CN108300681B - Streptomyces rochei and application thereof - Google Patents
Streptomyces rochei and application thereof Download PDFInfo
- Publication number
- CN108300681B CN108300681B CN201810357031.0A CN201810357031A CN108300681B CN 108300681 B CN108300681 B CN 108300681B CN 201810357031 A CN201810357031 A CN 201810357031A CN 108300681 B CN108300681 B CN 108300681B
- Authority
- CN
- China
- Prior art keywords
- fermentation
- liquid
- streptomyces rochei
- medium
- fertilizer
- 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.)
- Active
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
- C12R2001/465—Streptomyces
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Pest Control & Pesticides (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Agronomy & Crop Science (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a Streptomyces rochei Z331-A, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15348. The Streptomyces rochei Z331-A and its fermentation product can be used for preparing agricultural and medical antibiotics, bactericide and fertilizer. The streptococcus rochei Z331-A provided by the invention is easy to culture, and the fermentation product can resist bacteria and plant pathogenic fungi in a broad spectrum; especially has excellent effects on marine vibrio parahaemolyticus and methicillin-resistant staphylococcus aureusBiological activity; and the fermentation product is resistant to ultraviolet radiation, acid and alkali, heat and stable, and has industrial development value.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to Streptomyces rochei and application thereof in preventing and controlling pathogenic bacteria and fungi.
Background
The current antibiotic pharmacy market, antibiotics from microorganisms account for 67%, with actinomycetes producing 2/3. Because the antibiotics produced by the microorganisms are mostly natural and green medicaments, the antibiotics have the characteristics of various resistance mechanisms, safety to the environment and the like, and are less prone to generate drug resistance compared with artificial chemical preparations, so that the antibiotics are paid more attention to. MRSA is an abbreviation for methicillin-resistant staphylococcus aureus, called "superbug", has broad-spectrum resistance to aminoglycosides, macrolides, tetracyclines, fluoroquinolones, sulfonamides, rifampicin to varying degrees, except for vancomycin. Has become one of the important pathogenic bacteria of nosocomial and community infections. Since the first patient was discovered in the uk in 1961 and thereafter spread worldwide at an alarming rate, it is estimated that approximately one hundred thousand patients are hospitalized annually for serious infections. At present, the only antibiotics for clinically effective control and treatment are vancomycin, but with the use of antibiotics in a flooding way, the problem of drug resistance and drug resistance is caused. The 2002 center for disease control promulgates the world's first vancomycin-resistant staphylococcus aureus. There is a great need to develop drugs effective in treating MRSA.
At present, various agricultural antibiotics have been developed for killing bacteria, fungi, insects, mites, viruses, weeds, plant growth regulators and other fields. However, with the use of large doses of agricultural antibiotics, the development of drug resistance cannot be avoided, and therefore, the development of new and safer agricultural antibiotics becomes necessary.
Vibrio parahaemolyticus (Vp) is a gram-negative halophilic bacterium, widely exists in coastal seawater, sea river junctions and aquatic animals, can cause diseases of various cultured animals such as fish, shrimp, crab and shellfish, and brings serious economic loss to aquaculture industry. In addition, Vibrio parahaemolyticus can cause food poisoning and seriously harm human health. Human consumption of water products contaminated with Vibrio parahaemolyticus can cause acute gastroenteritis and, in severe cases, sepsis or even death. In recent years, there have been multiple outbreaks of food-borne diseases caused by Vp in asia, europe and north america. The statistics of food-borne diseases monitoring networks in China shows that food-borne diseases caused by Vp are the first of microbial food-borne diseases, and how to effectively prevent and control vibrio parahemolyticus becomes a problem to be solved urgently in the fields of aquaculture industry and food safety. At present, antibiotics are mainly used for killing vibrio parahaemolyticus, but the vibrio parahaemolyticus has different degrees of resistance to various antibiotics commonly used in aquaculture industry, the multidrug resistance situation is very serious, and the discovery of finding a new antibiotic to resist the vibrio parahaemolyticus becomes necessary.
Disclosure of Invention
Aiming at the problems of lack of antibiotics for effectively controlling MRSA and vibrio parahaemolyticus, lack of types of agricultural antibiotics and the like, the invention provides streptomyces rochei, the fermentation product of which has broad-spectrum antibacterial property and can inhibit a plurality of plant pathogenic fungi, gram-positive and gram-negative bacteria and drug-resistant MRSA strains.
In order to achieve the purpose, the invention adopts the following technical scheme.
Streptomyces rochei (S. (A)Streptomyces rochei) Z331-A, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15348; the 16S rRNA sequence is shown in SEQ number 1.
The application of the Streptomyces rochei Z331-A and the fermentation product thereof in preparing medical antibiotics, agricultural antibiotics, bactericides and fertilizers.
A preparation method of the Streptomyces rochei Z331-A fermentation product comprises the following steps:
(1) inoculating the preserved strain into a basic culture medium, and performing shake-flask fermentation to obtain an activated bacterium solution;
(2) inoculating the activated bacterial liquid into a seed culture medium, and performing shake-flask fermentation to obtain a seed liquid;
(3) inoculating the seed liquid into a fermentation culture medium, stirring and fermenting to obtain fermentation liquid containing fermentation products.
The inoculation amount in the step (1) is 1 percent; the inoculation amount in the step (2) is 3 percent; the inoculation amount in the step (3) is 3-5%.
In the steps (1) - (3), the fermentation temperature is 25-30 ℃; the rotating speed is 180-200 r/min.
In the steps (1) and (2), the culture time is 2-3 days; in the step (3), the culture time is 7-10 days.
The basic culture medium, the seed culture medium and the fermentation culture medium are respectively selected from any one of Gao's I, Ke's, Chao's, starch, glucose asparagine, potato glucose, yeast extract wort, nutrient agar, glycerol asparagine or ISP-2 (streptomyces 2) liquid culture medium; preferably a Gao's No. one liquid medium or an ISP-2 liquid medium.
Preferably, in the above preparation method, the step (3) further comprises a process of removing the microbial cells from the fermentation broth; preferably centrifugation or filtration; the centrifugal speed is 8000-10000 r/min.
A medicinal antibiotic, agricultural antibiotic and bactericide are prepared from the Streptomyces rochei Z331-A or its fermentation product.
The control objects of the antibiotic comprise plant pathogenic fungi, gram-negative bacteria and gram-positive bacteria of fusarium, colletotrichum, pythium and phytophthora. The plant pathogenic fungus is preferably Fusarium graminearum (F.graminearum)Fusarium graminearum) Fusarium of dragon fruit (A)Fusarium pitaya) Mango malformation bacteria (1)Fusarium mangiferae) Colletotrichum gloeosporioides (A), (B), (CColletotrichum gloeosporioides Penz), colletotrichum aculeatum (Cucurbitaceae) ((ii)Colletotrichum lagenarium) Pythium echinulatum (A) and (B)Pythium acanthicum Drechsler) and tobacco blight (Phytophtora parasiticavar. nicotianae Tucker); more preferably, the strain is Pityrosporum ovale, mango malformation bacteria, cucurbitaceae colletotrichum and tobacco blight. The gram-negative bacterium is preferably Vibrio parahaemolyticus ((V))Vibrio parahaemolyticus) Bacillus proteus: (A)Bacillus proteus) Escherichia coli (E.coli)Escherichia coil) (ii) a The gram-positive bacterium is preferably Bacillus subtilis (B.) (Bacillus subtilis) Streptococcus agalactiae (Streptococcus agalactiae) Staphylococcus aureus (S.) (Staphylococcus aureus) And methicillin-resistant Staphylococcus aureusStaphylococcus (MRSA), methicillin-resistantStaphylococcus aureus)。
A fertilizer is prepared from the Streptomyces rochei Z331-A or its fermentation product. The fertilizer is preferably bacterial manure and liquid fertilizer. The suitable target is preferably selected from Solanaceae and Cucurbitaceae crops, such as eggplant, Capsici fructus, fructus Lycopersici Esculenti, fructus Cucumidis Sativi and Cucurbitaceae.
The invention has the following advantages:
the streptococcus rochei Z331-A provided by the invention is easy to culture, and a fermentation product generated by fermentation can resist broad-spectrum bacteria and plant pathogenic fungi; especially has excellent biological activity to marine vibrio parahaemolyticus and methicillin-resistant staphylococcus aureus; and the fermentation product is resistant to ultraviolet radiation, acid and alkali, heat and stable, and has industrial development value.
Strain preservation information
Streptomyces rochei (S.) (Streptomyces rochei) Z331-A is preserved in China general microbiological culture collection management center in 2018, 2 months and 8 days, the preservation address is microbial research institute of Beijing China national academy of sciences, No. 3 of Xilu No.1 of Beijing, Chaoyang, the Chaoyang district, and the preservation number is CGMCC number 15348.
Drawings
FIG. 1 shows the shape (front and back) of Streptomyces rochei Z331-A on a high-grade medium;
FIG. 2 is a Z331-A phylogenetic tree constructed based on the 16S rRNA sequence;
FIG. 3 shows the inhibition zones of the fermentation broth Z331-A against different bacteria;
FIG. 4 shows the growth promoting effect of different concentrations of fermentation broth Z331-A on tomato seedlings (30 d);
FIG. 5 shows the inhibition zone size after different UV treatment times;
FIG. 6 is a graph of the effect of ultraviolet light on the activity of fermentation broth;
FIG. 7 shows the size of the zone of inhibition after different pH treatments;
FIG. 8 is a graph of the effect of pH on fermentation broth activity;
FIG. 9 shows the size of the zone of inhibition after different temperature treatments;
FIG. 10 is a graph showing the effect of heating on the activity of a fermentation broth.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 isolation and characterization of Z331-A
1.1 Strain isolation
Taking a soil sample collected from coastal sandy land of a sea area island in Haikou City in Hainan province in 2015 6 months, air-drying for 7 days, preparing diluents with different concentrations by using sterile water, adding 50 g/mL potassium dichromate into a Gao's I culture medium as a separation culture medium, and separating the soil sample by using a dilution plate method. Culturing at 28 deg.C for 4 weeks, selecting different suspected actinomycetes, and separating again on the plate to obtain pure culture Z331-A.
1.2 identification of the Strain
1.2.1 morphological characteristics
On the Gao's medium I, the Z331-A strain grows well, the colony is slightly raised, the texture is compact, dry, it is a little fine hair, opaque, the aerial hyphae are white or grey white, the substrate hyphae are light yellow, the growth is limited, does not produce pigment. Under an optical microscope, intrabasal hyphae have no transverse septa and are not broken, aerial hyphae are branched more, spore filaments are mostly long and straight, are partially bent and have chain spores, and some mature spore chains fall off to form spores which are cylindrical or short rod-shaped.
1.2.2 culture characteristics
The strains are respectively cultured on solid culture media such as Gao's I, Ke's, Chao's, starch, glucose asparagine, PDA, yeast extract wort, nutrient agar, glycerol asparagine and the like, and the growth condition and the pigment production condition of hyphae are observed, and the results are shown in a table 1: the strains grow well on the tested culture medium, and form abundant aerial hyphae and basal hyphae, wherein the aerial hyphae are white, light yellow to gray, and the basal hyphae are light yellow, orange, lemon yellow, orange yellow and golden yellow to gray. In addition to the production of orange pigment by the Cracker medium, no pigment production was observed in the other media above.
TABLE 1 culture characteristics of Strain Z331-A
1.2.3 physiological Properties
The carbon source of the strain Z331-A was selected, and the results are shown in Table 2: in addition to the unavailability of D-fructose, other sugars such as D-glucose, D-mannitol, D-galactose, sucrose, raffinose, inositol, and L-arabinose can be utilized. The strain can liquefy the gelatin, solidify and peptone milk, and hydrolyze starch; but can not reduce nitrate, has no capacity of degrading cellulose and can not generate hydrogen sulfide and melanin.
TABLE 2 physiological and biochemical characteristics of Strain Z331-A
Note: "+" is a positive result and "-" is a negative result.
1.2.416S rRNA analysis
And extracting the genome of Z331-A, wherein the used primers are universal primers (a forward primer: 5'-AGAGTTTGATCCTGGCTCAG-3' and a reverse primer: 5'-GGTTACCTTGT TACGACTT-3') of the 16S rRNA of the bacteria, amplifying the 16S rRNA gene, purifying and sequencing, and the sequence is shown as SEQ number 1. Similarity comparison was performed by the BLASTN program of NCBI, and sequences with higher similarity were selected, and phylogenetic trees were constructed using the MEGA 6.0 software adjacency method, as shown in FIG. 2. From the constructed phylogenetic tree, the strain Z331-A belongs to the genus Streptomyces (A)Streptomyces sp.) It and a model strainStreptomyces rocheiNBRC 12908 has the closest genetic distance, is positioned in the same branch on a phylogenetic tree, has the similarity of the 16S rRNA sequence of 99 percent and is identified as streptomyces rochei (S.) (Streptomyces rochei）。
Example 2 preparation of Z331-A fermentation product
(1) Inoculating the strain Z331-A frozen in a glycerol tube into a Gao's first liquid culture medium with the liquid loading amount of 40 mL/100 mL according to the inoculation amount of 1%, and performing shake culture at 28 ℃ and 180 r/min for 3d to obtain activated bacterial liquid;
(2) inoculating the activated bacterium liquid into a Gao's first liquid culture medium with the liquid loading amount of 40 mL/100 mL according to the inoculation amount of 3%, and performing shake-flask culture at 28 ℃ for 180 r/min for 3d to obtain a seed liquid;
(3) inoculating the seed solution into an ISP-2 culture medium with the liquid loading amount of 40 mL/100 mL according to the inoculation amount of 3%, and performing shake flask culture at 28 ℃ and 180 r/min for 7d to obtain a fermentation liquid;
(4) centrifuging the fermentation liquor at 9000 r/min, taking supernatant, and filtering with a 0.22 mu m filter membrane to obtain sterile fermentation liquor.
Example 3 biological Activity of Z331-A fermentation broths against phytopathogenic fungi
Taking the sterile fermentation liquid obtained in the example 2, adopting a medium-containing method to determine the resistance of the fermentation liquid to fusarium graminearum (Fusarium graminearum) ((Fusarium graminearum)、Rubber anthracnose disease (Colletotrichum gloeosporioides Penz）、Mango deformity bacteria (1)Fusarium mangiferae）、Colletotrichum gloeosporioides (cucurbitaceae) (colletotrichum gloeosporioides)Colletotrichum lagenarium) Fusarium of dragon fruit（Fusarium pitaya）、Pythium echinulatum (A), (B), (C)Pythium acanthicum Drechsler)、Tobacco blight disease (Phytophtora parasiticavar. nicotianae Tucker) 7 phytopathogens by the following method: melting PDA agar culture medium, cooling to about 55 deg.C, mixing the fermentation filtrate with PDA agar culture medium at a volume ratio of 1:9 to obtain fermentation liquid of 10%, and shaking to obtain flat plate containing medicine of Z331-A fermentation liquid. After cooling, 1 pathogenic bacteria cake (diameter 6 mm) to be tested is placed in the center of the plate and cultured in a constant temperature incubator at 28 ℃. PDA agar medium plates without fermentation filtrate were used as controls and repeated 3 times per treatment. Measuring the growth diameter of each fungus colony by adopting a cross method, and calculating the inhibition rate of the growth of hyphae by using a formula: hypha growth inhibition (%) = (control colony diameter-treated colony diameter)/control colony diameter × 100. The results are shown in Table 3: the fermentation liquor has inhibiting effect on 7 plant pathogenic bacteria, and mango malformation bacteria、The bacteriostatic effect of the anthracnose bacteria, the fusarium of the dragon fruit and the tobacco blight of the cucumber is better,the bacteriostasis rate reaches more than 50 percent, and particularly the bacteriostasis rate to the anthracnose pathogen of cucumber is nearly 70 percent.
TABLE 3 inhibition of 7 phytopathogenic fungi by Z331-A fermentation broths
Note: data are mean ± standard deviation.
Example 4 biological Activity of Z331-A fermentation broths on bacteria
Taking the sterile fermentation broth obtained in example 2, and measuring it by paper diffusion method for Vibrio parahaemolyticus: (Vibrio parahaemolyticus) Bacillus subtilis (A), (B) and (C)Bacillus subtilis) Bacillus proteus: (A)Bacillus proteus) Escherichia coli (E.coli)Escherichia coil) Staphylococcus aureus (S.) (Staphylococcus aureus) Streptococcus agalactiae (Streptococcusagalactiae) And 2 strains of methicillin-resistant Staphylococcus aureus (MRSA)Staphylococcus aureus) MRSA ATCC43300 and MRSA ATCC35951, by the following methods: the melted MH agar medium was poured into a petri dish having a diameter of 9cm to a thickness of 4mm, and cooled to room temperature to prepare a plate for use. Dripping 25 mu L of sample Z331-A to be tested on a circular filter paper sheet with the diameter of 6mm, and preparing a filter paper sheet with the medicine after water is volatilized. Diluting the bacterial suspension with physiological saline to 1 × 108cfu/mL, i.e., measured with a spectrophotometer, and measured at an absorbance value of 0.08 to 0.10 with a wavelength set to 625 nm. After dilution, some of the bacterial suspensions are respectively taken by cotton swabs within 15 minutes, redundant water is filtered on the walls, the bacterial suspensions are evenly coated on the whole sterile MH flat plate, prepared filter paper sheets with medicines are pasted at proper positions, meanwhile, positive controls (gentamicin (10 microgram/sheet) is pasted on gram-dyeing negative bacteria as a positive control, and sulfamethoxazole drug sensitive paper sheets (300 microgram/sheet) are pasted on gram-dyeing positive bacteria as a positive control), and the bacterial suspensions are cultured for 18-24 hours at 35 ℃. The parallel test was repeated 3 times, and the inhibition zone diameter was measured by the cross method, and the results are shown in fig. 3 and table 4: 8 strains of Z331-A fermentation brothThe bacteria have inhibiting effect.
TABLE 4 inhibitory Effect of Z331-A fermentation broth on 8 bacteria
Note: "+" is a positive result and "-" is a negative result. Data are mean ± standard deviation.
Example 5 Effect of Z331-A fermentation broth on tomato growth
The OD value of the supernatant of the sterile fermentation obtained in example 2 was measured and adjusted to 0.08 using sterile water to obtain stock solutions, which were then diluted 100-fold, 500-fold, and 1000-fold respectively for future use.
5.1 Effect of fermentation broths on seed Germination and immature embryo growth
Selecting full and uniform tomato seeds, cleaning with sterile water to remove dust, sterilizing with 70% ethanol for 15 s, sterilizing with 1% sodium hypochlorite for 3 min, and washing with sterile water for 6 times. Taking appropriate amount of seeds, respectively adding into a triangular flask containing sterile fermentation supernatant stock solution of strain Z331-A, 100 times dilution, 500 times dilution, 1000 times dilution and sterile distilled water (CK), and soaking seeds for 12-18 h at room temperature. Placing proper amount of sterile quartz sand into the lower layer of the culture dish under aseptic operation to make the height about 4mm, laying a layer of sterile filter paper on the upper layer, placing 50 seeds with different concentrations into each dish, dripping sterile water until the upper layer of filter paper is wetted, repeating each dish for 3 times, placing the culture dish in an illumination incubator for culture, and measuring the germination rate after 3 days. After 7d, 10 seedlings are respectively selected for different concentration treatment, the length of the hypocotyl and the radicle is measured, and software SAS is used for statistical analysis. The summary vitality index was calculated by referring to the method of Duan et al (2013): concise vigor index = germination (%) × radicle length (mm). The results are shown in Table 5: compared with the control treatment, the 100-time diluent has the most obvious effects on the germination of tomato seeds and the growth promotion of hypocotyls and radicles; the 500-fold and 1000-fold dilution liquid also has obvious influence on the germination of tomato seeds and the increase of hypocotyls and radicles, has obvious difference, and has the highest concise vitality index; the stock solution has negative effects on the germination of tomato seeds and the growth of hypocotyls and radicles, and plays a role in inhibiting.
TABLE 5 Effect of Strain Z331-A fermentation broth on tomato seed germination and immature embryo growth
Note: different capital letters after the same column of data indicate significant differences (P < 0.05)
5.2 growth promoting effect of fermentation broth for root irrigation on tomato potted plant
When the tomato seedlings grow to 4-5 true leaves, the tomato seedlings with the same growth vigor and size are planted in pots filled with 200g of sterilized soil, and 1 plant is fixedly planted in each pot. The fermentation supernatant stock solution, the 100-fold diluent, the 500-fold diluent and the 1000-fold diluent are respectively poured into corresponding nutrition bowls, and only distilled water is poured as a Control (CK). Each treatment was 10 replicates. The culture was carried out in a 25 ℃ climatic chamber. After 30 d, the plant height, root length, fresh weight and dry weight of the tomato seedlings are measured, and the results are shown in table 6 and fig. 4: the 100-time dilution liquid has the best growth promotion activity on tomato seedlings, and compared with distilled water (CK) only, the plant height, the root length, the fresh weight and the dry weight of the tomato seedlings are respectively increased by 115.29%, 113.33%, 115.52% and 110.26%. Compared with the control treatment, the 500-time diluent and the 1000-time diluent have obvious growth promotion effect and obvious difference. The growth promoting effect of each treatment is as follows: 100 fold dilution >500 fold dilution >1000 fold dilution. The fermented supernatant stock solution shows inhibition effect completely, the inhibition effect on each index is obvious, and the inhibition rate reaches 38.46-41.82%.
TABLE 6 growth promoting action of different concentrations of fermentation broth of strain Z331-A on tomato seedlings (30 d)
Note: different capital letters in the same column in the table indicate that the LSD test is significantly different at a P <0.05 level.
Example 6 stability of fermentation broth Z331-A
And (3) respectively taking 10mL of the fermentation liquor obtained in the step (3) in the example 2, filling the fermentation liquor into 3 groups of triangular bottles with the volume of 50mL, processing the fermentation liquor in the following various modes, respectively taking 1mL of the processed fermentation liquor, filling the fermentation liquor into a 1.5mL centrifuge tube, filtering the fermentation liquor by using a 0.22 mu L microporous filter membrane, and filling the fermentation liquor into a sterile 1.5mL new centrifuge tube to obtain a sterile processed substance. The activity of the corresponding sterile treatment was determined by the paper diffusion method described in example 4 to determine the effect of different treatment modalities on the active substance. The corresponding 25 μ L of treated fermentation broth was added to a sterile filter paper sheet, with MRSA ATCC43300 as the test bacteria and fludarxin (ceftazidime) as the positive control.
5.1 Effect of UV light on the biological Activity of fermentation broths
Placing the fermentation liquor at 40cm of ultraviolet ray with power of 20W, respectively irradiating for 30min, 1h, 2h, 3h, 4h, 5h and 6h, then respectively taking 1mL of treatment liquid for standby, and measuring the activity change by a paper sheet method together with the untreated fermentation liquor, wherein the results are shown in a graph 4 (wherein A is ultraviolet irradiation for 0.5h, B is ultraviolet irradiation for 1h, C is ultraviolet irradiation for 2h, D is ultraviolet irradiation for 3h, E is ultraviolet irradiation for 4h, F is ultraviolet irradiation for 5h, G is ultraviolet irradiation for 6h, and Z is redaxsin) and a graph 5 (wherein the same letter represents no significant difference in LSD test, different letters represent LSD test, and p is significant difference when < 0.05): compared with a control, the antibacterial activity of the fermentation liquor has no significant difference within 1h of ultraviolet treatment, after 6h of ultraviolet treatment, the antibacterial zone still has 16.60mm which is 97.65% of the control, and the result shows that Z331-A has good tolerance to ultraviolet irradiation.
5.2 Effect of pH on the biological Activity of the fermentation broths
10/50mL of Z331-A fermentation liquid in a triangular flask was adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 with 1mol/L NaOH and 1mol/L HCL, respectively, and after 1 hour of treatment, the pH was adjusted back to the original pH, and the change in activity was measured by a paper sheet method, as shown in FIG. 6 (wherein A is pH 2, B is pH 3, C is pH 4, D is pH 5, E is pH 6, F is pH 7, G is pH 8, H is pH 9, J is pH 10, and Z is redaxane) and FIG. 7 (wherein the same letter represents no significant difference in LSD assay, different letters represent LSD assay, and p <0.05 is significant difference): treating the fermentation liquor for 1h under the condition that the pH value is 5-8, wherein the activity has no significant difference, and the diameter of the inhibition zone is the largest (17.00 mm); but the diameter of the inhibition zone is reduced along with the increase of acidity and alkalinity of the pH value of the treatment. When the fermentation liquor is treated under the conditions of strong acidity at pH 2 and strong alkalinity at pH 10, the diameters of inhibition zones are respectively 16.80mm and 16.10mm, which are respectively 98.82% and 94.70% of the control. The fermentation liquor is shown to be very stable under neutral, weak acid and weak alkaline conditions, and the acid resistance is stronger than the alkali resistance, so that the fermentation liquor is more stable in an acid-base environment.
5.3 Effect of heating on the biological Activity of the fermentation broths
The fermentation solutions of Z331-A were treated at 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C, 100 deg.C, 120 deg.C for 30min, cooled to room temperature, and measured for activity change by a paper sheet method, as shown in FIG. 8 (where A is 50 deg.C treatment, B is 60 deg.C treatment, C is 70 deg.C treatment, D is 80 deg.C treatment, E is 90 deg.C treatment, F is 100 deg.C treatment, G is 120 deg.C treatment, and Z is Fudaxin) and FIG. 9 (where the same letter represents no significant difference in LSD test, different letters represent LSD test, and p <0.05 represents significant difference): compared with the control, the treatment temperature is 50-70 ℃, the activity of the fermentation liquor has no significant difference, and the diameter of the inhibition zone is 17.00 mm. However, the activity of the fermentation broth tends to decrease with increasing treatment temperature. When the treatment temperature is 80 deg.C, 90 deg.C, and 100 deg.C, the diameter of the inhibition zone is 16.50mm, 13.92mm, and 8.26mm, and the activity is 97.60%, 81.88%, and 48.59% of the control respectively. The activity is zero only when the temperature reaches 120 ℃. This indicates that the strain fermentation broth active substance has good temperature tolerance.
Sequence listing
<110> university of Hainan
<120> Streptomyces rochei strain and application thereof
<130> 20180411
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1431
<212> DNA
<213> Streptomyces rochei
<400> 1
ttgaagtgcg cgtgcttacc atgcaagtcg aacgatgaac cacttcggtg gggattagtg 60
gcgaacgggt gagtaacacg tgggcaatct gccctgcact ctgggacaag ccctggaaac 120
ggggtctaat accggatact gatcctcgca ggcatctgcg aggttcgaaa gctccggcgg 180
tgcaggatga gcccgcggcc tatcagctag ttggtgaggt aacggctcac caaggcgacg 240
acgggtagcc ggcctgagag ggcgaccggc cacactggga ctgagacacg gcccagactc 300
ctacgggagg cagcagtggg gaatattgca caatgggcga aagcctgatg cagcgacgcc 360
gcgtgaggga tgacggcctt cgggttgtaa acctctttca gcagggaaga agcgaaagtg 420
acggtacctg cagaagaagc gccggctaac tacgtgccag cagccgcggt aatacgtagg 480
gcgcaagcgt tgtccggaat tattgggcgt aaagagctcg taggcggctt gtcacgtcgg 540
ttgtgaaagc ccggggctta accccgggtc tgcagtcgat acgggcaggc tagagttcgg 600
taggggagat cggaattcct ggtgtagcgg tgaaatgcgc agatatcagg aggaacaccg 660
gtggcgaagg cggatctctg ggccgatact gacgctgagg agcgaaagcg tggggagcga 720
acaggattag ataccctggt agtccacgcc gtaaacggtg ggcactaggt gtgggcaaca 780
ttccacgttg tccgtgccgc agctaacgca ttaagtgccc cgcctgggga gtacggccgc 840
aaggctaaaa ctcaaaggaa ttgacggggg cccgcacaag cggcggagca tgtggcttaa 900
ttcgacgcaa cgcgaagaac cttaccaagg cttgacatac accggaaaac cctggagaca 960
gggtccccct tgtggtcggt gtacaggtgg tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttgtccc gtgttgccag caggcccttg 1080
tggtgctggg gactcacggg agaccgccgg ggtcaactcg gaggaaggtg gggacgacgt 1140
caagtcatca tgccccttat gtcttgggct gcacacgtgc tacaatggcc ggtacaatga 1200
gctgcgatac cgcgaggtgg agcgaatctc aaaaagccgg tctcagttcg gattggggtc 1260
tgcaactcga ccccatgaag tcggagtcgc tagtaatcgc agatcagcat tgctgcggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacgtca cgaaagtcgg taacacccga 1380
agccggtggc ccaacccctt gtgggaggga gcttcgaagg tgactcagtt t 1431
Claims (11)
1. Streptomyces rochei (S. (A)Streptomyces rochei) Z331-A, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 15348.
2. The use of the streptomyces rochei Z331-a and the fermentation broth thereof as defined in claim 1 in the preparation of medical antibiotics, agricultural bactericides or fertilizers.
3. A medical antibiotic, agricultural antibiotic or agricultural fungicide prepared from the streptomyces rochei Z331-a or its fermentation broth as defined in claim 1.
4. The medical antibiotic, agricultural antibiotic or agricultural fungicide according to claim 3, wherein the control subjects include phytopathogenic fungi, gram-negative bacteria and gram-positive bacteria;
the plant pathogenic fungi is fusarium graminearum (F.graminearum)Fusarium graminearum) Fusarium of dragon fruit (A)Fusarium pitaya) Mango malformation bacteria (1)Fusarium mangiferae) Rubber colletotrichum (B), (B) and (C)Colletotrichum gloeosporioidesPenz), colletotrichum aculeatum (Cucurbitaceae) ((ii)Colletotrichum lagenarium) Pythium echinulatum (A) and (B)Pythium acanthicumDrechsler) and Phytophthora nicotianae (Phytophtora parasitica var . nicotianae Tucker)；
The gram-negative bacterium is vibrio parahaemolyticus (Vibrio parahaemolyticus) Bacillus proteus (A), (B), (C)Bacillus proteus) And Escherichia coli (Escherichia coil)；
Gram-positive bacteriaIs Bacillus subtilis (B.subtilis)Bacillus subtilis) Streptococcus agalactiae (1)Streptococcus agalactiae) Staphylococcus aureus (1)Staphylococcus aureus) And methicillin-resistant Staphylococcus aureus (MRSA)Staphylococcus aureus)。
5. A fertilizer prepared from the streptomyces rochei Z331-a of claim 1 or a fermentation broth thereof.
6. The fertilizer of claim 5, wherein the fertilizer is a bacterial fertilizer and a liquid fertilizer; the applicable objects are crops of solanaceae and cucurbitaceae.
7. A process for preparing streptomyces rochei Z331-a fermentation broth as claimed in claim 1, comprising the steps of:
(1) inoculating the preserved strain into a basic culture medium, and performing shake-flask fermentation to obtain an activated bacterium solution;
(2) inoculating the activated bacterial liquid into a seed culture medium, and performing shake-flask fermentation to obtain a seed liquid;
(3) inoculating the seed liquid into a fermentation culture medium, stirring and fermenting to obtain fermentation liquid containing fermentation products.
8. The method according to claim 7, wherein the amount of the inoculum in the step (1) is 1%; the inoculation amount in the step (2) is 3 percent; the inoculation amount in the step (3) is 3-5%; in the steps (1) - (3), the fermentation temperature is 25-30 ℃; the rotating speed is 180-; in the steps (1) and (2), the culture time is 2-3 days; in the step (3), the culture time is 7-10 days.
9. The method according to claim 7, wherein the basal medium and the seed medium are Gauss No. one liquid medium, and the fermentation medium is ISP-2 liquid medium.
10. The method according to claim 7, wherein the step (3) is followed by a process of removing the microbial cells from the fermentation broth.
11. The method according to claim 10, wherein the step of removing the microbial cells is centrifugation or filtration; the centrifugal speed is 8000-10000 r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810357031.0A CN108300681B (en) | 2018-04-20 | 2018-04-20 | Streptomyces rochei and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810357031.0A CN108300681B (en) | 2018-04-20 | 2018-04-20 | Streptomyces rochei and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108300681A CN108300681A (en) | 2018-07-20 |
| CN108300681B true CN108300681B (en) | 2021-07-23 |
Family
ID=62848552
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810357031.0A Active CN108300681B (en) | 2018-04-20 | 2018-04-20 | Streptomyces rochei and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108300681B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2799848B2 (en) * | 2019-06-18 | 2021-05-18 | Univ Valladolid | Inclusion complex with Streptomyces antimicrobial metabolites and biofertilizer comprising it |
| CN110218685B (en) * | 2019-06-28 | 2020-05-08 | 江苏省农业科学院 | Biocontrol actinomycetes and application thereof |
| CN113234635B (en) * | 2021-05-31 | 2022-09-16 | 元和生物科技(德州)有限公司 | Compound microbial agent for preventing and treating damping off of hot peppers |
| CN113528388A (en) * | 2021-07-19 | 2021-10-22 | 海南大学 | Streptomyces corallini symbiosis, method for producing actinomycin D by fermentation and application |
| CN114456973B (en) * | 2022-01-11 | 2022-08-05 | 湖北省生物农药工程研究中心 | Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases |
| CN114657068A (en) * | 2022-04-11 | 2022-06-24 | 广西地源之本肥业有限公司 | Preparation method of Kangtu weed-inhibiting bacteria |
| CN114854631B (en) * | 2022-05-12 | 2023-06-23 | 中国热带农业科学院热带生物技术研究所 | Sponge-derived biocontrol streptomyces ITBB-ZK-a5 and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6133196A (en) * | 1996-08-13 | 2000-10-17 | Regents Of The University Of Minnesota | Biological control of plant disease on roots of conifer seedlings |
| ES2158769A1 (en) * | 1999-02-03 | 2001-09-01 | Smithkline Beecham Sa | Set of altemycidin derivatives combating bacterial infections consists of products of fermentation of streptomyces rochei as enzyme inhibitors |
| CN104195085A (en) * | 2014-09-03 | 2014-12-10 | 北京林业大学 | Streptomycete with broad-spectrum antibacterial activity and application thereof |
| CN105969686A (en) * | 2016-05-18 | 2016-09-28 | 贵州省烟草科学研究院 | Bacterial-wilt-resistant Streptomyces rochei strain and bio-organic fertilizer thereof |
| CN106967638A (en) * | 2017-03-27 | 2017-07-21 | 陕西博秦生物工程有限公司 | A kind of application of Lou Che Shi streptomycetes in parasitics weeds are prevented and kill off |
| CN107652064A (en) * | 2017-11-01 | 2018-02-02 | 海南大学 | A kind of biological pesticide for promoting plant growth, improving immunity and preparation method thereof |
-
2018
- 2018-04-20 CN CN201810357031.0A patent/CN108300681B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6133196A (en) * | 1996-08-13 | 2000-10-17 | Regents Of The University Of Minnesota | Biological control of plant disease on roots of conifer seedlings |
| ES2158769A1 (en) * | 1999-02-03 | 2001-09-01 | Smithkline Beecham Sa | Set of altemycidin derivatives combating bacterial infections consists of products of fermentation of streptomyces rochei as enzyme inhibitors |
| CN104195085A (en) * | 2014-09-03 | 2014-12-10 | 北京林业大学 | Streptomycete with broad-spectrum antibacterial activity and application thereof |
| CN105969686A (en) * | 2016-05-18 | 2016-09-28 | 贵州省烟草科学研究院 | Bacterial-wilt-resistant Streptomyces rochei strain and bio-organic fertilizer thereof |
| CN106967638A (en) * | 2017-03-27 | 2017-07-21 | 陕西博秦生物工程有限公司 | A kind of application of Lou Che Shi streptomycetes in parasitics weeds are prevented and kill off |
| CN107652064A (en) * | 2017-11-01 | 2018-02-02 | 海南大学 | A kind of biological pesticide for promoting plant growth, improving immunity and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| "Effect of different cultural, physiological and nutritional parameters on Streptomyces rochei (MTCC 10109) antimicrobial metabolites production.";Reddy, N. G.等;《Journal of Pharmacy Research》;20111231;第4卷(第8期);第2675-2678页 * |
| "Optimization of culture conditions of Streptomyces rochei (MTCC 10109) for the production of antimicrobial metabolites";N,Gopi Reddy等;《Egyptian Journal of Biology》;20111231;第13卷;第21-29页 * |
| "响应面试验优化链霉菌Z331-A的发酵条件";范丽霞等;《中国酿造》;20180514;第37卷(第4期);第77-82页 * |
| "放线菌Z331-A的鉴定、培养条件优化及发酵液活性研究";范丽霞;《中国优秀硕士学位论文全文数据库（电子期刊）工程科技Ⅰ辑》;20200815(第8（2020）期);B018-49 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108300681A (en) | 2018-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108300681B (en) | Streptomyces rochei and application thereof | |
| CN110257290B (en) | Plant pathogenic bacteria inhibitor, and strain and application thereof | |
| CN107974427B (en) | Marine streptomyces with bacteriostatic activity | |
| CN111647522B (en) | Entomopathogenic nematode symbiotic bacterial strain for inhibiting various plant fungal diseases and application thereof | |
| CN107075459B (en) | Novel bacterium belonging to the genus Bacillus and use thereof | |
| CN112646735B (en) | Metarhizium anisopliae, microbial insecticide, preparation method and application | |
| CN113061554A (en) | Bacillus belgii for preventing and treating banana wilt and application thereof | |
| CN108641989A (en) | One plant of Methylotrophic bacillus and its application | |
| CN114437964B (en) | Bacillus belicus strain and application thereof | |
| CN115261233B (en) | Biocontrol fungus for stem rot of saffron crocus and application thereof | |
| CN116751715A (en) | Paenibacillus polymyxa and application thereof in field of plant pathogenic bacteria control | |
| CN113862156B (en) | Fusarium oxysporum (Fusarium oxysporum) K2018-1418 and application thereof | |
| CN107699526A (en) | One plant of actinomycetes strain for preventing and treating gray mold and its application | |
| CN108913625B (en) | Salt-tolerant streptomycete, microbial inoculum thereof and application of microbial inoculum thereof in promoting plant growth | |
| CN107629985B (en) | Plant endophytic bacterium with antagonistic effect on plant pathogenic fungi | |
| CN113755389A (en) | Bacillus belgii and application thereof | |
| CN107517788A (en) | A kind of method for preventing and treating rice blast | |
| CN116083266B (en) | Streptomyces flagelliformis, biocontrol microbial inoculum and application | |
| CN108587969B (en) | Preparation and application of verticillium dahliae strain HCX-01 capable of preventing and treating cotton verticillium wilt | |
| CN114032182B (en) | Fungus with functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth | |
| CN116240126A (en) | Multifunctional bacillus belgium SB10 and application thereof | |
| CN100560716C (en) | Grass green streptomycete | |
| CN113817642A (en) | Bacillus belgii YJ02, and microbial preparation and application thereof | |
| CN105670954A (en) | Endophytic bacillus subtilis and application thereof | |
| CN113462612B (en) | Streptomyces hygroscopicus DL5C and application thereof in preventing and treating powdery mildew |
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 |