CN110684684B - High-temperature-resistant biocontrol streptomyces and application thereof - Google Patents
High-temperature-resistant biocontrol streptomyces and application thereof Download PDFInfo
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C05—FERTILISERS; MANUFACTURE THEREOF
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- C05F3/00—Fertilisers from human or animal excrements, e.g. manure
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- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a high-temperature-resistant biocontrol streptomyces and application thereof, belonging to the technical field of biology. The Streptomyces sp DL70H is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO.M 2019486 and the preservation date of 2019, 06 and 25. The high-temperature resistant biocontrol streptomyces is obtained by mutagenesis screening, keeps good growth and metabolism under the high-temperature condition of 50 ℃, and meets the requirement that the effective viable count in zymogen liquid is more than or equal to 2.0 multiplied by 109cfu/mL, and meanwhile, the Streptomyces sp DL70H and the fermentation product or the zymocyte liquid thereof have obvious antagonistic effect on various plant pathogenic fungi, and have obvious biological control advantages and industrial production potential.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-temperature-resistant biocontrol streptomyces and application thereof.
Background
The biological fertilizer is a representative biological living product which leads crops to obtain a specific fertilizer effect by using the life activities of microorganisms and products thereof, and has irreplaceable effects on the aspects of fertilizing soil, improving the utilization rate of chemical fertilizers, inhibiting the absorption of nitrate nitrogen, heavy metals and pesticides by the crops, purifying and repairing soil, reducing crop diseases, promoting the decomposition and utilization of livestock excrement and crop straws, protecting the environment, improving the quality of crop products, improving food safety and the like. Among them, various important functional microorganisms play a key role in bioremediation.
The streptomycete is a gram-positive filamentous actinomycete widely distributed in soil, is suitable for growth at the temperature of about 28-32 ℃, has the pH value of about 7.0, contains various antibiotic, auxin, phenylacetic acid, succinic acid, cytokinin and other antibacterial active substances and plant growth essential regulators in fermentation metabolites, and plays an important role in the fields of biological prevention and resource utilization of livestock and poultry manure and the like. On one hand, the propagation of various plant pathogenic fungi is inhibited, diseases are prevented, seedlings are protected, the division of plant cells is stimulated, the rooting, the germination and the maturation of crops are promoted, and the yield and the quality of the crops are effectively improved; on the other hand, the accumulation of nitrogen, phosphorus and potassium nutrient elements in the composting process is improved, the titer of the biological fertilizer and the soil fertility are improved, and the using amount of the fertilizer is obviously reduced.
At the high-temperature stage of compost fermentation of poultry and livestock manure, streptomycete commonly used at normal temperature is inactivated due to high temperature, various organic matters cannot be efficiently decomposed in the composting process, pathogenic microorganisms such as ascarid eggs and coliform are killed, and therefore the streptomycete has high-temperature-resistant physiological characteristics and is significant for improving the recycling and harmless treatment process of the poultry and livestock manure. At present, streptomycete mutant strains are mostly finished by protoplast fusion or protoplast physicochemical mutagenesis, but different streptomycete protoplast preparation processes have great difference and are complex to operate, and the breeding efficiency of excellent strains needs to be improved urgently.
Disclosure of Invention
In order to improve the environmental adaptability and biological prevention and control effects of streptomyces at a high temperature stage in the production process of biofertilizer, the invention provides a high temperature resistant biocontrol streptomyces strain and simultaneously provides application of the strain in preventing and controlling plant diseases.
The invention provides a high-temperature resistant biocontrol Streptomyces strain, which is obtained by mutagenesis screening and is named as Streptomyces (Streptomyces sp.) DL70H, and the strain is preserved in China center for type culture Collection (address: eight-way 299 in Wuchang district, Wuhan city, Hubei province, postfix 430072) in 2019, 6 and 25 months, and the preservation number is as follows: CCTCC No. m 2019486.
The second aspect of the invention provides application of Streptomyces sp DL70H in preventing and treating plant diseases.
In the above application, the plant disease is caused by plant pathogenic fungi, and the plant pathogenic fungi include cucumber fusarium wilt bacteria, cucumber black spot bacteria, tomato ralstonia solanacearum, phytophthora capsici, potato rhizoctonia solani or sclerotinia sclerotiorum.
The third aspect of the invention provides a microbial inoculum for controlling plant diseases, wherein the effective components of the microbial inoculum are Streptomyces sp DL70H and fermentation products thereof or zymogen liquid of Streptomyces sp DL 70H.
In a fourth aspect, the invention provides the use of Streptomyces sp DL70H in the composting of poultry manure.
A screening method of high temperature resistant Streptomyces sp DL70H comprises the following steps:
a. inoculating Streptomyces sp DL70 as original strain to slant culture medium, culturing at 28-32 deg.C for 3-5 days, collecting spore on the slant culture medium with physiological saline preheated to 35-40 deg.C, and making into spore with concentration of 108-1010Spore suspension per mL;
b. immediately mutagenizing the spore suspension by using ultraviolet light for 120s, then mutagenizing the spore suspension by using microwaves for 60s in a dark room at the temperature of 35-40 ℃, and standing and incubating for 45min at the temperature of 35-40 ℃ to obtain mutagenized spore suspension;
c. inoculating the spore suspension subjected to the mutagenesis treatment into a liquid activation culture medium, carrying out shaking culture at the temperature of 35-40 ℃ and 200rpm for 48-120h, and carrying out centrifugal enrichment and separation on thalli; transferring the obtained thallus into a fresh liquid activation culture medium, carrying out shaking culture at the temperature of 40-45 ℃ and the rpm of 200 for 48-120h, and carrying out centrifugal enrichment and separation on the thallus; transferring the obtained thallus into a fresh liquid activation culture medium, carrying out shaking culture at the temperature of 45-50 ℃ and 200rpm for 48-120h, concentrating the bacterial liquid by 1-10 times, uniformly coating the concentrated bacterial liquid on a solid activation culture medium, and carrying out standing culture at the temperature of 45-50 ℃ for 5-7 days; selecting a streptomycete single colony which grows rapidly, transferring the streptomycete single colony to a slant culture medium, culturing for 3-5 days at the temperature of 28-32 ℃ to obtain mature slant spores, and preserving the mature spores by using a 40% glycerol solution to obtain a plurality of high-temperature resistant streptomycete strains;
d. transferring the obtained mature spores of the high-temperature resistant streptomycete into a liquid activation culture medium, carrying out shaking culture at the speed of 150-200rpm for 24-48h at the temperature of 28-32 ℃, transferring the activated bacterial liquid into a liquid fermentation culture medium, and carrying out shaking culture at the speed of 150-200rpm for 3-5 days at the temperature of 28-32 ℃ to obtain a fermentation bacterial liquid of the high-temperature resistant streptomycete; and (3) carrying out plant pathogenic fungi antagonism experiments by using zymocyte liquid or fermentation products, and screening mutagenic strains with optimal bacteriostatic activity by using bacteriostatic zones as indexes to obtain the high-temperature resistant Streptomyces sp DL 70H.
The wavelength of the ultraviolet light in the step b is 260nm, the power of the ultraviolet light is 20W, the ultraviolet light processing time is 60-180s, the microwave processing frequency is 400MHZ, and the microwave processing time is 30-60 s.
The microwave mutagenesis treatment in step b is carried out in a dark room at 35-40 ℃.
The slant culture medium and the solid activation culture medium in the method comprise, by mass, 2% -5% of sucrose, 0.5% -1.0% of corn steep liquor, 0.01% -0.1% of monopotassium phosphate, 0.01% -0.1% of magnesium sulfate, 0.005% -0.05% of zinc sulfate, 0.0001% -0.001% of ferrous sulfate, 2% of agar and the balance of water, and the pH of the culture medium is adjusted to 7.0-7.5 by using 50% ammonia water solution.
The liquid activation culture medium and the fermentation culture medium in the method comprise, by mass, 2% -5% of sucrose, 0.5% -1.0% of corn steep liquor, 0.01% -0.1% of monopotassium phosphate, 0.01% -0.1% of magnesium sulfate, 0.005% -0.05% of zinc sulfate, 0.0001% -0.001% of ferrous sulfate and the balance of water, and the pH value of the culture medium is adjusted to 7.0-7.5 by using 50% ammonia water solution.
The invention has the beneficial effects that: the high-temperature resistant Streptomyces biocontrol Streptomyces (Streptomyces sp.) DL70H provided by the invention keeps good growth and metabolism under the high-temperature condition of 50 ℃, and the effective viable count in the zymocyte liquid is more than or equal to 2.0 multiplied by 109cfu/mL, and meanwhile, the Streptomyces sp DL70H and the fermentation product or the zymocyte liquid thereof have obvious antagonistic effect on various plant pathogenic fungi, and have important industrial application value in producing biofertilizer by utilizing livestock manure compost and biologically preventing and treating plant pathogenic fungi and diseases.
Drawings
FIG. 1 is a map of hypha and spore morphology of a starting strain Streptomyces (Streptomyces sp.) DL 70.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: obtaining and identifying original strain Streptomyces sp DL70
1. Isolation of Streptomyces sp DL70
Streptomyces sp DL70 is separated from deep soil of the rhizosphere of a healthy plant in the great continuous coastal beach, and the soil close to the end of the rhizosphere is subjected to gradient dilution and coating, and then is separated. The specific operation steps are as follows:
a. and (3) coating the soil of the healthy crop rhizosphere on the Dalianxing beach, wherein the sampling depth is 15-20 cm. 1g of rhizosphere soil is weighed, put into a triangular flask with 99mL of sterile water added with glass beads, and cultured for 60min under the conditions of 30 ℃ and 200 rpm.
b. Gradient dilution with pipette (10)-3、10-4、10-5) 100 mul of the dilution was spread on a Gao's first medium plate, 5 dishes were spread for each dilution, and the plate was incubated in an incubator at 30 ℃ for 3 to 5 days. Observing the growth condition of the formed actinomycete colony every day, selecting the actinomycete single colony formed earliest in the culture time, carrying out dilution, streaking, separation and culture, transferring to a Gao's first culture medium, streaking, purifying and preserving.
The composition of the Gao's first culture medium is (g/L): 20 parts of soluble starch, 1 part of potassium nitrate, 0.5 part of sodium chloride, 0.5 part of monopotassium phosphate, 0.5 part of magnesium sulfate, 0.01 part of ferrous sulfate and 20 parts of agar. The balance of water is made up to 1000mL, the pH is adjusted to 7.2-7.4, and the high-pressure moist heat sterilization is carried out for 15min at 121 ℃.
Wherein, the single colony of the actinomycete which grows fastest appears after 24 hours of culture on 15 culture dishes coated according to different dilution concentrations, and the strain is named as DL70 after purification and preservation.
2. Identification of Streptomyces sp DL70
The morphological characteristics of the strain DL70 are as follows: the colony is circular, compact and yellowish after being cultured on a Gao's first culture medium for 3-5 days at the optimal culture temperature of 30 ℃, hypha in the medium has no transverse septum, is not cracked and is hawksbill yellow, aerial hypha is branched and pink, spore silk is linear or wavy, spores are oval or circular, the surface is smooth, and the spores are pink and purple when being mature. According to the manual of Streptomyces identification, strain DL70 has typical Streptomyces morphological characteristics (FIG. 1).
Extracting the genomic DNA of the strain DL70 obtained by separation and purification, performing 16s rDNA sequencing analysis (the sequencing result is detailed in a sequence SEQ ID NO.1) by a company Limited in the biological engineering (Shanghai), selecting a mode strain with higher homology to perform multi-sequence comparison analysis with a strain sequence by using ClustalX1.81 software through BLAST homologous comparison in an NCBI database, preliminarily identifying and separating the purified strain into Streptomyces sp by combining morphological characteristics of bacterial colonies, storing the strain in a China center for type culture collection in 2019 and 20 days, and naming the strain as the Streptomyces sp DL70 with the preservation number of CCTCC NO. M2019475 and the unit address of eight-way 299 in the Wuhan Changchang region in Hubei province.
Example 2: preparation of spore suspension
(1) The slant culture medium and the solid activation culture medium are mixed according to the mass ratio: 2% of sucrose, 0.5% of corn steep liquor, 0.05% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.05% of zinc sulfate, 0.001% of ferrous sulfate, 2% of agar and the balance of water, uniformly mixing, adjusting the pH value of the culture medium to 7.5 by using a 50% ammonia water solution, sterilizing at 121 ℃ and 0.11MPa for 20min, and cooling to room temperature for later use;
(2) the original starting strain is Streptomyces (Streptomyces sp.) DL70, a small amount of spores are dipped by an inoculating needle under aseptic conditions and inoculated on a slant culture medium, after the culture is carried out for 5 days at the temperature of 30 ℃, mature spores on the slant culture medium are collected by using 0.9% physiological saline preheated to the temperature of 40 ℃, and 10mL Streptomyces spores with the concentration of 10mL are prepared8Spore suspension per mL.
Example 3: mutagenesis of spore suspensions
The spore suspension obtained in example 2 was treated sequentially by UV mutagenesis and microwave mutagenesis, first by mutagenesis for 120s with an UV lamp (wavelength 260nm, power of UV lamp 20W), then by mutagenesis for 60s with microwave (frequency 400MHZ) in a dark room at 37 ℃, and by incubation for 45min at 37 ℃ to obtain the mutagenized spore suspension.
Example 4: screening of high temperature resistant Streptomycete
(1) Inoculating the mutagenized spore suspension obtained in example 3 into 100mL of liquid activation medium, performing shaking culture at 37 ℃ and 180rpm for 24h, and centrifuging at 4000rpm for 1min to enrich thalli;
(2) resuspending the thallus enriched in the step (1) by using 10mL of liquid activation culture medium, transferring the thallus into 90mL of liquid activation culture medium, carrying out shake culture at 45 ℃ and 180rpm for 48h, and centrifuging at 4000rpm for 1min to enrich the thallus;
(3) and (3) resuspending the thallus enriched in the step (2) by using 10mL of liquid activation medium, transferring the thallus into 90mL of liquid activation medium, carrying out shake culture for 72h at 180rpm under the condition of 50 ℃, centrifuging at 4000rpm for 1min to enrich the thallus, concentrating the bacterial liquid by 10 times, uniformly coating the bacterial liquid on the solid activation medium, carrying out inversion culture at 50 ℃ for 5-7 days, and screening 3 rapidly-growing single colonies which are respectively numbered as DL701, DL702 and DL 703.
(4) And (3) selecting 3 streptomycete single colonies which grow rapidly in the step (3), transferring the single colonies to a slant culture medium, culturing for 3-5 days at the temperature of 30 ℃ to obtain mature slant spores, and preserving the mature spores by using a 40% glycerol solution to obtain the high-temperature resistant streptomycete.
Example 5: preparation of high-temperature resistant streptomyces fermentation broth
The streptomyces thermophilic streptomyces DL701, DL702 and DL703 obtained in the embodiment 4 are utilized to carry out spore suspension preparation, seed culture, fermentation culture and effective viable count determination in sequence, and the specific operation steps are as follows:
a. inoculating high temperature resistant streptomyces DL701, DL702 and DL703 to slant culture medium under aseptic condition, culturing at 30 deg.C for 3 days, collecting spores on the slant culture medium with sterile water, and preparing spore with concentration of 1010Spore suspension per mL;
b. inoculating the spore suspension obtained in the step a into a liquid seed culture medium under the aseptic condition, and carrying out shaking culture at 30 ℃ and 180rpm for 24 hours to prepare a liquid seed solution;
c. under the aseptic condition, the liquid seed solution in the step b is inoculated into a liquid fermentation culture medium according to the volume ratio of 10 percent, the temperature is 30 ℃, the rpm is 200, and the aseptic air is introduced for fermentation culture for 48 hours, so as to obtain the high temperature resistant streptomyces zymophyte liquid.
The seed culture medium or the fermentation culture medium comprises the following components in percentage by weight (g/L): 50 parts of sucrose, 10 parts of corn steep liquor, 5 parts of monopotassium phosphate, 1 part of magnesium sulfate, 0.5 part of zinc sulfate and 0.01 part of ferrous sulfate, and adjusting the pH value of the culture medium to 7.0-7.5 by using 50% ammonia water solution.
Example 6: screening of high-temperature-resistant biocontrol streptomyces and determination of antibacterial activity of high-temperature-resistant biocontrol streptomyces
Inoculating fresh cucumber Fusarium oxysporum f.sp.cuminerum, cucumber scab (Alternaria cuminerum), tomato ralstonia solanacearum, Phytophthora capsici (Phytophthora capsici leonian), potato verticillium solani (Rhizoctonia solani ktihn of potato) and Sclerotium sclerotiorum (Sclerotia sclerotiorum (Lib.) Bar) cakes (inoculated with a 5 mm-sized fungus cake by a puncher) in the center of a potato glucose agar medium (PDA) plate, fixing 5 mm-sized sterile filter paper at a distance of 5cm from the center cake of the plate, respectively inoculating 3 strains of the Streptomyces thermostabilis DL701, DL702 and DL703 fermentation stock solutions in example 5, respectively, setting a control group of starting strain (Streptomyces sp) and carrying out repeated culture at a constant temperature of 70 ℃ and observing the original strain after the culture at 30 ℃ and carrying out repeated treatments, the results were averaged. The result is shown in table 1, the Streptomyces thermotolerans DL702 has the best bacteriostatic activity on the six plant pathogenic fungi, namely the screened target strain, namely the Streptomyces thermotolerans, is named as Streptomyces sp DL70H, has the preservation number of CCTCC No. m 2019486, has bacteriostatic activity on the plant pathogenic fungi superior to that of the original starting strain, namely Streptomyces sp DL70, is resistant to high temperature of 50 ℃, and is suitable for being applied to livestock manure composting fermentation.
TABLE 1 antagonistic action of different Streptomyces thermotolerans on phytopathogenic fungi
Example 7: determination of effective viable count of high-temperature fermentation of Streptomyces DL70H (Streptomyces sp.)
The preparation of spore suspension, seed culture, fermentation culture and the determination of effective viable count are carried out sequentially by using the high temperature resistant Streptomyces sp DL70H obtained in example 6, and the specific operation steps are as follows:
a. inoculating high temperature resistant biocontrol Streptomyces sp DL70H into slant culture medium under aseptic condition, culturing at 30 deg.C for 3 days, collecting spores on the slant culture medium with sterile water, and making spore concentration 1010Spore suspension per mL;
b. under the aseptic condition, inoculating the spore suspension obtained in the step a into a liquid seed culture medium, and carrying out shaking culture at 30 ℃ and 180rpm for 24 hours to prepare a liquid seed solution;
c. under aseptic conditions, inoculating the liquid seed solution obtained in the step b into a liquid fermentation culture medium according to the volume ratio of 10%, introducing sterile air at 50 ℃ and 200rpm, and fermenting and culturing for 72-120h to obtain the high-temperature resistant Streptomyces biocontrol DL70H zymocyte solution.
d. Taking the Streptomyces thermophilic (Streptomyces sp.) DL70H zymocyte solution cultured for 72h, 96h and 120h in the step c under the aseptic condition, and performing gradient dilution by using a pipette gun (10)-6、10-7、10-8) 100 mul of the dilution was spread on a Gao's first medium plate, 5 dishes were spread for each dilution, and the plate colonies were counted after 3-5 days of culture in an incubator at 30 ℃ with the average effective viable count shown in Table 2.
The seed culture medium or the fermentation culture medium comprises the following components in percentage by weight (g/L): 50 parts of sucrose, 10 parts of corn steep liquor, 5 parts of monopotassium phosphate, 1 part of magnesium sulfate, 0.5 part of zinc sulfate and 0.01 part of ferrous sulfate, and adjusting the pH value of the culture medium to 7.0-7.5 by using 50% ammonia water solution.
The result shows that compared with the control group of Streptomyces sp DL70, the experimental group of high temperature resistant Streptomyces sp DL70H can keep good growth activity under the high temperature condition of 50 ℃, and the effective viable count is not less than 2.0 multiplied by 10 after fermentation for 72-96h9cfu/mL。
TABLE 250 ℃ effective viable count of Streptomyces sp DL70H fermented broth
It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Sequence listing
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<120> high-temperature-resistant biocontrol streptomyces and application thereof
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Claims (4)
1. A strain of Streptomyces biocontrol (Streptomyces sp.) is obtained by mutagenesis and screening, is named as Streptomyces DL70H, and has a preservation number of CCTCC NO. M2019486.
2. The use of streptomyces DL70H as claimed in claim 1 for controlling plant diseases, characterized in that: the plant diseases are caused by plant pathogenic fungi, and the plant pathogenic fungi are cucumber Fusarium oxysporum f.sp.cuminerinum, cucumber scab (Alternaria cucumerina), tomato ralstonia solanacearum, Phytophthora capsici (Phytophthora capsici leonian), potato verticillium solani (Rhizoctonia solani ktithin of potatoo) or Sclerotinia sclerotiorum (sclerotium rolfsii (Lib.) de Bar).
3. A microbial inoculum for controlling plant diseases, which is characterized in that the effective components of the microbial inoculum are the streptomyces DL70H and the fermentation product thereof or the zymocyte liquid of the streptomyces DL70H in claim 1.
4. Use of the streptomyces DL70H as claimed in claim 1 in composting livestock manure.
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