CN110484463B - Bacillus mojavensis B282 and application thereof - Google Patents
Bacillus mojavensis B282 and application thereof Download PDFInfo
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- CN110484463B CN110484463B CN201910641326.5A CN201910641326A CN110484463B CN 110484463 B CN110484463 B CN 110484463B CN 201910641326 A CN201910641326 A CN 201910641326A CN 110484463 B CN110484463 B CN 110484463B
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- 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
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- 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
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- 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
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Abstract
The invention discloses a Bacillus mojavensis (Bacillus mojavensis) B282 with the preservation number of CGMCC NO. 17656; the invention also discloses application of the Bacillus mojavensis (Bacillus mojavensis) B282 in preparation of an antifungal microbial preparation and an antifungal compost; the invention also discloses a method for preparing the antifungal compost, which is used for preparing the high-efficiency antifungal compost by adding the antifungal activity microorganisms into the secondary fermentation compost pile and depending on the proliferation of the microorganisms. The Bacillus mojavensis B282 provided by the invention has no ecological toxicity, high safety, wide antibacterial spectrum and simple culture, can be proliferated in a compost secondary fermentation heap, and improves the antifungal effect of the compost; the method for preparing the antifungal compost, provided by the invention, is simple to operate and low in cost, can effectively improve the antifungal drug effect of compost products, and has a very wide application prospect in biological control of plant diseases.
Description
Technical Field
The invention belongs to the technical field of environmental biotechnology and biological control of plant diseases, and particularly relates to the technical field of microorganisms for inhibiting fungi.
Background
The application of the chemical fertilizer and the pesticide plays an important role in the intensive planting of agricultural production, and the development of agriculture is promoted to a certain extent. However, in recent years, problems of soil hardening, salinization, soil fertility reduction and the like are increasingly exposed due to excessive use of chemical fertilizers and pesticides, and excessive chemical fertilizers and pesticides cause soil microflora to be disordered, beneficial organisms to be reduced, and crop soil-borne diseases to be aggravated year by year. Soil-borne diseases refer to diseases caused by invasion of crops from roots or stem bases of crops by pathogens living in soil under appropriate conditions, and commonly include damping-off, root rot, melon wilt and the like, and the diseases cause huge economic loss to agricultural production every year and severely restrict agricultural development. Pathogenic bacteria causing the diseases are generally multi-host microorganisms, have strong living capacity and infection capacity, are identified as one of the most difficult diseases to control by the plant kingdom, and the existing main control methods of the diseases have great limitations, so that a new control strategy is urgently needed to be developed.
Nowadays, a biological control method which accords with the strategic concept of modern agricultural sustainable development becomes a first choice measure for controlling soil-borne diseases, a plurality of biological control strains are discovered in succession, and the biological control strains are widely applied to production and obtain obvious effects. The biocontrol bacteria are various in types and mainly comprise groups such as trichoderma, bacillus, pseudomonas and streptomyces. The research on the biocontrol bacteria is still going deep, and the microorganisms have wide application prospect.
On the other hand, with the enlargement of the scale of the breeding industry, the generated mass of livestock and poultry manure is abandoned everywhere, which not only seriously pollutes the environment, but also greatly wastes the raw materials which can be used as organic fertilizers and bio-organic fertilizer products, and obviously weakens the sustainable development capability of agriculture in China. The composting is a main way for realizing the harmless and resource utilization of organic wastes such as livestock and poultry manure and the like. In aerobic fermentation of compost, a stage in which the temperature rises until it starts to decrease is generally called a primary fermentation (main fermentation) stage, and a stage in which the temperature of the compost starts to decrease until it becomes thoroughly decomposed is called a secondary fermentation (after fermentation) stage. In the primary fermentation stage, easily-decomposed organic matters rich in the compost are quickly decomposed under the action of aerobic microorganisms, and a large amount of heat energy is released, and the temperature of the compost layer is continuously increased to 60-70 ℃ and is kept for a period of time due to the good heat preservation effect of the compost materials. By means of the high-temperature period, pathogenic bacteria, worm eggs and weed seeds in the compost are killed, harmlessness is achieved, and the compost is primarily decomposed. In the secondary fermentation stage, undecomposed easily-degradable organic matters and undecomposed organic matters in the primary fermentation are continuously decomposed and converted into stable organic matters such as humic acid, amino acid and the like, meanwhile, beneficial microorganisms in the compost are proliferated to generate various functional substances, and finally, the relatively stable and decomposed compost is formed. The effect of the organic fertilizer is directly influenced by stable organic matters and various active functional substances formed in the secondary fermentation stage.
Since the last 40 s, there has been a continuing need to add microorganisms or agents to compost to improve the quality of compost and to make compost products perform specific functions. The research on adding microorganisms or microbial inoculum once or more times in the primary fermentation stage is more, and the improvement of the quality of secondary fermentation compost products by adding microorganisms or microbial inoculum in the secondary fermentation stage is rarely reported. Meanwhile, the purpose of adding microorganisms or microbial inoculum in the compost is focused on shortening the fermentation period, increasing the compost temperature, accelerating the decomposition of compost matrix, eliminating odor, degrading antibiotics and the like, and relatively few researches on improving the antifungal drug effect of compost products by externally connecting antifungal active microorganisms are carried out.
Disclosure of Invention
The first purpose of the invention is to provide a bacillus mojavensis (bacillus (Bacillus) mojavensis) separated from the secondary fermentation compost of livestock and poultry wastesBacillusmojavensis）B282。
The bacillus mojavensis (A), (B) and (C)Bacillus mojavensis) B282 is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 4 and 29, with the preservation number of CGMCC NO.17656 and the preservation address of No. 3 Hospital No.1, Xilu, Beijing, the rising area.
The second object of the present invention is to provide Bacillus mojavensis (B.) (Bacillus mojavensis) The application of B282 in preparing antibacterial microbial preparation.
Further, the fungi include the plant pathogenic fungi banana wilt disease bacteria (A), (B), (C)Fusariumoxysporumf.sp. cubense) Cucumber fusarium wilt bacteria (Fusariumoxysporumf.sp.cucumeri num) Wheat root rot (C.sp.)Bipolarissorokiniana) Phytophthora capsici (a), (b), (c) and (c)Phytophthoracapsici) Botrytis cinerea (A), (B), (CBotrytis cinereal）。
The third object of the present invention is to provide Bacillus mojavensis (B.) (Bacillus mojavensis) B282 application in preparing microbial preparation for improving antifungal effect of compost.
The fourth purpose of the invention is to provide a method for improving the antifungal effect of compost, which comprises the following steps:
1) inoculating antifungal active microorganisms into an LB liquid culture medium, and performing liquid shake culture for 48 hours to obtain a culture bacterial liquid;
2) centrifuging the culture bacteria liquid for 15min at 7500rpm, discarding the upper layer culture medium, and resuspending the thallus with a small amount of sterile water to obtain compost additive bacteria liquid;
3) adding compost additive bacteria liquid into the mixed compost until the number of the antifungal activity microorganism colonies in the compost reaches 1 multiplied by 106 cfu/g, obtaining bacterial liquid compost;
4) and (3) performing secondary fermentation on the bacterial liquid compost in an aerobic composting manner for 20-40d, and evaporating the water content of the compost to be below 40% under the condition that the temperature is not higher than 35 ℃ to obtain secondary bacterial liquid fermented compost.
Further, the culture conditions are as follows: the initial natural pH, the culture temperature 30 ℃, shaking table 200rpm culture 48 h.
Further, the amount of bacteria or spores contained in the culture solution is more than 1 × 108cfu/ml。
Further, the preparation method of the LB liquid culture medium comprises the following steps: adding tryptone 10g, yeast extract 5g, and sodium chloride 10g into 950 ml deionized water, diluting to 1000ml with deionized water, and sterilizing at 121 deg.C for 20min under natural pH.
Further, the aerobic composting method comprises the following steps: and (3) intermittently supplying oxygen from the bottom of the compost body by using an air pump, and thoroughly turning the compost every 5 days to uniformly mix the composts at all levels.
The bacillus mojavensis (B) provided by the inventionBacillusmojavensis) B282 simple culture, short period, no ecological toxicity,High safety and stability, and can be used for treating banana wiltFusariumoxysporumf.sp. cubenseFu sariumoxysporumf.sp.cubense) Cucumber fusarium wilt bacteria (Fusariumoxysporumf.sp.cucumerinum) Wheat root rot (C.sp.)Bipolarissorokiniana) Phytophthora capsici (a), (b), (c) and (c)Phytophthoracapsici) Botrytis cinerea (A), (B), (CBotrytis cinereal) And various plant pathogenic fungi have good antagonistic action and wide antibacterial spectrum. Meanwhile, the strain can be proliferated in a secondary fermentation pile body, and the antifungal effect of the compost can be improved. In particular Bacillus mojavensis (B.), (B.)Bacillusmojavensis) B282 has wide development and application prospect in biological control of plant diseases and improvement of antifungal effect of compost, and is beneficial to pollution-free production and sustainable development of agriculture.
Drawings
FIG. 1 shows Bacillus mojavensis (B.)Bacillus mojavensis) Microscopic morphological characteristics of B282.
FIG. 2 shows Bacillus mojavensis (M.), (B.), (BBacillusmojavensis) B282 plant pathogenic fungi. In the figure, CK group is non-inoculated Bacillus mojavensis (B.), (Bacillus mojavensis) Control group B282, group B282 inoculated with Bacillus mojavensis: (Bacillusmojavensis) Experimental group of B282.
FIG. 3 shows Bacillus mojavensis (M.), (B.), (BBacillusmojavensis) B282 dynamic change of thallus in the compost secondary fermentation pile.
FIG. 4 is a graph of a plate test of the inhibitory effect of the secondary fermentation compost leaching solution on plant pathogenic fungi. In the figure, CK group is a control group without compost leaching liquor, and EC group is a control group without Bacillus mojavensis (Bacillus mojavensis)Bacillus mojavensis) B282, wherein B282 is a leaching liquor group added with Bacillus mojavensis: (B282)Bacillusmojavensis) B282 secondary fermentation compost leaching group.
FIG. 5 shows the diameters of the colonies of phytopathogenic fungi of the respective treatment groups. In the figure, CK group is a control group without compost leaching liquor, and EC group is a control group without Bacillus mojavensis (Bacillus mojavensis)Bacillus mojavensis) B282 secondary fermentation compost leaching group, B282 groupAdding Bacillus mojavensis (B)Bacillus mojavensis) B282 secondary fermentation compost leaching group.
Detailed Description
Example 1
Bacillus mojavensis (B.), (BBacillus mojavensis) Isolation and characterization of Strain B282
Collecting cow dung compost secondary fermentation sample, adopting beef extract peptone culture medium to separate bacteria, adopting counter-culture to primarily separate banana wilt bacteria: (Fusariumoxysporumf.sp. cubenseFusariumoxysporumf.sp. cubense) A strain with inhibiting effect.
The preparation method of the beef extract peptone medium comprises the following steps (taking preparation of 1L medium as an example): 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000ml of water, wherein the pH is 7.4-7.6, and the beef extract is sterilized at 121 ℃ for 30 minutes.
The isolated strain was named strain B282, and its morphology is shown in FIG. 1, and it belongs to a rod-shaped gram-negative bacterium and forms spores. The optimal growth temperature of the strain is about 37 ℃, and the proper growth pH range is 5.0-8.8. Extracting the genomic DNA of the strain and using 16s rDNA andgyrAprimers Polymerase Chain Reaction (PCR) was performed on the genomic DNA to propagate a specific DNA sequence, and strain alignment was performed using National Center for Biotechnology Information (NCBI) database. The detailed information is as follows:
1) 16S rDNA sequence analysis:
16s rDNA forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively;
16s rDNA reverse primer 27F: 5'-GGTTACCTTGTTACGACTT-3', respectively;
the sequence of the 16S rDNA obtained by amplification is shown as SEQ ID NO.1, and the total length is 1390 bp. Comparing the 16S rDNA sequence obtained by amplification with the gene sequence of related strain at NCBI, finding out strain B282 and model strainBacillus mojavensisNBRC 15718 homology up to 99.93%.
2）gyrAGene sequence analysis:
gyrAa forward primer: 5'-CAGTCAGGAAATGCGTACGTCCTT-3', respectively;
gyrAreverse primer: 5'-CAAGGTAATGCTCCAGGCATTGCT-3', respectively;
amplification resultgyrAThe gene sequence is shown in SEQ ID NO.2, and the total length is 1017 bp. Subjecting the amplified product to amplificationgyrAThe gene sequence is compared with the gene sequence of related strain on NCBI, and the strain B282 and the model strain are foundBacillus mojavensisNRRL B-14698 has 97.31% homology.
Therefore, the strain B282 can be judged to be a Bacillus mojavensis strain (B) ((B))Bacillusmojavensis). After identifying and confirming the species of the bacillus mojavensis (B.) (Bacillus mojavensis) B282 is preserved in China general microbiological culture Collection center (CGMCC for short, No. 3 Hospital No.1 Xilu, Beijing City, the rising district, Beijing) in 2019, 4 months and 29 days; the preservation number is CGMCC NO. 17656; this biomaterial has been tested for survival and passes the test.
Example 2
Antifungal Effect of Strain B282
Respectively punching Botrytis cinerea with a 5mm punch (Botrytis cinereal) Banana wilt bacteria (Fusariumoxysporumf.sp. cubenseFusariumoxysporumf.sp. cubense) Cucumber fusarium wilt bacteria (Fusariumoxysporumf.sp.cucumerinum) Phytophthora capsici (a), (b), (c) and (c)Phytophthoracapsici) Wheat root rot (C.sp.)Bipolarissorokiniana) A fungus cake round piece is inoculated in the center of a PDA culture medium plate, and bacillus mojavensis (Bacillus mojavensis) is inoculated at a position 1.5 cm away from the centerBacillus mojavensis) The strain B282 and the control group were not inoculated with B282, and photographs were taken after culturing at 28 ℃ in the dark for 4 days. Whether the strain B282 has antagonistic effect on the plant pathogenic fungi is determined by observing the zone of inhibition, and the strain B282 is found to have good antagonistic effect on the plant pathogenic fungi. (FIG. 2)
The experimental results show that: bacillus mojavensis (B.), (BBacillus mojavensis) B282 has good antagonistic effect on the pathogenic fungi, and has a wide antibacterial spectrum.
The preparation method of the PDA culture medium is as follows (taking 1L of culture medium as an example): peeling 200g potato, cutting into small pieces, decocting in water, boiling, decocting for 30min, filtering with gauze, adding 20g glucose into the filtrate, adding deionized water to 1000ml, adding 20g agar, naturally adjusting pH, and sterilizing at 115 deg.C for 30 min.
Example 3
Method for improving antifungal drug effect of compost and verification 1) inoculating antifungal activity microorganism (such as strain B282) in LB liquid culture medium, and performing liquid shake culture for 48h to obtain compost with bacteria or spore content more than 1 × 108cfu/ml of culture broth.
The culture conditions are as follows: the initial natural pH, the culture temperature 30 ℃, shaking table 200rpm culture 48 h. At this time, the bacterial liquid contains more than 1 × 10 bacteria or spores8cfu/ml；
The preparation method of the LB liquid culture medium comprises the following steps (taking 1L of culture medium as an example): adding tryptone 10g, yeast extract 5g, and sodium chloride 10g into 950 ml deionized water, diluting to 1000ml with deionized water, and sterilizing at 121 deg.C for 20min under natural pH.
2) Preparing chicken manure, cow manure and mushroom residue after primary fermentation (mass ratio 6: 5: 5) and (5) mixing and composting.
Preparing culture bacteria solution of antifungal active microorganism (such as strain B282) in step 1, centrifuging at 7500rpm for 15min, removing upper layer culture medium, and re-suspending the bacteria with small amount of sterile water to obtain compost additive bacteria solution.
Adding compost additive bacteria solution into the mixed compost until the colony number of antifungal active microorganism (such as strain B282) in the compost reaches 1 × 106 cfu/g, and obtaining bacterial liquid compost.
And performing secondary fermentation on the bacterial liquid compost in an aerobic composting manner for 20-40 d.
And (3) adopting an aerobic composting mode in the whole secondary fermentation process, intermittently supplying oxygen from the bottom of the compost by using an air pump, thoroughly turning the compost every 5 days, uniformly mixing the composts at all levels, and finally evaporating the water content of the compost product to be below 40% under the condition that the temperature is not higher than 35 ℃ to obtain the secondary bacterial liquid fermented compost.
3) Samples are collected from all levels of the secondary bacterial liquid fermentation compost body every 10 days by a five-point sampling method and are uniformly mixed, and the samples are placed at 4 ℃ and are reserved for flat plate counting.
Respectively taking 10g of samples collected at each sampling time point, placing in 100ml of sterile water with glass strains, standing for 20min, and shaking fully for 30min at 200rpm of a shaking table to prepare mother liquor bacterial suspension.
Taking 100 mul of mother liquor, adding the mother liquor into a centrifuge tube containing 900 mul of sterile water, oscillating and mixing uniformly to fully disperse the sample to obtain 10 mul of mother liquor-1And (4) diluting the solution. Then diluting with sterile water to obtain 10-2、10-3、10-4、10-5And 10-6 dilution.
100 mul of diluent is uniformly coated on a beef extract peptone medium plate, 3 plates are coated on each gradient diluent, then the number of the antifungal activity microorganisms (such as the strain B282) in the plates is counted after the plates are cultured in an incubator at 37 ℃ for 36 hours, the colony number of the antifungal activity microorganisms (such as the strain B282) in a compost secondary fermentation pile body at different time points is calculated, the content of the antifungal activity microorganisms (such as the strain B282) in each gram of compost sample is calculated according to the colony number on the diluent plates, and the results are shown in table 1 and figure 3.
The results show that: an antifungal activity of a microorganism (e.g., strain B282) is required to survive the secondary composting and to proliferate in the compost secondary fermentation compost in an amount of 1.0X 10 from the initial addition6cfu/g to at least 5X 107cfu/g。
The preparation method of the beef extract peptone medium comprises the following steps (taking preparation of 1L medium as an example): 3g of beef extract, 10g of peptone, 5g of NaCl, 20g of agar and 1000ml of water, wherein the pH is 7.4-7.6, and the beef extract is sterilized at 121 ℃ for 30 minutes.
TABLE 1 dynamic behavior of strain B282 in compost secondary fermentation heap
| Time of composting | 10 |
20 |
30 |
40 d |
| Degree of dilution | 10-4 | 10-5 | 10-5 | 10-5 |
| Colony count/ |
10/8/5 | 10/10/12 | 15/10/10 | 10/5/13 |
| Thallus content cfu (× 107/g) | 0.77±0.26 | 10.67±1.15 | 11.67±2.89 | 9.30±4.04 |
Example 4
Bacteriostatic effect of compost extract
Adding 10g of the product after the secondary fermentation of the compost into 80 ml of sterile water with glass beads, fully shaking for 1 h at 200rpm of a shaking table, filtering the obtained suspension by using sterile double-layer gauze, centrifuging the filtrate at 7500rpm for 15min, taking the supernatant, adding the supernatant into a non-scalding PDA culture medium, and uniformly mixing to ensure that the volume ratio of the leaching solution to the PDA culture medium is 1:50, and adding the same amount of the control group into the PDA culture medium without the need of hand scalding to ensure that the volume ratio of the leaching solution toReplacing the bacteria water. Equal amount of medium was poured into a dish. After the mixture solidified, the mixture was punched with a 5mm punch to obtain Botrytis cinerea: (Botrytis cinereal) Banana wilt bacteria (Fusariumoxysporumf.sp. cubenseFusariumoxysporumf.sp. cubense) Cucumber fusarium wilt bacteria (Fusariumoxysporumf.sp.cucumerinum) Phytophthora capsici (a), (b), (c) and (c)Phytophthoracapsici) Wheat root rot (C.sp.)Bipolarissorokiniana) The fungus cake discs are inoculated in the center of a PDA culture medium plate, 1 disc is arranged in each dish, 3 dishes are processed in each dish, and the dishes are cultured under the dark condition at the temperature of 28 ℃.
After inoculation of banana vascular wilt bacteria (Fusariumoxysporumf.sp. cubenseFusariumoxysporumf.sp. cubense) Wheat root rot (C.sp.)Bipolarissorokiniana) After 90 hours; inoculation of cucumber wilt bacteria: (Fusariumoxysporumf.sp. cucumerinum) After 137.5 h; inoculating Phytophthora capsici (A), (B), (C) and (C)Phytophthoracapsici) After 161.5 h; inoculating Botrytis cinerea (A)Botrytis cinereal) After 245h, the plates inoculated with different groups of phytopathogens were photographed separately and the colony diameters were measured by the cross method, and the results are shown in FIGS. 4 and 5.
The experimental results show that:
without addition of Bacillus mojavensis (B.) (Bacillus mojavensis) B282 leaching liquor (EC group) of secondary fermentation compost and adding Bacillus mojavensis (B)Bacillus mojavensis) The secondary fermentation compost leaching liquor (B282 group) of B282 has certain inhibitory effect on plant pathogenic fungi.
The inhibition rate of the two compost leaching solutions on plant pathogenic fungi is reflected by (the diameter of the control group bacterial colony-the diameter of the experimental group bacterial colony)/(the diameter of the control group bacterial colony). Two compost leaching solutions to Botrytis cinerea: (Botrytiscinereal) The inhibition rates of (A) and (B) are respectively 38.75% and 83.56%, and the inhibition rate of (A) is higher than that of banana vascular wilt bacteriumFusariumoxysporumf.sp. cubenseFusariumoxysporumf.sp. cubense) The inhibition rates of the compounds are 31.45 percent and 62.11 percent respectively, and the compounds have the effect on cucumber fusarium wilt bacteria (A), (B), (C)Fusariumoxysporumf.sp.cucumerinum) The inhibition rates of the compounds are respectively 31.88 percent and 52.01 percent, and the compounds have the inhibition rate on phytophthora capsici (Phytophthora capsici) ((II))Phytophthoracapsici) Is inhibited byThe rates are respectively 62.42 percent and 83.39 percent, and the rate is higher than that of the rhizoctonia solani (of wheat)Bipolarissorokiniana) The inhibition rates of (a) were 57.08% and 78.14%, respectively. Less than the addition of Bacillus mojavensis (Bacillus mojavensis) B282 secondary fermentation compost leaching liquor, adding Bacillus mojavensis (Bacillus (Moghai) (R))Bacillus mojavensis) The leaching liquor of the secondary fermentation compost of B282 has better inhibiting effect on plant pathogenic fungi.
The preparation method of the PDA culture medium is as follows (taking 1L of culture medium as an example): peeling 200g potato, cutting into small pieces, decocting in water, boiling, decocting for 30min, filtering with gauze, adding 20g glucose into the filtrate, adding deionized water to 1000ml, adding 20g agar, naturally adjusting pH, and sterilizing at 115 deg.C for 30 min.
Sequence listing
<110> institute of microbiology of Chinese academy of sciences
<120> bacillus mojavensis B282 and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1390
<212> DNA
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 1
taaaggttac ctcaccgact tcgggtgtta caaactctcg tggtgtgacg ggcggtgtgt 60
acaaggcccg ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc 120
ttcacgcagt cgagttgcag actgcgatcc gaactgagaa cagatttgtg ggattggctt 180
aacctcgcgg tttcgctgcc ctttgttctg tccattgtag cacgtgtgta gcccaggtca 240
taaggggcat gatgatttga cgtcatcccc accttcctcc ggtttgtcac cggcagtcac 300
cttagagtgc ccaactgaat gctggcaact aagatcaagg gttgcgctcg ttgcgggact 360
taacccaaca tctcacgaca cgagctgacg acaaccatgc accacctgtc actctgcccc 420
cgaaggggac gtcctatctc taggattgtc agaggatgtc aagacctggt aaggttcttc 480
gcgttgcttc gaattaaacc acatgctcca ccgcttgtgc gggcccccgt caattccttt 540
gagtttcagt cttgcgaccg tactccccag gcggagtgct taatgcgtta gctgcagcac 600
taaggggcgg aaacccccta acacttagca ctcatcgttt acggcgtgga ctaccagggt 660
atctaatcct gttcgctccc cacgctttcg ctcctcagcg tcagttacag accagagagt 720
cgccttcgcc actggtgttc ctccacatct ctacgcattt caccgctaca cgtggaattc 780
cactctcctc ttctgcactc aagttcccca gtttccaatg accctccccg gttgagccgg 840
gggctttcac atcagactta aggaaccgcc tgcgagccct ttacgcccaa taattccgga 900
caacgcttgc cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg 960
gttaggtacc gtcaaggtac cgccctattc gaacggtact tgttcttccc taacaacaga 1020
gctttacgat ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga ctttcgtcca 1080
ttgcggaaga ttccctactg ctgcctcccg taggagtctg ggccgtgtct cagtcccagt 1140
gtggccgatc accctctcag gtcggctacg catcgttgcc ttggtgagcc gttacctcac 1200
caactagcta atgcgccgcg ggtccatctg taagtggtag ccgaagccac cttttatgtt 1260
tgaaccatgc ggttcaaaca agcatccggt attagccccg gtttcccgga gttatcccag 1320
tcttacaggc aggttaccca cgtgttactc acccgtccgc cgctaacatc agggagcaag 1380
ctcccatctg 1390
<210> 2
<211> 1017
<212> DNA
<213> Bacillus mojavensis (Bacillus mojavensis)
<400> 2
aatgcgtacg tcctttttgg attatgcgat gagcgttatc gtatcccggg cgcttccgga 60
tgtgcgtgac ggtttaaaac cggttcacag acggatttta tacgcgatga atgatttagg 120
catgacgagt gacaagccgt ataaaaaatc ggctcgtatc gtcggagaag ttatcgggaa 180
ataccatcct cacggtgatt cagcggtata tgaatctatg gtcagaatgg cgcaggattt 240
caactaccgt tatatgctgg tggacggtca cggaaacttt ggttctgtag acggagactc 300
agccgctgcg atgcggtaca ccgaagccag aatgtctaag atcgccatgg aaattctgcg 360
agacatcaca aaagacacga ttgattatca agataactat gatggttcag agagagaacc 420
tgttgtcatg ccgtcaagat tcccaaacct gcttgtaaat ggagcagccg gtattgcagt 480
cggaatggct acaaatatac cgccgcacca gcttggagaa attattgacg gagtgcttgc 540
cgtaagcgag aacaaggaca taacaatcca agagctgatg gaattcattc ctggaccgga 600
tttcccgact gccggacaaa ttttaggcag aagcggaatt cgcaaggcat atgaatccgg 660
aagaggttct attacgatta gggcaaaagc agaaattgaa gaaacttcat cagggaaaga 720
aagaatttta gtaacagaac tcccttatca ggtgaataaa gcgcgtctaa ttgagaagat 780
tgctgatctt gtcagagata agaaaattga aggaataact gatttgcgtg atgaatctga 840
ccgtaacggt atgcggattg tcattgaaat cagacgagat gccaatgcgc atgttatcct 900
gaacaatctt tacaaacaaa cggccctgca aacttccttc gggattaaca tgctggcgct 960
tgttgacggt gagccgaagg tattaaattt aaagcaatgc ctggagcatt accttga 1017
Claims (8)
1. Bacillus mojavensis (A)Bacillus mojavensis) B282, the preservation number is CGMCC NO. 17656.
2. The Bacillus mojavensis of claim 1, (b) CBacillus mojavensis) B282 use for the preparation of antimicrobial microbial agents against fungi, including phytopathogenic fungi: banana vascular wilt bacterium (Fusarium oxysporum f.sp. cubense）、Cucumber fusarium wilt bacteria (Fusarium oxysporum f.sp.cucumerinum) Wheat root rot (C.sp.)Bipolaris sorokiniana) Phytophthora capsici (a), (b), (c) and (c)Phytophthora capsici) Botrytis cinerea (A), (B), (CBotrytis cinereal）。
3. The Bacillus mojavensis of claim 1, (b) CBacillus mojavensis) B282 application in preparing microbial preparation for improving antifungal effect of compost, wherein the fungi comprise plant pathogenic fungi: banana vascular wilt bacterium (Fusarium oxysporum f.sp. cubense) Cucumber fusarium wilt bacteria (Fusarium oxysporum f.sp.cucumerinum) Wheat root rot (C.sp.)Bipolaris sorokiniana) Phytophthora capsici (a), (b), (c) and (c)Phytophthora capsici) Botrytis cinerea (A), (B), (CBotrytis cinereal）。
4. A method for improving the antifungal effect of compost is characterized by comprising the following steps:
1) the Bacillus mojavensis of claim 1, (b) CBacillus mojavensis) B282 is inoculated in an LB liquid culture medium, and liquid shake culture is carried out for 48 hours to obtain a culture bacterial liquid;
2) centrifuging the culture bacteria liquid for 15min at 7500rpm, discarding the upper layer culture medium, and resuspending the thallus with a small amount of sterile water to obtain compost additive bacteria liquid;
3) adding compost additive bacteria liquid into the mixed compost until the number of the antifungal activity microorganism colonies in the compost reaches 1 multiplied by 106cfu/g, obtaining bacterial liquid compost;
4) and (3) performing secondary fermentation on the bacterial liquid compost in an aerobic composting manner for 20-40d, and evaporating the water content of the compost to be below 40% under the condition that the temperature is not higher than 35 ℃ to obtain secondary bacterial liquid fermented compost.
5. The method of claim 4, wherein the culturing conditions are: the initial natural pH, the culture temperature 30 ℃, shaking table 200rpm culture 48 h.
6. The method according to claim 4, wherein the amount of bacteria or spores in the culture broth is more than 1X 108cfu/ml。
7. The method of claim 4, wherein the LB liquid medium is prepared by: adding tryptone 10g, yeast extract 5g, and sodium chloride 10g into 950 ml deionized water, diluting to 1000ml with deionized water, and sterilizing at 121 deg.C for 20min under natural pH.
8. The method of claim 4, wherein the aerobic composting process comprises: and (3) intermittently supplying oxygen from the bottom of the compost body by using an air pump, and thoroughly turning the compost every 5 days to uniformly mix the composts at all levels.
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| CN108863675A (en) * | 2018-09-03 | 2018-11-23 | 内蒙古蒙苒生物科技有限公司 | A kind of microbiological fermented organic fertilizer and preparation method thereof |
| CN109182219A (en) * | 2018-10-19 | 2019-01-11 | 青海大学 | One plant of Mo Haiwei bacillus for promoting the growth of Henry David Thoreau grass and its application |
| CN109734488A (en) * | 2019-03-27 | 2019-05-10 | 山东博华高效生态农业科技有限公司 | A kind of high-quality biological fertilizer and preparation method thereof |
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