CN112812993B - Bacillus belgii capable of preventing and treating root rot of salvia miltiorrhiza and application thereof - Google Patents
Bacillus belgii capable of preventing and treating root rot of salvia miltiorrhiza and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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Abstract
The invention discloses a Bacillus beiLeisi capable of preventing and treating root rot of red sage root, the serial number of the strain is Bacillus beiLeisi Bv1-1, the strain has been preserved in China center for type culture Collection (CCTCC for short), the preservation address is: the preservation number of the registered book is CCTCC M2020925, and the preservation date is 2020, 12 and 21 days. The Bacillus belgii has a good inhibiting effect on root rot pathogenic bacteria of salvia miltiorrhiza, has a control effect on root rot diseases of salvia miltiorrhiza of more than 55%, can effectively reduce the incidence of root rot diseases of salvia miltiorrhiza, and has an effect superior to that of the existing common chemical pesticide. The Bacillus belgii screened by the invention can not generate the common problems of chemical pesticides such as drug resistance, pesticide residue, environmental pollution and the like after being used, is healthy and environment-friendly to people and livestock, and meets the requirements of people on ecological agriculture.
Description
Technical Field
The invention relates to Bacillus belgii capable of preventing and treating root rot of salvia miltiorrhiza and application thereof, belonging to the technical field of microorganisms.
Background
Salvia miltiorrhiza Salvia milithioriza bge is a perennial herb of Labiatae, is taken as a medicine by drying roots and rhizomes, and is one of the first-choice medicines for treating cardiovascular and cerebrovascular diseases. In recent years, the planting of salvia has become an important measure for the poverty elimination and the countryside revivification in poverty-stricken areas in western Henan province. In order to ensure the yield of the salvia miltiorrhiza, most medicinal material growers widely use chemical fertilizers and chemical agents in the planting process of the salvia miltiorrhiza, so that the quality of the salvia miltiorrhiza is reduced to a certain extent, fertilizer damage and chemical damage can also occur under severe conditions, even chemical agent residue exceeds the standard, and meanwhile, the ecological environment of a planting area is polluted. The utilization of beneficial antagonistic bacteria for biological prevention and soil control is a research hotspot which is concerned at present. At present, the biological mode of microorganisms is mainly relied on to control plant diseases, including bacteria, fungi, actinomycetes and the like. The root rot of salvia miltiorrhiza is the most important disease in the production of salvia miltiorrhiza, and the yield of salvia miltiorrhiza is seriously reduced. The root rot pathogens most threatening salvia miltiorrhiza mainly include Fusarium proliferatum, Fusarium solani, Fusarium oxysporum and Alternaria tenuissima. Biological control has been paid attention by students in the aspect of controlling root rot diseases of salvia miltiorrhiza, biocontrol strain screening of pathogenic bacteria F.solani of the root rot disease of salvia miltiorrhiza and microbial inoculum and application thereof have been widely researched, and it has been disclosed that biocontrol strains mainly comprise bacillus subtilis, bacillus amyloliquefaciens, trichoderma and the like, and have fewer effective biocontrol strains on pathogenic bacteria F.proliferum, F.oxysporum and Alternaria sp, so that the screening of biocontrol strains effective simultaneously on different pathogenic bacteria of the root rot disease of salvia miltiorrhiza has important theoretical and practical significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the Bacillus beijerinckii capable of preventing and treating the root rot of the salvia miltiorrhiza, and the screening method and the application of the strain.
The technical scheme adopted by the invention for solving the technical problems is as follows:
the Bacillus beiLeisi capable of producing root rot of red sage root is Bacillus velezensis Bv1-1, which has been preserved in China Center for Type Culture Collection (CCTCC) and has the preservation address: the preservation number of a registered volume is CCTCC M2020925, and the preservation date is 2020, 12 months and 21 days.
The Bacillus beilesensis capable of producing the root rot disease of the salvia miltiorrhiza comprises the following screening methods: collecting salvia miltiorrhiza rhizosphere soil in a salvia miltiorrhiza field, weighing 1g of soil, adding 10ml of sterile water, fully oscillating to obtain a soil suspension, shaking the soil suspension uniformly, diluting, coating 0.2ml of diluent on an LB solid plate, culturing, separating and purifying after bacterial colonies grow out, screening out a strain with an inhibiting effect on salvia miltiorrhiza root rot by using a plate antagonism test, and identifying to obtain bacillus beilesiensis Bv 1-1.
The Bacillus beiLeisi capable of producing the bacillus beiensis for preventing and treating root rot of salvia miltiorrhiza is applied to preventing and treating root rot of salvia miltiorrhiza.
The Bacillus beilesiensis capable of producing the bacterial agent for preventing and treating the root rot of the salvia miltiorrhiza bunge can be applied to preparation of the bacterial agent for preventing and treating the root rot of the salvia miltiorrhiza bunge.
The preparation method of the microbial inoculum comprises the following steps: inoculating Bacillus velezensis (Bacillus velezensis) Bv1-1 into LB culture medium, performing shake culture at 28 deg.C and 180rpm for 12h to prepare seed bacteria, inoculating seed bacteria into LB culture medium at an inoculum size of 2%, and fermenting at 28 deg.C and 180rpm for 48h to form fermentation liquid, i.e. the microbial inoculum.
The concentration of live Bacillus belief (Bacillus velezensis) Bv1-1 in the fermentation liquor is 10 7 ～10 8 CFU/mL。
The LB culture medium is as follows: 10g of tryptone, 5g of yeast extract and 10g of sodium chloride, adding distilled water to a constant volume of 1000mL, uniformly mixing, subpackaging in triangular flasks, and sterilizing at 121 ℃ for 30 min.
The using method of the microbial inoculum comprises the following steps: the microbial inoculum is diluted by 10 times by sterile water to ensure that the viable bacteria concentration of the diluted solution is 10 6 ～10 7 CFU/ml, irrigating root around root of Saviae Miltiorrhizae radix; the root irrigation is carried out 1 time every 7 days, and the root irrigation is carried out 2 times.
The invention has the beneficial effects that: the invention aims to prevent and treat root rot of salvia miltiorrhiza by using microorganisms, so that Bv1-1 capable of preventing and treating root rot of salvia miltiorrhiza is separated from rhizosphere soil of salvia miltiorrhiza through separation identification, plate antagonism and disease-preventing potting experiments and is identified as Bacillus belief (Bacillus velezensis). Experiments show that the Bacillus belgii has a good inhibiting effect on root rot pathogens of salvia miltiorrhiza, has a control effect on root rot diseases of salvia miltiorrhiza of more than 55%, can effectively reduce the morbidity of the root rot diseases of salvia miltiorrhiza, and has an effect superior to that of the existing common chemical pesticide. The Bacillus belgii screened by the invention can not generate the common problems of chemical pesticides such as drug resistance, pesticide residue, environmental pollution and the like after being used, is healthy and environment-friendly to people and livestock, and meets the requirements of people on ecological agriculture.
Drawings
FIG. 1 is a graph comparing the results of plate antagonism tests of strain Bv1-1 against Fusarium heterosporum, Fusarium solani, Fusarium oxysporum and Alternaria tenuis.
FIG. 2 is a colony morphology of the strain Bv 1-1.
FIG. 3 is a graph comparing the results of the allergy test of strain Bv1-1 to tobacco leaves, wherein the left side of the graph is strain Bv1-1, and the right side of the graph is a positive control.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
Separation of biocontrol bacteria
Collecting salvia miltiorrhiza rhizosphere soil in a salvia miltiorrhiza field, weighing 1g of soil, adding 10ml of sterile water, fully oscillating to obtain a soil suspension, shaking the soil suspension uniformly, diluting, taking 200 mu L of diluent, coating the diluent on an LB solid flat plate, culturing until bacterial colonies grow out, picking bacterial colonies with different sizes, colors or forms, streaking, separating and purifying on the surface of an LB solid culture medium to obtain 1 strain, which is named as Bv 1-1.
(II) screening of biocontrol bacteria (Flat plate antagonism test)
Screening biocontrol bacteria by using a plate confrontation method: respectively inoculating fusarium, fusarium solani, fusarium oxysporum and alternaria tenuis on the root rot pathogenic bacteria layer of salvia miltiorrhiza on a PDA culture medium, culturing for 5 days at 28 ℃, preparing cultured root rot pathogenic bacteria of salvia miltiorrhiza into bacterial cakes by using a perforator with a sterilized diameter of 8mm, connecting the bacterial cakes to one side of a new PDA flat plate (the diameter is 90mm), sterilizing a paper disc ring with the diameter of 8mm, placing the paper disc ring on the other side of the PDA culture medium, sucking 10 mu l of biocontrol bacteria suspension, and slowly dripping the biocontrol bacteria and the paper disc ring into the paper disc ring, wherein the biocontrol bacteria and the paper disc ring are positioned on the same straight line during inoculation. Sealing with sealing film, culturing at 28 deg.C, recording the size of the antibacterial band when the control pathogenic bacteria grow to the paper disc, and measuring the colony radius of the corresponding pathogenic bacteria on the plate. The contrast is the culture of Fusarium, Fusarium solani, Fusarium oxysporum and Alternaria gracilis on the root rot pathogenic bacteria layer of the salvia miltiorrhiza and the culture of inoculated sterile water paper discs on PDA, the colony treatment is repeated for at least 3 times, and the result of the culture of confrontation is checked after 5 days, and the result is shown in table 1 and figure 1, wherein Bv1-1 has effects on 4 pathogenic bacteria.
Table 1: bacteriostatic effect of Bacillus belgii (Bv1-1) on root rot pathogenic bacteria of salvia miltiorrhiza
| Pathogenic bacteria | Antibacterial belt (mm) | The antibacterial rate is% |
| Fusarium proliferatum | 5.0 | 65.2 |
| Fusarium solani | 4.6 | 57.6 |
| Fusarium oxysporum | 3.3 | 63.5 |
| Alternaria gracilis | 6.3 | 56.6 |
(III) identification of biocontrol bacteria
And performing morphological identification on the screened strains with good antagonistic effect, and determining the strains to be the Bellis bacillus subtilis through morphological, physiological and biochemical analysis (see table 2) and molecular biological identification. The Bacillus belgii forms milky white colonies on the culture medium, is opaque, has folds on the surface, slightly bulges in the middle, uneven edges and is stiff when lifted (see figure 2).
Table 2: bv1-1 physiological and biochemical characteristics
(IV) sequencing of biocontrol bacteria
Sequencing: the strain Bv1-1 is sequenced, and the identification result is Bacillus belgii (Bacillus velezensis).
The 16SrDNA gene sequencing result is as follows:
<110> institute of plant protection, institute of agricultural sciences, Henan province
<120> Bacillus belgii capable of preventing and treating root rot of salvia miltiorrhiza and application thereof
<141>2021-01-07
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(V) preparation of microbial inoculum
Inoculating Bacillus subtilis Bv1-1 into LB culture medium, performing shake culture at 28 deg.C and 180rpm for 12h to prepare seed bacteria, inoculating seed bacteria into LB culture medium at an inoculum size of 2%, and fermenting at 28 deg.C and 180rpm for 48h to obtain fermentation liquid, i.e. microbial inoculum. The concentration of live Bacillus belief (Bacillus velezensis) Bv1-1 in the fermentation liquor is 10 7 ～10 8 CFU/mL。
(VI) test of tobacco leaf allergy caused by microbial inoculum
Using prepared microbial inoculum to treat tobaccoThe lower epidermis was inoculated subcutaneously with 10 mM MgSO 4 As a negative control, the wild tobacco fire was used as a positive control (YH). Under the conditions of 24 ℃ and 60% -80% high humidity, allergic necrotic spots appear after 24-48h as positive reaction, and yellow spots appear after 3d as negative reaction. As a result, as shown in FIG. 3, the Bacillus bleekii Bv1-1 anaphylaxis test is a negative reaction, so that the Bacillus bleekii Bv1-1 is proved to be non-pathogenic and can be used as a biocontrol bacterium for preventing and treating root rot of salvia miltiorrhiza.
(VII) disease prevention experiment
Germinating Saviae Miltiorrhizae radix seed on a tray filled with sterilized soil, transferring into nutrition pot, culturing for 30 days, inoculating biocontrol bacteria by root irrigation method, wetting soil with small amount of clear water before root irrigation, inoculating Bacillus beijerinckii Bv1-1 bacterial agent (viable bacteria concentration of 10) at rhizosphere 7 ～ 10 8 CFU/mL), diluting the fermentation broth 10 times with sterile water, 10mL for each plant, and inoculating 10mL again at intervals of 7 d; the control group is inoculated with an equal amount of LB culture medium (CK) by the same method, in addition, a medicament positive control group is arranged, the control medicament is 1000-time diluent of 50 percent carbendazim wettable powder, each salvia miltiorrhiza seedling is 10mL, and after 7 days, the roots are irrigated for 1 time and 2 times in total. After the root is irrigated for 3d for the last time in each group, the fusarium solani which is the pathogen of the root rot of salvia miltiorrhiza is inoculated. The inoculation method comprises the following steps: irrigating root, inoculating, collecting 10mL of root rot pathogen spore suspension (concentration is 1 × 10) 6 root/mL) of the seedlings of the salvia miltiorrhiza, watering for 1 time after 3 days, placing the seedlings in a constant temperature incubator at 28 ℃ for culture, and regularly observing the disease condition of the salvia miltiorrhiza. And after culturing for 30 days, investigating and calculating the morbidity, disease index and prevention and treatment effect. The grading standard of the root rot disease of salvia miltiorrhiza is carried out according to related documents, and the result is shown in table 3.
TABLE 3 Bacillus belgii Bv1-1 disease prevention effect on salvia miltiorrhiza root rot pot culture
The result shows that the control effect of the Bacillus beilesensis Bv1-1 on the root rot of the salvia miltiorrhiza is 59.7%, and the effect is obvious.
The foregoing description is only a preferred embodiment of the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> institute of plant protection, institute of agricultural sciences, Henan province
<120> Bacillus belgii capable of preventing and treating root rot of salvia miltiorrhiza and application thereof
<141> 2021-01-07
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gttagggggt ttccgcccct tagtgctgca gctaacgcat taagcactcc gcctggggag 900
tacggtcgca agactgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 960
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc 1020
tagagatagg acgtcccctt cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct 1080
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgatc ttagttgcca 1140
gcattcagtt gggcactcta aggtgactgc cggtgacaaa ccggaggaag gtggggatga 1200
cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg gacagaacaa 1260
agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt tcggatcgca 1320
gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag catgccgcgg 1380
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac cacgagagtt tgtaacaccc 1440
gaagtcggtg aggtaacctt tatggagcca gccgccgaag gtgggacaga tgattggggt 1500
gaagtcgtaa caaggtagcc gta 1523
Claims (6)
1. Bacillus belgii capable of preventing and treating root rot of red sage root, the strain number is Bacillus belgii ((R))Bacillus velezensis) Bv1-1, which is preserved in China center for type culture Collection, and the preservation address is as follows: the preservation number of the registered book is CCTCC M2020925, and the preservation date is 2020, 12 and 21 days.
2. The use of Bacillus belgii according to claim 1 for the control of root rot in Salvia miltiorrhiza.
3. The application of the Bacillus belgii of claim 1 in preparing a microbial inoculum for preventing and treating root rot of Salvia miltiorrhiza.
4. The use of claim 3, wherein the preparation method of the microbial inoculum comprises the following steps: bacillus belgii (B.), (B.), (B.), (B.)Bacillus velezensis) Bv1-1 is inoculated in LB culture medium, the seed bacteria is prepared by shaking culture for 12h under the conditions of 28 ℃ and 180rpm, the seed bacteria is inoculated in the LB culture medium by 2 percent of inoculum size, and the fermentation is carried out for 48h under the conditions of 28 ℃ and 180rpm, thus forming fermentation liquor, namely the microbial inoculum.
5. The method for producing the microbial preparation according to claim 4, wherein Bacillus belgii (Bacillus subtilis) is used as the microbial preparationBacillus velezensis) The concentration of Bv1-1 viable bacteria is 10 7 ～10 8 CFU/mL。
6. The use of claim 3, wherein the microbial inoculum is used by a method comprising: the microbial inoculum is diluted by 10 times by sterile water to ensure that the viable bacteria concentration of the diluent is 10 6 ～10 7 CFU/ml, irrigating roots around roots of Salvia miltiorrhiza; the root irrigation is carried out 1 time every 7 days, and the root irrigation is carried out 2 times.
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