CN114456986A - Broad-spectrum antibacterial Bacillus aryabhattai and application thereof - Google Patents
Broad-spectrum antibacterial Bacillus aryabhattai and application thereof Download PDFInfo
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Abstract
The invention discloses a broad-spectrum bacteriostatic Bacillus aryabhattai and application thereof. The strain is Bacillus aryabhattai (Bacillusaryabhattai) AU0102, which is preserved in China general microbiological culture Collection center in 2018, 01, 15 and the preservation number is CGMCC NO.15218, the strain has broad-spectrum antibacterial activity, has antagonistic effect on 18 pathogenic fungi including pine needle brown spot pathogen and 4 pathogenic bacteria, particularly has the inhibition effect on pine needle brown spot pathogen of 93.90 percent, has the prevention and treatment effects on pine needle brown spot by 300 times and 500 times of diluent of fermentation liquor of 95.42 percent and 92.86 percent, and can effectively control the occurrence of pine needle brown spot.
Description
Technical Field
The invention relates to broad-spectrum bacteriostatic Bacillus aryabhattai and application thereof, belonging to the technical field of microorganisms.
Background
Bacillus aryabhattai (Bacillus aryabhattai) belongs to the kingdom of bacteria, the phylum firmicutes, the phylum sporotrichum, the class of bacilli, the order of bacillales, the family of bacillaceae, and is a new reporter bacterium of the genus Bacillus. The Bacillus aryabhattai has wide application, is reported to be used for preventing and treating diseases such as tobacco black shank (CN201310559441.0), rice blast, blight and potato scab (CN201910375594.7), can also be used for inhibiting rhizopus microsporus (201910836765.1), aspergillus flavus (CN201510296912.2), generating auxin, siderophore, ammonia, protease, salt tolerance, antagonism and phosphate dissolution (CN201610036163.4 and CN202111159072.7), degrading cypermethrin (CN201611136508.X), effectively degrading polyethylene plastics (CN202111159072.7) and the like, but has no report that the Bacillus aryabhattai is used for preventing and treating pine needle brown spot.
Pine needle brown spot is a common disease on pine caused by trichotheca pine (Lecunosticta acicola), is mostly seen in new needle leaves, not only causes the area of a pine forest to be greatly reduced, but also causes huge loss to ecology. At present, the prevention and treatment of pine needle brown spot mainly adopt prevention and treatment methods such as chemical agent prevention and treatment, cultivation measures and the like. The chemical prevention and control has good prevention and control effect, but has the problems of environmental pollution, easy generation of drug resistance and the like. And the prevention and control effect is poor by adopting cultivation measures. Biological control is increasingly favored because of the advantages of high control efficiency, strong specificity, good drug effect durability, environmental friendliness, difficult generation of drug resistance and the like. But at present, few reports are provided for biological control of pine needle brown patch.
Disclosure of Invention
Aiming at the problems, the invention provides a broad-spectrum bacteriostatic Bacillus aryabhattai AU0102 and application thereof, wherein the Bacillus aryabhattai AU0102 has a suppression effect on plant diseases of different families, has a broad bacteriostatic spectrum, particularly has a best suppression effect on pine needle brown spot germs, can be developed for a microbial pesticide for pine needle brown spot, and realizes green and efficient prevention and control on pine needle brown spot.
The invention screens Bacillus aryabhattai (Bacillus aryabhattai) AU0102 with stronger inhibition effect on pine needle brown spot from pine forest land soil in the ring green region of Weihai city in Shandong province, the strain is preserved in the China general microbiological culture Collection center in 2018, 01 and 15 days, and the preservation number is CGMCC NO. 15218. Experiments show that: the strain AU0102 and metabolites thereof can effectively inhibit the growth of pine needle brown spot pathogen, and have wide application prospect in prevention and treatment of pine needle brown spot.
The invention also provides application of the Bacillus aryabhattai AU0102 in inhibiting pathogenic fungi such as pine needle brown spot pathogen, pine wilt fungus, poplar canker pathogen, apple ring rot pathogen and the like and pathogenic bacteria such as citrus canker pathogen, ginger blast pathogen, walnut black spot pathogen, watermelon soft rot pathogen and the like.
The invention also provides a fermentation method of the Bacillus aryabhattai AU0102, which is characterized in that after the Bacillus aryabhattai AU0102 is used for preparing seed liquid, the seed liquid is inoculated into a fermentation culture medium and is fermented and cultured for 20-30 hours at the temperature of 26-30 ℃ to obtain fermentation liquid.
The formula (mass ratio) of the seed liquid culture medium is as follows: peptone 1%, beef powder 0.3%, sodium chloride 0.5%, and water in balance, adjusting pH to 7.3 + -0.1.
The formula (mass ratio) of the liquid fermentation culture medium is as follows: 2.0-2.5% of soluble starch, 3.0-3.5% of fish peptone, 0.1-0.2% of magnesium sulfate, 0.5-0.8% of sodium chloride and the balance of water, and adjusting the pH value to 7.1 +/-0.1.
The invention also provides application of the Bacillus aryabhattai AU0102 fermentation broth in prevention and treatment of pine needle brown spot.
The invention has the beneficial effects that:
1. broad spectrum antibacterial property
The Bacillus aryabhattai AU0102 has antagonistic broad spectrum, and has different degrees of inhibition effects on the growth of 18 pathogenic fungi (pine needle brown spot pathogen, pine wilt disease pathogen, poplar rot pathogen, apple ring rot pathogen and the like) and 4 pathogenic bacteria (citrus canker pathogen, ginger blast pathogen, walnut black spot pathogen and watermelon soft rot pathogen); particularly, the inhibition effect on pine needle brown spot pathogen and pine wilt disease fungus is obvious, and the inhibition effect is 93.90% and 91.19% respectively.
2. Good effect of preventing and controlling pine needle brown spot
Experiments show that: the strain AU0102 and its metabolite can effectively inhibit the growth of pine needle brown spot pathogen. The liquid preparation (fermentation liquor) prepared by taking the strain as a raw material is diluted by 300 times and 500 times and then directly sprayed, the prevention and treatment effect is up to 95.42 percent and 92.86 percent, and the strain has wide application prospect in prevention and treatment of pine needle brown spot.
3. The strain is easy to ferment and produce, simple in use mode and suitable for large-area popularization and use.
Drawings
FIG. 1 shows the inhibitory effect of Bacillus aryabhattai AU0102 on pine needle brown spot pathogen;
FIG. 2 shows the inhibitory effect of Bacillus aryabhattai AU0102 fermentation supernatant on pine needle brown spot pathogen;
FIG. 3 shows the single colony morphology of Bacillus aryabhattai AU 0102;
FIG. 4 shows the form of Bacillus aryabhattai AU0102 cells (simple staining);
FIG. 5 shows the gram-staining results of Bacillus aryabhattai AU 0102;
FIG. 6 shows the result of staining of Bacillus aryabhattai AU0102 spores.
Detailed Description
The effects of the present invention will be described below with reference to examples.
Example 1: isolation and purification of Bacillus aryabhattai (Bacillus aryabhattai) Strain AU0102
The Bacillus aryabhattai (Bacillus aryabhattai) AU0102 is separated from soil by adopting a dilution plate method and a plate marking method, and the specific separation method comprises the following steps:
(1) soil sample collection
The sampling place is a black pine forest farm in the green region of Weihai city, Shandong province; the sampling method is a five-point sampling method, namely, respectively collecting appropriate amount of soil around the plot and in the depth range of 10-15cm in the center, uniformly mixing the soil in equal amount, putting the mixture into a freshness protection package, marking the collection place, time and the collection person to immediately separate or putting the mixture into a refrigerator at 4 ℃ for storage.
(2) Soil suspension preparation
Accurately weighing 1g of soil sample in a triangular flask filled with 99mL of sterile water, placing the triangular flask containing glass beads in a shaking table at 28 ℃ and uniformly shaking at 120rpm for 20-30 min, and then placing the triangular flask in a water bath kettle at 55-60 ℃ to incubate for 20-30 min to obtain the soil suspension.
(3) Dilution coating
Sequentially diluting the bacterial sample to 10 times by using a 10-fold dilution method-1～10-6A diluted bacterial sample; are respectively from 10-4、10-5And 10-6The diluted samples were pipetted 100. mu.L onto plates in NA solid medium, three replicates for each gradient.
(4) Culture and purification
After culturing for 36h at 28 ℃, selecting microbial colonies with different forms on an NA culture medium to streak on an NA culture medium plate, and regularly observing the growth condition of the colonies; then selecting single bacteria for purification by adopting a plate marking method, numbering and storing respectively.
By the method, 12 strains of bacteria are preliminarily separated according to different colony forms and are respectively numbered as AU 0101-AU 0112.
Example 2: high-efficiency antagonistic strain screening of pine needle brown spot
(1) Preliminary screening
Screening antagonistic strains which are high-efficiency antagonistic to pine needle brown spot disease pathogen aschersonia pine needle (Lecunostta acicola) by using a plate-opposing culture method.
Carrying out streak culture on the 12 strains to be detected separated and purified in the example 1 on an NA culture medium, and carrying out constant-temperature culture at 28 ℃ for 24h to obtain a bacterial liquid of the strain to be selected; selecting a pine needle brown spot germ strain to carry out activated culture on a PDA culture medium, and carrying out constant temperature culture at 25 ℃ for 60 h; punching a fungus cake with the diameter of 5mm on the edge of the activated pine needle brown spot pathogen by using a puncher, and inoculating the fungus cake in the center of a flat plate; inoculating and activating bacterial liquid of the strains to be selected by using an inoculating loop point at the periphery 1.5cm away from the center; only 5mm pine needle brown spot pathogen was inoculated in the center of the PDA plate in the control group; each treatment was repeated 3 times; after the treatment is finished, culturing at constant temperature of 28 ℃, observing the inhibition effect of the strains to be selected on the pathogenic bacteria day by day, measuring the size of the disease spots of the experimental group after the pathogenic bacteria of the control group overgrow the flat plate, and calculating the bacteriostasis rate.
The inhibition rate was ═ 100% (control colony diameter-cake diameter) - (treated colony diameter-cake diameter) ]/(control colony diameter-cake diameter) ×.
The results showed that 5 of the 12 isolated strains of bacteria of example 1 had inhibitory effects on the growth of pine needle brown spot pathogen, AU0102, AU0104, AU0107, AU0109 and AU0112 (table 1); statistics of the bacteriostasis rate shows that the inhibition effect of the strain AU0102 on pine needle brown spot fungus is 90.54%, and the bacteriostasis rate is obviously higher than that of other strains (Table 1 and figure 1).
TABLE 1 inhibitory Effect of different strains on pine needle brown spot pathogen
(2) Double sieve
And performing fermentation culture on AU0102, AU0104, AU0107, AU0109 and AU0112 which are obtained by primary screening and have an inhibiting effect on the growth of pine needle brown spot pathogen. The specific method comprises the following steps: transferring a ring by using a strain inoculating ring, putting the ring into a triangular flask (50mL) filled with 10mL of NA liquid culture medium, and carrying out constant-temperature shaking culture at 28 ℃ and 120r/min for 24h to obtain a seed solution; inoculating the seed solution into a triangular flask (250mL) filled with 100mL of NA liquid culture medium according to the inoculation amount of 1%, and performing constant-temperature shaking culture at 28 ℃ and 120r/min for 24h to obtain a fermentation solution; filtering the fermentation liquid with a 0.22 μm bacterial filter to obtain fermentation supernatant; uniformly mixing the fermentation supernatant with a PDA culture medium at the temperature of 45-50 ℃ in a ratio of 1:19, pouring the mixture into a culture dish, cooling, and then putting a pathogenic bacteria cake with the diameter of 5mm in the center of a flat plate; adding sterile water to the control group according to the same proportion to replace the fermentation supernatant; each treatment was repeated 3 times; after the treatment is finished, culturing at the constant temperature of 28 ℃, observing the inhibition effect of the fermented supernatant of 5 strains on pathogenic bacteria day by day, measuring the size of the lesion of the experimental group after the pathogenic bacteria of the control group overgrow the flat plate, and calculating the bacteriostasis rate.
The results show that the AU0102, AU0104, AU0107, AU0109 and AU0112 fermentation supernatants all have inhibitory effects on the growth of pine needle brown spot pathogen, and the inhibitory rate of the AU0102 fermentation supernatants is up to 88.30% (see Table 2 and figure 2).
TABLE 2 inhibitory effect of fermentation supernatants of different strains on pine needle brown spot pathogen
The results show that the strain AU0102 and the metabolite thereof can effectively inhibit the growth of pine needle brown spot pathogen and have wide application prospects in prevention and treatment of pine needle brown spot.
Example 3: strain AU0102 identification
(1) Microbiological characteristics: the colony morphology of the strain on an NA culture medium or an LB culture medium is regular and circular, and the surface at the early stage is moist, smooth, viscous and semitransparent milk; the surface of the later stage is provided with folds and bulges, is milk white and has a diameter of 3-4 mm (refer to fig. 3). Culturing in NA culture medium at 28 deg.C for 24 hr, simply staining with crystal violet, and performing microscopic examination to obtain short rod-shaped thallus (see FIG. 4); gram staining was positive (see FIG. 5). Spore staining revealed that the spores were oval.
(2) Molecular biological Properties
The sequence determination result of the 16S rDNA gene of the strain is as follows (SEQ-1):
CTAGCTCCTTACGTGTATACTCTCCGACTTCGGGTGTTACAAACTCTCGTGGTGTGA CGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTAC TAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTT ATGGGATTGGCTTGACCTCGCGGTCTTGCAGCCCTTTGTACCATCCATTGTAGCACGTGT GTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGT CACCGGCAGTCACCTTAGAGTGCCCAACTAAATGCTGGCAACTAAGATCAAGGGTTGCG CTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCAC CTGTCACTCTGTCCCCCGAAGGGGAACGCTCTATCTCTAGAGTTGTCAGAGGATGTCAA GACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGG GCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTA ATGCGTTAGCTGCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTA CGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGT CAGTTACAGACCAAAAAGCCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTC ACCGCTACACGTGGAATTCCGCTTTTCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGA CCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGCGCGCT TTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACG TAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACGAGCAGTTACTCTCGTACTT GTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTG CTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTG GGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTATGCATCGTTGCC TTGGTGAGCCGTTACCTCACCAACTAGCTAATGCACCGCGGGCCCATCTGTAAGTGATAG CCGAAACCATCTTTCAATCATCTCCCATGAAGGAGAAGATCCTATCCGGTATTAGCTTCG GTTTCCCGAAGTTATCCCAGTCTTACAGGCAGGTTGCCCACGTGTTACTCACCCGTCCGC CGCTAACGTCATAGAAGCAAGCTTCTAATCAGTTCGCTCGACTGCATTATAGTCTGC。
through microbiological characteristics and molecular biological characteristics, the strain AU0102 is identified as Bacillus aryabhattai, which is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 01, 15 and with the preservation address of Beijing China and the preservation number of CGMCC NO. 15218.
Example 4: bacillus aryabhattai AU0102 antibacterial broad-spectrum study
The plate confronting method is adopted to measure the bacteriostatic ability of the bacillus aryabhattai AU0102 on pathogenic bacteria (18 pathogenic fungi and 4 pathogenic bacteria) to be tested, and the specific method is as follows:
control group: the activated pathogenic bacteria to be tested are inoculated in the center of a PDA or NA plate, and 2 mu L of sterile water is inoculated at the periphery of 1.5cm away from the center.
Experimental groups: inoculating the activated pathogenic bacteria to be tested to the center of a PDA or NA plate, and inoculating 2 mu L of the activated Bacillus aryabhattai AU0102 bacterial solution (the Bacillus aryabhattai AU0102 is subjected to streak culture on an NA culture medium and is subjected to constant temperature culture at 28 ℃ for 24h to obtain the bacterial solution) to the periphery of 1.5cm away from the center.
Each treatment was repeated three times. Culturing the above treatments in an incubator at 28 deg.C, photographing after pathogenic bacteria of control group overgrow the plate, detecting the size of lesion of experimental group, and calculating the antibacterial rate.
As a result, the analysis shows that the Bacillus aryabhattai AU0102 has different degrees of inhibitory effects on the growth of 18 pathogenic fungi and 4 pathogenic bacteria (refer to tables 3 and 4); particularly, the inhibition effect on pine needle brown spot pathogen and pine wilt disease fungus is obvious, and the inhibition effect is 93.90% and 91.19% respectively. The result shows that the Bacillus aryabhattai AU0102 has inhibition effect on plant diseases of different families and has broad antibacterial spectrum; the microbial pesticide has the best effect of inhibiting pine needle brown spot germs, can be developed for pine needle brown spot microbial pesticides, and realizes green and efficient prevention and treatment of pine needle brown spot.
TABLE 3 inhibitory Effect of AU0102 on pathogenic fungi
TABLE 4 inhibitory Effect of AU0102 on pathogenic bacteria
Note: "+" indicates significant inhibition.
Example 5: process for preparing liquid preparation of Bacillus aryabhattai AU0102
The formula of the seed liquid culture medium comprises: peptone 1%, beef powder 0.3%, sodium chloride 0.5%, and water in balance, adjusting pH to 7.3 + -0.1.
The fermentation medium formula comprises: soluble starch 2.2%, fish peptone 3.2%, magnesium sulfate 0.15%, sodium chloride 0.6%, and water in balance, and adjusting pH to 7.1 + -0.1.
The preparation process of the liquid preparation of the Bacillus aryabhattai AU0102 comprises the following steps:
(1) obtaining a seed liquid: a small amount of bacteria of Bacillus aryabhattai AU0102 is picked from the test tube slant, transferred into a seed liquid culture medium, and subjected to constant temperature shaking culture at 28 ℃ and 120r/min for 24h to obtain a seed liquid.
(2) Large-scale fermentation culture: sterilizing the fermentation medium material in a fermentation tank (at the temperature of 121-124 ℃ for 30 min); and (3) pouring the cultured seed liquid (according to the inoculation volume ratio of 5-10%) into a fermentation tank under the aseptic condition. Fermenting and culturing for 24-28 h at the temperature of 28 ℃. The number of bacteria can reach about 500 hundred million/ml, and the spore transformation rate can reach about 85%. The fermentation liquid is AU0102 liquid preparation.
Example 6: prevention and treatment effect of Bacillus aryabhattai AU0102 liquid preparation on pine needle brown spot
This example provides relevant experiments with bacillus aryabhattai AU0102 liquid formulation against pine needle brown patch.
(1) Reagent for testing
AU0102 liquid formulation (prepared in example 5); wettable powder of 75% carbendazim (commercially available).
(2) Test article and control object
The crop to be tested is black pine; the control object is pine needle brown patch.
(3) Conditions of the test
The test site is arranged in a black pine forest field in the green region of cigarette terrace city in Shandong province, and pine needle brown spot diseases are serious in the site in the past year; and (4) performing a forest test for preventing and treating pine needle brown spot in 2019, 4 and 15 days. The cultivation conditions and management measures of all the test cells are consistent.
(4) Test design and arrangement
The test designs 500 times, 700 times and 1000 times dilution treatment of Bacillus aryabhattai AU0102 liquid preparation, and 5 treatments of 75% carbendazim 700 times and no-drug application clear water as control, wherein each cell is 30m3The above steps are repeated 4 times, and all cells are randomly arranged. The application (pine needle spraying) is carried out respectively at 15 days in 4 months, 30 days in 4 months, 15 days in 5 months and 30 days in 5 months, the application amount (diluted) is 50 kg/cell, and the disease condition is investigated at the end of 6 months.
(5) Test investigation and calculation method
And counting the disease incidence after the interval of 30d after the last time of medication, and calculating the prevention and treatment effect.
The disease progression of pine needle brown spot is shown in table 5.
TABLE 5 grading criteria for pine needle brown spot disease
Disease index DI (%) [ Σ (number of disease stages) × number of diseased plants in the stage) ]/[ (highest number of disease stages × total number of investigated plants) ]/(100) control effect (%) (control disease stage index-treatment disease index)/control disease index × 100
The obtained data were subjected to statistical analysis using DPS data processing software and Excel software.
(6) Results
TABLE 6 disease index and prevention and treatment effect of pine needle brown spot after each treatment
After 30 days of treatment, the disease incidence is investigated, the disease incidence index and the disease prevention effect are counted, and the results are shown in a table 6. The analysis shows that the incidence index of the clear water control reaches 52.67 percent, and the incidence index of each treatment is lower than that of the clear water control. The morbidity indexes of 300-fold, 500-fold and 700-fold diluted solutions of AU0102 liquid preparation are respectively 2.42%, 3.76% and 5.67%, and the morbidity index of 700-fold diluted solution of 75% carbendazim is 8.19%; AU0102 has control effects of 95.42%, 92.86% and 89.24% respectively, wherein the control effect of 300 times diluted preparation is the best, and has significant difference with other treatments on the level P < 0.05; the control effect of the 75% carbendazim 700-fold diluent is 84.45%. In conclusion, the liquid preparation containing the bacillus aryabhattai AU0102 can effectively control the harm of pine needle tan disease, and the control effect is better than that of 75% carbendazim wettable powder.
SEQUENCE LISTING
<110> Shandong Guavi forestry prevention and control Co Ltd
<120> bacillus aryabhattai with broad-spectrum bacteriostasis and application thereof
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1427
<212> DNA
<213> 16S rDNA Gene sequence of Bacillus aryabhattai (Bacillus aryabhattai) AU0102
<400> 1
ctagctcctt acgtgtatac tctccgactt cgggtgttac aaactctcgt ggtgtgacgg 60
gcggtgtgta caaggcccgg gaacgtattc accgcggcat gctgatccgc gattactagc 120
gattccagct tcatgtaggc gagttgcagc ctacaatccg aactgagaat ggttttatgg 180
gattggcttg acctcgcggt cttgcagccc tttgtaccat ccattgtagc acgtgtgtag 240
cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg gtttgtcacc 300
ggcagtcacc ttagagtgcc caactaaatg ctggcaacta agatcaaggg ttgcgctcgt 360
tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca ccacctgtca 420
ctctgtcccc cgaaggggaa cgctctatct ctagagttgt cagaggatgt caagacctgg 480
taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540
tcaattcctt tgagtttcag tcttgcgacc gtactcccca ggcggagtgc ttaatgcgtt 600
agctgcagca ctaaagggcg gaaaccctct aacacttagc actcatcgtt tacggcgtgg 660
actaccaggg tatctaatcc tgtttgctcc ccacgctttc gcgcctcagc gtcagttaca 720
gaccaaaaag ccgccttcgc cactggtgtt cctccacatc tctacgcatt tcaccgctac 780
acgtggaatt ccgcttttct cttctgcact caagttcccc agtttccaat gaccctccac 840
ggttgagccg tgggctttca catcagactt aagaaaccgc ctgcgcgcgc tttacgccca 900
ataattccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960
gtggctttct ggttaggtac cgtcaaggta cgagcagtta ctctcgtact tgttcttccc 1020
taacaacaga gttttacgac ccgaaagcct tcatcactca cgcggcgttg ctccgtcaga 1080
ctttcgtcca ttgcggaaga ttccctactg ctgcctcccg taggagtctg ggccgtgtct 1140
cagtcccagt gtggccgatc accctctcag gtcggctatg catcgttgcc ttggtgagcc 1200
gttacctcac caactagcta atgcaccgcg ggcccatctg taagtgatag ccgaaaccat 1260
ctttcaatca tctcccatga aggagaagat cctatccggt attagcttcg gtttcccgaa 1320
gttatcccag tcttacaggc aggttgccca cgtgttactc acccgtccgc cgctaacgtc 1380
atagaagcaa gcttctaatc agttcgctcg actgcattat agtctgc 1427
Claims (9)
1. A broad-spectrum bacteriostatic Bacillus aryabhattai (Bacillus aryabhattai) AU0102, wherein the preservation number of the strain is CGMCC NO. 15218.
2. Use of bacillus aryabhattai AU0102 according to claim 1 for the inhibition of pathogenic fungi.
3. The use according to claim 2, wherein the pathogenic fungi is any one or more of pine needle brown spot pathogen, pine wilt disease pathogen, poplar canker pathogen, apple ring rot pathogen, tomato gray mold pathogen, tobacco brown spot pathogen, tomato early blight pathogen, peanut verticillium wilt pathogen, strawberry gray mold pathogen, tobacco black shin pathogen, wheat red mold pathogen, pepper anthracnose pathogen, broad bean withered disease pathogen, watermelon wilt pathogen, grape anthracnose pathogen, peach brown rot pathogen, grape white rot pathogen, and walnut brown spot pathogen.
4. Use of bacillus aryabhattai AU0102 according to claim 1 for the inhibition of pathogenic bacteria.
5. The use according to claim 4, wherein the pathogenic bacteria is any one or more of citrus canker pathogen, ginger bacterial wilt pathogen, walnut black spot pathogen and watermelon soft rot pathogen.
6. The method for fermenting the bacillus aryabhattai AU0102, according to claim 1, wherein after the bacillus aryabhattai AU0102 is used for preparing the seed solution, the seed solution is inoculated into a fermentation medium and is subjected to fermentation culture at 26-30 ℃ for 20-30 hours to obtain the fermentation broth.
7. The method for fermenting bacillus aryabhattai AU0102 according to claim 6, wherein said fermentation medium is, by weight: 2.0-2.5% of soluble starch, 3.0-3.5% of fish peptone, 0.1-0.2% of magnesium sulfate, 0.5-0.8% of sodium chloride and the balance of water, and adjusting the pH value to 7.1 +/-0.1.
8. A fermentation broth of Bacillus aryabhattai AU0102 produced by the fermentation process of claim 6 or 7.
9. Use of the bacillus aryabhattai AU0102 fermentation broth of claim 8 for the control of pine needle tan disease.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114891679A (en) * | 2022-05-19 | 2022-08-12 | 山东省林业科学研究院 | Pseudomonas fluorescens and application thereof in prevention and treatment of cherry branch diseases |
| CN116218724A (en) * | 2023-02-02 | 2023-06-06 | 河北省科学院生物研究所 | Bacillus aryabhattai, microbial agent and application thereof |
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| NL2031759B1 (en) * | 2022-05-02 | 2023-11-13 | Guangxi Academy Agricultural Sciences | Bacillus and its Application and Multi-strain Fermentation Compound Fermentation Plant Enzyme |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942505A (en) * | 2010-04-28 | 2011-01-12 | 南京农业大学 | Pathogenic aeromonas hydrophila assay kit |
| CN101993836A (en) * | 2010-06-13 | 2011-03-30 | 河南省农业科学院 | Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof |
| CN105993273A (en) * | 2016-05-31 | 2016-10-12 | 山东胜伟园林科技有限公司 | Method for carrying out original soil cultivation on mild saline-alkali soil through needle-leaved tree fallen leaf organic matter containing nutrient soil |
| CN108841744A (en) * | 2018-06-15 | 2018-11-20 | 保定微控生物科技有限公司 | A kind of bacillus subtilis with diseases prevention and degradation Phos double action |
-
2022
- 2022-03-09 CN CN202210230797.9A patent/CN114456986B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942505A (en) * | 2010-04-28 | 2011-01-12 | 南京农业大学 | Pathogenic aeromonas hydrophila assay kit |
| CN101993836A (en) * | 2010-06-13 | 2011-03-30 | 河南省农业科学院 | Bacillus subtilis strain YB-81, fungicide and preparation method and application thereof |
| CN105993273A (en) * | 2016-05-31 | 2016-10-12 | 山东胜伟园林科技有限公司 | Method for carrying out original soil cultivation on mild saline-alkali soil through needle-leaved tree fallen leaf organic matter containing nutrient soil |
| CN108841744A (en) * | 2018-06-15 | 2018-11-20 | 保定微控生物科技有限公司 | A kind of bacillus subtilis with diseases prevention and degradation Phos double action |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2031759B1 (en) * | 2022-05-02 | 2023-11-13 | Guangxi Academy Agricultural Sciences | Bacillus and its Application and Multi-strain Fermentation Compound Fermentation Plant Enzyme |
| CN114891679A (en) * | 2022-05-19 | 2022-08-12 | 山东省林业科学研究院 | Pseudomonas fluorescens and application thereof in prevention and treatment of cherry branch diseases |
| CN114891679B (en) * | 2022-05-19 | 2023-06-20 | 山东省林业科学研究院 | Pseudomonas fluorescens and application thereof in preventing and controlling cherry branch diseases |
| CN116218724A (en) * | 2023-02-02 | 2023-06-06 | 河北省科学院生物研究所 | Bacillus aryabhattai, microbial agent and application thereof |
| CN116574642A (en) * | 2023-04-19 | 2023-08-11 | 吉林农业大学 | Lecaniella albopictus YZ-151 fermentation preparation and preparation method and application thereof |
| CN116574642B (en) * | 2023-04-19 | 2024-05-28 | 吉林农业大学 | Lecaniella albopictus YZ-151 fermentation preparation and preparation method and application thereof |
| CN117025444A (en) * | 2023-06-29 | 2023-11-10 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Polystyrene plastic degrading bacterium and method for degrading plastic by using same |
| CN117025444B (en) * | 2023-06-29 | 2024-04-02 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Polystyrene plastic degrading bacterium and method for degrading plastic by using same |
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