CN118406620A - Bacillus amyloliquefaciens strain and application thereof - Google Patents
Bacillus amyloliquefaciens strain and application thereof Download PDFInfo
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- CN118406620A CN118406620A CN202410870758.4A CN202410870758A CN118406620A CN 118406620 A CN118406620 A CN 118406620A CN 202410870758 A CN202410870758 A CN 202410870758A CN 118406620 A CN118406620 A CN 118406620A
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and discloses bacillus amyloliquefaciens and application thereof. Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain ZB with the preservation number of CGMCC No:29399. the bacillus amyloliquefaciens can grow and be used at the water temperature of 8-30 ℃, can purify water quality, inhibit the breeding of aquatic mildew and fusarium, and can be widely applied to aquaculture.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus amyloliquefaciens strain and application thereof.
Background
The saprolegniasis is also called Fuscoporia or white hair disease, and is caused by parasitic fungi on the body surface of aquatic animals, wherein the fungi are mainly phylum Eumycota, phylum flagelliform mycotina, class of algae fungus, order of saprolegniales, genus saprolegniasis of saprolegniaceae, genus saprolegniasis, and the like, and the propagation temperature of the saprolegniasis is between 13 and 18 ℃. The water mould belongs to the field of fresh water, is popular in both domestic and foreign cultivation areas, has wide temperature application range, can grow and reproduce at 5-26 ℃ and can sometimes grow and reproduce even at the water temperature of 30 ℃.
When the hydromycete is initially parasitic, no abnormal symptoms of the aquatic animal can be generally seen; when the condition is seen, the mycelium has invaded the animal wound, growing out the outer mycelium, like an off-white cotton wool. Because the water mold can secrete a large amount of protein decomposing enzyme, the organism can secrete a large amount of mucus after being stimulated, the sick fish starts to be dysphoric and rubs with other solid matters, and then the fish body is overloaded, moves slowly, has poor appetite and finally is lean and dead. In the hatching process of fish eggs, the disease also frequently occurs, and internal hyphae invade human egg membranes, and a large amount of external hyphae are clustered outside the egg membranes, so the disease is called as 'egg silk disease'. In addition, crabs, shrimps, tortoise and soft-shelled turtles and the like also suffer from saprolegniasis.
The saprolegnia is a conditional pathogen, especially in spring and autumn, the water body environment is deteriorated, the water body flora is disordered or the culture density is too high, when physical damage exists on the surface of the fish body, the saprolegnia has the characteristics of no host selectivity, wide epidemic range, difficult healing and the like, the explosion of the saprolegnia often easily leads to the death of the fish in a large area, and the aim of preventing can be achieved only by inhibiting the saprolegnia from the source. Therefore, the saprolegniasis must be actively treated by a method of combining prevention and control.
There are many chemical means for preventing and curing water mildew, such as malachite green, potassium permanganate, formaldehyde, chlorine dioxide and hydrogen peroxide, but these chemical reagents all have chemical residues and environmental pollution problems, especially malachite green has high residues in aquatic animals because of its difficult degradation, and can have risks of teratogenesis, carcinogenesis and the like for mammals and human beings, and is now forbidden in fishery production by various countries.
CN101985605A discloses a water mould antagonistic bacterium and application thereof, which is characterized in that a bacillus amyloliquefaciens strain Sh1 is separated from soil near a culture water body, and a colony is circular, milky white, smooth in surface and serrated in edge; strain Sh1 was inoculated into a plate containing aqueous mold, and the strain was found to have distortion of the hydromold at the edge of the zone of inhibition, multiple breaks or mid-constriction of the hyphae of hydromold, and the hyphae growth was significantly inhibited, which was administered by intraperitoneal injection. The literature (separation and identification of grass carp pathogenic bacteria AvX and screening research of antagonistic bacteria thereof, aquatic science, 2018) reports that a bacillus amyloliquefaciens strain which can inhibit the growth of aeromonas veronii is obtained by separating and purifying pond water and sediment. CN101792723B discloses a bacillus amyloliquefaciens and its application in aquaculture, which is separated from the sediment of river channel to obtain bacillus amyloliquefaciens, and the thallus cells are added into water to rapidly degrade the nitrate nitrogen, nitrite nitrogen and COD content in the aquaculture water.
Therefore, a green, environment-friendly and efficient technology for inhibiting the water mildew becomes particularly important, and the microbial control is to utilize the interaction between microorganisms to inhibit the growth of pathogenic bacteria, so as to protect a host from being infected by the pathogenic bacteria.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, an object of the present invention is to provide a strain of bacillus amyloliquefaciens and use thereof, which are intended to solve the problems of the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
One of the purposes of the present invention is to protect a strain of Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens) strain ZB.
The second object of the present invention is to protect a fermentation culture of Bacillus amyloliquefaciens, obtained by inoculating the strain as described above in a culture medium for cultivation.
The third object of the present invention is to protect a microbial agent comprising a strain as described above or a fermentation culture as described above.
The fourth object of the present invention is to protect the use of a strain as described above or a fermentation culture as described above or a microbial agent as described above in at least one of the following: a1 Nitrogen-containing wastewater denitrification treatment; a2 Preparing a product for preventing or treating mycosis of aquatic animals.
Compared with the prior art, the invention has the following beneficial effects:
The bacillus amyloliquefaciens strain ZB can efficiently inhibit the growth of saprolegnia and fusarium; the method can grow well at the water temperature of 10 ℃, and provides a novel microbial method for preventing and controlling fish saprolegniasis. In addition, the bacillus amyloliquefaciens strain ZB can effectively degrade the ammonia nitrogen and nitrite content in the nitrogen-containing wastewater, and has good application value and application prospect in wastewater treatment.
Drawings
FIG. 1 is a diagram showing the morphology of Bacillus amyloliquefaciens ZB in LB plate culture in example 1 of the present invention.
FIG. 2 is a graph showing the growth results of Bacillus amyloliquefaciens ZB at 8℃at 10℃at 12℃at 15℃at 26℃in example 2 of the present invention.
FIG. 3 is a graph showing the results of inhibition of water mold by Bacillus amyloliquefaciens ZB in example 2 of the present invention.
FIG. 4 is a graph showing the result of inhibiting Fusarium by Bacillus amyloliquefaciens ZB in example 2 of the present invention.
FIG. 5 is a graph showing the results of the treatment of saprolegniasis with the lyophilized powder containing Bacillus amyloliquefaciens ZB of example 4 of the present invention.
FIG. 6 is a graph showing the second result of the treatment of saprolegniasis with the lyophilized powder containing Bacillus amyloliquefaciens ZB in example 4 of the present invention.
FIG. 7 is a graph showing the results of the treatment of saprolegniasis with the lyophilized powder containing Bacillus amyloliquefaciens ZB in example 4 of the present invention.
Detailed Description
One of the purposes of the present invention is to protect a strain of Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens) strain ZB. The strain ZB protected by the invention is obtained by separating and culturing the surface of a fish body suffering from saprolegniasis, and is identified as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) through morphological observation and 16S rRNA gene sequencing, and is named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain ZB, and the preservation number is CGMCC No:29399, deposited in China general microbiological culture Collection center, with the address: the korean district North Star, beijing city, part No. 1, no. 3.
The morphological characteristics of the bacillus amyloliquefaciens strain ZB on the LB plate are large colony, in a wrinkled state, milky white and opaque.
The bacillus amyloliquefaciens strain ZB has strong inhibition effect on the genus Saprolegnia (Saprolegnia) and the genus Fusarium (Fusarium), and the inhibition zone of the bacillus amyloliquefaciens strain ZB on the genus Saprolegnia is 18-26 mm; in addition, the zone of inhibition for Fusarium (Fusarium) was 18mm. The genus Hypocrea (Saprolegnia) is selected from the group consisting of Hypocrea salmonida (Saprolegnia salmonis), hygromyces parasiticus (Saprolegnia parasitica); the Fusarium (Fusarium) is selected from Fusarium cornea (Fusarium keratoplasticum).
Wherein, the bacillus amyloliquefaciens strain ZB has a 16S rDNA sequence shown in SEQ ID NO. 1.
Wherein the bacillus amyloliquefaciens strain ZB tolerates temperatures as low as 8 ℃. Bacillus amyloliquefaciens is generally considered to have a temperature tolerance range of 20-37 ℃, and the strain ZB of the application can well grow at 8 ℃, 10 ℃, 12 ℃, 15 ℃ and 26 ℃, and is particularly suitable for seasons of the outbreak of saprolegniasis in spring and autumn.
The second object of the present invention is to protect a fermentation culture of Bacillus amyloliquefaciens, obtained by inoculating the strain as described above in a culture medium for cultivation.
Wherein the temperature of the culture may be 8 to 37℃or 8 to 16℃or 14 to 22℃or 20 to 34℃or 26 to 37℃or 10℃12℃15℃26 ℃.
Wherein the culture medium comprises tryptone, yeast powder and sodium chloride.
Further, the culture medium comprises the following raw materials in parts by weight based on the total volume of the culture medium: 5-15 g/L of tryptone, 1-10 g/L of yeast powder and 5-15 g/L of sodium chloride. In a specific embodiment, the yeast extract is prepared from 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 1L of water.
The third object of the present invention is to protect a microbial agent comprising a strain as described above or a fermentation culture as described above.
Typically, the effective viable count of the microbial agent is at least 1.0X10 8 CFU/g. CFU/g is interpreted in the art in the usual sense and indicates the number of colonies of microorganisms contained in 1g of the test sample, and CFU indicates colony forming units.
Wherein, the dosage form of the microbial agent comprises freeze-dried powder, capsules, tablets or granules. In one specific example, it is a lyophilized powder.
Wherein the microbial agent further comprises auxiliary materials.
Further, the auxiliary material comprises skimmed milk powder or hydrolyzed starch. In one specific example, it is skimmed milk powder.
The application also provides a preparation method of the microbial agent, which comprises the following steps:
1) Inoculating bacillus amyloliquefaciens strain ZB into a culture medium for culture to obtain fermentation liquor;
2) Mixing the fermentation liquor with auxiliary materials, and then freeze-drying to obtain the microbial agent.
Further, the bacillus amyloliquefaciens strain ZB is subjected to an activation treatment, wherein the activation treatment is to inoculate a strain of a strain collection center to an LB plate and place the LB plate in a constant temperature incubator at 12 ℃ for activation.
Further, the mass-volume ratio of the fermentation liquid to the auxiliary materials can be (50-100) g:1L may be (50 to 74) g:1L, may be (60 to 85) g:1L may be (82-100) g:1L, 50 g/1L.
Further, the culture medium comprises the following raw materials in parts by weight: 5-15 g/L of tryptone, 1-10 g/L of yeast powder and 5-15 g/L of sodium chloride. In one specific example, the yeast extract is 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 1L of water.
Further, the temperature of the culture is 8-37 ℃. In one specific example, 30 ℃.
Further, the time of the culture is 1 to 10 days. In one specific example, 3d.
The fourth object of the present invention is to protect the use of a strain as described above or a fermentation culture as described above or a microbial agent as described above in at least one of the following: a1 Nitrogen-containing wastewater denitrification treatment to reduce the content of water mold in water; a2 Preparing a product for preventing or treating mycosis of aquatic animals.
Wherein the pathogenic bacteria of the mycosis are selected from one or more of the genera Saprolegnia (Saprolegnia), mirabilis (Achlya) and Fusarium (Fusarium).
Wherein the genus Hypocrea (Saprolegnia) is selected from Hypocrea salmonida (Saprolegnia salmonis) or Hygromyces parasiticus (Saprolegnia parasitica).
Wherein the Fusarium (Fusarium) is selected from Fusarium cornea (Fusarium keratoplasticum).
Wherein the aquatic animal is a fish, and the fish is a freshwater fish.
Wherein the nitrogen-containing means that the nitrogen-containing agent contains more than one of ammonia nitrogen, nitrate or nitrite.
Ammonia nitrogen and nitrite nitrogen are the most critical factors affecting water quality. The lethal or near-lethal effects of high concentrations of ammonia nitrogen, nitrate on marine and freshwater organisms are also reported in many documents. Therefore, maintaining low concentrations of ammonia nitrogen, nitrite and nitrate is an important goal to achieve sustainable development in aquaculture.
When the bacillus amyloliquefaciens strain ZB is used for degrading ammonia nitrogen according to the inoculation amount of 10v/v%, ammonia nitrogen content can be degraded from 50mg/L to 1.2mg/L only in 3 days, and the degradation rate is reduced by 97.6%.
When the bacillus amyloliquefaciens strain ZB is used for degrading nitrite according to the inoculation amount of 10v/v%, the nitrite content can be degraded from 22mg/L to 0.05mg/L only in 3 days, and the nitrite content is reduced by 99.8%.
Therefore, the bacillus amyloliquefaciens strain ZB has the nitrification capacity for efficiently removing ammonia nitrogen and the denitrification capacity for efficiently removing nitrite. By utilizing the characteristics of the strain ZB, the difficult problems of removing ammonia nitrogen, nitrite and nitrate nitrogen in wastewater can be well solved.
The bacillus amyloliquefaciens strain ZB is applied to preventing or treating fish saprolegniasis, can effectively reduce the saprolegniasis incidence of freshwater fish caused by saprolegniasis or mucormycosis, and has obvious effect of preventing and treating fish saprolegniasis.
The pathogenic bacteria Fusarium are widely present in soil, fresh water and sea water and can endanger plants, lower animals and up to mammals. When aquatic animals, particularly fish, become ill, the epidermis of the head, back, dorsal fin and tail of the ill fish begins to become inflamed and then ulcerated, growing a large number of fine filaments, resembling a hydromold. The bacillus amyloliquefaciens strain ZB can also be applied to preventing or treating fish fusarium.
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
In the embodiment of the application, the culture medium comprises the following formula:
LB liquid medium: 10g/L of tryptone, 5g/L of yeast powder, 10g/L of sodium chloride and 1L of purified water; 2% of agar powder is added in the solid.
PDA medium: 200g/L of potato, 20g/L of glucose, 1L of purified water and 2% of agar powder when solid.
In the embodiment of the application, the water mold is obtained through conventional separation culture, and specifically comprises the following steps: scraping a small amount of white floccules from the surface of fish body suffering from saprolegniasis, placing the fish body in 100mL of sterile physiological saline, then taking 100 mu L of the fish body, coating the fish body on a PDA flat plate, placing the fish body in a constant temperature incubator at 26 ℃ for culture, separating and purifying, and carrying out species identification on the fish body by Shanghai Saprolegnia Hemsl Biotechnology limited company to obtain parasitic Saprolegnia (Saprolegnia) D-SM-ITS, namely obtaining fungus genome DNA, and comparing the fungus genome DNA with ITS to find that the similarity between the fungus genome DNA and salmon Saprolegnia (Saprolegnia salmonis) is 100%.
In the embodiment of the application, fusarium is obtained through conventional separation culture, and specifically comprises the following steps: scraping a small amount of sticky matters from the surface of a fish body suffering from Fusarium, placing the fish body in 100mL of sterile normal saline, then taking 100 mu L of the fish body, coating the fish body on a PDA flat plate, placing the fish body in a constant temperature incubator at 26 ℃ for culture, separating and purifying, and performing species identification on the fish body to obtain Fusarium (Fusarium) SM-ITS by Shanghai Seisaria, namely obtaining fungus genome DNA, and comparing the fungus genome DNA with ITS to find that the similarity between the fungus genome DNA and Fusarium cornea (Fusarium keratoplasticum) is 99.81%.
EXAMPLE 1 screening and isolation of Bacillus amyloliquefaciens
In this example, bacillus amyloliquefaciens is obtained by screening and separation. The method comprises the following steps:
1.1, sample collection
1) Scraping a small amount of mucus from the surface of a fish body, putting the fish body into 100mL of sterile normal saline for gradient dilution by 100 times, then taking 100 mu L of the fish body, coating the fish body onto an LB and PDA flat plate, placing the fish body on a constant temperature incubator at 15 ℃ for 3d, separating and purifying to obtain a plurality of bacterial colonies, respectively inoculating each purified bacterial strain into 100mL of LB culture medium, and placing the bacterial colonies on a normal temperature shaking table for shake culture for 3d to obtain single bacterial colony fermentation bacterial liquid.
2) Meanwhile, inoculating the single colony of the saprolegnia D-SM-ITS into a PDA liquid culture medium, placing the single colony on a normal-temperature shaking table for shake culture for 3D, coating 100 mu L of saprolegnia liquid fermentation liquor onto the whole PDA flat plate, and then taking a sterile filter paper sheet with the diameter of 6mm to be stuck on the surface of the PDA flat plate for compaction.
3) And (2) respectively taking 20 mu L of the single colony fermentation broth in the step (1) and dripping the single colony fermentation broth on the sterile filter paper sheet in the step (2), then placing the sterile filter paper sheet in a constant temperature incubator for culture, repeating three groups of single colonies, and finally screening to obtain a colony capable of inhibiting the growth of saprolegnia.
The morphological features of this colony on LB plates were: the colony is large, in a wrinkled state, milky white and opaque, as shown in FIG. 1.
1.2, 16S rDNA Gene sequencing to determine species
The purified strain was submitted to 16S rDNA sequencing by Haisenno Gene technologies Inc., and the obtained sequences were aligned in the national center for biological information (NCBI) （https://blast.ncbi.nlm.nih.gov/Blast.cgiPROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome） database, which found that the 16S rDNA sequence was 99.72% homologous to the model strain Bacillus amyloliquefaciens Bacillus amyloliquefaciens in GenBank.
The sequence of 16S rDNA is as follows:
TGCTCCTGGAGCGGGCAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAACCTAGCGAGTAGT（SEQ ID NO.1）
Comprehensive, strain morphology and 16S rDNA, the strain is Bacillus amyloliquefaciens. The strain is classified as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain ZB, and the preservation number is CGMCC No:29399, deposited in China general microbiological culture Collection center, with the address: the korean district North Star, beijing city, part No. 1, no. 3.
EXAMPLE 2 Low temperature resistance and antibacterial Property study of Strain ZB
The strain ZB is inspected for low temperature resistance and antibacterial performance. Comprises the following steps:
2.1 Low temperature resistance study
The isolated Bacillus amyloliquefaciens strain ZB was inoculated into LB plates and cultured in a constant temperature incubator at 8℃and 10℃and 12℃and 15℃and 26℃respectively for 3 days. After 3 days of cultivation, the cultivation was observed, and the results are shown in FIG. 2.
As is clear from FIG. 2, the strain ZB of the present application can grow even under the low-temperature 8℃culture condition, and the growth is still good under the 12℃culture condition.
2.2 Antibacterial investigation of Water mould
(1) Inoculating the separated bacillus amyloliquefaciens strain ZB to an LB plate, placing the LB plate in a constant temperature incubator at 12 ℃ for activation, then inoculating the activated bacillus amyloliquefaciens strain ZB to 100mL of LB liquid medium, and fermenting and culturing for 3 days at 12 ℃, wherein the content of the strain in a fermentation broth after fermenting and culturing is 5 multiplied by 10 8 cfu/mL for later use.
(2) The aquatic mould D-SM-ITS is inoculated on a PAD plate for activation, bacterial colonies on a PDA plate are white, have longer hyphae, and are placed in an incubator at 4 ℃ after the bacterial colonies grow over the whole plate and serve as inoculation sources for subsequent aquatic mould experiments. Simultaneously, 200mL of PDA water mould liquid is cultivated for standby.
(3) The liquid of the water mould in the step (2) is coated on the whole new PDA plate, three sterile drug-sensitive tablets soaked by the fermentation liquid of the bacillus amyloliquefaciens strain ZB are placed at the center of the new PDA plate, and the new PDA plate is cultured for 2 to 3 days, and three groups are repeated.
The experimental results are shown in fig. 3, and a larger transparent ring appears, so that the ZB strain has a stronger inhibition effect on the hydromycete, and the diameter of the inhibition ring is shown in table 1.
TABLE 1
As can be seen from Table 1, the bacterial strain ZB has an obvious bacteriostatic effect on the water mold.
2.3 Antibacterial investigation of Fusarium
The difference from step 2.1 is that: the saprolegnia D-SM-ITS is replaced by fusarium SM-ITS, and the rest experimental operations are the same as those of the step 2.1. The bacteriostasis circle after 3 days is shown in fig. 4, wherein the left graph is a graph of the result of soaking the bacterial strain ZB in the sterile drug-sensitive tablet, and the right graph is a graph of the result of soaking the sterile drug-sensitive tablet in sterile distilled water. The diameter of the inhibition zone is shown in Table 2.
TABLE 2
From Table 2, it is clear that the bacterial strain ZB has an obvious antibacterial effect on Fusarium.
EXAMPLE 3 Performance study of bacterial strain ZB to degrade ammonia nitrogen and nitrite
In this example 3, the nitrifying performance test and the denitrifying performance test were performed by using strain ZB, and specifically include the following:
3.1 test of nitration Performance
Taking the fermentation broth containing the strain ZB in the step 2.2.1 in the example 2, inoculating the fermentation broth into a nitrifying culture medium according to the inoculum size of 10v/v%, placing the fermentation broth into a constant-temperature shaking table, and culturing at the rotating speed of 150 r/min. The nitrifying culture medium is as follows: ammonium chloride 0.2mg/L, sodium citrate 2mg/L, potassium dihydrogen phosphate 0.2mg/L, ferrous sulfate 0.02mg/L, magnesium sulfate 0.1mg/L, and purified water 1L. 10 replicates were made.
Fermenting and culturing at 26 ℃ for 3 days, and detecting the ammonia nitrogen content in the culture medium before inoculation and after fermenting and culturing for 3 days respectively.
Then adopting Nahner reagent photometry to detect. 5mL of the fermentation product was taken, placed in a 50mL cuvette, diluted to the standard, 1.0mL of potassium tartrate solution was added, and the absorbance was measured at 420nm, see specifically the method for Navier reagent photometry (A) in Water and wastewater monitoring analysis method (fourth edition).
The detection principle is as follows: an alkaline solution of mercury iodide and potassium iodide of the Naviet reagent reacts with ammonia to form a reddish brown colloidal compound, which is strongly absorbed over a broad wavelength.
As a result, the ammonia nitrogen concentration was degraded from 50mg/L to 1.2mg/L, which was reduced by 97.6%.
3.2 Denitrification Performance test
Taking the fermentation broth containing the strain ZB in the step 2.2.1 in the example 2, inoculating the fermentation broth into a denitrification medium according to the inoculum size of 10v/v%, placing the fermentation broth into a constant-temperature shaking table, and culturing at the rotating speed of 150 r/min. The denitrification medium comprises: sodium nitrite 0.2mg/L, sodium citrate 2mg/L, potassium dihydrogen phosphate 0.2mg/L, ferrous sulfate 0.02mg/L, magnesium sulfate 0.1mg/L, and purified water 1L. 10 replicates were made.
Fermenting and culturing at 26 ℃ for 3 days, and detecting the nitrite content in the culture medium before inoculation and after fermenting and culturing for 3 days respectively.
Then the detection is carried out by adopting an N- (1-naphthyl) -ethylenediamine photometry method (A). 5mL of the fermentation product was taken, added to a 50mL cuvette, diluted to the standard, 1.0mL of a color reagent was added, and absorbance was measured at 540nm, see specifically "Water and wastewater monitoring analysis method (fourth edition)".
The detection principle is as follows: in a phosphoric acid medium, when the pH value is 1.8+/-0.3, nitrite reacts with p-aminobenzene sulfonamide to generate diazonium salt, and then the diazonium salt is coupled with N- (1-naphthyl) -ethylenediamine to generate red dye. Has a maximum absorption at a wavelength of 540 nm.
As a result, it was found that the nitrite concentration was degraded from 22mg/L to 0.05mg/L, which was reduced by 99.8%.
Example 4 microbial preparation and its use in saprolegniasis
Preparing the fermentation broth of the strain ZB into freeze-dried powder, and carrying out experiments for preventing and treating saprolegniasis.
4.1 Preparation of lyophilized powder
The bacillus amyloliquefaciens strain ZB activated in the step 2.2.1 in the example 2 is inoculated into 100mL of LB liquid medium, and fermentation culture is carried out for 3 days at the temperature of 30 ℃ to obtain a fermentation broth.
Adding skimmed milk powder into the fermentation broth; the mass volume ratio of the fermentation liquid to the skimmed milk powder is 50g:1L, and then preparing freeze-dried powder by a freeze dryer.
The content of viable bacteria in the freeze-dried powder is 8 multiplied by 10 8 cfu/g.
4.2 Preventing saprolegniasis
And (3) carrying out a splash mode drug administration saprolegniasis prevention experiment on the fancy carp. Setting 3 sprinkling bacteria powder concentrations: the amount of the freeze-dried powder added into each ton of water is 1g, 3g and 5g respectively, three parallel comparison cylinders are arranged for each concentration of the bacterial powder, and three blank parallel comparison cylinders are arranged at the same time. Each control cylinder was set to 10 feet and the water temperature was set to 12 ℃. One week of observation, the results are shown in Table 3.
TABLE 3 Table 3
As can be seen from Table 3, about 70% of the fish in the control jar was infected with saprolegniasis, and the addition amounts of the lyophilized powder were respectively 1 g/ton of water, 3 g/ton of water, 5 g/ton of water, and the ratio of the fish infected with saprolegniasis was respectively 30%, 13.3% and 3.3%; the mortality rate of the control fish in the group of 3 g/ton water and 5 g/ton water is 43.3%, and the bacterial strain ZB can well prevent saprolegniasis.
4.3 Treatment of saprolegniasis
4.3.1 Treatment experiments on fancy carp infected with saprolegniasis
The koi is divided into 2 varieties, wherein the variety 1 is red and white koi in a certain farm in Jiangsu su, and the variety 2 is tea carp in a certain farm in Shanghai.
2 Groups are arranged in each fancy carp variety, 1 group is a bacterial powder experimental group, and 1 group is a control group; each group had 3 replicates of 30 fish.
The fungus powder experimental group is: the freeze-dried powder is added into water according to the proportion of 5g of freeze-dried powder added into each ton of water at 9 points in the morning.
The water temperature was set at 12 ℃. The results are shown in Table 4 after observation for 7 days.
Marking one fish in the bacterial powder experimental group of the variety 1, and observing and photographing before and after the experiment, wherein the result is shown in fig. 5; one fish in the bacterial powder experimental group of variety 2 is marked, and is observed and photographed before and after the experiment, and the result is shown in fig. 6.
TABLE 4 Table 4
As can be seen from Table 4, only 10% of the fish recovered health after 7 days of fish culture in the control group, whereas about 92% of the fungus powder experimental group recovered, and saprolegniasis was effectively treated.
As can be seen from FIGS. 5 and 6, when the lyophilized powder containing Bacillus amyloliquefaciens was added to water, the mold on the fish body was almost eliminated, and the fish was in good condition.
4.3.2 Pharmaceutical treatment experiments on Loongfish infected with saprolegniasis
According to the feedback of the king of the Hangzhou dragon fish fan, white floccules grow on the body surface of the cultured dragon fish, the swimming is slow, the appetite is reduced, and the disease is diagnosed as saprolegniasis for 15 days.
9 Hours a day, 3g of freeze-dried powder is added into each ton of water, and the temperature for cultivating the dragon fish is 28 ℃.
Over 5 days, the hydromold of the Loongopus had substantially disappeared as shown in FIG. 7.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (10)
1. The bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain ZB has a preservation number of CGMCC No:29399.
2. The strain of claim 1, wherein the strain' S16S rDNA sequence comprises the sequence set forth in SEQ ID No. 1;
And/or, the strain tolerance temperature is not lower than 8 ℃.
3. A fermentation culture of bacillus amyloliquefaciens, characterized in that it is obtained by inoculating the strain according to claim 1 or 2 in a culture medium for cultivation.
4. A fermentation culture according to claim 3, wherein the cultivation temperature is 8-30 ℃.
5. A microbial agent comprising the strain of claim 1 or 2 or the fermentation culture of claim 3 or 4.
6. The microbial agent of claim 5, wherein the microbial agent comprises an effective viable count of at least 5 x 10 8 CFU/mL;
And/or the dosage form of the microbial agent comprises freeze-dried powder, capsules, tablets or granules.
7. The microbial agent of claim 6, further comprising an adjuvant.
8. Use of a strain according to claim 1 or 2 or a fermentation culture according to claim 3 or 4 or a microbial agent according to any one of claims 5 to 7 in at least one of the following: a1 Nitrogen-containing wastewater denitrification treatment; a2 Preparing a product for preventing or treating mycosis of aquatic animals.
9. The use according to claim 8, wherein the nitrogen-containing means containing one or more of ammonia nitrogen, nitrate or nitrite.
10. The use according to claim 8, wherein the pathogenic bacteria of the mycoses are selected from one or more of the genera Saprolegnia (Saprolegnia), myceliophthora (Achlya) and Fusarium (Fusarium).
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