CN113736690B - Streptomyces alboflavus, microbial inoculum containing streptomyces alboflavus and application of streptomyces alboflavus and microbial inoculum in prevention and treatment of plant diseases - Google Patents
Streptomyces alboflavus, microbial inoculum containing streptomyces alboflavus and application of streptomyces alboflavus and microbial inoculum in prevention and treatment of plant diseases Download PDFInfo
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Abstract
The invention relates to a plant control technology, and discloses streptomyces alboflavus and a microbial inoculum and application thereof in plant disease control. The preservation number of the streptomyces albidoflavus (streptomyces albideflaviniger) provided by the invention is CCTCC NO: m2021913. The culture method of streptomyces alboflavus provided by the invention comprises the following steps: activating streptomyces alboflavus to obtain a seed solution, and performing fermentation culture on the seed solution to obtain a fermentation liquid. The microbial inoculum provided by the invention contains the streptomyces alboflavus or the fermentation liquor prepared by the culture method. The method for controlling plant diseases provided by the invention comprises the following steps: contacting the streptomyces alboflavus and/or the microbial inoculum with plants. The streptomyces alboflavus and the fermentation product thereof can effectively inhibit the growth of hypha of plant pathogenic fungi, thereby achieving the purpose of prevention and control.
Description
Technical Field
The invention relates to a plant control technology, in particular to a streptomyces alboflavus, a microbial inoculum containing the streptomyces alboflavus and application of the streptomyces alboflavus and the microbial inoculum in plant disease control.
Background
Plant diseases are one of natural disasters seriously damaging crops, about 80 percent of the plant diseases are caused by plant pathogenic fungi, and the plant diseases can cause the symptoms of death, rot, wilting and the like of local cells and tissues of the crops, further influence the yield and the quality of the crops, have adverse effects on the development of national economy and the living level of people, and seriously threaten the economy of agricultural products.
At present, for plant diseases caused by the infection of plant pathogenic fungi, the main prevention and treatment means is to use chemical pesticides and cultivate disease-resistant varieties. The chemical pesticide is used in successive years, the drug resistance of plant pathogenic fungi is enhanced, serious pollution problem is caused to the ecological environment, and the chemical pesticide is easy to accumulate or remain in human bodies along with the ingestion and contact of food, thus harming the health and safety of human beings. The cultivation of crop varieties capable of resisting diseases is considered as an economic and effective way for preventing and treating plant diseases, but no high-quality and high-resistance variety can be popularized in a large area at present.
Disclosure of Invention
The invention aims to solve the problems of serious chemical pesticide pollution residue and high difficulty in cultivating antiviral varieties in the prior art, and provides the streptomyces alboflavus, the microbial inoculum containing the streptomyces alboflavus and the application of the streptomyces alboflavus and the microbial inoculum in preventing and treating plant diseases.
In order to achieve the above object, the present invention provides a Streptomyces alboflavus, which has a preservation number of CCTCC NO: m2021913.
The second aspect of the invention provides a culture method of streptomyces alboflavus, which comprises the following steps:
activating streptomyces alboflavus to obtain a seed solution, and performing fermentation culture on the seed solution to obtain a fermentation liquid, wherein the preservation number of the streptomyces alboflavus is CCTCC NO: m2021913.
Preferably, the activation adopts an LB culture medium, and a liquid culture medium of the fermentation culture contains glycerol, dextrin, soybean protein, yeast powder, ammonium sulfate and calcium carbonate;
preferably, in the liquid culture medium for fermentation culture, the concentration of the glycerol is 18-20g/L, the concentration of the dextrin is 18-20g/L, the concentration of the soybean protein is 8-12g/L, the concentration of the yeast powder is 2-3g/L, the concentration of the ammonium sulfate is 0.4-0.5g/L, and the concentration of the calcium carbonate is 2-3g/L; the pH value of the liquid culture medium for fermentation culture is 7.2-7.4.
Preferably, the conditions of activation at least satisfy: the temperature is 25-28 ℃, the rotating speed is 160-200rpm, and the time is 48-168h;
the conditions of the fermentation culture at least meet the following conditions: the inoculation amount is 5-15 vol%, the temperature is 25-28 deg.C, the rotation speed is 160-200rpm, and the time is 48-168h.
The third aspect of the invention provides a microbial inoculum, which contains the streptomyces alboflavus or fermentation liquor prepared by the culture method of the streptomyces alboflavus.
The fourth aspect of the invention provides the application of the streptomyces alboflavus and/or the microbial inoculum in preventing and treating plant diseases.
Preferably, the plant disease is selected from plant diseases caused by at least one of fusarium graminearum, pyricularia pyricularis, alternaria nicotianae, rhizoctonia solani, colletotrichum capsici and fusarium oxysporum.
Preferably, the plant disease is selected from at least one of wheat scab, pear rot, tobacco brown spot, rice sheath blight, pepper anthracnose and watermelon fusarium wilt.
In a fifth aspect, the present invention provides a method for controlling plant diseases, which comprises: contacting the streptomyces alboflavus and/or the microbial inoculum with plants.
Preferably, the plant disease of the plant is selected from plant diseases caused by at least one of fusarium graminearum, pyricularia piricola, alternaria tabacum, rhizoctonia solani, colletotrichum gloeosporioides and fusarium oxysporum;
preferably, the plant is selected from at least one of wheat, bergamot pear, tobacco, rice, capsicum and watermelon.
Through the technical scheme, the invention has the beneficial effects that:
the Streptomyces alboflavus (Streptomyces albiflaviniger) provided by the invention can form an effective growth inhibition effect on various plant pathogenic fungi such as fusarium graminearum, pyrus pyricularis, alternaria alternata, rhizoctonia solani, colletotrichum gloeosporioides, fusarium oxysporum and the like, and continuously has a remarkable inhibition effect in a certain period of time; the streptomyces alboflavus can be used as a green, safe and environment-friendly biological control medium, and provides a new choice for biological control of crops such as wheat, bergamot pear, tobacco, rice, pepper, watermelon and the like.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Biological preservation
The strain provided by the invention is Streptomyces alboflavus (Streptomyces alboflaviniger) and is preserved in China center for type culture collection (CCTCC for short, the address is located in the China, wuhan university, zip code: 430072) in 20 months in 2021, the preservation number is CCTCC No: m2021913.
Drawings
FIG. 1 is a colony morphology of the PESY23 strain obtained in example 1;
FIG. 2 is an aerial hyphal map of the strain PESY23 obtained in example 1;
FIG. 3 is a phylogenetic tree of the strain PESY23 obtained in example 1;
FIG. 4 is the results of experiments showing the antagonism of the PESY23 obtained in example 1 with plant pathogenic fungi, wherein Fusarium graminearum No.1, pyrus pyrifolia Hutch, alternaria alternate No. 3, rhizoctonia solani No. 4, colletotrichum capsici Hu, and Fusarium oxysporum niveum No. 5, and Fusarium oxysporum niveum No. 6.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For numerical ranges, each range between its endpoints and individual point values, and each individual point value can be combined with each other to give one or more new numerical ranges, and such numerical ranges should be construed as specifically disclosed herein.
The invention provides a Streptomyces alboflavus, the preservation number of the Streptomyces alboflavus (Streptomyces albiflaviniiger) is CCTCC NO: m2021913.
Streptomyces alboflavinis (Streptomyces albiflaviniger) of the present invention is isolated from a healthy pepper plant sample.
According to the present invention, the method for screening Streptomyces alboflavus can adopt a method for screening a new strain which is conventional in the art, and for example, the screening method can comprise the following steps:
(1) Shearing off lateral roots of healthy plants collected from a multi-year continuous cropping hot pepper field, cleaning silt with clear water, drying in a superclean workbench, then putting into disodium hydrogen phosphate for ultrasonic treatment to further remove surface impurities, taking out roots, sequentially disinfecting the surfaces of the roots with absolute ethyl alcohol, 4% sodium hypochlorite and sodium thiosulfate, cleaning for 3 times with sterile water (coating the water for the last 1-time cleaning on a flat plate, culturing at 25-35 ℃, checking the surface disinfection effect), and drying the thoroughly disinfected roots in the superclean workbench;
(2) Drying the thoroughly-sterilized root system obtained in the step (1), cutting into small sections, placing on a separation culture medium, culturing at 25-35 ℃, picking actinomycetes on root system tissues on the separation culture medium after a single colony grows out, scribing, separating and purifying to obtain a strain to be selected, adding glycerol, and storing at-80 ℃;
(3) Respectively carrying out an antagonism experiment on the strains to be selected obtained in the step (2), wherein the specific process comprises the following steps: adding pepper anthracnose pathogen into a PDA solid culture medium for activation, after hyphae grow out, punching a bacterial cake by using a puncher, placing the bacterial cake at the center of a flat plate of another fresh PDA solid culture medium, placing the bacterial cake upside down, inoculating the to-be-selected bacterial strain separated in the step (2) to the periphery of the bacterial cake, inoculating 2 or 4 bacterial cakes on each flat plate, culturing at the constant temperature of 25-35 ℃, and judging the antagonistic effect according to the existence of a bacteriostatic band and/or a bacteriostatic ring;
(4) And (4) performing a second antagonism experiment on the candidate strain with the antagonism obtained in the step (3) according to the antagonism experiment process in the step (3), and screening out a strain with stronger antagonism.
Through the screening, the Streptomyces alboflavinis (Streptomyces albiflaviniger) is obtained and is preserved in China center for type culture collection (CCTCC No): m2021913, the nucleotide sequence of 16s rDNA is shown in SEQ ID No:1 is shown.
According to the invention, the separation culture medium is a Gauss No.1 culture medium added with bacteriostatic agent, and the bacteriostatic agent added in the Gauss No.1 culture medium contains nalidixic acid (the final concentration is 20-30 mg/L), potassium dichromate (the final concentration is 120-180 mg/L) and benomyl (the final concentration is 20-30 mg/L); the culture medium of Gauss No.1 may be a commercially available product or may be prepared by itself, and illustratively, the culture medium of Gauss No.1 contains 20g/L of soluble starch and KNO 3 1g/L、K 2 HPO 4 0.5g/L、MgSO 4 ·7H 2 O 0.5g/L、NaCl 0.5g/L、FeSO 4 ·7H 2 O0.01 g/L, agar 20g/L, pH =7.4-7.6.
The streptomyces alboflavus provided by the invention can generate a large amount of live bacteria of the streptomyces alboflavus after being cultured, and the culture method has no special requirement as long as the streptomyces alboflavus can be proliferated.
The second aspect of the invention provides a culture method of streptomyces alboflavus, which comprises the following steps: activating streptomyces alboflavus to obtain a seed solution, and performing fermentation culture on the seed solution to obtain a fermentation liquid, wherein the preservation number of the streptomyces alboflavus is CCTCC NO: m2021913.
According to the invention, in the culture process of the streptomyces alboflavus, a single colony of the streptomyces alboflavus is inoculated into an activated liquid culture medium to be activated to obtain a seed solution, and then the seed solution is transferred into a fermentation culture medium to be cultured to obtain a fermentation liquid, so that the multiplication of the streptomyces alboflavus is realized.
In order to improve the proliferation rate of the streptomyces alboflavus and increase the yield of the fermentation product of the streptomyces alboflavus, preferably, an LB culture medium is adopted for activation; the liquid culture medium for fermentation culture contains glycerol, dextrin, soybean protein, yeast powder, ammonium sulfate and calcium carbonate.
Illustratively, the LB medium contains peptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, natural pH; in the liquid culture medium for fermentation culture, the concentration of glycerol is 18-20g/L, the concentration of dextrin is 18-20g/L, the concentration of soybean protein is 8-12g/L, the concentration of yeast powder is 2-3g/L, the concentration of ammonium sulfate is 0.4-0.5g/L, and the concentration of calcium carbonate is 2-3g/L; the pH value of the liquid culture medium for fermentation culture is 7.2-7.4.
According to the invention, the conditions of activation are at least such that: the temperature is 25-28 deg.C, specifically 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, or any value between the above two values; the rotation speed is 160-200rpm, specifically 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the two values; the time is 48-168h, specifically 48h, 96h, 120h, 144h, 168h, or any value between the two values; the conditions of the fermentation culture at least meet the following conditions: the inoculation amount is 5-15 vol%, specifically 5 vol%, 7 vol%, 9 vol%, 11 vol%, 13 vol%, 15 vol%, or any value between the two values; the temperature is 25-28 deg.C, specifically 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, or any value between the above two values; the rotation speed is 160-200rpm, specifically 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, or any value between the two values; the time is 48-168h, and specifically can be 48h, 96h, 120h, 144h, 168h, or any value between the two values.
The present invention can further separate the viable bacteria of streptomyces alboflavus in the fermentation broth, and the separation method is not particularly limited as long as the bacteria can be enriched from the fermentation broth, and for example, the separation method can be realized by a centrifugation and/or filtration method, and the centrifugation and filtration conditions can be known conditions, which are not described herein again.
The third aspect of the invention provides a microbial inoculum, which contains the streptomyces alboflavus or the fermentation liquor prepared by the culture method of the streptomyces alboflavus.
According to the invention, the concentration of streptomyces alboflavus in the microbial inoculum is not particularly limited, and can be specifically selected according to specific conditions, which is not described in detail herein.
In addition, according to different preset applications, the microbial inoculum provided by the invention can be prepared into different formulations, and corresponding components such as an excipient and the like which do not influence the activity of the streptomyces alboflavus are added. For example, the microorganism may be a fermentation broth of Streptomyces alboflavus, or a microorganism obtained by removing a supernatant from a fermentation broth of Streptomyces alboflavus. The preparation method of the microbial inoculum comprises the following steps of mixing the microbial inoculum, and adding auxiliary materials into the microbial inoculum. Illustratively, the microbial inoculum can be prepared into a biological agent, biological soil, biological fertilizer, or the like containing the streptomyces alboflavus or the fermentation broth prepared by the culture method of the streptomyces alboflavus.
The fourth aspect of the invention provides the application of the streptomyces alboflavus and/or the microbial inoculum in preventing and treating plant diseases.
According to the invention, the plant disease is selected from plant diseases caused by at least one of fusarium graminearum, pyricularia pyricularis, alternaria alternata, rhizoctonia solani, colletotrichum capsici and fusarium oxysporum. The streptomyces alboflavus provided by the invention can effectively inhibit the growth of fusarium graminearum, pyricularia aromatica, alternaria alternata, rhizoctonia solani, colletotrichum gloeosporioides and fusarium oxysporum kuhn, and can be further applied to plant diseases caused by any one or more of fusarium graminearum, pyricularia aromatica, alternaria alternata, rhizoctonia solani, colletotrichum gloeosporioides and fusarium oxysporum kuhn.
According to the invention, the carrier of the plant disease can be any plant, preferably, the streptomyces alboflavus provided by the invention is used for preventing and treating the plant disease of wheat, bergamot pear, tobacco, rice, pepper and watermelon, wherein the plant disease is formed by fusarium graminearum, pyrus pyricularis, alternaria alternata, rhizoctonia solani, pepper anthracnose and watermelon fusarium oxysporum.
According to the present invention, the plant disease is at least one selected from wheat scab, pear rot, tobacco brown spot, rice sheath blight, pepper anthracnose and watermelon fusarium wilt.
In a fifth aspect, the present invention provides a method for controlling plant diseases, which comprises: contacting the streptomyces alboflavus and/or the microbial inoculum with plants.
According to the present invention, the form of Streptomyces alboflavus to be brought into contact with a plant is not particularly limited as long as it is ensured that the Streptomyces alboflavus can effectively inhibit a plant pathogenic fungus causing a plant disease after the addition, and for example, the form may be an activated fungus body (fermentation broth or cell precipitate) cultured up to the logarithmic phase, a dried fungus body powder after freeze-drying, or a microbial inoculum obtained by compounding, and preferably the form is an activated fungus body cultured up to the logarithmic phase.
The present invention also has no particular limitation on the amount of Streptomyces alboflavus to be added, which may be determined depending on the plant and its growing environment.
According to the invention, the plant diseases of the plants are selected from plant diseases caused by at least one of fusarium graminearum, pyricularia pyricularis, alternaria alternata, rhizoctonia solani, colletotrichum capsici and fusarium oxysporum;
according to the present invention, the plant is selected from at least one of wheat, bergamot pear, tobacco, rice, capsicum, and watermelon.
The present invention will be described in detail below by way of examples.
In the following examples, raw materials, reagents, instruments, equipment, and the like are commercially available or can be prepared by existing methods;
the preparation method of the LB culture medium comprises the following steps: mixing 10g of peptone, 5g of yeast powder and 5g of sodium chloride, supplementing distilled water to 1000mL, naturally adjusting pH, and sterilizing with high-pressure steam at 121 ℃ for 30min;
the preparation method of the PDA solid culture medium comprises the following steps: mixing 200g of potatoes, 20g of glucose and 18g of agar, supplementing distilled water to 1000mL, naturally adjusting the pH value, and sterilizing for 30min by high-pressure steam at 115 ℃;
the preparation method of the SM14 liquid culture medium comprises the following steps: mixing 19g of glycerol, 19g of dextrin, 10g of soybean protein, 2.5g of yeast powder, 0.4g of ammonium sulfate and 2.5g of calcium carbonate, supplementing distilled water to 1000mL, adjusting the pH value to 7.2-7.4, and sterilizing for 30min by high-pressure steam at 121 ℃;
the preparation method of the separation culture medium comprises the following steps: mixing 20g soluble starch and 1g KNO 3 0.5g of K 2 HPO 4 0.5g of MgSO 5 4 ·7H 2 O, 0.5g NaCl, 0.01g FeSO 4 ·7H 2 Mixing O, 20g of agar, 0.03g of nalidixic acid, 0.15g of potassium dichromate and 0.03g of benomyl, supplementing distilled water to 1000mL, adjusting the pH value to 7.4-7.6, and sterilizing for 30min by high-pressure steam at 121 ℃;
the preparation method of the Gao's 2 liquid culture medium comprises the following steps: 5g of peptone, 5g of NaC1 and 10g of glucose were mixed, and then distilled water was added thereto to 1000mL, followed by natural pH and autoclaving at 115 ℃ for 30min.
Example 1
(1) Shearing off lateral roots of healthy plants collected from a multi-year continuous cropping hot pepper field, cleaning silt with clear water, drying the plants in a superclean workbench, then putting the plants in disodium hydrogen phosphate for ultrasonic treatment for 1min to further remove surface impurities, taking out roots, sequentially disinfecting the surfaces of the roots with absolute ethyl alcohol, 4% sodium hypochlorite and sodium thiosulfate, cleaning the surfaces of the roots with sterile water for 3 times (coating the water for cleaning the last 1 time on a flat plate, culturing at 28 ℃, and checking the surface disinfection effect), and drying the thoroughly disinfected roots in the superclean workbench;
(2) Drying the thoroughly-sterilized root system obtained in the step (1), cutting into small sections, placing on a separation culture medium, culturing at 28 ℃, picking actinomycetes on root system tissues to streak, separating and purifying on the separation culture medium after a single colony grows out to obtain a strain to be selected, adding glycerol, and storing at-80 ℃;
(3) Respectively carrying out an antagonism experiment on the strains to be selected obtained in the step (2), wherein the specific process comprises the following steps: adding pepper anthracnose pathogen into a PDA solid culture medium for activation, after hyphae grow out, punching a fungus cake by using a puncher, placing the fungus cake at the center of a flat plate of another fresh PDA solid culture medium, placing the fungus cake upside down, inoculating the separated strains to be selected around the fungus cake, inoculating 2 or 4 strains on each flat plate, culturing at a constant temperature of 28 ℃, and judging the antagonistic effect according to the existence of a bacteriostatic band and/or a bacteriostatic ring;
(4) And (4) performing a second antagonistic experiment on the to-be-selected strain with antagonistic action obtained in the step (3) by referring to the antagonistic experiment process in the step (3), and screening out a strain with strong antagonistic capability, namely PESY23.
Morphological identification of the strain PESY23
The strain PESY23 is inoculated on a solid culture medium of No. 2 Gao's, streak culture is carried out, the strain is inverted to the condition of 28 ℃, the culture is carried out for 96 to 120h, and the colony morphology of the strain PESY23 after the culture on the solid culture medium of No. 2 Gao's is shown in figure 1. The observation shows that the bacterial colony of the strain PESY23 is small, compact, dry and opaque, has smooth surface, tidy edge and monotonous color when young, is not easy to pick up, and then develops into villiform, powdery on the surface, gray or black, and has different colors on the front side and the back side.
FIG. 2 shows aerial hyphal pattern of the strain PESY23, and FIG. 3 shows phylogenetic dendrogram of the strain PESY23.
And carrying out molecular experiment identification on the PESY23.
Selecting a single colony of the strain PESY23, placing the single colony in a triangular flask filled with 20mL of Gao's No. 2 liquid culture medium, carrying out shake culture at the temperature of 26 ℃ and the rotating speed of 180rpm for 3d, taking 1mL of bacterial liquid, centrifuging at the rotating speed of 10000rpm for 1min, and collecting thalli; grinding the thalli into uniform slurry under the aseptic condition, extracting total DNA of the genome by referring to the UNIQ-10 column type bacterial genome extraction kit specification, diluting the total DNA to about 50ng/uL as a template, and carrying out 16S rDNA gene amplification.
PCR amplification was performed with reference to the following amplification system: after mixing 25. Mu.L of 2 × EasyTaq PCR Supermix (+ dye), 1.5. Mu.L of the forward primer, 1.5. Mu.L of the reverse primer, and 1.5. Mu.L of the total DNA template, the mixture was made up to 50. Mu.L with sterile water;
in this embodiment, 2 × easy taq PCR SuperMix (+ dye) is purchased from beijing holotype gold biotechnology limited, wherein common reagents required for PCR amplification, such as DNA polymerase, dNTPs, reaction buffer, electrophoresis buffer, and the like, are pre-contained, and those skilled in the art can directly select other PCR amplification kits according to needs, or perform PCR amplification by using independent DNA polymerase, dNTPs, and reaction buffer according to needs;
the upstream primer adopts 27F, and the nucleotide sequence of the upstream primer is shown as SEQ ID No:2, the downstream primer adopts 1492R, and the nucleotide sequence of the downstream primer is shown as SEQ ID No:3 is shown in the figure;
SEQ ID No.2(27F)：5′-TCCTCCGCTTATTGATATGC-3′；
SEQ ID No.3(1492R)：5′-CAAACTTGGTCATTAGAGGA-3′。
the conditions for PCR amplification were: pre-denaturation at 95 ℃ for 10min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and repeating for 35 cycles; extension was continued for 10min at 72 ℃.
The amplified product obtained by the PCR amplification is separated by 1% agarose gel electrophoresis, an obvious band is formed near 1500bp, the amplified product is subjected to bidirectional sequencing, and the gene sequence obtained by the sequencing is shown as SEQ ID NO. 1. The sequence of the gene obtained by sequencing is compared with the nucleotide sequence in an EZ BioCloud database (https:// www. Ezbiocloud. Net), and the result shows that the relationship between the PESY23 strain and Streptomyces albicans strain NRRL B-1356 (accession number: AJ 391812) is nearest, and the homology reaches 99.57%.
By combining morphological structure characteristics of the PESY23 strain and phylogenetic tree analysis, the PESY23 strain is determined to be Streptomyces alboflavus (Streptomyces albifyingiger), the Streptomyces alboflavus PESY23 is preserved in the China center for type culture collection at 7 months and 20 days 2021, and the preservation number is CCTCC NO: m2021913.
Example 2
S1, selecting a single colony of the PESY23 strain obtained in the embodiment, placing the single colony in a triangular flask filled with 20mL of LB liquid culture medium, and performing shake culture at the temperature of 26 ℃ and the rotating speed of 180rpm for 120 hours to prepare seed liquid;
s2, inoculating the seed solution obtained in the step S1 into a fermentation tank filled with SM14 liquid culture medium in an inoculation amount of 10 volume percent, and performing shaking culture at the temperature of 26 ℃ and the rotating speed of 180rpm for 120h to prepare a fermentation liquid of the strain PESY23.
Example 3
S1, selecting a single colony of the PESY23 strain obtained in the embodiment, placing the single colony in a triangular flask filled with 20mL of LB liquid culture medium, and performing shake culture at the temperature of 25 ℃ and the rotating speed of 200rpm for 48 hours to prepare seed liquid;
s2, inoculating the seed solution obtained in the step S1 into a fermentation tank filled with SM14 liquid culture medium in an inoculation amount of 15 volume percent, and performing shake culture at the temperature of 25 ℃ and the rotation speed of 200rpm for 48 hours to prepare a fermentation liquor of the PESY23 strain.
Example 4
S1, selecting a single colony of the PESY23 strain obtained in the embodiment, placing the single colony in a triangular flask filled with 20mL of LB liquid culture medium, and performing shake culture at the temperature of 28 ℃ and the rotating speed of 160rpm for 168 hours to prepare seed liquid;
s2, inoculating the seed liquid obtained in the step S1 into a fermentation tank filled with SM14 liquid culture medium in an inoculation amount of 5 vol%, and performing shaking culture at the temperature of 28 ℃ and the rotating speed of 160rpm for 168h to prepare a fermentation liquid of the strain PESY23.
Test example 1
Fusarium graminearum is inoculated on a PDA solid culture medium for activation, a cake is inoculated in the center of a fresh PDA solid culture medium by a puncher after hyphae grow out, a single colony of the PESY23 strain obtained in example 1 is inoculated at a point 3cm away from the Fusarium graminearum cake, the culture is carried out for 6 days at the temperature of 25 ℃, the radius of a bacteriostatic circle is measured, and the result is shown in Table 1. Fig. 4 shows an experiment chart of antagonism between the strain PESY23 provided in example 1 and fusarium graminearum on a PDA solid medium, and as can be seen from fig. 4, no.1 and table 1, the strain PESY23 has a good antagonistic effect on fusarium graminearum.
TABLE 1 antagonistic action of the strain PESY23 on the inhibition zone radius of Fusarium graminearum on PDA solid Medium
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 13.0 | 13.5 | 12.9 | 13.2 | 13.15 |
Test example 2
Inoculating the bergamot pear rot germs on a PDA solid culture medium for activation, taking a fungus cake after hypha growth by using a puncher and inoculating the fungus cake in the center of a fresh PDA solid culture medium, taking a PESY23 strain obtained in the example 1, inoculating the single fungus drop at a position 3cm away from the bergamot pear rot fungus cake, culturing for 6 days at 25 ℃, measuring the radius of an inhibition zone, and obtaining the result shown in a table 2. In FIG. 4, no. 2 is an antagonistic experiment chart of the PESY23 strain provided in example 1 and the bergamot pear rot pathogen on the PDA solid medium, and as can be observed from No. 2 in FIG. 4 and Table 2, the PESY23 strain has good antagonistic effect on the bergamot pear rot pathogen, and the inhibition capability of the PESY23 strain on the bergamot pear rot pathogen is kept at a high level within one week.
TABLE 2PESY23 antagonistic Pyria rot pathogen inhibition zone radius on PDA solid medium
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 10.0 | 11.1 | 8.3 | 10.3 | 9.93 |
Test example 3
Inoculating alternaria alternata on a PDA solid culture medium for activation, taking a fungus cake by a puncher after hypha grows out, inoculating the fungus cake at the center of a fresh PDA solid culture medium, taking a PESY23 strain obtained in example 1, inoculating the single fungus drop at a position 3cm away from the alternaria alternata fungus cake, culturing for 6 days at 25 ℃, measuring the radius of a bacteriostatic circle, and obtaining the result shown in table 3. Fig. 4 is an experimental graph of the antagonism of the PESY23 strain provided in example 1 with the alternaria alternata on the PDA solid medium, and it can be observed from fig. 4, no. 3 and table 3 that the PESY23 strain has a good antagonism against the alternaria alternata, and the inhibition ability of the PESY23 strain against the alternaria alternata is kept at a high level within one week.
TABLE 3 radius of inhibition zone of PESY23 strain on PDA solid medium for antagonizing alternaria alternata
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 11.0 | 11.2 | 11.5 | 11.2 | 11.23 |
Test example 4
(1) Taking 2mL of fermentation liquor of the PESY23 strain obtained in the embodiment 2, centrifuging for 1min at the rotating speed of 10000rpm, collecting thalli, filtering the thalli by using a 0.22 mu m disposable bacterial filter, and placing the obtained filtrate at-20 ℃ for later use;
(2) Inoculating rhizoctonia solani on a PDA solid culture medium for activation, taking a fungus cake by a puncher after hypha grows out, and inoculating the fungus cake in the center of a fresh PDA solid culture medium to prepare a rhizoctonia solani antagonistic test plate; four holes were punched at a distance of 3cm from the cake, and then 100. Mu.L of the filtrate obtained in step (1) was added to the holes, incubated at 25 ℃ for 6 days, and the radius of zone of inhibition was measured, and the results are shown in Table 4. FIG. 4 is an experiment chart of antagonism between the fermentation liquor of the PESY23 strain obtained in example 2 and Rhizoctonia solani on PDA solid medium, and as can be seen from FIG. 4 and Table 4, the fermentation liquor of the PESY23 strain has good antagonism to Rhizoctonia solani, and the inhibitory activity of the fermentation liquor of the PESY23 strain to Rhizoctonia solani is kept at a high level within one week.
TABLE 4PESY23 Strain antagonizes the zone radius of inhibition of Rhizoctonia solani on PDA solid Medium
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 9.0 | 9.2 | 8.9 | 9.0 | 9.00 |
Test example 5
(1) Taking 2mL of the fermentation liquor of the PESY23 strain obtained in the example 3, centrifuging at the rotation speed of 10000rpm for 1min, collecting thalli, filtering the thalli by using a 0.22 mu m disposable bacterial filter, and placing the obtained filtrate at-20 ℃ for later use;
(2) Inoculating pepper anthracnose pathogen on a PDA solid culture medium for activation, taking a fungus cake by using a puncher after hypha grows out, and inoculating the fungus cake in the center of a fresh PDA solid culture medium to prepare a pepper anthracnose pathogen antagonistic test panel; four holes were punched at a distance of 3cm from the cake, 100. Mu.L of the filtrate obtained in step (1) was added to the holes, incubated at 25 ℃ for 6 days, and the radius of zone of inhibition was measured, the results are shown in Table 5. FIG. 4 shows the experiment chart of the antagonism between the fermentation liquid of the PESY23 strain obtained in example 2 and the Colletotrichum capsici on the PDA solid medium, and it can be observed from FIG. 4, no. 5 and Table 5 that the fermentation liquid of the PESY23 strain has good antagonism to the Colletotrichum capsici.
TABLE 5PESY23 radius of zone of inhibition of antagonism of pepper anthracnose pathogen on PDA solid medium
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 13.0 | 14.0 | 12.9 | 13.6 | 13.38 |
Test example 6
(1) Taking 2mL of the fermentation liquor of the PESY23 strain obtained in the example 4, centrifuging at the rotation speed of 10000rpm for 1min, collecting thalli, filtering the thalli by using a 0.22 mu m disposable bacterial filter, and placing the obtained filtrate at-20 ℃ for later use;
(2) Inoculating watermelon wilt pathogen on PDA solid culture medium for activation, growing hypha, inoculating a bacterial cake in the center of fresh PDA solid culture medium with a puncher, and making into an antagonistic test plate for watermelon wilt pathogen; four holes were punched at a distance of 3cm from the cake, 100. Mu.L of the filtrate obtained in step (1) was added to the holes, incubated at 25 ℃ for 6 days, and the radius of zone of inhibition was measured, the results are shown in Table 6. Please refer to fig. 4, 6 is an experimental chart of antagonism between fermentation broth of the PESY23 strain obtained in example 3 and fusarium oxysporum f.sp.cubense on a PDA solid medium, and it can be observed from fig. 4, 6 and table 6 that the fermentation broth of the PESY23 strain has good antagonism to the fusarium oxysporum f.sp.cubense.
TABLE 6 inhibition zone radius of PESY23 strain antagonizing watermelon fusarium wilt on PDA solid culture medium
| Days of culture | 1 | 2 | 3 | 4 | Mean value of |
| Radius of bacteriostatic circle (mm) | 17.2 | 17.0 | 16.9 | 17.2 | 17.10 |
Test example 7
Culturing alternaria alternate in a glass greenhouse at a constant temperature of 26 ℃ for 4-5d in a potting inoculation mode, pricking 4 needles on the middle leaves of tobacco seedlings in a 4-leaf stage by using a No. 5 syringe needle, then sticking fungus cakes of the alternaria alternate to the pricked positions, dipping sterile absorbent cotton in sterile water to cover the fungus cakes on the pricked positions, preserving moisture, repeating the steps for 3 times after 15 strains are treated, culturing at a high temperature of 28-30 ℃, and removing the fungus cakes after 48 hours of moisture preservation;
the tobacco seedlings inoculated with the alternaria alternata are divided into 3 groups, 50-time diluent (treatment 1) of the fermentation liquid of the PESY23 strain obtained in example 1, 500-time diluent (treatment 2) of 50% carbendazim WP and clear water (a control group) are respectively and uniformly sprayed on the reverse front sides of the tobacco seedling leaves of the corresponding group, the disease index is investigated after inoculation for 7 days by taking the dropping water on the leaf surfaces, the prevention and treatment effect is calculated, and the results are shown in table 7.
The common antifungal agent carbendazim is used as a positive control, and the result of a pot experiment shows that the fermentation liquid of the PESY23 strain obtained by adopting the optimized fermentation condition has good prevention and treatment effect on the alternaria alternate, the disease index after treatment is 25.86, which is obviously lower than that of the control, and the prevention and treatment effect can reach 58.58 percent and is equivalent to that of 50 percent carbendazim WP.
TABLE 7
| Treatment of | Index of disease condition | Control effect (%) |
| Control group | 62.43 | - |
| Process 1 | 25.86 | 58.58 |
| Treatment 2 | 23.45 | 62.43 |
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
SEQUENCE LISTING
<110> institute of microorganisms of Hunan province
<120> Streptomyces alboflavus, microbial inoculum containing same and application of Streptomyces alboflavus and microbial inoculum in plant disease control
<130> 2021.8.3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1426
<212> DNA
<213> unknown
<400> 1
cgacggcggc ggcttaccat gcagtcgaac gatgaaccgg tttcggccgg ggattagtgg 60
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gggtctaata ccggatacga ctgccgaccg catggtctgg tggtggaaag ctccggcggt 180
gcaggatgag cccgcggcct atcagcttgt tggtggggtg atggcctacc aaggcgacga 240
cgggtagccg gcctgagagg gcgaccggcc acactgggac tgagacacgg cccagactcc 300
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cgcaagcgtt gtccggaatt attgggcgta aagagctcgt aggcggcttg tcgcgtcgga 540
tgtgaaagcc cggggcttaa ctccgggtct gcattcgata cgggcaggct agagttcggt 600
aggggagatc ggaattcctg gtgtagcggt gaaatgcgca gatatcagga ggaacaccgg 660
tggcgaaggc ggatctctgg gccgatactg acgctgagga gcgaaagcgt ggggagcgaa 720
caggattaga taccctggta gtccacgccg taaacgttgg gaactaggtg tgggcgacat 780
tccacgttgt ccgtgccgca gctaacgcat taagttcccc gcctggggag tacggccgca 840
aggctaaaac tcaaaggaat tgacgggggc ccgcacaagc ggcggagcat gtggcttaat 900
tcgacgcaac gcgaagaacc ttaccaaggc ttgacataca ccggaaaact ctggagacag 960
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Claims (10)
1. The Streptomyces alboflavus is characterized in that the preservation number of the Streptomyces alboflavus (Streptomyces albiflaviniger) is CCTCC NO: m2021913.
2. A method for culturing Streptomyces alboflavus is characterized by comprising the following steps:
activating streptomyces alboflavus to obtain a seed solution, and performing fermentation culture on the seed solution to obtain a fermentation liquid, wherein the preservation number of the streptomyces alboflavus is CCTCC NO: m2021913.
3. The method for culturing Streptomyces alboflavus as claimed in claim 2, wherein the activation is carried out by using LB medium, and the liquid medium for fermentation culture contains glycerol, dextrin, soybean protein, yeast powder, ammonium sulfate and calcium carbonate.
4. The method for culturing Streptomyces alboflavus according to claim 3, wherein the concentration of glycerol in the liquid culture medium of the fermentation culture is 18 to 20g/L, the concentration of dextrin is 18 to 20g/L, the concentration of soybean protein is 8 to 12g/L, the concentration of yeast powder is 2 to 3g/L, the concentration of ammonium sulfate is 0.4 to 0.5g/L, and the concentration of calcium carbonate is 2 to 3g/L;
the pH value of the liquid culture medium for fermentation culture is 7.2-7.4.
5. The method of culturing Streptomyces alboflavus according to claim 2, wherein the activation conditions are at least: the temperature is 25-28 ℃, the rotating speed is 160-200rpm, and the time is 48-168h;
the conditions of the fermentation culture at least meet the following conditions: the inoculation amount is 5-15 vol%, the temperature is 25-28 ℃, the rotating speed is 160-200rpm, and the time is 48-168h.
6. A microbial inoculum comprising the Streptomyces alboflavus according to claim 1 or a fermentation broth obtained by the culture method of the Streptomyces alboflavus according to any one of claims 2 to 5.
7. The use of the Streptomyces alboflavus of claim 1 and/or the microbial agent of claim 6 for controlling plant diseases, wherein the plant diseases are selected from plant diseases caused by at least one of Fusarium graminearum, pyrus pyrifolia, alternaria alternata, rhizoctonia solani, colletotrichum capsici and Fusarium oxysporum.
8. The use according to claim 7, wherein the plant disease is selected from at least one of wheat scab, bergamot pear rot, tobacco brown spot, rice sheath blight, pepper anthracnose and watermelon fusarium wilt.
9. A method for controlling a plant disease, comprising: contacting the Streptomyces alboflavus of claim 1 and/or the microbial inoculum of claim 6 with a plant; wherein the plant diseases of the plants are selected from the plant diseases caused by at least one of fusarium graminearum, pyricularia pyricularis, alternaria alternata, rhizoctonia solani, colletotrichum capsici and fusarium oxysporum.
10. The method for controlling plant diseases according to claim 9, wherein the plant is selected from at least one of wheat, bergamot pear, tobacco, rice, capsicum, and watermelon.
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