pathogenic bacteria is published in 4 months of 2023, and the widely distributed pathogenic bacteria at present, namely article name ："Mechanisms of Surfactin from Bacillus subtilis SF1 against Fusarium foetens:A Novel Pathogen Inducing Potato Wilt". in Journal of Fungi, cause serious yield reduction on potato planting, are objects needing to be mainly controlled, but the green and safe bacterial antagonist on the market at present has poor control effect on the pathogenic bacteria.

If the stress resistance of the potatoes to diseases is fundamentally improved, the yield of the potatoes can be greatly improved, the dosage of various bactericides in fields is reduced, and the method is more scientific and environment-friendly. However, no effective agent for improving fusarium diseases of potatoes exists at present.

Accordingly, there is a need for further improvements in the art.

Disclosure of Invention

In order to solve the problems, the invention provides the application of bacillus belicus SL-K2 in preparing the guanine extract, the preparation method and the application of the guanine extract, and the guanine extract is successfully extracted from bacillus belicus SL-K2 for the first time, has the effects of promoting plant growth and improving plant disease resistance, and can be used as an active ingredient of a medicament to play a role in potato planting industry.

In order to solve the problems, the application provides the following technical scheme:

In a first aspect, the application provides an application of bacillus beleiensis in preparing guanine extracts, wherein bacillus beleiensis SL-K2 is adopted, and the preservation number of the bacillus beleiensis is CGMCC 28155.

The bacillus beleiensis is separated and purified from sludge at the offshore river mouth of the urban wetland park in the sea city by the applicant, is named as bacillus beleiensis SL-K2 strain, and is preserved in China general microbiological culture collection center (CGMCC 28155) at the 8 th month 11 of 2023. The strain is facultative aerobic bacteria, gram positive bacteria, and spores are positioned in the middle or at the partial ends of the bacteria, so that the bacteria do not expand when the spores are generated, and the bacteria are in a rod shape. The strain is identified as a novel bacillus belay strain by sequencing and analyzing a 16S rDNA gene of the strain (Bacillus velezensis).

The inventor discovers that guanine extract can be extracted from the bacillus beijerinus SL-K2 strain for the first time, and the bacillus beijerinus has great application potential.

In a second aspect, the present application also provides a process for the preparation of guanine extracts, which process is: guanine extracts were extracted from fermentation broth of Bacillus bailii SL-K2.

Preferably, specifically, the preparation method of the guanine extract comprises the following steps:

s1, centrifuging fermentation liquor of bacillus belicus SL-K2, removing thalli, and reserving a fermentation liquor supernatant;

s2, drying and grinding the supernatant of the fermentation broth into powder to obtain dry powder of a fermentation broth mixture;

S3, carrying out alcohol precipitation on the dry powder by 10 times of volume of ethanol, keeping the temperature at 4 ℃ for 24 hours, filtering with filter paper, separating filtrate and precipitate, and precipitating to obtain a primary extract;

S4, removing ethanol in the precipitate obtained in the previous step, preparing dry powder, dissolving the dry powder in DMSO (dimethyl sulfoxide), performing dialysis treatment, and removing macromolecules above 500Da to obtain a secondary extract;

S5, performing HPLC (high Performance liquid chromatography) purification on the secondary extract, and separating to obtain 1 refined extract, wherein the refined extract is guanine extract.

Preferably, in the preparation method of the guanine extract, the preparation method of the fermentation broth of bacillus belgium SL-K2 in the step S1 comprises the following steps:

s0, inoculating bacillus belicus SL-K2 into a liquid culture medium, and shake culturing for 40-55 h, wherein the rotation speed of a shaking table is 120-200 rpm/min to obtain a culture solution; the liquid culture medium is added with fusarium microspore inactivating liquid of the malus spectabilis.

Preferably, the liquid culture medium comprises the following components in percentage by mass:

1 to 10 percent of fusarium microspore inactivating liquid with the concentration of 10 ^3～10⁵/ml, 0.05 to 0.5 percent of beef extract, 0.1 to 1 percent of peptone and 0.1 to 1 percent of bean cake powder; corn steep liquor with the concentration of 0.5-3%; 0.05 to 0.3 percent of CaCO ₃; 0.01-0.05% of MgSO ₄ and the balance of water.

Preferably, the concentration of the fusarium oxysporum virens inactivating liquid is 10 ⁴/ml.

Further preferably, the liquid culture medium comprises the following components in percentage by mass: the liquid culture medium comprises the following components in percentage by mass: 5% of 10 ⁴/ml fusarium oxysporum liquid, 0.1% of beef extract, 0.5% of peptone and 0.5% of bean cake powder; corn steep liquor 1.0%; caCO ₃0.1％;MgSO₄ 0.03% and water as the rest.

Preferably, the preparation method of the fusarium oxysporum f.sp.begoniae inactivating liquid comprises the following steps: the microspore suspension of Fusarium roseum is treated at 55-65 deg.c for 20-40 min to maintain the integrity of spore cell morphology and lose germination activity, so as to obtain deactivated spore liquid.

Preferably, in the step S5, the chromatographic column is a Hypersil GOLD C18 column, the column temperature is 23-28 ℃, and the secondary extracting solution is carried out in a two-dimensional nano liquid phase system at the flow rate of 1.0ml/min at the flow rate of 0:3,1:9, 14:90, 13:7 and 18:5; eluent A adopts acetic acid (90%), B is an apodization system of acetonitrile and methanol (acetonitrile 8%, methanol 2%), and the chromatographic retention time is 1.5-1.6min.

In a third aspect, the present application also provides a guanine extract, which is prepared by the aforementioned preparation method.

Guanine is a major base constituent of nucleic acids and has important physiological functions. Meanwhile, guanine also has a plurality of medicinal values, is a medical intermediate of traditional Chinese medicines, such as a compound antiviral drug of acyclovir, ganciclovir and other zolo Wei Jilie drugs, and has great industrial production value.

However, at present, guanine is chemically synthesized by adopting a traditional production process and taking methyl cyanoacetate as a starting material, and mainly undergoes the following five chemical reactions in sequence:

(1) Condensation reaction: cyclizing methyl cyanoacetate with guanidine nitrate (or guanidine hydrochloride) to obtain 2, 4-diamino-6-hydroxypyrimidine; (2) nitrosation reaction: nitrosation reaction is carried out with sodium nitrite to obtain 2, 4-diamino-5-nitroso-6-hydroxypyrimidine. (3) hydrogenation reduction reaction: the 2,4, 5-triamino-6-hydroxy pyrimidine is obtained through hydrogenation or iron powder reduction. (4) salt formation reaction: reacting with sulfuric acid to obtain 2,4, 5-triamino-6-hydroxy pyrimidine sulfate. (5) cyclization reaction: reflux with sodium formate or formamide in formic acid to give guanine formate. And (3) decoloring and refining the guanine formate obtained by the cyclization reaction in dilute hydrochloric acid to obtain guanine hydrochloride, and then, performing free reaction in dilute alkaline water to obtain a guanine finished product.

In the traditional method, the third-step iron powder reduction reaction is forbidden, and the hydrogenation reduction has higher safety threshold due to the hydrogen and pressure, and in addition, the byproduct treatment difficulty is higher and the environmental protection treatment cost is high due to the use of liquid alkali, sulfuric acid and formic acid.

The guanine extract obtained by the preparation method provided by the invention is used as a bacterial fermentation broth extract, has good fusarium resistance and plant growth promotion effects, is extremely low in cost, strong in process suitability and high in safety, and more importantly, can be used as a plant immune elicitor to activate plant immune response, so that the disease resistance of crops is improved; the compound can become a novel medicament with green, safe and environment-friendly property, and has wide application prospect.

In a fourth aspect, the present application also provides a microbial immunity inducer, the active ingredient of which comprises the guanine extract. Experiments show that guanine has no bacteriostasis on fusarium in vitro, but can induce the disease resistance of plants and promote plant growth after acting on the plants.

In a fifth aspect, the application also provides the application of the guanine extract and the microbial immunity inducer in promoting plant growth.

In a sixth aspect, the application also provides an application of the guanine extract and the microbial immunity inducer in improving disease resistance of plants to Fusarium head blight. In the examples of the present application, the plants are exemplified by potatoes.

Experiments prove that the guanine extract prepared by the method provided by the application has obvious seedling strengthening and stress resisting effects on potatoes, promotes the growth of the potato seedlings, improves the disease resistance of the potato seedlings, and reduces the probability of infection of potato plants by fusarium, so that the guanine extract can be used as an active ingredient of a microbial immunity inducer and applied to the aspects of plant growth promotion and stress resistance improvement.

The invention has the following beneficial effects:

1. the application discovers a method for separating guanine extract from bacillus bailii SL-K2 which is automatically screened, and the guanine extract is used as a bacterial fermentation broth extract, has the effects of resisting fusarium and promoting plant growth, and has the advantages of extremely low cost, strong process suitability, high safety and no pollution to the environment, and more importantly, the guanine extract can be used as a plant immunity elicitor to activate plant immunity reaction so as to improve the disease resistance of crops, and can be applied to preparing microbial pesticides or related preparations.

2. The guanine extract has good application prospect, fully develops the application value of bacillus bailii and improves the utilization rate of bacillus bailii.

3. Compared with the traditional mode of producing guanine by using methyl cyanoacetate as a starting material through a chemical synthesis process, the microbial fermentation production method provided by the application is more environment-friendly, and solves the problems of higher byproduct treatment difficulty, high environmental protection treatment cost, higher safety threshold and the like in the traditional chemical synthesis method.

Drawings

FIG. 1 shows the results of bacteriostasis of Bacillus belicus SL-K2 on Fusarium roseum wilt of Malus;

FIG. 2 is an electron microscope image of vegetative cell and spore morphology of Bacillus bailii SL-K2; a is a vegetative cell, B is a spore;

FIG. 3 is a chemical structure diagram of guanine and xanthine;

FIG. 4 is a chromatogram of the negative ion of guanine extract;

FIG. 5 is a mass spectrum of negative ions of guanine extract;

FIG. 6 is a positive ion chromatogram of guanine extract;

FIG. 7 is a positive ion mass spectrum of guanine extract;

FIG. 8 is a comparison of the above-ground growth of potato seedlings treated in each experimental group of example 3;

FIG. 9 is the total weight of potato seedlings treated in each experimental group in example 3;

FIG. 10 is a comparison of chlorophyll a content of potatoes treated in each experimental group of example 3;

FIG. 11 is a comparison of soluble sugar content of potato leaf treated in each experimental group of example 3;

FIG. 12 is the malondialdehyde content of leaves treated in each experimental group in example 3.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. In the present invention, the equipment, materials, etc. used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.

For a sufficient disclosure, information on the self-isolated Bacillus bailii SL-K2 is provided below. EXAMPLE 1 isolation and characterization of Bacillus bailii SL-K2

1. Isolation and screening of strains

1.1 Experimental method:

Collecting silt at the offshore river mouth of the urban wetland park in the sea city, putting into a sterile plastic bag, feeding into a laboratory for experiment, weighing 10g of soil (uniformly mixing in advance), adding 90ml of sterile water, oscillating for 15min on a shaking table at 190r/min to prepare 10 ^-1 bacterial suspension, filling 9 test tubes into 9ml of sterile water, sucking 1ml of 10 ^-1 bacterial suspension by a microsampler, injecting into a first test tube, marking the test tube as 10 ^-2, fully shaking to prepare 10 ^-2 bacterial suspension, and serially diluting to 10 ^-3,10^-4,10^-5,10^-6,10^-7,10^-8,10^-9,10^-10 bacterial suspension. Respectively sucking 100 μm from 10 ^-2,10^-4,10^-6,10^-8,10^-10 bacterial suspension, injecting into LB solid culture medium prepared in advance, uniformly coating with a coating rod, and culturing in a 37 deg.C incubator for 1-2d. And (3) selecting single colonies with different culture characteristics, purifying by a plate streak separation method, and carrying out antagonism experiments on the purified strains and Fusarium roseum. And screening antagonistic bacteria by adopting a flat plate counter method, and finally observing whether a bacteria inhibition zone appears between two bacterial colonies.

1.2 Results and analysis

As shown in figure 1, a strain of bacteria is separated, has good antibacterial effect on Fusarium roseum, and well prevents the expansion of Fusarium roseum when being cultivated in a counter manner, and is named as: SL-K2 strain.

2. Observation of morphological characteristics of SL-K2 strain morphological antagonistic bacteria

2.1 Experimental methods

(1) Observation of colony appearance: the antagonistic bacteria obtained by screening are streaked by a flat plate and cultured for 24 hours in a constant temperature incubator at 37 ℃, the morphological characteristics of the observed colony are referred to the handbook of the identification of common bacterial systems, and the analysis of gram staining and physiological and biochemical characteristics are referred to the experiments of microbiology.

(2) Spore staining: picking and culturing the antagonistic bacteria smear for 48 hours, dyeing the antagonistic bacteria smear with saturated malachite green water solution for 10 minutes, flushing the antagonistic bacteria smear with tap water, then dyeing the antagonistic bacteria smear with safranine staining solution for 1 minute, washing the antagonistic bacteria smear with water, and performing microscopic examination after the antagonistic bacteria smear is sucked by absorbent paper.

2.2 Results and analysis

The strain is facultative aerobic bacteria, gram positive bacteria, and spores are positioned in the middle or at the partial ends of the bacteria, so that the bacteria do not expand when the spores are generated, and the bacteria are in a rod shape. As can be seen from FIG. 1, the colony surface of the strain is opaque, dirty white or yellowish. Single cells were observed under electron microscopy to be 1.3-2.7 microns long without capsules (see FIG. 1). The spores are 0.8-1.5 microns long, oval, centered or slightly biased in the cell (FIG. 3).

In addition, the SL-K2 strain was sent to the Optimago company for sequencing, and analysis and comparison result identification of the 16SrDNA gene of the SL-K2 strain revealed that the SL-K2 strain was a novel Bacillus bailii (Bacillus velezensis).

EXAMPLE 2 preparation and identification of guanine extracts of Bacillus bailii

In this example, an experimental group and a control group were set, and the experimental group was operated according to the following steps, while the control group was prepared from the same extraction material and steps as the experimental group, except that the step of induction treatment of fusarium spores of malus spectabilis was omitted.

1. Experimental method

(1) Culture medium configuration

The formula of the liquid culture medium comprises: 5% of 10 ⁴/ml of fusarium oxysporum liquid, 0.1% of beef extract, 0.5% of peptone, 0.5% of bean cake powder, 1.0% of corn steep liquor, 0.1% of CaCO ₃, 0.03% of MgSO ₄ and the balance of water.

The preparation method of the fusarium oxysporum inactivated solution for the malus spectabilis comprises the following steps: the microspores of Fusarium roseum are treated for 30min at 60 ℃ to keep the spore cell morphology intact but lose germination activity, so as to prepare the inactivated spore liquid. Spores in the inactivated liquid lose germination activity, do not generate toxin, but can induce bacillus bailii to generate antibacterial substances.

(2) Culturing and fermenting bacillus bailii

Mycelia of Bacillus bailii SL-K2 were inoculated into 50ml of the above liquid medium, subjected to shaking culture at 37℃at 150rpm/min for 48 hours to obtain a seed solution, and then inoculated into 300ml of the above liquid medium at 8% of the inoculum size, subjected to shaking culture at 37℃at 150rpm/min for 48 hours to obtain a fermentation broth.

(3) Preparation of fermentation broth dry powder

Centrifuging the fermentation liquor after pathogen spore induction culture, separating thalli from the fermentation liquor, removing thalli, ventilating and drying the supernatant of the fermentation liquor at 50 ℃ in a drying box, and grinding the dried mixture into powder to obtain dry powder of the fermentation liquor. In other embodiments, the freeze-drying process can be used to obtain the freeze-dried powder of the fermentation broth, so that the effect is equivalent, and the processing efficiency of the drying process is higher, thus being more suitable for mass production.

(4) The dry powder was subjected to ethanol precipitation with 10 volumes of ethanol, kept at 4 ℃ for 24 hours, and then filtered with filter paper to separate the filtrate from the precipitate.

(5) Dialysis treatment of primary extract

Removing ethanol in the precipitate by rotary evaporator to obtain dry powder, dissolving the dry powder in DMSO (dimethyl sulfoxide), dialyzing, and removing macromolecules above 500Da to obtain secondary extractive solution.

(6) Chromatographic purification treatment

The secondary extract was subjected to high performance liquid chromatography purification using a Hypersil GOLD C18 column (2.1X100 mm,5 μm; thermo FISHER SCIENTIFIC, waltham, USA) at a column temperature of 25deg.C in a two-dimensional nano-liquid phase system at flow rates of 1.0ml/min at 0:3,1:9, 14:90, 13:7 and 18:5. In the elution process, acetic acid (90%) is adopted as eluent A, B is a system for removing acetonitrile and methanol (8% of acetonitrile and 2% of methanol), the chromatographic retention time is 1.5-1.6min, and finally 1 refined extract of bacillus belicus is obtained by purification.

(7) Mass spectrometry analysis

Carrying out mass spectrometry on the refined extract, and carrying out full scanning on positive ions and negative ions in a nuclear mass ratio range of 50-1500m/z by electrospray mass spectrometry, wherein nitrogen is used as atomizing gas, and the flow rate is 6L/min; the temperature is 180 ℃; pressure was 1.0Bar and data acquisition and processing used LC/MS data analysis software supplied with the instrument (version 4.1). The nuclear mass ratio data corresponding to a particular elemental composition is calculated using formula prediction software provided with the instrument. It is required that the error between the measured nuclear mass ratio and the standard nuclear mass ratio of the substance is not more than 5ppm.

2. Experimental results and analysis

The result of the chromatograms of the refined extract of bacillus belicus is shown in fig. 4 and 6, and the positive ion mass spectrum and the negative ion spectrum of the refined extract are shown in fig. 7 and 5 respectively.

By analyzing the negative ion mass spectrum of FIG. 5, the maximum peak is a molecular ion peak, the M/Z value is 150.0148, and the unique molecular formula is C ₅H₅N₅ O (negative ion hydrogenation: C ₅H₄N₅ O is C ₅H₅N₅ O after H is added) given by LC/MS data analysis software (version 4.1) provided by an instrument. Meanwhile, the analysis of the positive ion spectrum chart of fig. 7 shows that the unique molecular formula is also C ₅H₅N₅ O (the positive ion is reduced in hydrogen, namely C ₅H₆N₅ O is added with H and then is C ₅H₅N₅ O).

Furthermore, the retention time of the material signal of the positive and negative ions is within 1.5-1.6 min. The guanine chemical structure is 2-amino-6-hydroxy purine, the signal is also analyzed by anion chromatography because of hydroxy, and the signal of positive ion mass spectrum is strong because guanine has amino as alkalescence, and the prior mass spectrum characteristic related report about guanine and the precursor peak in mass spectrum are combined: xanthine (see FIG. 3), the refined extract compound was inferred to be guanine.

Whereas no compound C ₅H₅N₅ O was found in the control extract, this indicates: without induction treatment of pathogenic bacteria, fusarium roseum spores, bacillus belicus SL-K2 did not express the compound: guanine.

EXAMPLE 3 use of guanine extract of Bacillus bailii

1. Experimental method

Since it has been found in the past that guanine does not directly inhibit the growth of pathogenic bacteria, the applicant speculates that guanine induces plant autoimmune reactions as an elicitor, thereby inhibiting the invasion and spread of plant pathogenic bacteria. Therefore, in this example, a potting method, i.e., a potting potato, was used to determine the indirect bacteriostatic effect of guanine extract of SL-K2 strain on Fusarium roseum.

(1) Culturing of indicator bacteria:

Fusarium roseum is inoculated in PDA solid culture medium and cultured at 28 deg.c for 7 days.

Then the spores of the fungus are washed from the surface of the mycelium by sterile physiological saline to prepare spore suspension, and the concentration is adjusted to 10 ⁶/ml by the counting of a blood cell counting plate for standby.

(2) Sample set arrangement:

The experimental and control groups were each set with at least 3 replicates according to the following procedure:

Water control group (CK): filling with equal amount of sterile distilled water.

Treatment group a: basically the same as the method of the experimental group 2, only the step of fusarium spore induction treatment is omitted and is used as a sample;

Treatment group B: the guanine refined extract prepared according to the complete experimental steps is taken as a sample;

treatment group C: and centrifuging the fermentation liquor of bacillus belicus subjected to pathogen-fusarium spore induction treatment to remove thalli, and separating the thalli by a molecular sieve to remove substances above 500D as a sample, wherein the pretreatment is the same as the pretreatment of the refined extract.

The samples of each experimental group were dissolved in sterile distilled water to prepare 10mg/ml solutions, and filtered through a 0.22 μm sterile filter.

(3) Potting experiment

Root irrigation treatment is carried out on each pot of potato seedlings by using equal amounts of sterile water (CK) and A, B, C treatment groups of sample diluents (the concentration is 10 mg/ml), 1500ml of each pot is put into light for 1-2 days, 10ml of activated fusarium oxysporum spore suspension (10 ⁶/ml) with the same amount is inoculated, and the growth condition of plants is observed and recorded. Each set of experiments was set up with 3 replicates and the results were analyzed and plotted for statistically significant differences.

Seedling growth vigor and total weight measurements were performed for each group, and indexes such as chlorophyll content (chlorophyll a, chlorophyll b, and total chlorophyll content) of plants, soluble sugar, malondialdehyde content (markers of cell senescence degree) were measured, and the measurement method was as follows: spectrophotometric assay. Each set of experiments was repeated 3 times.

Chlorophyll, which includes chlorophyll a, is the primary pigment that absorbs solar energy for photosynthesis. The chlorophyll a content is high, the photosynthetic capacity of the leaf is strong, and the stronger the resistance to strong light. The soluble sugar can help plant cells to maintain water balance, improve cell osmotic pressure and protect cell membranes, and can accumulate when plants are in adverse conditions, and when the content of the soluble sugar is high, the plants can attract small insects to gnaw plant leaves, and simultaneously, virus diseases can be transmitted, so the content of the soluble sugar is one of important indexes for reflecting the stress resistance of the plants.

Plant organs age or suffer injury in adverse circumstances, membrane lipid peroxidation often occurs, and Malondialdehyde (MDA) is the final breakdown product of membrane lipid peroxidation. Therefore, the lower the malondialdehyde content, the better the state of the plant and the good stress resistance. Malondialdehyde as a marker of the degree of cellular senescence can reflect stress resistance of plants. Therefore, in this embodiment, the plurality of indices are measured separately.

The obtained data are subjected to difference significance analysis and drawing by using SAS data processing software.

2. Experimental results and analysis

(1) From the results of FIGS. 8 and 9, it can be seen that the control inoculated with Fusarium roseum was frequently associated with wilting. The treatment B (guanine extract) has the best action on the potato seedlings against Fusarium roseum wilt, so that the seedlings grow best, and the total weight of the seedlings is far larger than that of other experimental groups; and the treatment C (induced fermentation broth filtrate) is inferior to the treatment A (fermentation broth filtrate induced by no pathogenic bacterial spore suspension), the capability of resisting Fusarium roseum is very weak, and seedlings are weak and sallow.

Compared with the control and other 2 treatments, the guanosine refined extract (treatment B) has the best effect of promoting the growth and resisting the stress of the potatoes, and the chlorophyll a content is the highest, so that the photosynthesis is stronger, the organic matters are more synthesized in unit time, and the plant growth is faster; meanwhile, the content of the soluble sugar and malondialdehyde is the lowest, the low content of the soluble sugar can reduce the harm of small insects, and many small insects are also transmission media of plant virus diseases, so that the probability of infection of potato plants by viruses can be reduced. The lowest content of malondialdehyde indicates that the B-treated potato seedlings have the strongest stress resistance.

(2) As can be seen from the comparison of the A group and the B, C group, the induction of the fusarium spore suspension of the malus spectabilis has a decisive effect on the generation of guanine in the fermentation liquor, and the fusarium which resists the malus spectabilis and is prepared by the method has the strongest effect and shows the effect of promoting the growth of seedlings. While the A treatment without spore suspension induction showed a weaker resistance to Fusarium, it is presumed that other substances in the fermentation broth also have a certain inhibition effect on Fusarium.

It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.

Claims

1. The application of bacillus beleiensis (Bacillus velezensis) SL-K2 in the preparation of guanine is characterized in that the preservation number of bacillus beleiensis SL-K2 is CGMCC No.28155.

2. A process for the preparation of guanine, characterized by: guanine is extracted from the fermentation broth of bacillus belgium SL-K2 according to claim 1.

3. The method for preparing guanine according to claim 2, characterized by comprising the following steps:

S3, performing alcohol precipitation on dry powder of the fermentation liquor mixture by 8-12 times of ethanol, keeping for 20-30 hours, filtering with filter paper, separating filtrate and precipitate, and precipitating to obtain a primary extract;

S4, removing ethanol in the precipitate obtained in the previous step, preparing dry powder, dissolving the dry powder, and performing dialysis treatment to remove macromolecules with a size of more than 500Da to obtain a secondary extract;

s5, performing HPLC (high Performance liquid chromatography) purification on the secondary extract, and separating to obtain a refined extract, wherein the refined extract is guanine.

4. The process for producing guanine according to claim 3, wherein the fermentation broth of Bacillus belicus SL-K2 in the step S1 is produced by:

Inoculating bacillus belicus SL-K2 into a liquid culture medium, and shake culturing for 40-55 h, wherein the rotation speed of a shaking table is 120-200 rpm, so as to obtain a culture solution; the liquid culture medium is added with fusarium microspore inactivating liquid of the malus spectabilis.

5. The preparation method of guanine according to claim 4, wherein the liquid culture medium comprises the following components in percentage by mass:

1-10% of fusarium oxysporum microspore inactivating liquid with the concentration of 10 ^3~10⁵/ml, 0.05-0.5% of beef extract, 0.1-1% of peptone and 0.1-1% of bean cake powder; 0.5-3% corn steep liquor; 0.05-0.3% CaCO ₃; 0.01-0.05% of MgSO ₄ and the balance of water.

6. The preparation method of guanine according to claim 5, wherein the preparation method of the fusarium oxysporum virens microspore inactivation solution of the malus spectabilis comprises the following steps: and (3) treating the microspore suspension of Fusarium roseum at 55-65 ℃ for 20-40 min.

7. The use of guanine as defined in claim 2 for promoting plant growth.

8. The use of guanine as defined in claim 2 for increasing disease resistance of potato against fusarium wilt of begonia.