CN111808783A - Saline-alkali tolerant enterobacter cloacae and production method and application of viable bacteria preparation thereof - Google Patents
Saline-alkali tolerant enterobacter cloacae and production method and application of viable bacteria preparation thereof Download PDFInfo
- Publication number
- CN111808783A CN111808783A CN202010766530.2A CN202010766530A CN111808783A CN 111808783 A CN111808783 A CN 111808783A CN 202010766530 A CN202010766530 A CN 202010766530A CN 111808783 A CN111808783 A CN 111808783A
- Authority
- CN
- China
- Prior art keywords
- enterobacter cloacae
- strain
- saline
- zts
- pepper
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
-
- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
- C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Pest Control & Pesticides (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Plant Pathology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a salt and alkali tolerant enterobacter cloacae and a production method and application of a live bacterial preparation thereof. The invention discloses a saline-alkali tolerant Enterobacter cloacae (Enterobacter cloacae) ZTS-10 separated from the rhizosphere soil of capsicum in saline-alkali soil, the preservation number of the strain in China general microbiological culture Collection center (CGMCC) is CGMCC No.20181, and the preservation date is 7, 3 and 2020. The strain has strong saline-alkali resistance, high planting rate in saline-alkali soil, IAA (International Association for the agricultural chemical of the pepper), phosphate and potassium production capacity, capability of promoting pepper growth, strong antagonistic effect on pathogenic bacteria of pepper phytophthora blight, capability of producing microbial fertilizer, particularly capability of producing microbial fertilizer special for pepper in saline-alkali soil, capability of reducing pepper phytophthora blight and capability of improving yield and quality.
Description
Technical Field
The invention relates to a salt and alkali tolerant enterobacter cloacae and a production method and application of a live bacterial preparation thereof, belonging to the technical field of agricultural biology.
Background
China is a large pepper producing country, the pepper planting area in China can reach 200 million hectares, and the pepper planting area accounts for more than 12% of the vegetable planting area. The pepper has strong adaptability, multiple varieties and high economic benefit, and becomes an important prop product for increasing the income of farmers and developing rural areas in many areas. The pepper phytophthora blight is the most serious disease currently threatening pepper production, and the disease has been widely generated in pepper growing areas around the world. If the disease condition is appropriate in the field, the disease can cause the yield reduction of about 30 percent, sometimes even up to 100 percent, and seriously restrict the economic development.
The pepper disease is mainly a devastating fungal disease caused by Phytophthora capsici (Phytophthora capsicii). Phytophthora capsici overwintering in plant seeds, soil and diseased residues of host plants mainly in the form of oospores and chlamydospores, germinates when the temperature and humidity are proper, and infects plants to cause the disease. The pepper phytophthora blight mostly occurs in the bud emergence and fruit bearing period of the pepper, the field is rapidly expanded, and large-area death is often caused.
For a long time, the control of pepper phytophthora blight is mainly chemical control, and technical measures such as optimized cultivation management, improved irrigation mode and the like are combined. The chemical control mainly uses a large amount of benamide bactericides such as agricultural metalaxyl, metalaxyl-M and the like, and although good control effect is obtained, the excessive use of chemical pesticides not only gradually generates a certain degree of drug resistance of phytophthora capsici, but also causes great potential threats to human health, food safety and environmental protection. Therefore, the development of biological control technology and the reduction of the use of chemical pesticides are inevitable trends in the development of modern agriculture.
Biological control refers to a measure for controlling the occurrence and development of plant diseases by using beneficial organisms or gene products. It has the advantages of no environmental pollution, safety to human and other living things, energy saving, ecological balance keeping and the like, and is an important means for developing green food and protecting ecological environment. The biological control measures of the pepper phytophthora blight mainly comprise screening and utilizing antagonistic microorganisms, inducing a host to generate disease resistance, adopting a gene engineering technology to control the pepper phytophthora blight, developing a biological source bactericide and the like. At present, antagonistic bacteria such as bacillus, pseudomonas fluorescens and the like, and antagonistic fungi such as trichoderma, aspergillus, pythium oligandrum and the like are used for biological control of pepper phytophthora blight, but because the control effect is limited, the growth of pepper is not obviously promoted, and the method cannot be popularized in large scale in agricultural production.
The saline-alkali soil is an important land resource in China, the total area of the saline-alkali soil and saline-alkali obstacle cultivated land in China exceeds 5 hundred million acres, wherein the total area of the saline-alkali soil and the saline-alkali obstacle cultivated land has the agricultural utilization potential of 2 hundred million acres, and accounts for about 10 percent of the area of the cultivated land in China. The pepper has certain saline-alkali resistance and is one of vegetable crops widely cultivated in saline-alkali soil. The biological control of the pepper phytophthora blight of the saline-alkali soil requires that the biological control strain has stronger saline-alkali tolerance and can be planted in the saline-alkali soil besides better disease-resistant growth promoting effect. Therefore, the screening of the saline-alkali tolerant disease-resistant growth-promoting strain has important significance.
In view of the current situation, the invention provides a saline-alkali tolerant enterobacter cloacae strain for promoting the growth of hot pepper and biological control of hot pepper phytophthora blight, and particularly for biological control of hot pepper phytophthora blight in saline-alkali soil cultivation.
Disclosure of Invention
The invention aims to provide an Enterobacter cloacae (Enterobacter cloacae) ZTS-10 and a preparation method and application of a live bacterial preparation thereof. The enterobacter cloacae ZTS-10 strain is separated from the rhizosphere soil of the pepper in the saline-alkali soil, has strong saline-alkali resistance, has the capabilities of producing IAA, dissolving phosphorus and potassium and antagonizing phytophthora capsici, can promote the growth of the pepper by using a live bacterial preparation, and has higher control effect on phytophthora capsici caused by the phytophthora capsici.
A salt and alkali tolerant Enterobacter cloacae (Enterobacter cloacae) ZTS-10 separated from saline-alkali soil pepper rhizosphere soil is disclosed, wherein the preservation number of the strain in China general microbiological culture Collection center (CGMCC) is CGMCC No. 20181; the preservation address is No. 3 Xilu No.1 of Beijing, Chaoyang, and the preservation date is 7 months and 3 days in 2020.
The colony and thallus characteristics of the Enterobacter cloacae ZTS-10 strain are as follows: culturing in NA culture medium at 37 deg.C for 48h to obtain colony diameter of 0.5-0.8mm, white semitransparent, round, smooth surface, swelling, moistening, viscous, glossy, uniform texture, and uniform edge; the thallus is in a short rod shape.
The physiological and biochemical characteristics of the Enterobacter cloacae ZTS-10 strain are as follows: gram-negative bacilli; positive catalase test, positive citrate utilization test, positive malonate utilization test, positive nitrate reduction test and positive V-P test; the hydrolysis test of starch is negative, the fermentation test of D-glucose is positive, the fermentation test of D-mannitol is positive, the fermentation test of lactose is positive, and the fermentation test of maltose is positive.
The invention also provides a live bacterial preparation taking the Enterobacter cloacae strain ZTS-10 as a main effective component.
The preparation method of the viable bacteria preparation of the Enterobacter cloacae ZTS-10 strain is characterized in that the viable bacteria preparation is prepared by the steps of carrying out amplification culture on Enterobacter cloacae ZTS-10, inoculating the cultured product to a fermentation medium, culturing the cultured product at 37 ℃ for 36 to 48 hours, adding 0.01 to 0.02 percent (w/w) of Propyl Gallate (PG), 4 to 6 percent (w/w) of yeast extract dry powder and 0.01 to 0.03 percent (w/w) of carbazone, and uniformly stirring the mixture.
The preparation method of the viable bacteria preparation of the Enterobacter cloacae ZTS-10 strain comprises the following steps:
1) activating strains: transferring the enterobacter cloacae ZTS-10 strain preserved at low temperature to the test tube slant of NA culture medium, and culturing at 37 deg.C for 24-36h for activation;
2) preparing seeds in a triangular flask: scraping activated Enterobacter cloacae ZTS-10 lawn with inoculating loop, inoculating in NB culture medium, and culturing at 37 deg.C for 24-36 hr;
3) preparing strains in a seeding tank: transferring the seeds in the triangular flask into a seeding tank according to the inoculation amount of 1-2% of the volume ratio, and culturing at 37 ℃ for 24-36 h;
4) fermentation culture: inoculating the strain in the seeding tank into a fermentation tank according to the inoculation amount of 1-2% by volume, and culturing at 37 ℃ for 36-48h to obtain fermentation liquor of enterobacter cloacae ZTS-10;
the formula of the fermentation medium is as follows: bran powder 5g, corn flour 10g, yeast extract powder 5g, potassium dihydrogen phosphate 1.5g, cellulase 0.1g, water 1000mL, initial pH 7.5.
5) Adding 0.01-0.02% (w/w) of Propyl Gallate (PG), 4-6% (w/w) of yeast extract dry powder and 0.01-0.03% of kasong into the Enterobacter cloacae ZTS-10 fermentation liquor, and stirring uniformly to obtain the viable bacteria preparation.
The invention also provides application of the salt and alkali tolerant enterobacter cloacae ZTS-10 strain in producing IAA, dissolving phosphorus and potassium and antagonizing phytophthora capsici.
The invention also provides application of the saline-alkali tolerant enterobacter cloacae ZTS-10 live bacteria preparation in preventing and treating pepper phytophthora blight and promoting pepper growth.
The use method of the live Enterobacter cloacae ZTS-10 preparation comprises the following steps: the enterobacter cloacae ZTS-10 viable bacteria preparation is diluted by 100-500 times with water, and is applied with water during root irrigation or watering.
The invention has the beneficial effects that: the enterobacter cloacae ZTS-10 is separated from saline-alkali soil, has strong saline-alkali resistance, high planting rate in the saline-alkali soil, IAA production, phosphate and potassium dissolving capacity, can promote the growth of hot pepper, has strong inhibiting effect on pathogenic bacteria of hot pepper epidemic disease, can be used for producing biological fertilizer, particularly special microbial fertilizer for the saline-alkali soil, is used for biological control of hot pepper epidemic disease, can reduce the occurrence of disease, and improves the yield and quality of hot pepper.
Drawings
FIG. 1 shows the form of Enterobacter cloacae ZTS-10 strain;
FIG. 2 is the IAA-producing ability of Enterobacter cloacae ZTS-10 strain; note: from left to right are: CK (positive control, adding 50. mu.L 50mg/L IAA), inoculating ZTS-10 strain; the results show that: the color turns red after the ZTS-10 strain is inoculated, which shows that the strain has the capability of producing IAA;
FIG. 3 is a phosphate solubilizing ring of Enterobacter cloacae ZTS-10 strain on an organophosphorus culture medium, wherein the inner ring is a bacterial colony and the outer ring is a phosphate solubilizing transparent ring;
FIG. 4 shows the growth of Enterobacter cloacae ZTS-10 strain on a silicate bacterial culture medium to form a smooth transparent oil droplet-shaped colony.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1: screening of Enterobacter cloacae ZTS-10
(1) Screening of saline-alkali tolerant strains
Enterobacter cloacae ZTS-10 was isolated from the rhizosphere soil of Capsicum annuum. The soil was sampled in the Binzhou city district of Shandong province in 2018 in 9 months, and was moderate saline-alkali soil. The specific separation method comprises the following steps: the soil sample was mixed well, 5g was weighed and placed in a triangular flask containing 95mL of sterile water and 10 beads, and shaken at 37 ℃ and 180rpm for 30 min. Taking 1mL of soil suspension for 10-1—10-7Serial concentration gradient dilutions were made and then 10 taken-5、10-6、10-7Three dilutions were plated onto plates containing selection medium and cultured in an inverted format at 37 ℃ for 2 d. Single colonies were picked individually and streaked onto plates containing selection medium and cultured in an inverted format at 37 ℃ for 2 d. Picking single colony, transferring to the slant of the preservation culture medium test tube, culturing at 37 deg.C for 2d, growing with thallus Porphyrae, and storing in 4 deg.C refrigerator.
The formula of the selective culture medium is as follows: 10g of peptone, 5g of yeast extract, 20g of NaCl, 20g of agar, 1000mL of distilled water, pH8.0, and sterilizing at 121 ℃ for 20 min.
The formula of the preservation culture medium is as follows: 10g of peptone, 5g of yeast extract, 10g of NaCl, 20g of agar, 1000mL of distilled water, pH7.5, and sterilizing at 121 ℃ for 20 min.
(2) IAA (indoleacetic acid) -producing bacterium screening
Inoculating the functional strain screened in the step (1) into an LB liquid culture medium containing L-tryptophan (200mg/L), culturing at 28 ℃ and 180r/min for 4d by shaking. 50. mu.L of the bacterial suspension was dropped on a white ceramic plate, and 50. mu.L of Salkowski colorimetric solution was added thereto. A positive control was prepared by adding 50. mu.L of 50mg/L IAA to the sample. The white ceramic plate was left at room temperature in the dark for 30min and observed, and the red-colored one indicated that IAA was secreted.
The LB liquid culture medium comprises the following components in percentage by weight: 10.0g of peptone, 5.0g of yeast extract, 10.0g of NaCl and 1000mL of distilled water; sterilizing at 121 deg.C for 20 min; pH 7.0.
The formula of the Salkowski colorimetric solution is as follows: 35% HClO450mL、0.5mol/L FeCl31mL。
(3) Screening of phosphate solubilizing Strain
Respectively transferring the strains with the IAA production capability screened in the step (2) to an organophosphorus bacteria culture medium, and determining whether the strains have the capability of dissolving phosphorus.
The formula of the organophosphorus bacteria culture medium is as follows: glucose 10.0g, (NH)4)2SO40.5g、MgSO4·7H2O 0.3g、MnSO4·4H2O 0.03g、KCl 0.3g、FeSO4·7H2O 0.03g、NaCl 0.3g、CaCO35.0g, lecithin 0.2g, agar 1.6%, and distilled water 1000mL, and sterilizing at 121 deg.C for 20min at pH 7.0-7.5. Note: dissolving lecithin in anhydrous ethanol, adding water after completely dissolving, and heating until ethanol is completely evaporated.
(4) Screening of Potassium-decomposing Strain
Inoculating the strains with the phosphate-solubilizing capability screened in the step (3) to a silicate bacteria culture medium plate by a three-zone streaking method, culturing at a constant temperature of 37 ℃ for 72h, and selecting strains which can grow and form colonies on the plate, namely primary screening strains. And repeating the steps for the primary screened strains for re-screening, and selecting strains with fast growth and large bacterial colonies.
The formula of the silicate bacteria culture medium is as follows: sucrose 5g, MgSO40.5g、CaCO30.1g、Na2HPO42g、FeCl30.005g, 1g of glass powder, 1.6% of agar and 1000mL of distilled water, and sterilizing at 121 ℃ for 15min, wherein the pH value is 7.0.
(5) Screening phytophthora capsici antagonistic bacteria by using confrontation culture method
Inoculating phytophthora capsici blocks with the diameter of 5mm to the center of a PDA culture medium flat plate, inoculating the strains screened in the steps at equal intervals at the position 2.5cm away from the center of the flat plate, repeating the treatment for 3 times by taking the flat plate without the screened strains as a control, culturing for 10 days at 28 ℃, selecting the strains with larger antibacterial zone diameter, and storing for later use.
Through the multiple screening, the growth capacity, IAA production capacity, phosphate dissolving capacity, potassium dissolving capacity and phytophthora capsici antagonistic bacteria of phytophthora capsici pathogenic bacteria with strong saline-alkali resistance and IAA production capacity, phosphate dissolving capacity and potassium dissolving capacity are comprehensively considered, and the antagonistic bacteria is numbered ZTS-10. As shown in FIG. 2, the color turned red after inoculation with ZTS-10 strain, indicating its ability to produce IAA; as shown in FIG. 3, the phosphate solubilizing ring of Enterobacter cloacae ZTS-10 strain on the organophosphorus culture medium, in which the inner ring is a bacterial colony and the outer ring is a phosphate solubilizing transparent ring, shows that the strain ZTS-10 has strong capability of solubilizing organophosphorus; as shown in FIG. 4, ZTS-10 strain can grow and form colonies on silicate bacteria medium, which indicates its potassium-solubilizing ability.
Example 2 identification of Enterobacter cloacae ZTS-10
(1) Morphological and physiological biochemical characteristics
The colony and thallus characteristics of the Enterobacter cloacae ZTS-10 strain are as follows: culturing in NA culture medium at 37 deg.C for 48h to obtain colony diameter of 0.5-0.8mm, white semitransparent, round, smooth surface, swelling, moistening, viscous, glossy, uniform texture, and uniform edge; the cells were in the form of short rods (as shown in FIG. 1). The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and 7.0 of pH.
The physiological and biochemical characteristics of the Enterobacter cloacae ZTS-10 strain are as follows: gram-negative bacilli; positive catalase test, positive citrate utilization test, positive malonate utilization test, positive nitrate reduction test and positive V-P test; the hydrolysis test of starch is negative, the fermentation test of D-glucose is positive, the fermentation test of D-mannitol is positive, the fermentation test of lactose is positive, and the fermentation test of maltose is positive.
(2)16S rDNA sequence analysis
The ZTS-10 strain is inoculated into NB medium and shake-cultured at 37 ℃ and 180r/min for 24 h. The NB culture medium comprises the following components in percentage by weight: 3g of beef extract, 10g of peptone, 5g of NaCl, 1000ml of water and pH 7.0. Collecting thallus, extracting total DNA, and performing PCR amplification of 16S rDNA gene under the guide of universal primers F27: 5'-AGA GTT TGA TCA TGG CTC AG-3' and F27: 5'-AGA GTT TGA TCA TGG CTC AG-3' of prokaryotic 16S rRNA gene by using the total DNA as a template. After the amplification product is separated by 1% agarose gel electrophoresis, the amplification product is recovered by a gel recovery kit and handed over to Shanghai Biotechnology Limited company for sequencing. The sequence of the 16S rDNA is shown in SEQ No. 1.
Through morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain is Enterobacter cloacae (Enterobacter cloacae) named as Enterobacter cloacae ZTS-10. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 7-3.2020, with the preservation number of CGMCC No. 20181.
Example 3 preparation of Enterobacter cloacae ZTS-10 bacterial agent
The preparation method of the enterobacter cloacae ZTS-10 strain microbial inoculum comprises the following steps:
1) activating strains: transferring the strain of the enterobacter cloacae ZTS-10 preserved at low temperature to the test tube slant of the NA culture medium, and culturing at 37 ℃ for 24h for activation. The NA culture medium is a nutrient agar culture medium, and the formula of the NA culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar, 1000mL of water and pH7.0.
2) Preparing seeds in a triangular flask: scraping activated Enterobacter cloacae ZTS-10 lawn with inoculating loop, inoculating into NB culture medium, and culturing at 37 deg.C for 24 hr. The NB culture medium is a nutrient broth culture medium, and the formula of the NB culture medium is as follows: 3g of beef extract, 10g of peptone, 5g of NaCl, 1000mL of water and pH7.0.
3) Preparing strains in a seeding tank: inoculating the seeds in the triangular flask into a 10L seeding tank filled with 6-7L NB medium at an inoculation amount of 2% by volume, culturing at 37 ℃ for 24h, stirring at a rotation speed of 200 and 220rpm for the whole process, and ventilating at a ventilation rate of 3-4L/min for 0-10h, wherein the ventilation rate is 6-9L/min.
4) Fermentation culture: inoculating the seed tank strain into 500L fermentation tank with inoculum size of 2% by volume. The fermentation medium is 300-350L in the fermentation tank, and the fermentation is carried out for 36-48h at 37 ℃ to obtain the fermentation liquid of the Enterobacter cloacae ZTS-10. The whole stirring speed is 150-. After the fermentation is finished, the viable count of the enterobacter cloacae ZTS-10 in the fermentation liquor is (1-2) x 1010cfu/ml。
The formula of the fermentation medium in the step 4) is as follows: bran powder 5g, corn flour 10g, yeast extract powder 5g, potassium dihydrogen phosphate 1.5g, cellulase 0.1g, water 1000mL, initial pH 7.5. The cellulase is purchased from Tai' an Xin Deli biological technology limited company, and the product specification is 10 ten thousand U/g. The definition and detection method of the enzyme activity unit of the cellulase execute the light industry standard QB 2583-.
The preparation method of the fermentation medium in the step 4) comprises the following steps: weighing raw materials according to the formula, dissolving in water, heating to 50-55 deg.C, maintaining the temperature for 1-2h, heating to 121 deg.C, maintaining the temperature for 20-30min, and sterilizing.
5) Preparation of the viable bacteria preparation: adding 0.01% (w/w) of Propyl Gallate (PG), 5% (w/w) of yeast extract dry powder and 0.01% of kasons into the Enterobacter cloacae ZTS-10 fermentation liquor, and uniformly stirring to obtain a viable bacteria preparation with viable bacteria content of (1.0-2.0) x 1010cfu/mL. The yeast extract dry powder is produced by Angel Yeast Co.
Example 4 prevention and treatment Effect of Enterobacter cloacae ZTS-10 on Phytophthora capsici
Soil for pot culture experiments is taken from the coastal urban area of Binzhou, Shandong province, and the soil type is saline-alkali soil. And (3) pot culture test fertilizing amount: the usage amount of the organic fertilizer is 1 percent (dry weight of the organic fertilizer/dry weight of soil), the organic fertilizer is produced by Shandong Zongtian Biotech limited company, and the nutrient indexes of the organic fertilizer are 55.6 percent of organic matters, 2.1 percent of N and P2O52.2％、K2O 1.6％。
The potting test was performed 3 times by setting 1 treatment group, 1 control group, 10 pots each, and culturing 2 peppers per pot. The pepper plant grows to the four-leaf stageMixing with six leaves, and mixing with live Enterobacter cloacae ZTS-10 preparation (1 × 10)10cfu/mL) to 100 times, and irrigating 50mL of roots per pot of treated seedlings and 50mL of phytophthora capsici suspension at the same time. Control (CK) only 50mL of Phytophthora capsici suspension per pot was irrigated. After 10 days of root irrigation treatment, symptoms are observed and disease severity and control effect are counted. The preparation method of the phytophthora capsici suspension comprises the following steps: inoculating Phytophthora capsici to PDA plate, culturing at 28 deg.C for 8-10 days, and making spore suspension with spore concentration of 1 × 106cfu/mL. The PDA culture medium formula comprises: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH value is natural.
Disease grading standard, grade 0: no disease at all; level 1: local disorders (dark brown to black, overflowed) in the outer leaf and stem; and 2, stage: the color of the stem part of the outer leaf is below 1/3, or the outer leaf has a few wilting (falling off); and 3, level: the whole plant 1/3 is overflowed, shrunk and discolored, or falls down, leaves fall off and withers. The correlation index calculation formula is as follows.
The incidence rate is (number of diseased plants/total number of investigated plants) × 100%.
The control effect is (number of non-diseased plants/total investigated plants) × 100%.
The disease index ∑ (number of plants at each stage × representative value at each stage)/(total number of investigated plants × highest value) × 100%.
The test results are shown in table 1, and during the experimental investigation period, the disease index of the pepper phytophthora blight of the control group reaches 81.27%, which indicates that the pepper phytophthora blight pathogenic bacteria adopted in the experiment have strong pathogenicity to the tested pepper. Meanwhile, the disease index of the pepper phytophthora blight in the treatment group inoculated with the enterobacter cloacae ZTS-10 live bacteria preparation and the pepper phytophthora capsici is obviously reduced, and the prevention and treatment effect is 56.39%. This shows that the live Enterobacter cloacae ZTS-10 preparation has good control effect on phytophthora blight of capsicum.
TABLE 1 prevention and treatment of Phytophthora capsici by Enterobacter cloacae ZTS-10 viable bacteria preparation
Note "-" indicates none. Different lower case letters after the same column of data indicate that the difference between different treatments is statistically significant at a level of P < 0.05.
Example 5 growth promoting action of Enterobacter cloacae ZTS-10 on Capsicum plants
Soil for pot culture experiments is taken from the coastal urban area of Binzhou, Shandong province, and the soil type is saline-alkali soil. And (4) selecting the pepper seeds with full seeds and uniform sizes, and disinfecting the surfaces. And uniformly spreading the sterilized seeds in a preservation box paved with sterilization gauze, covering a layer of sterilization gauze, adding a proper amount of sterile water to moisten the gauze, sealing, accelerating germination at 28 ℃ in the dark, sowing the seeds in a seedling pot after the seeds germinate white root tips, and keeping the seeds for later use when the seeds grow to the heart of six leaves.
Taking a pepper seedling with six leaves and one core, transplanting the pepper seedling into a flowerpot containing 200.0g of soil for a pot culture test, and taking 15mL of a 500-time diluted enterobacter cloacae ZTS-10 viable bacteria preparation to irrigate roots and inoculate the roots in the soil around the roots of the pepper seedling. Meanwhile, setting a control, adding 100 times of sterilized enterobacter cloacae ZTS-10 viable bacteria preparation (1 × 10) diluent into rhizosphere soil of pepper seedlings8cfu/mL)15 mL. Each treated 20 pots, 1 pepper seedling per pot. The experiment was repeated 3 times. The treated pepper plants are cultured in a small greenhouse under the conditions of 26 ℃, 14 hours of illumination time and 10 hours of darkness. The pots were watered with sterile distilled water once a day, approximately 5mL per pot. After 20 days, healthy plants are selected to measure the plant height, root length, stem thickness, fresh weight, dry weight, water content and other data of the pepper plants.
The test results are shown in Table 2, and the ZTS-10 has a remarkable growth promoting effect on the growth of the pepper, which is shown in that compared with a control group, the ZTS-10 suspension treatment obviously increases the height, the root length, the stem thickness, the fresh weight and the dry weight of the pepper plant, and the Enterobacter cloacae ZTS-10 can remarkably promote the growth of the pepper plant and increase the substance accumulation.
TABLE 2 growth promoting effect of viable Enterobacter cloacae ZTS-10 preparation on Capsici fructus plants
Note that different lower case letters after the same column of data indicate that the difference between different treatments is statistically significant at a level P < 0.05.
SEQUENCE LISTING
<110> Shandong Zongtian Biotech Co., Ltd
<120> saline-alkali tolerant enterobacter cloacae and production method and application of viable bacteria preparation thereof
<130>0
<160>1
<170>PatentIn version 3.3
<210>1
<211>1407
<212>DNA
<213> 16S rRNA sequence of Enterobacter cloacae (Enterobacter cloacae) ZTS-10
<400>1
agtcgaacgg tagcacagag agcttgctct cgggtgacga gtggcggacg ggtgagtaat 60
gtctgggaaa ctgcctgatg gagggggata actactggaa acggtagcta ataccgcata 120
atgtcgcaag accaaagagg gggaccttcg ggcctcttgc catcagatgt gcccagatgg 180
gattagctag taggtggggt aacggctcac ctaggcgacg atccctagct ggtctgagag 240
gatgaccagc cacactggaa ctgagacacg gtccagactc ctacgggagg cagcagtggg 300
gaatattgca caatgggcgc aagcctgatg cagccatgcc gcgtgtatga agaaggcctt 360
cgggttgtaa agtactttca gcggggagga aggtgttgtg gttaataacc acagcaattg 420
acgttacccg cagaagaagc accggctaac tccgtgccag cagccgcggt aatacggagg 480
gtgcaagcgt taatcggaat tactgggcgt aaagcgcacg caggcggtct gtcaagtcgg 540
atgtgaaatc cccgggctca acctgggaac tgcattcgaa actggcaggc tggagtcttg 600
tagagggggg tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg aggaataccg 660
gtggcgaagg cggccccctg gacaaagact gacgctcagg tgcgaaagcg tggggagcaa 720
acaggattag ataccctggt agtccacgcc gtaaacgatg tcgatttgga ggttgtgccc 780
ttgaggcgtg gcttccggag ctaacgcgtt aaatcgaccg cctggggagt acggccgcaa 840
ggttaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900
cgatgcaacg cgaagaacct tacctggtct tgacatccac agaactttcc agagatggat 960
tggtgccttc gggaactgtg agacaggtgc tgcatggctg tcgtcagctc gtgttgtgaa 1020
atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag cggttaggcc 1080
gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga cgtcaagtca 1140
tcatggccct tacgaccagg gctacacacg tgctacaatg gcgcatacaa agagaagcga 1200
cctcgcgaga gcaagcggac ctcataaagt gcgtcgtagt ccggattgga gtctgcaact 1260
cgactccatg aagtcggaat cgctagtaat cgtagatcag aatgctacgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa gaagtaggta 1380
gcttaacctt cgggagggcg ctaccac 1407
Claims (7)
1. The salt and alkali tolerant Enterobacter cloacae strain is Enterobacter cloacae (Enterobacter cloacae) ZTS-10, and the preservation number is CGMCC No. 20181.
2. The strain of claim 1, wherein the strain is used for producing IAA, dissolving phosphorus and potassium and antagonizing phytophthora capsici.
3. A live bacterial preparation comprising the Enterobacter cloacae strain ZTS-10 according to claim 1 as an active ingredient.
4. The method for preparing the viable bacteria preparation according to claim 3, wherein the viable bacteria preparation is obtained by performing expanded culture on the Enterobacter cloacae strain ZTS-10, inoculating the strain to a fermentation medium, culturing the strain at 37 ℃ for 36 to 48 hours, adding 0.01 to 0.02% (w/w) of propyl gallate, 4 to 6% (w/w) of yeast extract dry powder and 0.01 to 0.03% (w/w) of Kathon into the fermentation broth, and uniformly stirring.
5. The method of preparing the viable bacteria preparation according to claim 4, wherein the fermentation medium is: bran powder 5g, corn flour 10g, yeast extract powder 5g, potassium dihydrogen phosphate 1.5g, cellulase 0.1g, water 1000mL, initial pH 7.5.
6. The live bacterial preparation according to claim 3, for use in controlling phytophthora blight of capsicum and promoting the growth of capsicum.
7. The use method of the viable bacteria preparation of claim 3, wherein the viable bacteria preparation is diluted by 100 to 500 times with water and is applied along with water during root irrigation or watering.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010766530.2A CN111808783B (en) | 2020-08-03 | 2020-08-03 | Enterobacter cloacae and production method and application of viable bacteria preparation thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010766530.2A CN111808783B (en) | 2020-08-03 | 2020-08-03 | Enterobacter cloacae and production method and application of viable bacteria preparation thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111808783A true CN111808783A (en) | 2020-10-23 |
| CN111808783B CN111808783B (en) | 2022-03-04 |
Family
ID=72864192
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010766530.2A Active CN111808783B (en) | 2020-08-03 | 2020-08-03 | Enterobacter cloacae and production method and application of viable bacteria preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111808783B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760264A (en) * | 2021-02-01 | 2021-05-07 | 山东大学 | Enterobacter cloacae strain and application thereof |
| CN112961807A (en) * | 2021-03-30 | 2021-06-15 | 中国科学院成都生物研究所 | Microbial composition and application thereof in promoting germination and growth of highland barley seeds |
| CN116286542A (en) * | 2023-04-12 | 2023-06-23 | 河北省科学院生物研究所 | Enterobacter cloacae CBY-9 and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543801A (en) * | 2003-11-22 | 2004-11-10 | 西北农林科技大学 | Preparation for biological prevention and cure of hot pepper epidemic disease and method for producing the same |
| KR20100082143A (en) * | 2009-01-08 | 2010-07-16 | 대한민국(농촌진흥청장) | An anti-phytophthora gene cluster specifically inhibiting the growth of phytophthora spp. and the prevention method of phytophthora using thereof |
| US20110077158A1 (en) * | 2009-09-29 | 2011-03-31 | Slininger Patricia J | Biocontrol of Storage Maladies of Potatoes by Bacterial Antagonists Produced in Co-Culture |
| CN105255769A (en) * | 2015-11-03 | 2016-01-20 | 湖南中烟工业有限责任公司 | Enterobacter cloacae and application thereof |
| CN108587959A (en) * | 2018-04-27 | 2018-09-28 | 福建农林大学 | Raw dissolving phosphor and dissolving potassium fixed nitrogen clostridium perfringen and its application in one plant of moso bamboo |
| CN108795797A (en) * | 2018-05-24 | 2018-11-13 | 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) | Raw enterobacter cloacae and its application in one plant of maize root system |
| CN109280628A (en) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | A kind of enterobacteria strain and its application in promotion Bamboo Growth |
| CN109503228A (en) * | 2018-11-30 | 2019-03-22 | 山东省科学院生态研究所 | A kind of liquid bacterial manure and preparation method thereof using Ludwig enterobacteria fermented garlic stalk |
| CN110616171A (en) * | 2019-11-20 | 2019-12-27 | 山东佐田氏生物科技有限公司 | Saline-alkali-resistant Pacific bacillus and viable bacteria preparation and application thereof |
| CN111117910A (en) * | 2019-12-27 | 2020-05-08 | 江西农业大学 | Enterobacter ludwigii PN6 and application thereof |
-
2020
- 2020-08-03 CN CN202010766530.2A patent/CN111808783B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1543801A (en) * | 2003-11-22 | 2004-11-10 | 西北农林科技大学 | Preparation for biological prevention and cure of hot pepper epidemic disease and method for producing the same |
| KR20100082143A (en) * | 2009-01-08 | 2010-07-16 | 대한민국(농촌진흥청장) | An anti-phytophthora gene cluster specifically inhibiting the growth of phytophthora spp. and the prevention method of phytophthora using thereof |
| US20110077158A1 (en) * | 2009-09-29 | 2011-03-31 | Slininger Patricia J | Biocontrol of Storage Maladies of Potatoes by Bacterial Antagonists Produced in Co-Culture |
| CN105255769A (en) * | 2015-11-03 | 2016-01-20 | 湖南中烟工业有限责任公司 | Enterobacter cloacae and application thereof |
| CN108587959A (en) * | 2018-04-27 | 2018-09-28 | 福建农林大学 | Raw dissolving phosphor and dissolving potassium fixed nitrogen clostridium perfringen and its application in one plant of moso bamboo |
| CN108795797A (en) * | 2018-05-24 | 2018-11-13 | 珠海市现代农业发展中心(珠海市金湾区台湾农民创业园管理委员会、珠海市农渔业科研与推广中心) | Raw enterobacter cloacae and its application in one plant of maize root system |
| CN109280628A (en) * | 2018-08-08 | 2019-01-29 | 江西农业大学 | A kind of enterobacteria strain and its application in promotion Bamboo Growth |
| CN109503228A (en) * | 2018-11-30 | 2019-03-22 | 山东省科学院生态研究所 | A kind of liquid bacterial manure and preparation method thereof using Ludwig enterobacteria fermented garlic stalk |
| CN110616171A (en) * | 2019-11-20 | 2019-12-27 | 山东佐田氏生物科技有限公司 | Saline-alkali-resistant Pacific bacillus and viable bacteria preparation and application thereof |
| CN111117910A (en) * | 2019-12-27 | 2020-05-08 | 江西农业大学 | Enterobacter ludwigii PN6 and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| B. JASIM • C等: "《Plant growth promoting potential of endophytic bacteria isolated from Piper nigrum》", 《PLANT GROWTH REGUL》 * |
| IRABOR A AND MMBAGA等: "《Evaluation of Selected Bacterial Endophytes for Biocontrol Potential against Phytophthora Blight of Bell Pepper ( Capsicum annuum L.)》", 《JOURNAL OF PLANT PATHOLOGY & MICROBIOLOGY》 * |
| J.F.麦克法丁著，林万明译: "《医学细胞生化试验鉴定手册》", 31 August 1985, 人民卫生出版社 * |
| TOH, S. C.,等: "《Screening for antifungal-producing bacteria from Piper nigrum plant against Phytophthora capsici》", 《INTERNATIONAL FOOD RESEARCH JOURNAL》 * |
| 袁红莉等主编: "《农业微生物学及实验教程》", 28 February 2009 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112760264A (en) * | 2021-02-01 | 2021-05-07 | 山东大学 | Enterobacter cloacae strain and application thereof |
| CN112760264B (en) * | 2021-02-01 | 2022-04-22 | 山东大学 | Enterobacter cloacae strain and application thereof |
| CN112961807A (en) * | 2021-03-30 | 2021-06-15 | 中国科学院成都生物研究所 | Microbial composition and application thereof in promoting germination and growth of highland barley seeds |
| CN112961807B (en) * | 2021-03-30 | 2023-01-20 | 中国科学院成都生物研究所 | Microbial composition and application thereof in promoting germination and growth of highland barley seeds |
| CN116286542A (en) * | 2023-04-12 | 2023-06-23 | 河北省科学院生物研究所 | Enterobacter cloacae CBY-9 and application thereof |
| CN116286542B (en) * | 2023-04-12 | 2023-08-15 | 河北省科学院生物研究所 | Enterobacter cloacae CBY-9 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111808783B (en) | 2022-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111808783B (en) | Enterobacter cloacae and production method and application of viable bacteria preparation thereof | |
| CN112175888B (en) | Bacillus belgii Hsg1949 and application thereof | |
| CN110616171B (en) | Saline-alkali-resistant Pacific bacillus and viable bacteria preparation and application thereof | |
| CN113151062B (en) | Bacillus belgii LJBV19 and application thereof | |
| CN113621532B (en) | Composite microbial agent containing saline-alkali tolerant side bacillus licheniformis and preparation method thereof | |
| CN107136122B (en) | Biocontrol microbial inoculum for preventing and treating potato late blight | |
| CN111073825B (en) | Bacterium with plant soil-borne disease resistance effect and application thereof | |
| CN111690578B (en) | Salt and alkali resistant Siamese bacillus and production method and application of viable bacteria preparation thereof | |
| CN112608869B (en) | Saline-alkali-tolerant Argentina pseudomonads as well as viable bacteria preparation and application thereof | |
| CN112342173B (en) | Bacillus belgii and application thereof | |
| CN113755382A (en) | Bacillus aryabhattai NDFY-1 and application thereof | |
| CN109182219A (en) | One plant of Mo Haiwei bacillus for promoting the growth of Henry David Thoreau grass and its application | |
| CN108913625B (en) | Salt-tolerant streptomycete, microbial inoculum thereof and application of microbial inoculum thereof in promoting plant growth | |
| CN112111432B (en) | Double-source humic acid biofertilizer and preparation method and application thereof | |
| CN111808776B (en) | Saline-alkali-tolerant air bacillus and preparation method and application of viable bacteria preparation thereof | |
| CN110791459B (en) | Bacillus subtilis for preventing and controlling continuous cropping lily soil-borne blight and application thereof | |
| CN109957535B (en) | Bacillus simplex, microbial agent prepared by using bacillus simplex, biological fertilizer and application | |
| CN117467557A (en) | Paenibacillus strain with growth promoting and biocontrol functions and application thereof | |
| CN114350559B (en) | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN112501084B (en) | Rhizosphere probiotic Klebsiella ZH07 and application thereof | |
| CN114196598B (en) | Suspension fertilizer and preparation method thereof | |
| CN117106668B (en) | Saline-alkali tolerant growth promoting pseudomonas P522 and application thereof | |
| CN116426445B (en) | Pseudomonas bacteria NJAU-T102 and application thereof | |
| CN115895972B (en) | Composite microbial inoculum and application thereof in prevention and control of tobacco soil-borne diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Production method and application of a strain of Enterobacter cloacae and its live bacterial preparation Effective date of registration: 20230808 Granted publication date: 20220304 Pledgee: Postal Savings Bank of China Limited Pingyin County sub branch Pledgor: SHANDONG ZOETICLAND BIOLOGICAL TECHNOLOGY CO.,LTD. Registration number: Y2023980051283 |