CN114214231A - Rice endophytic streptomyces griseofulvin Ahn75 and application thereof - Google Patents

Abstract

The invention discloses a rice endophytic streptomyces griseofulvin Ahn75 strain and application thereof. The strain is classified and named as: streptomyces griseofulensis (Streptomyces griseobrunneus) with a accession number: CCTCC NO: M2019890. The streptomyces griseofulvin Ahn75 can effectively inhibit the growth of 12 different physiological race rice blast pathogenic bacteria in vitro. The fermentation filtrate of the strain can inhibit the hypha growth and spore germination of Magnaporthe grisea simultaneously, and contains more active compounds with sizes below 3kD, 10kD-30kD and above 30kD, and the small molecular active compound contains valinomycin. Meanwhile, the strain has growth promoting properties of siderophores production, potassium dissolution, nitrogen fixation and the like. These results indicate that the strain has the potential of developing a broad-spectrum and environment-friendly novel rice pesticide fertilizer.

Description

Rice endophytic streptomyces griseofulvin Ahn75 and application thereof

Technical Field

The invention belongs to the field of microorganisms, and relates to a rice endophytic streptomyces griseofulvus and application thereof in rice blast resistance, rice growth promotion and the like.

Background

The rice blast is caused by infection of fungus Pyricularia oryzae (Magnaporthe oryzae), and is one of the most serious rice diseases in rice regions all over the world. The rice blast only needs about 20 days from infection, formation of disease spots to diffusion of new spores, can infect most tissues of rice and occurs in rice planting areas all over the world. The rice yield loss caused by rice blast accounts for about 10% -30% of the total yield every year, and nearly sixty million of people can be cultivated. At present, the cultivation of new disease-resistant varieties and the spraying of chemical pesticides are main means for preventing and treating rice blast, but the breeding of the new disease-resistant varieties is long in period and has the problems that the resistance is easy to lose, and the large use of the chemical pesticides not only pollutes the environment, but also causes harmful substances to remain in rice, and influences the health of human beings. Therefore, the search for new methods for controlling rice blast has been increasingly focused, and among them, biological control has become an important research field.

Endophytes are microbial flora that can stably colonize the interior of a plant throughout or part of the plant's growth cycle. Endophytes grow inside plants without competition for survival with soil bacteria, as compared to rhizosphere and soil microorganisms. Moreover, the endophyte can survive in the plant body without generating obvious adverse effect on the host plant, and forms a mutual and beneficial relationship with the plant in the long-term co-evolution process. Recent studies have shown that rice plants themselves also have some endophytes that inhibit rice blast fungi. As one of the components of the current largest antibiotic production group, Streptomyces endophyticus has higher biological control potential obviously.

Disclosure of Invention

The invention aims to provide endophytic streptomycete for preventing and treating rice blast, the strain can efficiently inhibit 12 different physiological races of rice blast germs, and antibacterial active compounds in fermentation liquor are various and have different sizes. The strain belongs to endophyte, and is obtained by screening uninvolved rice stem tissues in a rice blast disease area of Hainan rice.

The strain obtained by the invention is combined with the morphological characteristics of colony thallus and a phylogenetic tree based on 16S rRNA, gyrB, atpD, recA, rpoB and trpB gene sequences, and is identified as Streptomyces griseobrunius Ahn 75. The strain is preserved by China center for type culture Collection (preservation address: Wuhan university) in 2019, 11/4, with the preservation number of CCTCC NO: M2019890.

The second purpose of the invention is to provide the application of the streptomyces griseofulvin Ahn75 in preventing and treating rice blast.

The third purpose of the invention is to provide the application of the streptomyces griseofulvin 75 fermentation liquor in preventing and treating rice blast.

Further, the fermentation broth inhibits hypha growth and/or spore germination of Pyricularia oryzae.

The obtained strain can effectively inhibit 12 different physiological races of rice blast pathogenic bacteria, and has the highest inhibition rate of 61.93% on No. 42 of rice blast pathogenic bacteria.

Further research shows that: the bacteriostatic activity of the biomass and fermentation liquor of the rice endophytic streptomyces griseofulvin Ahn75 in the ISP2 culture medium with the initial pH of 7-9 is higher than that of the initial pH of 4-6. The highest biomass is 1190.63mg/50mL, and the highest bacteriostasis rate of 100 mu L fermentation liquor to the growth of rice pear spore hypha is 72.83%.

The activity of the rice endophytic streptomyces griseofulvin 75 fermentation liquor is stable below 50 ℃, the activity is gradually reduced after the fermentation liquor is treated at the temperature of above 80 ℃, and the fermentation liquor is stable to proteinase K.

The fourth purpose of the invention is to provide the application of the active substance in the fermentation liquor of the streptomyces griseofulvin 75 in preventing and treating rice blast.

Furthermore, the active substances in the fermentation liquor of the rice endophytic streptomyces griseofulvin Ahn75 are distributed below 3kD and 10kD-30kD, and the active substances above 30kD and below 3kD contain valinomycin.

The inhibition rate of valinomycin on rice blast hypha reaches 52.27% when the concentration of valinomycin is 15 mu g/mL.

The fifth purpose of the invention is to provide the application of the streptomyces griseofulvin Ahn75 in promoting the growth of rice.

Furthermore, the rice endophytic streptomyces griseofulvin Ahn75 has the functions of producing at least one of siderophores, decomposing potassium and fixing nitrogen.

The obtained strain can stably colonize rice root, stem and leaf tissues, can effectively inhibit the growth of rice blast germs, can fix nitrogen, dissolve potassium and produce iron carriers, and has good development and application prospects.

Drawings

FIG. 1: the colony and strain morphology of strain Ahn 75;

FIG. 2: the strain Ahn75 is based on a phylogenetic tree of 5 housekeeping genes such as 16S rRNA and gyrB;

a is 16S rRNA; b, 5 gene;

FIG. 3: growth of strain Ahn75 in ISP2 medium at different pH values;

FIG. 4: the bacterial strain Ahn75 fermentation liquor inhibits the growth of rice pear spore hypha and spore germination;

A. growth state of hyphae on the fermentation filtrate flat plate containing different concentrations; B. the inhibition level of the fermentation filtrate on the growth of the rice pear spore hyphae; C. the level of inhibition of the fermentation filtrate on the germination of pyricularia oryzae spores.

FIG. 5: the stability of the fermentation liquor of the strain Ahn75 to temperature and proteinase K;

a, heat treatment; and B, treating by proteinase K.

FIG. 6: mass spectrometric identification of valinomycin in the fermentation broth of the strain Ahn 75;

FIG. 7: colonization analysis of the strain Ahn75 in rice root, stem and leaf tissues.

A. Control rice roots stained with trypan blue and rice roots sprayed with Ahn 75-GFP;

the colonization amount of Ahn75 in different tissues of rice.

The specific implementation mode is as follows:

the present invention will be further illustrated with reference to the following examples, which should not be construed as limiting the invention thereto.

Example 1 screening and purification of Ahn75 Strain

Collecting rice tissues with mild morbidity and no morbidity from a rice field in the city of Susan Hainan, cleaning, sterilizing the surface, cutting a stem into small segments with the length of about 1cm, placing the small segments in a TWYE culture medium, culturing at 37 ℃, picking out endophytic actinomycetes to be separated out from the tissues, transferring the endophytic actinomycetes to an 1/2PDA culture medium, streaking, separating and purifying to obtain a pure culture of the endophytic actinomycetes. The purified strain was stored in suspension in 25% glycerol to-80 ℃ freezer.

Inoculating fresh fungus cakes of different physiological race rice blast germs at the center of an ISP2 culture medium with the diameter of 90mm, then inoculating actinomycete strains at the position 3cm away from the center of the fungus cakes by using a sterile toothpick, setting sterile strain treatment as a control, repeating the treatment for 3 times, culturing at the constant temperature of 28 ℃ for 7d, measuring the colony radius of the pathogenic bacteria, and calculating the bacteriostasis rate. The antagonistic strain inhibition rate (%) is (control group colony radius-treatment group colony radius)/(control group colony radius-cake radius) × 100.

The composition of the TWYE medium used was: 0.25g of yeast extract, 0.5g of dipotassium phosphate, 18g of agar powder and 1000mL of pure water, wherein the pH value is 7.2 +/-0.2, and the mixture is sterilized at the high temperature and the high pressure of 121 ℃ for 25 min. When the culture medium is cooled to about 55 ℃, benomyl with the final concentration of 50 mu g/mL and nalidixic acid with the final concentration of 25 mu g/mL are added under the aseptic condition and are fully and evenly mixed, and then the plate is inverted. 1/2PDA medium composition: 100g of potato, 10g of glucose and 18g of agar, wherein the pH is natural, 1000mL of water is used, and the potato is sterilized at the high temperature of 121 ℃ and the high pressure for 25 min. ISP2 medium composition used: 10g of malt extract, 4g of yeast extract, 4g of glucose, pH 7.2, 1000mL of water, and high-temperature and high-pressure sterilization at 115 ℃ for 30 min.

Example 2 identification of Ahn75 Strain

1. And (3) observing the strain morphology: the bacterial strain is cultured on an ISP2 culture medium, the colony at the initial stage is round, the air silk is yellow and white, the spore at the early stage is gray green, the spore at the later stage is earthy yellow, the base silk is light yellow brown, and the red brown pigment is produced for a long time. The strain cultured on the ISP2 culture medium by inserting sheets is observed under a phase contrast microscope, the spore silks are straight or wavy, the conidia are spherical to elliptical, and the surface of the spore is smooth. The early and late colony morphology and hyphal and spore morphology of the Ahn75 strain are shown in FIG. 1.

2. And (3) strain identification:

the genome DNA of actinomycete Ahn75 is extracted by adopting a bacterial total DNA extraction kit of Shanghai bioengineering GmbH, the 16s rRNA sequence of the Ahn75 strain is amplified by taking the genome DNA as a template and 27F/765R and 704F/1492R as primers (the primer sequences are shown in table 1), and meanwhile, the conserved sequences of gyrB, atpD, recA, rpoB and trpB genes of the Ahn75 strain are obtained by PCR amplification (the primers are shown in table 1) and sequenced. The 5 conserved gene sequences are connected into a sequence in an end-to-end mode, and on the basis of the sequence, a phylogenetic tree (figure 2) is constructed by using a Neighbor-Joining method (NJ) by using software Culstal W2 and Mega7.0. An evolutionary tree based on 16s rRNA showed that the Ahn75 strain had the highest homology of 99.48% with the standard strain streptomyces griseofulnenus ATCC 4.1838. The phylogenetic tree of the 5 gene shows that Ahn75 is identical to streptomyces griseofulensis ATCC 4.1838 and streptomyces baculosus (s.bacillus) CGMCC 4.1584. Studies have shown that streptomyces griseofulvus and streptomyces baculosus are the same species. The strain is preserved in China center for type culture Collection in 2019, No. 11/4, with the preservation number: CCTCC NO: M2019890, preservation Unit Address: china, wuhan university.

TABLE 1 primer sequences for PCR amplification

Example 3 bacteriostatic ratio of the strains on different physiological races of rice blast

The bacteriostatic activity of the Ahn75 strain on physiological races of rice blast with different sources was tested by a plate confrontation test (the operation process is the same as that in example 1), and the results are shown in Table 2. The Ahn75 has higher bacteriostatic activity on 40 different physiological races of rice blast pathogenic bacteria, and the bacteriostatic rate on 10 Pyricularia oryzae strains exceeds 50 percent.

TABLE 2 inhibitory Activity of Ahn75 on mycelial growth of different physiological races of Pyricularia oryzae

EXAMPLE 4 growth Performance of the strains under different pH conditions

Inoculating Ahn75 strain into ISP2 solid culture medium, culturing for 3-5 days to produce spores, scraping spore powder, suspending into 0.5% Tween aqueous solution, and making into 1 × 10⁸The spore suspension of CFU/mL was inoculated at 2% inoculum size into ISP2 medium pH4-9, and cultured at 28 ℃ and 180rpm for 7 days, each pH was repeated 3 times. Collecting fermentation liquor, centrifuging at 10000rpm for 10min, precipitating on an ultraclean workbench, blow-drying water, weighing, measuring biomass, filtering the supernatant with a 0.22 μm sterile microporous filter membrane, adding 100 μ L sterile filtrate into an Oxford cup 2.5cm away from the center of a Pyricularia oryzae cake on an ISP2 solid culture medium, and performing an antibacterial test. The bacteriostatic rate was calculated according to the formula of example 1, and the result is shown in FIG. 3. The Ahn75 can not grow under acidic condition of pH4-5, can grow slowly in culture medium of pH6, has low antibacterial activity, can grow rapidly under pH7-9, and can obtain maximum biomass under pH9And the fermentation liquor has the highest activity under the condition of pH 8. However, the biomass and the bacteriostatic activity of the strain under the condition of pH7-9 are analyzed by SPSS19.0 one-way Duncan (D), and no significant difference exists.

EXAMPLE 5 Strain fermentation broth Activity and stability

Effect of the sterile fermentation filtrate of Ahn75 Strain on the growth of Magnaporthe grisea hyphae

After culturing Ahn75 in ISP2 at pH7 according to the method of example 4, the fermentation filtrate was collected after 7 days. Diluting the sterile fermentation liquor by 2, 4, 10, 50 and 100 times, respectively taking 1mL of stock solution and diluent with different concentrations, respectively mixing the stock solution and the diluent with 9mL of ISP2 culture solution cooled to 50 ℃, preparing culture medium plates respectively containing 10%, 5%, 2%, 1% and 0% of the sterile fermentation liquor, then inoculating fresh rice blast fungus cakes in the centers of the plates, repeating the treatment for 3 times, using sterile water to replace the sterile fermentation liquor as a control, culturing at the constant temperature of 28 ℃ for 7d, measuring the colony diameter of pathogenic bacteria by a cross method, and calculating the hypha growth inhibition rate (the inhibition rate (%) (the diameter of the control colony-the diameter of the treatment group colony)/(the diameter of the control colony-the diameter of the fungus cakes). times.100). The inhibition of the fermentation liquor of the Ahn75 strain on the pyricularia oryzae hypha shows concentration dependence, and when the concentration of the fermentation liquor is increased from 1% to 10%, the inhibition rate is increased from 25.05% + -0.22% to 80.88% + -1.54%, and the results are shown in FIGS. 4A and 4B.

Effect of the sterile fermentation filtrate of Ahn75 Strain on the Germination of Magnaporthe grisea spores

Preparation of rice blast spore suspension: washing Pyricularia oryzae spores stored on sorghum grains with sterile water, coating 100 mu L of Pyricularia oryzae spores on a fresh ISP2 plate, culturing at 28 ℃ for 3d, selecting a single colony to be inoculated on another fresh ISP2 plate, culturing at 28 ℃ for 6d, selecting mycelia to be inoculated on an oat culture medium (30g of oatmeal is boiled by pure water for 1h, four layers of gauze are filtered to remove precipitates, 100mL of squeezed fresh tomato juice, 15g of agar powder, pure water to reach a constant volume of 1000mL, pH 7.2 and 121 ℃ for 20min) is added, after the mycelia on the plate are fully grown (about 7-10 d), brushing off aerial mycelia by using a brush under an aseptic condition, and placing the plate in a 28 ℃ artificial climate incubator for culturing in light and dark alternation (14: 10 h). After the spores are fully produced (about 3-4 d), the spores are washed down by 5mL of sterile water in each dish, and sterilized wipes are usedFiltering with mirror paper to obtain the product 10⁵Perilla spore suspension/mL.

A sterile fermentation broth of Ahn75 was proportioned to a rice blast fungus spore suspension (10) in a 1.5mL Ep tube⁵one/mL) and sterile water, preparing sterile fermentation liquor with the concentration of 5%, 10%, 20% and 50%, repeating each treatment for 5 times, setting the sterile water and the pyricularia oryzae spore suspension to be mixed as a contrast, performing static culture in an incubator at 28 ℃ for 16h, detecting the spore germination condition by adopting a microscope under a lens of 10 multiplied by 20 times, counting the spore germination rate of each treatment, and calculating the spore germination inhibition rate according to the following formula, wherein the result is shown in figure 4C. The results showed that when the concentration of the Ahn75 filtrate was 50%, the inhibition rate of spore germination was 79.15% + -7.18%.

Spore germination inhibition (%) - (control spore germination rate-treated spore germination rate)/control spore germination rate × 100

Stability of the sterile fermentation filtrate of Ahn75 Strain to temperature and proteases

The Ahn75 sterile fermentation filtrate is respectively treated at different temperatures for 30min, protease K is added into the separately packaged Ahn75 sterile fermentation filtrate, the mixture is respectively treated at 50 ℃ for 30-120min, the obtained treatment liquid is respectively detected to inhibit the growth of the rice pear spore hyphae according to the method, and the result shows that after the Ahn75 fermentation filtrate is treated at 40 ℃ and 50 ℃ for 30min, the activity of the fermentation filtrate is basically unchanged with the untreated filtrate, the bacteriostatic activity of the fermentation filtrate is gradually reduced along with the increase of the treatment temperature, and the bacteriostatic activity is basically completely lost after the fermentation filtrate is treated at 121 ℃ for 30min, which indicates that the active substances generated by the strain are unstable at the high temperature of more than 80 ℃ and stable below 50 ℃ (figure 5A). After the Ahn75 fermentation broth is respectively treated by proteinase K for 30-120min, the bacteriostatic activity is still 93.99% -105.35%, and no significant difference is generated from the control (figure 5B).

Example 6 analysis and characterization of antibacterial active ingredients in Ahn75 Strain fermentation broth

1. Active compound size analysis

The method comprises the steps of respectively adopting 3kD, 10kD and 30kD ultrafiltration tubes to carry out ultrafiltration on Ahn75 fermentation filtrate, collecting ultrafiltration tube filtrate and supernatant, detecting the bacteriostatic activity of a treatment solution on the growth of rice pear spore hyphae according to example 4, and finding a result shown in Table 3, wherein the size of an antibacterial active substance synthesized by Ahn75 is mainly concentrated on more than 3kD, the bacteriostatic activity of the compound is 6.5 times of that of a compound below 3kD, the sizes of the compound are 3-10kD, the 10-30kD and the above 30kD are distributed, and the filtrate below 3kD also shows 7.45% of bacteriostatic rate, which indicates that the bacterial strain also synthesizes antibacterial compounds with the molecular weight of less than 3 kD.

TABLE 3 bacteriostatic activity of Ahn75 strain fermentation filtrate after ultrafiltration membrane treatment

2. Identification of active Compounds

Extracting sterile Ahn75 fermentation filtrate with n-butanol in equal volume, rotary evaporating organic phase, concentrating to obtain antibacterial crude extract, re-dissolving with sterile ultrapure water, and analyzing with liquid chromatography mass spectrometer (Agilent Poroshell 120HILIC-Z connected with Thermo LTQ Orbitrap XL mass spectrometer). The obtained secondary mass spectra were aligned in the GNPS Global Natural Product database (http:// GNPS. ucsd. edu). The results show that the mass M/z 1149.59, 1133.63 matches the mass-to-charge ratios of [ M + K ] +, [ M + Na ] +, of Valinomycin (Valinomycin) in the database NIST14, and that the MS2 patterns of the two precursor ions also match the secondary mass spectrum of Valinomycin (fig. 6). The molecular weight of valinomycin is about 1kD, and it was found that valinomycin has inhibitory activity against rice blast disease by analyzing the inhibitory activity of the purchased standard valinomycin against Pyricularia oryzae hyphae (method performed in example 5). From this, it is presumed that the Ahn75 strain synthesized valinomycin active against rice blast.

Analysis of Secondary metabolite biosynthesis Gene Cluster in genome of Ahn75 Strain

Extracting Ahn75 strain genome DNA according to example 2, fragmenting the genome DNA to construct an Illumina PE library (400bp library), carrying out Illumina Hiseq sequencing on the library through bridge PCR, shearing and splicing the sequence by using a bioinformatics analysis means after carrying out quality control on the obtained sequencing data to obtain a whole genome scanning sequence of the strain, and then carrying out gene prediction and annotation on the genome sequence by using each big data center. The biosynthetic gene cluster of the secondary metabolites of the genome is predicted by using anti-marsh software, and the genome of the strain is found to possibly contain the biosynthetic gene clusters of 40 secondary metabolite organisms, including 3 lactone antibiotics (lactones),7 non-ribosomal polypeptides (non-ribosomal peptide synthases, NRPS),2 similar non-ribosomal polypeptides (Nrps-like),3 non-ribosomal polypeptide-polyketides (Nrps-PKS),4 siderophores (siderophores),8 polyketide antibiotics (polyketide antibiotics, PKS),5 terpenoids (terpen), 6 antibacterial peptides and 2 other compounds (Table 4).

TABLE 4 Secondary metabolite biosynthesis Gene Cluster of Strain Ahn75

EXAMPLE 7 detection of growth promoting factor and synthetic Gene (Gene Cluster) of Ahn75

Spore powder of the Ahn75 strain is spotted on an Abira sinensis (Ashby) nitrogen-free solid culture medium, an inorganic phosphorus and organic phosphorus decomposing culture medium (PKO and Monkina culture medium), a potassium decomposing culture medium (Alexander bauov culture medium) and an iron carrier chromogenic culture medium (CAS culture medium), the Ahn75 strain is cultured for 7d at 28 ℃, the growth condition of the colony is detected, the diameters of the colony and a corresponding hydrolysis ring are measured by a cross method, and the result shows that the Ahn75 strain has the capabilities of fixing nitrogen, decomposing potassium and producing iron carriers (Table 5). The genome of Ahn75 strain was analyzed, and it was found that the strain contained genes (clusters) related to siderophores, nitrogen fixation and trehalose, IAA and growth-promoting factors such as arginine and potassium (Table 6).

TABLE 5 growth-promoting factor levels of Strain Ahn75

The used arbuscular fritillary (Ashby) nitrogen-free solid medium: 10g of glucose, KH₂PO₄ 0.2g，MgSO₄·7H₂O 0.2g，CaSO₄·2H₂O 0.1g，CaCO₃5g, NaCl 0.2g, agar 18.0g, deionized water 1000mL, pH 7.0.

PKO medium: tricalcium phosphate 5.0g, sucrose 10.0g, (NH)₄)₂SO₄0.5g yeast powder 0.5g, NaCl 0.2g, MgSO₄·7H₂0.1g of O, 0.2g of KCl, 3.0mL of 1% manganese sulfate, 3.0mL of 1% ferrous sulfate, pH7.0, 18.0g of agar and 1000mL of deionized water.

Monkina culture medium: glucose 20.0g, (NH)₄)₂SO₄ 0.5g，NaCl 0.3g，KCl 0.3g，MgSO₄·7H₂O 0.3g，FeSO₄·7H₂O 0.03g，CaCO₃ 5.0g，MnSO₄·4H₂O 0.03g，(NH₄)₃PO₄0.5g, 10mL of egg yolk liquid (the egg yolk liquid is prepared from sterile physiological saline and egg yolk 1: 1), 7.0-7.5 of pH and 1000mL of deionized water.

Alexander bauov culture medium: sucrose 5.0g, Na₂HPO₄ 2.0g，MgSO₄·7H₂O 0.5g，FeCl₃ 5.0mg，CaCO₃0.1g, 1.0g of potassium feldspar powder, 7.0-7.5 of pH, 18.0g of agar and 1000mL of deionized water.

CAS medium: 1mL of 20% glucose solution, 3mL of 10% casamino acid and 1mmol/L of CaCl₂ 0.1mL，1mmol/L MgSO₄2mL, 10mL CAS staining solution, 10mL phosphate buffer solution, 1.8g agar and 74mL deionized water.

Preparation of CAS dye solution (200 ml): solution A: 0.12g of Chrome Azure (CAS) was dissolved in 100mL of double distilled water and mixed with 1mmol/L FeCl₃20mL of the solution is mixed evenly to obtain 120mL of A solution. And B, liquid B: 0.15g of cetyltrimethylammonium bromide was dissolved in 80mL of double distilled water to obtain solution B. Slowly adding the solution A into the solution B, and fully and uniformly mixing to obtain the CAS dyeing solution.

Phosphate buffer solution: na (Na)₂HPO₄·12H₂O 2.427g，NaH₂PO₄·2H₂O 0.590g，KH₂PO₄ 0.075g，NH₄0.250g Cl, 0.125g NaCl, 100.0mL deionized water, pH 6.8.

TABLE 6 Strain Ahn75 growth-related genes (Cluster)

Example 8 colonization of Ahn75 in Rice

Spraying Ahn75 spore suspension in the 3-4 leaf stage of rice, spraying sterile water in a contrast mode, collecting and washing rice roots after 1-3 days, treating and dyeing the rice roots by adopting a trypan blue dyeing method, observing the colonization condition of endophytes under a phase contrast microscope, and finding that the actinomycetes can well grow on the rice roots, and the control roots without spraying Ahn75 have no hypha growth (figure 7A). Collecting different tissues of rice on days 1-27 after spraying the Ahn75 spore suspension, re-separating and counting the colonized Ahn75 by adopting an endophytic actinomycete separation method, counting the colonized quantity of the Ahn75 in different tissues of the rice at different times, and finding that the colonized quantity of the Ahn75 in each tissue of the rice is rapidly increased after spraying and then gradually decreased to a gentle state along with the time. Wherein the amount of colonization in the root tissue is maximal on day 3, while the amount of colonization in the stem and leaves peaks on day 5. Ahn-75 was more colonized in stem tissue than in roots and leaves, with higher colonizations from day 5 to day 27 than in roots and leaves (fig. 7B).

Claims (10)

1.A strain of Streptomyces griseobrunnus Ahn75 with the preservation number of CCTCC NO: M2019890 is generated in rice.

2. The application of the streptomyces griseofulvin Ahn75 in rice to prevention and treatment of rice blast as claimed in claim 1.

3. The application of the Streptomyces griseofulvin Ahn75 fermentation broth in rice to prevention and treatment of rice blast as claimed in claim 1.

4. The use according to claim 3, wherein the fermentation broth inhibits hyphal growth and/or spore germination of Pyricularia oryzae.

5. The use according to claim 3, wherein the bacteriostatic activity of the biomass and fermentation broth of the rice endophytic streptomyces griseofulvin 75 in the ISP2 culture medium with the initial pH of 7-9 is higher than that in the initial pH of 4-6.

6. The use according to claim 3, wherein the activity of the fermentation broth of Streptomyces griseofulvin 75 in rice is stable below 50 ℃, and gradually decreases after treatment at 80 ℃ or above, and the fermentation broth is stable to proteinase K.

7. The application of the active substance in the fermentation broth of streptomyces griseofulvin Ahn75 in rice to preventing and treating rice blast of rice as claimed in claim 1.

8. The use according to claim 7, wherein the active substances in the fermentation broth of streptomyces griseofulvin Ahn75 are distributed below 3kD, and 10kD-30kD, and above 30kD, and the active substances below 3kD comprise valinomycin.

9. The use of the streptomyces griseofulvin Ahn75 in rice as claimed in claim 1 for promoting the growth of rice.

10. The use according to claim 9, wherein the streptomyces griseofulvin Ahn75 has at least one of siderophore production, potassium release and nitrogen fixation.

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