CN110331112B

CN110331112B - Pseudomonas fluorescens for preventing and treating rice blast and application thereof

Info

Publication number: CN110331112B
Application number: CN201910695393.5A
Authority: CN
Inventors: 沙月霞; 董国菊; 魏照清; 沈瑞清; 哈学虎; 张昂; 伍顺华
Original assignee: Institute of Plant Protection of Ningxia Academy of Agriculture and Forestry Sicience
Current assignee: Institute of Plant Protection of Ningxia Academy of Agriculture and Forestry Sicience
Priority date: 2019-07-30
Filing date: 2019-07-30
Publication date: 2022-03-18
Anticipated expiration: 2039-07-30
Also published as: CN110331112A

Abstract

The invention discloses Pseudomonas fluorescens (Pseudomonas fluorescens) for preventing and treating rice blast and application thereof, belonging to the technical field of microorganisms. The Pseudomonas fluorescens (Pseudomonas fluorescens) S149 comprises CGMCC (China general microbiological culture collection center) which is preserved in 3-1.2019, and the preservation number is CGMCC NO. 17278. The pseudomonas fluorescens provided by the invention can prevent and treat the rice blast, has good prevention effect on the rice blast, has obvious bacteriostatic action on various plant pathogenic bacteria, has high prevention and treatment efficiency, has good rice blast prevention and treatment effects on different strains of rice in the south and the north of China, and can reduce the yield loss of the rice industry caused by sudden diseases in the near-harvest period to a greater extent.

Description

Pseudomonas fluorescens for preventing and treating rice blast and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to Pseudomonas fluorescens (Pseudomonas fluorescens) for preventing and treating rice blast and application thereof.

Background

Rice is one of the important food crops in the world, the rice is staple food which more than half of the population in the world depends on, the rice yield accounts for more than 1/4 of the total yield of the food in the world, and the total yield of the food in China is more than half. The rice blast is the most main disease of rice, occurs in 85 countries all over the world, and is one of rice diseases with the most serious harm to rice regions in the south and north of China. The rice blast causes 20-30% of rice yield loss every year, the serious year reaches more than 50%, and the yield loss every year can nourish 6 million people. Rice Blast (Rice Blast) is a worldwide fungal disease which is caused by ascomycetes Magnaporthe oryzae (T.T.Hebert) Yaegashi & Udagawa (amorphous: Pyricularia oryzae) and has strong outbreak and is easy to spread. The disease can cause damage to rice all the year round, and the damage extends to various parts of the rice, such as seedling rice blast, leaf occipital blast, knot rice blast, neck blast, branch blast, grain blast and the like.

The breeding of disease-resistant rice varieties, agronomic measures and chemical pesticide prevention are the most common measures in the current production, and the breeding of the disease-resistant rice varieties is generally long due to the easy variation of physiological small varieties of rice blast germs; the residue of chemical pesticide is easy to pollute the ecological environment and threaten the human health, and pathogenic bacteria are easy to generate drug resistance. Therefore, the search for a novel biocontrol bacterium which is friendly to human and environment and has good control effect is very important for the sustainable development of the rice industry.

Pseudomonas fluorescens (Pseudomonas fluorescens) can effectively control plant diseases, has the advantages of difficult generation of resistance of plant pathogenic bacteria, promotion of plant growth and the like, and is an important biocontrol bacterium for preventing and treating rice blast. Pseudomonas fluorescens JKD-2 separated from straw stubbles such as Xiyu spring secretes an extracellular protein which can dissolve rice blast germ mycelium and inhibit spore germination, and the prevention effect under greenhouse conditions reaches 60%. Pseudomonas aeruginosa (P.aeruginosa) SUB8 separated from Liaoxianlan, etc. has a width of 4.53cm for inhibiting zone of Magnaporthe grisea, generates antibiotic active substances of phenazine substances, and has obvious antagonistic effect on Magnaporthe grisea.

By utilizing the molecular biology technology, the research and the application of the novel pseudomonas fluorescens biocontrol preparation are developed, and the rice quality and the yield enhancement of the rice are improved. The research and application of pseudomonas fluorescens for preventing and treating the rice blast are the requirements of sustainable development of rice production, can meet the requirements of food organization in the united nations on safe development of food, but the pseudomonas fluorescens strain which has broad-spectrum, high-efficiency prevention and effect on the rice blast and high yield of antibacterial active substances is still lacking in the field.

Disclosure of Invention

In order to solve the technical problems in agricultural production and the defects in the prior art, the invention provides the pseudomonas fluorescens strain which has good prevention effect on rice blast and has obvious bacteriostatic action on various plant pathogenic bacteria.

To achieve the purpose, the invention adopts the following technical scheme.

In a first aspect, the invention provides Pseudomonas fluorescens (Pseudomonas fluorescens) S149, which is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation date is 3 and 1 days in 2019, and the preservation number is CGMCC NO. 17278.

In a second aspect, the present invention provides a microbial inoculum comprising pseudomonas fluorescens S149 as described in the first aspect.

In a third aspect, the present invention provides a method for controlling rice blast, which comprises applying Pseudomonas fluorescens S149 as described in the first aspect or the microbial agent as described in the second aspect to a rice crop.

In the method for controlling rice blast of the present invention, the microbial inoculum is a culture solution of the pseudomonas fluorescens S S149.

In the method for preventing and treating rice blast, the rice blast is one or more of seedling rice blast, leaf occipital blast, knot rice blast, panicle neck blast, branch and stalk blast and grain blast.

In a fourth aspect, the present invention provides the use of Pseudomonas fluorescens S149 as described in the first aspect or a microbial agent as described in the second aspect for controlling rice blast.

In the application of the invention for preventing and treating rice blast, the rice blast is one or more of seedling rice blast, leaf occipital blast, knot rice blast, panicle neck blast, branch and stalk blast and grain blast.

The invention has the following beneficial effects:

the pseudomonas fluorescens S149 provided by the invention has good control effect on rice blast, has obvious bacteriostatic action on various plant pathogenic bacteria, and has high control efficiency.

The pseudomonas fluorescens S149 provided by the invention has broad-spectrum and high-efficiency antibacterial and antibacterial capabilities on rice blast, has good rice blast control effects on different lines of rice in China, and can reduce the yield loss of rice industry caused by sudden diseases in the near-harvest period to a greater extent.

The pseudomonas fluorescens S149 provided by the invention has good effects of promoting the growth and the production of rice.

The pseudomonas fluorescens S149 provided by the invention is insensitive to various antibiotics, and can still keep the control effect after being applied with antibiotic medicines or fertilizers.

The pseudomonas fluorescens S149 provided by the invention can systematically induce main defense enzymes of rice, so that the activity of the defense enzymes is obviously improved.

Preservation information

The Pseudomonas fluorescens (Pseudomonas fluorescens) which is separated and identified in the invention is named as Pseudomonas fluorescens S149, is preserved in China general microbiological culture Collection center (CGMCC) 3.1.3.7.2019 (the address: China institute of sciences institute of microbiology No. 3 of Nacio-West Lu 1 Hopkins of Chaoyang province, Beijing), and has the preservation number of CGMCC N0.17278.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows the culture pattern of Pseudomonas fluorescens S149 strain of the present invention on KB medium plate.

FIG. 2 is a plate-like picture of Pseudomonas fluorescens S149 strain of the present invention and Pyricularia oryzae P131.

FIG. 3 is a plate-like picture of Pseudomonas fluorescens S149 fermentation culture fluid and crude extracellular metabolic extract against Pyricularia oryzae P131.

FIG. 4 is a picture showing the inhibition of the structure of rice blast fungus infection by Pseudomonas fluorescens S149 fermentation culture solution and extracellular metabolic crude extract of the present invention.

FIG. 5 shows the effect of Pseudomonas fluorescens S149 of the present invention on the rice seedling defense enzyme POD.

FIG. 6 shows the effect of P.fluorescens S149 of the present invention on rice seedling defense enzyme PPO.

FIG. 7 shows the effect of P.fluorescens S149 of the present invention on rice seedling defense enzyme SOD.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be arbitrarily combined with each other without conflict.

Example 1

The method for separating and screening the pseudomonas fluorescens S149 strain comprises the following steps:

(1) collecting samples: the collected sample is rice variety Ning-Ning No. 48 rhizosphere soil, the site is Ningxia Hui Shazhou village of Yao On county of Ningxia Hui nationality, the collected sample and the root system of the rice are pulled out together, the collected sample is packaged by a collecting bag and put in an ice box to be brought back to a laboratory for separation.

(2) The separation method comprises the following steps: weighing 1g of rhizosphere soil, putting into 100mL of sterile water, uniformly mixing under vibration at normal temperature for 1h, and mixing with 10, 10 percent of sterile water²And 10³Gradient dilution, absorbing the gradient dilution solution, coating on KB agar medium plate, drying, and inverting in 30 deg.C constant temperature incubatorCulturing for 48 h. Picking single colony, carrying out gradient streaking on a KB agar culture medium plate to obtain single colony again, storing the obtained single colony in 40% glycerol, and storing in an ultralow temperature refrigerator at-80 ℃ for later use.

(3) Screening of Pseudomonas fluorescens: placing Magnaporthe grisea P131 fungus cake in the center of the flat culture medium of tomato and oat, inoculating Pseudomonas fluorescens (2 cm in streak) at the distance of 2cm from the two sides of the fungus cake after 1d for opposite culture, setting blank control, and placing in an incubator for dark culture at 28 ℃. Each treatment was repeated with 10 dishes. After 5d, the colony diameter and zone of the pathogenic bacteria were measured, and the results are shown in Table 1 and FIG. 2.

King's B agar medium (KB): peptone 20g, Glycerol 10mL, K₂HPO₄ 1.5g， MgSO₄·7H₂O1.5g, agar 20.0g, pH 7.2 + -0.1, 121 ℃ sterilization for 20 min.

Tomato and oat culture medium: 30g of oatmeal, 1000mL of water is added, the mixture is heated for 1h in a boiling water bath, 0.4g of anhydrous calcium carbonate and 150mL of tomato juice are added after gauze filtration, the water is added to complement 1000mL, 17-20g of agar powder is added, subpackaging and sterilization are carried out (121 ℃, 20 min).

The pseudomonas fluorescens strain is characterized by the following characteristics: white and opaque, the cells are gram-negative bacteria, rod-shaped, (0.7-0.8) mum multiplied by (2.3-2.8) mum, and a plurality of polar flagella exist, do not produce spores, and can secrete yellow-green fluorescent pigment to emit fluorescence.

The inventor has preserved the strain in China general microbiological culture Collection center (CGMCC), wherein the preservation date is 3 months and 1 day in 2019, and the preservation number is CGMCC No. 17278.

Example 2

Determination of the biological Activity of Pseudomonas fluorescens S149 Strain:

(1) and (3) performing bacteriostatic determination on rice blast germs by using pseudomonas fluorescens S149 fermentation culture solution: placing a magnaporthe grisea P131 fungus cake in the center of a tomato oat plate culture medium, placing sterile oxford cups at the positions 2cm away from the two sides of the fungus cake, respectively adding fermentation liquor of pseudomonas fluorescens S149 into the cups to culture in opposition, and placing the cups in an incubator for dark culture at 28 ℃ by taking a sterile KB liquid culture medium as a control. Each treatment was repeated with 10 dishes. After 5d, the colony diameter and zone of the pathogenic bacteria were measured, and the results are shown in Table 1 and FIG. 3.

(2) And (3) performing bacteriostatic determination on the rice blast bacteria by using a pseudomonas fluorescens S149 extracellular metabolite crude extract: placing a rice blast bacterium P131 fungus cake in the center of a tomato oat plate culture medium, placing sterile oxford cups at the positions 2cm away from the two sides of the fungus cake, respectively adding crude extracts of extracellular metabolites into the cups to culture in opposition, and placing the cups in an incubator for dark culture at 28 ℃ by taking a sterile KB liquid culture medium as a control. Each treatment was repeated with 10 dishes. After 5d, the colony diameter and zone of the pathogenic bacteria were measured, and the results are shown in Table 1 and FIG. 3.

King's B liquid agar medium (KB): peptone 20g, Glycerol 10mL, K₂HPO₄ 1.5 g，MgSO₄·7H₂O1.5g, pH 7.2. + -. 0.1, sterilized at 121 ℃ for 20 min.

Preparation of crude extract of extracellular metabolite: the single colonies of the test strain S149 which have been activated for 24 hours are respectively inoculated into 500mL triangular flasks filled with 100mL King' S B culture solution, and after shaking culture is carried out for 24 hours at 28 ℃ and 180rpm/min, the single colonies are used as secondary seed solutions for standby. And then inoculating the secondary seed solution into a 500mL triangular flask filled with 100mL King's B culture solution according to the inoculation amount of 1%, and performing shake culture at 28 ℃ and 180rpm/min for 72h to obtain a bacterium-carrying culture solution of the bacterial strain. And finally, centrifuging the obtained culture solution with bacteria at 4 ℃ and 12000rpm/min for 15min to remove bacteria, filtering the supernatant (0.22 mu m) for sterilization, and obtaining the crude extract of the extracellular metabolite.

TABLE 1 culture of Pseudomonas fluorescens S149 strain against Pyricularia oryzae

Note that the experimental data in the table are the mean. + -. standard error of triplicates.

(3) Biocontrol effects of P.fluorescens S149 strain under greenhouse potting conditions: selecting healthy rice variety G19, sterilizing with 1% sodium hypochlorite solution for 10min, soaking in 70% ethanol for 1cm, washing with sterile water for 5 times, germinating in a climatic chamber at 28 deg.C, and sowing in a small plastic bucket (the bottom of the bucket has a diameter of 18 cm)X 21cm in height x 24.5cm in diameter of the upper part of the water bucket). Spraying fermentation liquor of pseudomonas fluorescens S149 to be detected after planting for 28 days, wherein the thallus concentration is 1 multiplied by 10⁸CFU/mL, 20mL per bucket, 4 replicates per treatment. Inoculating rice blast fungus spore suspension after 24h, wherein the spore concentration is 1 × 10⁶CFU/mL. After 7 days, the occurrence condition of the leaf plague is investigated, and the disease index and the control effect are calculated, and the results are shown in table 2.

The investigation method comprises the following steps of according to pesticide field efficacy test criteria (I), rice blast disease leaf pest investigation grading standard, grade 0: no disease; level 1: brown spots of only pinpoint size; and 3, level: a brown necrotic gray spot which is small and round and slightly long, and the diameter of the gray spot is 1-2 mm; and 5, stage: typical rice blast scab, the damaged area is less than 10%; and 7, stage: the damaged area of the typical rice blast scab is 26 to 50 percent; and 9, stage: all leaves die. Rice blast neck blast investigation grading standard, grade 0: without neck blast, granuloblast and branch blast; level 1: has granule blast and branch blast, the number of shrivelled granules is less than 5 percent; and 2, stage: the number of the shrunken grains is 6 to 10 percent; and 3, level: the number of the shrunken grains is 11 to 20 percent; 4, level: has the particle blast and the branch and stalk blast, the number of the shrunken particles is 21 percent to 30 percent; and 5, stage: has spike neck plague, granuloblast and branch stalk plague, the number of shrivelled grains is 21-30 percent; and 6, level: the shrunken grains caused by the neck blast are more than 80 percent.

Biocontrol effect calculation formula:

TABLE 2 determination of the controlling Effect of P.fluorescens S149 on leaf blast under greenhouse conditions

Treatment of	Index of disease condition	Control effect (%)
			S149	6.67±0.56b	79.98±0.42
Magnaporthe grisea P131 control	33.61±2.46a	/

Note: the significance analysis adopts a minimum error of polarization (LSD) (P is less than or equal to 0.05 +/-standard error).

(4) Biocontrol effect of pseudomonas fluorescens S149 strain under field test conditions: activating a test strain for 48h, inoculating the test strain into 1000mL of seed culture medium, performing shake culture at 37 ℃ and 200r/min for 12 h to prepare seed solution, inoculating the test strain into a fermentation tank according to 6% of inoculation amount, performing shake culture at 37 ℃ and 200r/min for 48h, and then preparing test strain fermentation liquor. A field efficacy test of pseudomonas fluorescens S149 is carried out on YaoOZHEN in Pinuo county, 10 days, 35 days, 60 days, 75 days and 95 days of fermentation culture solution of test microbial inoculum are respectively sown on rice, 60 days of planting are used for investigating the occurrence of rice leaf blast, and 110 days of planting are used for investigating the occurrence of panicle blast. Investigation of leaf blast: randomly selecting 3 points per test cell, investigating 20 points per point, and investigating 4 fallen leaves per plant. Investigation of neck blast: the number of plants was measured at 5 points per cell, and 20 plants were investigated at each point, thereby obtaining 100 plants. The area of each test cell is 50m²The cells are randomly arranged, each processing 4 repetitions. The results are shown in Table 3.

TABLE 3 field control effect of Pseudomonas fluorescens S149 strain on rice blast

Note: the significance analysis adopts a minimum error of polarization (LSD) (P is less than or equal to 0.05 +/-standard error). The rice variety is Ningjing No. 47.

(5) Growth promoting capacity of pseudomonas fluorescens S149 strain on rice seedlings: selecting healthy and consistent rice seeds G19, putting the seeds into a fermentation culture solution of pseudomonas fluorescens S149, soaking the seeds for 0.5h, taking out the seeds, putting the seeds into a culture dish paved with filter paper, culturing the seeds in an artificial climate box with the RH of 70% at 28 ℃, and investigating the white exposure quantity of the seeds after 48 h. Each treatment was repeated for 3 dishes. Healthy and consistent rice seeds are taken and put into a fermentation culture solution of pseudomonas fluorescens S149 to be soaked for 0.5h, then the rice seeds are sown in a rice seedling raising pond, after the rice seeds grow for 10 days, the root length and the plant height of the rice seedlings are investigated, and the results are shown in table 4.

TABLE 4 growth promoting ability of P.fluorescens S149 on rice seedlings

(6) Antagonistic action of pseudomonas fluorescens S149 strain on various phytopathogens:

fusarium oxysporum, Fusarium solani, Fusarium moniliforme, Fusarium oxysporum, Botrytis cinerea, Colletotrichum fragrans, Fusarium oxysporum, Fusarium chaetomium cupreum, tobacco phytophthora parasitica, Fusarium oxysporum and rhizoctonia solani cakes (1cm) are placed in the center of a PDA culture medium plate, pseudomonas fluorescens S149 (2 cm in streaked lines) are respectively inoculated at the positions of 2cm away from the two sides of the colony edge, and the colony edge is placed in an incubator for dark culture at 28 ℃ and repeated for 5 times. The diameters of the colonies of the pathogenic bacteria and the zone of inhibition were measured after 2 to 5 days, and the results are shown in Table 5.

TABLE 5 antagonistic Effect of Pseudomonas fluorescens S149 strains on various plant pathogenic bacteria

Pathogenic bacteria	Relative hypha growth inhibition ratio (%)
		Fusarium oxysporum	33.33±1.32
Fusarium solani	43.33±2.25
		Fusarium moniliforme	16.67±0.85
Blight of watermelon	23.08±1.52
		Botrytis cinerea	22.22±1.40
Strawberry anthracnose pathogen	59.26±1.24
		Leaf blight of apple	55.26±1.15
Leaf blight of crabapple	31.25±1.45
		Tobacco black shank fungus	46.43±2.53
Strawberry leaf blight bacterium	57.55±1.25

(7) The pseudomonas fluorescens S149 strain has the inhibition effect on the rice blast pathogen infection structure:

the inhibition effect of the strain S149 fermentation culture solution on the germination and the formation of attachment cells of rice blast fungus conidium: 72h of culture of the strain S149 fermentation broth and a suspension of Magnaporthe grisea P131 spores (1X 10)⁶CFU/mL) in a ratio of 1: 1, and observing morphological changes of hypha, conidia and anchorage of rice blast germs by using an optical microscope when the mixed solution is oppositely cultured in a 2mL sterile centrifuge tube at 28 ℃ for 24 h. The results are shown in Table 6 and FIG. 4.

The inhibition effect of the strain S149 on the germination and the formation of attachment cells of conidia of rice blast fungus is as follows: 72h of culture of a sterile supernatant of the strain S149 and a suspension of Magnaporthe grisea P131 spores (1X 10)⁶CFU/mL) in a ratio of 1: 1, and observing morphological changes of hypha, conidia and anchorage of rice blast germs by using an optical microscope when the mixed solution is oppositely cultured in a 2mL sterile centrifuge tube at 28 ℃ for 24 h. The results are shown in Table 6 and FIG. 4.

TABLE 6 bacteriostatic effect of P.fluorescens S149 on spore germination and appressorium formation of Pyricularia grisea

(8) Sensitivity of pseudomonas fluorescens S149 to antibiotics:

pseudomonas fluorescens S149 was tested for sensitivity to 11 commonly used antibiotics, ampicillin, chloramphenicol, erythromycin, kanamycin, rifampin, gentamicin, neomycin, nalidixic acid, streptomycin sulfate, tetracycline, and spectinomycin. The S149 strain was cultured with shaking at 28 ℃ and 180r/min overnight, and spread on LB plates containing antibiotics at different concentrations of 15. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 75. mu.g/mL and 100. mu.g/mL, respectively, and after 24 hours, it was observed whether the strain grew or not, 10 replicates per treatment. The results are shown in Table 7.

TABLE 7 sensitivity of P.fluorescens S149 to antibiotics

Note: "+" indicates that strain S149 is not sensitive to the antibiotic; "-" indicates that strain S149 is sensitive to the antibiotic.

(9) Inducing pseudomonas fluorescens S149 rice disease-resistant defense enzyme:

when the greenhouse potted rice plants grow to the tillering stage (14 days after sowing), the cell concentration is respectively set to be 1 multiplied by 10 according to the experimental design⁸The rice plants are subjected to spray inoculation treatment by CFU/mL test strain culture solution 50 mL/plastic box (15 nutrition pots), and then water is sprayed to the rice plants every morning (until water films and water drops exist on leaves) and is sprayed to the air, so that the test environment is kept to have higher humidity. The enzyme activity was measured on rice leaves at 1d, 2d, 3d, 5d and 7d after inoculation, respectively. Putting 1.0g of each treated rice leaf into a mortar, adding 5mL of 0.05M phosphate buffer (pH6.8), 0.1g of polyvinylpyrrolidone (PVP) and a very small amount of quartz sand, grinding into homogenate in liquid nitrogen, grinding on ice, centrifuging the mixed solution at 7000rpm at 4 ℃ for 25min, and storing the centrifuged supernatant, namely the crude enzyme solution to be detected, in a refrigerator at-80 ℃ for later use.

When POD activity was measured, the reaction mixture contained 2.65mL of 0.05M phosphate buffer (pH 6.8); 0.1mL of 0.18M guaiacol, 0.2mL of 0.03M H₂O₂(when the test was performed, the enzyme solution was added) in 0.05 mL, reacted at 20 ℃ for 5min, and the OD was measured at 470nm, where the enzyme activity was measured in U/g/min. These processes are all performed on ice, preventing enzyme inactivation. During measurement, an ultraviolet spectrophotometer is used, and H is added finally₂O₂Immediately, every 30s for 3min, the results are shown in FIG. 5.

When PPO activity is measured, 4.4mL of 0.05M phosphate buffer (pH6.8), 0.5mL of 150. mu.M pyrogallol and 0.1mL of enzyme solution are added into the reaction system, and 0.5mL of 15% H is added into the reaction system for reaction at 20 ℃ for 10min₂SO₄Stopping the reaction, measuring the OD value at 420nm, and performing the whole operation process at 0-4 ℃ with U/g/min as the unit of enzyme activity, wherein the result is shown in figure 6.

Wherein, Δ OD₄₇₀The difference value of the optical density of the illumination control tube and the sample tube at 470 nm; delta OD₄₂₀The difference value of the optical density of the illumination control tube and the sample tube at the position of 420 nm; t: time of enzyme action (min); VT: total sample volume (mL); v1: measuring the dosage (mL) of the sample; FW fresh weight of sample (g).

Superoxide dismutase oxidase (SOD) assay: taking 1.0g of each treated rice leaf, putting the rice leaf into a mortar, adding 5mL of 0.05M phosphate buffer (pH7.8), 0.1g of PVP and a small amount of quartz sand, grinding the mixture into homogenate in liquid nitrogen, and centrifuging the homogenate at 4 ℃ and 13000rpm for 20min to obtain supernatant, namely the crude enzyme solution to be detected. For the assay, 2.2mL of 0.05M phosphate buffer was aspirated, followed by 0.1mL of 6X 10^-5M Riboflavin, 0.3mL 0.13M methionine, 0.1mL 3X 10^-6M EDTA，0.2mL 1.25 ×10^-3M NBT, and finally 0.1mL of enzyme solution (0.1 mL of phosphate buffer was added as a control instead). Immediately after the reaction was stopped in the dark at 25 ℃ for 25min under 4000 Lux light (i.e., fluorescent lamp), the OD was measured at 560nm to inhibit 50% reduction of NBT photochemical reaction as one unit of enzyme activity, and the results are shown in FIG. 7.

Wherein, CK control OD value: the optical density value of the illumination control tube; processing OD value: optical density value of sample tube.

(10) Detecting the biocontrol active substances of the pseudomonas fluorescens S149 strain:

detection of amylase: inoculating a newly activated single colony on an LB (lysogeny broth) plate containing 0.2% of soluble starch, culturing for 48h to form obvious colonies, dropwise adding a Lugomorph iodine solution on the plate, dyeing for 10min, washing the plate with 70% ethanol to obtain a strain capable of producing amylase, wherein a colorless transparent ring can be formed around the growth of the colony under a black background, if the transparent ring is formed, the strain can produce the amylase, and each treatment is repeated for 3 times, and the results are shown in Table 8.

And (3) detection of protease: activated strains to be tested are punctured and inoculated on a 1% skim milk agar plate, the generation of peripheral transparent rings is observed after 24, 48 and 72 hours of culture at 30 ℃, the generation of the transparent rings indicates that protease is generated, each treatment is repeated for 3 times, and the result is shown in table 8.

And (3) detecting glucanase: the bacteria to be tested are inoculated on a plate containing an ABP culture medium, after the culture at 30 ℃ for 48 and 72 hours, whether a digestion circle appears in the plate or not is observed, if the digestion circle appears, the generation of glucanase is shown, each treatment is repeated for 3 times, and the results are shown in table 8.

Detecting siderophore: detecting the siderophin by using a CAS culture medium, inoculating the activated bacteria to be detected on a CAS detection culture medium plate, culturing at 30 ℃ for 72h, observing whether an orange halo is generated in the plate or not, if the orange halo is generated, indicating that the siderophin is generated, and repeating each treatment for 3 times, wherein the result is shown in Table 8.

And (3) detection of cellulase: activating the separated strain, inoculating to a cellulose screening culture medium plate, performing inverted culture at a constant temperature of 28 ℃ for 2d, dip-dyeing with 0.1% Congo red dyeing solution for 10min, and decolorizing with 1mol/L NaCl solution for 5 min. If the strain produces cellulase, clear transparent circles appear around the colonies, and each treatment was repeated 3 times, and the results are shown in Table 8.

Preparation of ABP medium: grinding Poria into powder with mortar, and sieving with 120 mesh sieve (KH)₂PO₄ 6.8g，K₂PHO₄·12H₂17.9g of O, 6.7g of yeast extract, 5.0g of tuckahoe powder (or beta-1.3-glucan), 120mg of aniline blue, 12g of agar powder, 6.8 of pH value and 1L of water.

Preparation of CAS Medium: 9g of tyrose peptone, 5g of yeast extract, 10g of trisodium citrate, 1g of disodium hydrogen phosphate, 1g of sodium dihydrogen phosphate, 10g of glucose and 20g of agar, and dissolving in 1L of water.

Cellulose enrichment Medium (1L) NaCl 6g, MgSO₄ 0.1g，KH₂PO₄ 0.5g，CaCl₂ 0.1 g,(NH₄)SO₄ 2.0g，K₂HPO₄2.0g of agar, 15g of agar and 5g of CMC-Na, and the pH is adjusted to 7.0; cellulose screening medium (1L) 1g of yeast powder was added to the cellulase-enriched medium.

Congo red staining solution, namely dissolving Congo red by distilled water, wherein the final concentration is 0.1% (w/v).

Congo red decoloration liquid is NaCI solution with the final concentration of 1 mol/L.

TABLE 8 Pseudomonas fluorescens S149 strain biocontrol active substance detection

Note: data are three replicates of test results; "+" indicates the detection result, and "-" indicates no detection.

It should be noted that: in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

The above examples are only for illustrating the technical solutions of the present invention, and are not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A fluorescent Pseudomonas (Pseudomonas fluorescens) S149 is characterized by comprising CGMCC (China general microbiological culture collection center) with the preservation date of 2019, 3 and 1 and the preservation number of CGMCC NO. 17278.

2. A microbial inoculum comprising pseudomonas fluorescens S149 of claim 1.

3. A method for controlling rice blast, which comprises applying Pseudomonas fluorescens S149 as set forth in claim 1 or the microbial agent as set forth in claim 2 to a rice crop.

4. The method of claim 3, wherein the microbial inoculum is a culture broth of Pseudomonas fluorescens S149.

5. The method of claim 3 or 4, wherein the blast disease is one or more of Misgurni oryzae, leaf blast, leaf occipital blast, knot blast, panicle blast, branch blast and grain blast.

6. Use of Pseudomonas fluorescens S149 as claimed in claim 1 or a microbial inoculum as claimed in claim 2 for controlling rice blast.

7. The use of claim 6, wherein the blast disease is one or more of Misgurni oryzae, leaf blast, leaf occipital blast, knot blast, panicle blast, branch blast and grain blast.