CN108795827B - Bacillus megaterium BM22, and water dispersible granule and application thereof - Google Patents
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Abstract
The invention discloses Bacillus megaterium (Bacillus megaterium) BM22, a water dispersible granule and an application thereof, wherein the strain is preserved in China general microbiological culture collection management center in 2017, 12 and 15 months, and the preservation number is CGMCC No. 15070. The water dispersible granule taking bacillus megaterium BM22 as an active ingredient is obtained by culturing seed liquid, culturing and fermenting in a mixture of a carrier, a wetting agent, a dispersing agent, a binder and a disintegrating agent, and then drying. The bacillus megaterium BM22 and the water dispersible granules thereof provided by the invention have special effect on the black spot of eucommia, are suitable for commercial production, have good affinity to the environment, and have the characteristics of sustainability and low cost.
Description
Technical Field
The invention belongs to the technical field of microorganisms, relates to bacillus megaterium BM22, and a water dispersible granule and application thereof, and particularly relates to a bacillus megaterium BM22 strain, a water dispersible granule and application thereof in black spot of eucommia ulmoides.
Background
Eucommia bark, a special tree species and famous medicinal materials in China, is a plant of the family eucommia, also called bakelite, silk cotton tree, Enzhong, silver silk tree, Chinese diabolo and the like, is a unique two-class protection plant which is cherished or endangered in China, and is called China's magic tree abroad. Eucommia bark has strong adaptability and wide distribution, is cultivated in Yangtze river and yellow river basin, and is mostly cultivated in Guizhou, Shaanxi, Hunan, Jiangxi, Henan, Sichuan and other provinces. It is treasure for the whole body, has wide application and great medicinal value and economic value. The eucommia leaves contain rich vitamin E, carotene and more than 10 elements needed by human body, can be drunk as eucommia tea, is a Chinese famous and precious health-care medicinal material, has the effects of reducing blood pressure, strengthening bones and muscles and tonifying liver and kidney, and has the effects of reducing blood fat, reducing blood sugar, losing weight, relaxing bowels, expelling toxin and promoting sleep; the leaves, the bark and the fruits of the eucommia ulmoides can be extracted to obtain hard rubber, which is also called gutta-percha, has high insulativity, can resist corrosion of acid, alkali, oil and chemicals, and is an excellent material for electric appliance insulation and submarine cables; the oil yield of the eucommia seeds reaches 27 percent, and the eucommia seeds are high-grade industrial oil; the bark of eucommia is a rare Chinese medicinal material, has the effects of tonifying liver and kidney, strengthening bones and muscles, benefiting waist and knees, removing soreness, reducing blood pressure and the like, is one of 42 rare Chinese medicinal materials which are mainly managed in China, and is also one of the traditional Chinese medicinal materials which are exported in large quantities at present. Obviously, the market demand at home and abroad is large, and the prices of the peels and leaves are gradually increased.
The black spot of eucommia bark is a common leaf disease, the disease spots on the leaves are firstly black small spots, then the disease spots are enlarged to be polygonal, the edges are not obvious, the centers of the disease spots are earthy yellow when the disease spots are dried, and the black small spots scattered in the later period are pathogen conidiospore devices. When the disease is serious, the leaves fall off, and the growth of eucommia is seriously influenced. The prevention and control of the eucommia black spot are not reported at home and abroad, and the biological prevention and control of protecting ecological balance, being environment-friendly, safe for people and livestock and sustainable utilization is not involved, so that the research on the biological prevention and control of the eucommia black spot and the preparation thereof is still blank.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art in preventing and treating the eucommia black spot disease, and provides a bacillus megaterium BM22 strain, a water dispersible granule, a preparation method of the water dispersible granule and application of the water dispersible granule in the eucommia black spot disease.
The invention is realized by the following technical scheme:
the Bacillus megaterium BM22 strain is separated from healthy eucommia leaves in Sichuan Mali invertebrate drug field by adopting a plate dilution coating method in 2017 at 6 and 15 days, has the bacteriostasis rate of more than 99 percent on eucommia black spot bacteria, is preserved in China general microbiological culture Collection center (CGMCC) of China institute of microbiology, institute of sciences and microbiology in 2017 at 12 and 15 days, has the address of No. 3 of North Chen Xilu No.1 of the sunward area in Beijing and has the preservation number of CGMCC No. 15070.
The single colony of the bacillus megaterium BM22 cultured on the NA plate culture medium for 2d is milky white, round, rough in surface, wavy in edge, opaque and non-adhesive.
The 16S rDNA sequence of the bacillus megatherium BM22 is shown in SEQ ID NO. 1.
The preparation method of the water dispersible granule of the bacillus megatherium BM22 comprises the following steps:
1) b, fermentation liquor of the bacillus megaterium: sterilizing the nutrient meat culture solution under high pressure, placing the nutrient meat culture solution in a triangular flask under the control of oxygen introduction, inoculating bacillus megaterium slant seeds in an aseptic state, and performing shaking culture to prepare bacillus megaterium fermentation liquor;
2) the carrier, the wetting agent, the dispersing agent, the binder and the disintegrating agent are filled into a wide-angle bottle according to a proportion under the control of oxygen introduction, bacillus megaterium fermentation liquor is inoculated in an aseptic state, the inoculation amount is 15 percent of the total amount, the mixture is kneaded, granulated by a rotary extrusion granulator, dried in a warm blast drying oven at 50 ℃ for 2 to 3 hours until the weight of a sample is unchanged, and sieved by a standard sieve of 5 to 10 meshes to obtain the bacillus megaterium water dispersible granule.
Further, the nutrient meat culture solution is as follows: 7g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of water.
Further, the wetting agent, the dispersing agent, the binder and the disintegrating agent comprise the following components in percentage by weight: wetting agent 5%, dispersing agent 8%, binder 4%, disintegrating agent 6%, and carrier to make up 100%.
Further, the carrier is diatomite, the wetting agent is nekal, the dispersing agent is sodium lignosulfonate, the binder is magnesium aluminum silicate, and the disintegrating agent is sodium carboxymethyl starch.
The performance indexes of the water dispersible granule prepared by the preparation method are as follows: the wetting time is 24s, the disintegration time is 60.28s, the suspension percentage is 86.15 percent, the ph is 6.65, the water content is 1.8 percent, the appearance is white columnar particles, and the continuous foamability is less than 25mL after 1 min.
The bacillus megaterium BM22 water dispersible granule prepared by the preparation method is also within the protection scope of the invention.
The content of live spores in the bacillus megatherium BM22 water dispersible granule is not lower than 1 × 109cfu/g。
The application of the bacillus megaterium BM22 in preventing and treating the eucommia black spot disease is in the protection scope of the invention, and the application of the bacterial suspension or the secondary metabolite of the bacillus megaterium BM22 in preventing and treating the eucommia black spot disease is also in the protection scope of the invention.
The application of the water dispersible granule taking the bacillus megaterium BM22 as an active ingredient in preventing and treating the eucommia black spot is to use the diluent of the water dispersant to carry out spray treatment in the plant growth process.
The invention has the beneficial effects that:
the bacillus megaterium BM22 and the water dispersible granules thereof have special effect on the black spot of eucommia ulmoides, have stress resistance, good storability, high temperature resistance, low temperature resistance and drying resistance, are suitable for commercial production, can avoid serious consequences of environmental pollution caused by chemical drugs (pesticides and chemical fertilizers), and have the characteristics of sustainability and low cost.
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FIG. 1 is an electrophoretogram of PCR amplification product of BM22rDNA-ITS sequence of Bacillus megaterium of the present invention;
FIG. 2 is a phylogenetic tree of strain BM22 constructed based on the 16S rDNA sequence.
Detailed Description
The technical solutions of the present invention are further described with reference to the following embodiments, which are merely preferred embodiments of the present invention, and not limiting the present invention in other forms, and any person skilled in the art may change or modify the technical contents disclosed above into equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the scope of the present invention, unless they depart from the technical spirit of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 isolation and characterization of Bacillus megaterium BM22
1.1 isolation of Bacillus megaterium BM22
Taking healthy eucommia leaves in the generation area of eucommia black spot in the Sichuan Mali hupehensis Uygur nest medicine field. The collected sample is washed cleanly by sterile water, 1.0g of tissue is weighed, the tissue is soaked in 70% alcohol for 1-2min, then sterilized by 1-3% sodium hypochlorite solution for 3-5min, after being washed for a plurality of times by sterile water, 100 mu L of the last washing liquid is absorbed to smear an NA flat plate (3 g of beef extract, 10g of peptone, 5g of sodium chloride, 15-20g of agar powder, 1000mL of distilled water and pH 7.0, the mixture is evenly mixed and subpackaged and then is sterilized at 121 ℃ for 30min under high pressure), and the mixture is cultured in the dark at 27 ℃ for 24h for comparison of surface sterilization and checking whether the surface sterilization is complete or not.
Cutting surface-sterilized leaves, placing into sterile mortar, adding sterilized quartz sand and 10mL sterile water, grinding, standing for 30min, and diluting for 10 min-1，10-2，10-3，10-4，10-5Total 5 gradients, 100. mu.L 10 aspirated-3，10-4，10-5The 3 gradient grinding liquids are respectively coated on NA plates (beef extract 3g, peptone 10g, sodium chloride 5g, agar powder 15-20g, distilled water 1000mL, pH 7.0, and after uniform mixing and subpackaging, autoclaving at 121 ℃ for 30min), each treatment is repeated for 3 times, culturing is carried out at 27 ℃ for 48h, and single colonies with obvious colony morphology difference are selected and enter a primary screen.
According to the difference of colony size, color, protrusion, edge characteristic, smooth surface, transparency, etc., selecting single colony, streaking and purifying on NA plate, and transferring the NA slant to 4 deg.c for storage. Inoculating a eucommia black spot pathogenic bacteria cake with the diameter of 5mm at the center of a PDA flat plate according to a five-point opposing method, inoculating the strains to be screened at equal intervals around the PDA flat plate, culturing at constant temperature of 25 ℃, and repeating 3 treatments each. The antagonism degree is determined according to the size of the inhibition zone generated by each screened bacterium, and a strain with excellent effect is reserved (BM 22).
1.2 identification of the strains
The endophyte BM22 was identified as Bacillus megaterium by morphological observation and combination of physiological and biochemical indicators (Table 1) and molecular biology.
The endophyte BM22 was identified as Bacillus megaterium by morphological observation and combination of physiological and biochemical indicators (Table 1) and molecular biology.
TABLE 1 physiological and biochemical indexes of strain BM22
A single colony of 2d BM22 strain cultured on NA plate medium was milky white, round, rough-surfaced, wavy-edged, opaque, and non-adherent.
Molecular biological identification (16S rDNA): extracting bacterial DNA to perform PCR amplification and electrophoresis, and recycling a product to a biotechnology company for sequencing; the determined sequences were subjected to homology BLAST analysis with sequences already reported in GenBank databases and multiple sequence comparisons with Clustalx (1.83) software, and phylogenetic trees were constructed using the adjacency method in Mega4.0 software to determine the phylogenetic status of the BM22 strain in microorganisms. The molecular biology identification obtains a DNA fragment with the length of 949bp (figure 1), the 16S rDNA sequence of the BM22 strain is submitted to a GenBank database for BLAST analysis, the bacterial 16S rDNA sequence with higher homology is selected and is subjected to multiple matching arrangement analysis by Clustalx software, and a phylogenetic tree is constructed by Mega analysis software (figure 2). It can be seen that the BM22 strain supports a poly-1 branch with 99% nucleotide sequence similarity of the 16SrRNA gene and a higher abduction value with JX286698 and is more distant from other Bacillus, indicating that BM22 has a closest relationship to Bacillus megaterium.
Based on the characteristics, the strain is classified and named as Bacillus megaterium (BM22), and has been stored in China general microbiological culture Collection center of China academy of sciences, China Committee of microbiology, 12 months and 15 days in 2017, with the collection number of CGMCC No. 15070.
Example 2 preparation of Bacillus megaterium BM22 Water dispersible granule
The bacillus megaterium BM22 obtained in example 1 is fermented and dried to prepare water dispersible granules, and the method specifically comprises the following steps:
1) fermentation liquor: the nutrient meat culture solution is sterilized under high pressure, bottled according to the proportion that 100mL of liquid is contained in a 300mL triangular flask (oxygen introduction control), inoculated with bacillus megaterium BM22 slant seeds in an aseptic state, inoculated with 2 slant seeds in each flask, and subjected to shaking culture at the temperature of 28-30 ℃, the initial pH value of 6.5 and 120r/min for 36 hours to prepare the bacillus megaterium BM22 fermentation liquid. Wherein the nutrient fleshy culture solution is as follows: 7g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of water.
2) Water dispersible granules: uniformly mixing 5% of nekal, 8% of sodium lignosulfonate, 4% of magnesium aluminum silicate, 6% of sodium carboxymethyl starch and 100% of diatomite, filling the mixture into a 300mL wide-angle bottle under the control of oxygen introduction, inoculating bacillus megaterium fermentation liquor in an aseptic state, wherein the inoculation amount is 15% of the total mass, and kneading the mixture by a mixer; granulating by a rotary extrusion granulator, continuously drying in a warm air drying oven at 50 ℃ for 2-3h until the weight of the sample is unchanged, sieving by a standard sieve of 5-10 meshes to prepare the bacillus megaterium water dispersible granules, and storing.
Water dispersible granule index of 1 × 109cfu/g live spore, the wetting time is 24s, the disintegration time is 60.28s, the suspension percentage is 86.15%, the ph is 6.65, the water content is 1.8%, the appearance is white columnar particles, and the continuous foamability is less than 25mL after 1 min.
And (3) preparation preservation: the prevention and treatment effect is not influenced after the bagged dry storage for 1 year at normal temperature or 2 years at low temperature (4 ℃).
Example 3 prevention effect experiment of Bacillus megaterium BM22 water dispersible granule on black spot of eucommia ulmoides
1) Preparing BM22 water dispersible granule diluent: the 1g of water dispersible granules prepared above was diluted with 50, 100, 200, 500, 800, 1000, 1500mL of each of them to prepare a diluted solution.
2) Preparation of spore suspension of Umbelliferae Peronospora Clarkeyi (Pestalotiopsis carpicola) by preparing conidia of Umbelliferae Peronospora Clarke (Pestalotiopsis Clarkei) cultured on PDA slant with water to obtain spore with concentration of 1 × 106cfu/mL spore suspension.
3) Selecting annual potted eucommia ulmoides seedlings as experimental objects, taking 1) water dispersible granule diluent to spray the seedlings by adopting a spraying method, and setting the following treatment: diluting stock solution 1g/mL by 50 times, 100 times, 200 times, 400 times, 800 times, 1200 times, 1600 times, spraying 1 time (3 times) every 7 days, 30 mL/time, and using sterile water as control. And after 15 days, inoculating the pathogenic bacteria spore suspension on eucommia ulmoides leaves by adopting a spraying method, inoculating 10 strains for each treatment, and moisturizing the inoculated parts, wherein each treatment is repeated for 3 times.
4) Disease investigation and statistics are carried out according to disease grading standards in Table 2 after the inoculation of pathogenic bacteria for 15 days, and the morbidity, disease index and control effect are calculated, and the results are shown in Table 3.
TABLE 2 Classification Standard of diseases of black leaf spot of eucommia ulmoides
Incidence (%) × 100 (number of diseased plants/total number of plants);
the disease index [ Σ (disease-grade number of plants × representative value)/total number of plants and × representative value of the most serious grade of disease ] × 100;
the preventing and treating effect (%) is [ (control disease index increasing rate-treatment disease index increasing rate)/control disease index increasing rate ] × 100.
TABLE 3 control Effect after 15d of Pot culture test
After the inoculation of pathogenic bacteria for 15d, the BM22 water dispersible granule diluent shows good protection effect on the black spot of eucommia ulmoides, and the control effect is reduced along with the increase of dilution times. As can be seen from Table 3, the control effect of the 1gBM22 water dispersible granule diluted into 50 times of solution is the highest, reaching 100 percent, which indicates that the colonization, growth and reproduction of pathogenic bacteria are completely controlled, and no diseases occur. The control effect is still over 60 percent after the solution is diluted to 800 times.
Example 4 Interforest control experiments
1) Reagent for testing
The bacillus megaterium BM22 water dispersible granule prepared in the embodiment 2 of the invention.
2) Disease treatment in field
1g of the water dispersible granules prepared above are prepared into stock solution and 50, 100, 200, 400, 800 and 1600-time diluent. The test area is divided into a mild occurrence area (below 5 percent), a moderate occurrence area (6-30 percent) and a severe occurrence area (above 30 percent) according to different degrees of morbidity, eucommia ulmoides with uniform growth potential for 1-2 years in the areas is selected, the preparation is sprayed along the leaf surfaces, the leaf surfaces are uniformly covered, water drops do not drop, and each plant is about 80 mL.
3) Test investigation
Survey statistics and calculation of control effect were carried out 15d after spraying, and the results are shown in table 4.
Disease investigation, incidence rate, disease index and prevention and treatment effect are calculated as above.
TABLE 4 forest control effect of the spray method on black spot of eucommia ulmoides
As can be seen from Table 4, the spray method is adopted to prevent and treat the black spot of eucommia ulmoides, the prevention effect of each concentration in a mild occurrence area is good, the stock solution is up to 96%, and the dilution time is more than 60% when the stock solution is diluted by 1600 times; the control effect of the stock solution in the moderate occurrence area exceeds 90 percent, and the dilution effect is 50-800 times better and can reach more than 72 percent; the control effect of the stock solution in the severe occurrence area is over 50 percent after dilution by 50 to 200 times. Therefore, the BM22 water dispersible granule has excellent effect on preventing and treating the eucommia black spot by spray treatment. Although the stock solution has the best control effect, in view of production cost, in forest control, the economic concentration is 1600 times that of moderate occurrence, 800 times that of moderate occurrence and 200 times that of 100 times that of severe occurrence.
In conclusion, the bacillus megaterium BM22 strain of the invention has good antagonistic action on the black spot of eucommia ulmoides, has the characteristics of strong stress resistance of spores, long commodity storage life and strong environmental suitability, is produced into a commodity biological agent, has good environmental compatibility, meets the strategic requirements of sustainable development, and has wide market prospect.
Sequence listing
<110> Sichuan university of agriculture
<120> Bacillus megaterium BM22 and water dispersible granule and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>949
<212>DNA
<213>Bacillus megaterium
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caggctgcgg ggctatacat gcagtcgagc gaactgatta gaagcttgct tctatgacgt 60
tagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg ataacttcgg 120
gaaaccgaag ctaataccgg ataggatctt ctccttcatg ggagatgatt gaaagatggt 180
ttcggctatc acttacagat gggcccgcgg tgcattagct agttggtgag gtaacggctc 240
accaaggcaa cgatgcatag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggct ttcgggtcgt aaaactctgt tgttagggaa 420
gaacaagtac gagagtaact gctcgtacct tgacggtacc taaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttatccgga attattgggc 540
gtaaagcgcg cgcaggcggt ttcttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactgggga acttgagtgc agaagagaaa agcggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcggctttt tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaaga 949
Claims (7)
1. The Bacillus megaterium BM22 is characterized in that the Bacillus megaterium BM22 is preserved in China general microbiological culture collection center in 2017, 12 and 15 days, and the preservation number is CGMCC No. 15070.
2. The preparation method of the water dispersible granule of the bacillus megaterium BM22 as claimed in claim 1, characterized by comprising the following steps:
1) bacillus megatherium BM22 fermentation broth: sterilizing the nutrient meat culture solution under high pressure, placing the nutrient meat culture solution in a triangular flask under the control of oxygen, inoculating bacillus megaterium BM22 slant seeds in an aseptic state, and performing shaking culture to obtain bacillus megaterium BM22 fermentation liquor; the nutrient meat culture solution is as follows: 7g of beef extract, 10g of peptone, 5g of NaCl and 1000mL of water;
2) water dispersible granules: loading a carrier, a wetting agent, a dispersing agent, a binder and a disintegrating agent into a wide-angle bottle in proportion under the control of oxygen introduction, inoculating bacillus megaterium BM22 fermentation liquor in an aseptic state, wherein the inoculation amount is 15% of the total amount, kneading the mixture, granulating the mixture by a rotary extrusion granulator, continuously drying the mixture for 2 to 3 hours in a warm blast drying oven at 50 ℃ until the weight of a sample is unchanged, and screening the mixture by a standard sieve of 5 to 10 meshes to obtain the bacillus megaterium BM22 water dispersible granule; the carrier is diatomite, the wetting agent is nekal, the dispersing agent is sodium lignosulfonate, the binder is magnesium aluminum silicate, and the disintegrating agent is sodium carboxymethyl starch.
3. The preparation method according to claim 2, wherein the wetting agent, the dispersing agent, the binder and the disintegrating agent comprise the following components in percentage by weight: wetting agent 5%, dispersing agent 8%, binder 4%, disintegrating agent 6%, and carrier to make up 100%.
4. The bacillus megaterium BM22 water dispersible granule prepared by the preparation method of claim 2.
5. The Bacillus megaterium BM22 water dispersible granule as claimed in claim 4, wherein the content of viable spores in the Bacillus megaterium BM22 water dispersible granule is not less than 1 × 109cfu/g。
6. The application of the bacillus megaterium BM22 as claimed in claim 1 in preventing and treating eucommia black spot.
7. The application of the bacillus megaterium BM22 water dispersible granule in preventing and treating eucommia black spot disease of claim 4 is characterized in that bacillus megaterium BM22 water dispersible granule diluent is used for spraying treatment in the plant growth process.
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| CN102816713B (en) * | 2012-06-12 | 2014-03-19 | 江苏农林职业技术学院 | Bacillus amyloliquefaciens and its application |
| CN103563991A (en) * | 2012-07-31 | 2014-02-12 | 江苏丘陵地区镇江农业科学研究所 | Preparation method of brevibacillus brevis water dispersible granules |
| CN105695353B (en) * | 2016-02-01 | 2020-08-25 | 山西省农业科学院农产品贮藏保鲜研究所 | Novel bacillus strain, preparation method of fermentation product of novel bacillus strain and application of fermentation product in preventing and treating pear black spot |
| CN107760631B (en) * | 2017-11-24 | 2021-04-09 | 四川农业大学 | Bacillus methylotrophicus B18, and granules and application thereof |
| CN108048357B (en) * | 2017-12-26 | 2021-01-05 | 武汉科诺生物科技股份有限公司 | Bacillus subtilis and culture method thereof |
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| CN107858304A (en) * | 2017-11-24 | 2018-03-30 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its liquid preparation and application |
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