CN103215210B - Bacillus siamensis and application of bacillus siamensis in preparation of microbial seaweed fertilizer - Google Patents
Bacillus siamensis and application of bacillus siamensis in preparation of microbial seaweed fertilizer Download PDFInfo
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Abstract
The invention provides bacillus siamensis and application of the bacillus siamensis in the preparation of a microbial seaweed fertilizer. The bacillus siamensis L13 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on 6th March 2013 under the CGMCC No: 7285. The screened high-activity bacillus siamensis fermentation liquor provided by the invention is used for preparing a disinfectant and a promoter, and also can be applied to preparing the efficient microbial seaweed fertilizer. The microbial seaweed fertilizer is not only complete in nutrition, but also prominent in stress resistance, disease and insect resistance, and growth promoting capacity, can replace a farm chemical, a fertilizer, and a hormone, improves soil properties and product quality and causes the product to reach the green or organic standard.
Description
Technical field
The present invention relates to microorganism and microbial fertilizer field, relate in particular to a strain Lyceum genus bacillus and the application in microorganism marine alga fertilizer thereof.
Background technology
China various places are the long-term chemical fertilizer that uses in leaf tobacco production, causes soil depletion, soil compaction, and nutrient imbalance, causes tobacco leaf interior quality to decline, aroma quality variation.In addition crop rotation year after year, disease and pest increases the weight of, and resistance enhancing, and vega disruption of ecological balance, has even had a strong impact on the Sustainable development of leaf tobacco production.The high speed development of agricultural and the problems such as the agriculture soil erosion, soil productivity decline, agricultural-food and the agricultural environment pollution that cause, environmental destruction, caused social extensive concern, Quality Safety Problems of Agricultural Products particularly, becomes the focus of public attention.For this reason; the Alternative Agriculture of the various ways such as Organic farming, biological husbantry, natural agriculture, the ecological agriculture and sustainable agriculture that in succession occur, by a lot of countries as solving food safety, protect species diversity, carry out the approach put into practice of the series of problems such as Sustainable development.And " nuisanceless ", " green ", " organic " are also becoming the new ideas that indicate agricultural product security, health, environmental protection.Organic tobacco leaf, along with the development of Organic farming, is constantly subject to people's attention and favors.
In the problem that microbial fertilizer brings at solution chemical fertilizer, there is original effect, and obtained good result of use, emphasis and the support of the agrotechnique development of Jiang Shi China.Beneficial microorganism kind in microbial fertilizer, whether vital movement is vigorous is the key of its validity, and high-activity microorganism and the meta-bolites thereof of finding antagonism pathogenic bacterium or pathogenic insect prevent and solve study hotspot and the breach that soil-borne disease is current microbial fertilizer.
Summary of the invention
In order to overcome the deficiency of traditional chemical fertilizer and common micro-organisms fertilizer, the object of this invention is to provide a kind of Lyceum genus bacillus and the application in preparation microorganism marine alga fertilizer thereof, the active Lyceum genus bacillus of a plant height that utilization of the present invention screens is for the preparation of efficient microorganism marine alga fertilizer.Not only nutrition is complete but also resistance, disease and insect resistance, growth promotion ability outstanding for this microorganism marine alga fertilizer, alternative agricultural chemicals, chemical fertilizer, hormone, and improvement soil property, improves product quality, makes product reach green or organic standard.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A Lyceum genus bacillus L13, its Classification And Nomenclature is Lyceum bacillus siamensis, its deposit number is CGMCC No.7285.
Further improvement to technique scheme: the Gram-negative of described Lyceum genus bacillus L13; Colony diameter 2-4mm, neat in edge, moistening smooth, high projection, sticky, be pure white; It is short and small shaft-like that thalli morphology is, raw or end life in gemma.
Further improvement to technique scheme: described Lyceum genus bacillus L13 has bacteriostatic action to tobacco alternaric bacteria, tobacco mosaic viruses disease, Alternaria alternate, black shank, sharp Fusariumsp oronoco bacterium.
The application of the bacterial strain fermentation liquor of Lyceum genus bacillus L13 described in the present invention also provides in preparing sterilizing agent.
Further improvement to technique scheme: the leaf-cutting that is used for sterilizing of described Lyceum genus bacillus L13 bacterial strain fermentation liquor is cut operating apparatus, viable count in fermented liquid >=200,000,000/mL.
The application of the bacterial strain fermentation liquor of Lyceum genus bacillus L13 described in the present invention also provides in preparing growth-promoting agent.
Further improvement to technique scheme: the bacterial strain fermentation liquor of described Lyceum genus bacillus L13 has growth-promoting functions, viable count in fermented liquid >=200,000,000/mL to the fresh weight of mung bean, root length, plant height.
The application of the bacterial strain fermentation liquor of Lyceum genus bacillus L13 described in the present invention also provides in preparation microorganism marine alga fertilizer.
Further improvement to technique scheme: Lyceum genus bacillus living bacteria count >=200,000,000/g in described microorganism marine alga fertilizer, organic >=35%.
Further improvement to technique scheme: the culture medium prescription for strain fermentation is as follows: Semen Maydis powder 6.0-8.0g/L, yeast powder 0.5-2.0g/L, glucose 3.0-5.0g/L, peptone 1.0-3.0g/L, soybean cake powder 10.0-12.0g/L, dipotassium hydrogen phosphate 0.5-1.0g/L, calcium carbonate 3.0-5.0g/L, magnesium sulfate heptahydrate 0.8-1.0g/L, manganous sulfate 0.1-0.5g/L, defoamer 1.5-3.0g/L, pH8.0-8.5.
Compared with prior art, advantage of the present invention and positively effect are: Lyceum Bacillus strain of the present invention has antimicrobial spectrum and very significant fungistatic effect very widely, mycelial growth to tobacco alternaric bacteria [Alternaria alternata (Fries) Keissler] has very strong restraining effect, and inhibiting rate is more than 80%; To the restraining effect of tobacco mosaic viruses sick (Tobacco mosaic virus, TMV) more than 85%; Alternaria alternate (Tobacco blown spot) and black shank (Tobacco black shank) are also had to good prevention effect, root rot, virus disease are also had to good result, and the fermented liquid of bacterial strain also can play to instrument the effect of sterilization on producing.
The microorganism marine alga fertilizer that the present invention produces is by marine alga activity extract, amino acid, organic humic acid, micronutrient boron, manganese, copper, zinc, prepare by a certain percentage living bacteria count >=200,000,000/g, the effectively compound microbial marine alga fertilizer of organic >=35%, this marine alga fertilizer not only nutrition is complete, and resistance, disease and insect resistance, growth promotion ability are very outstanding, crop yield amplitude reaches more than 25%, alternative agricultural chemicals, chemical fertilizer, hormone, avoid chemical pollution, improvement soil property, improve product quality, make product reach green or organic standard.
After the specific embodiment of the present invention, it is clearer that the other features and advantages of the invention will become.Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is described in further detail.
Embodiment 1
One, the separation screening of Lyceum genus bacillus
On July 4th, 2012 samples altogether more than 50 parts of rhizosphere soils from Yishui, Shandong, cigarette district, Zhucheng tobacco-growing soil, gets respectively 2g sample and is suspended in 20ml physiological saline, 180rpm, concussion 30min.Getting respectively 100 μ L sample suspension is evenly coated with at nutrient agar plate, in 30 ℃ of incubators, be inverted and cultivate 16h, single bacterium colony that picking comes in every shape is inoculated on nutrient agar plate by method of scoring, by repeated multiple times line, obtains pure bacterium, filters out altogether the pure bacterium of 88 strain.
Obtained strains is inoculated in liquid NB substratum with transfering loop, and 28 ℃, 180rpm, cultivates 64h, and the fermented liquid of cultivation is in 10000rpm, centrifugal 10min, and most of thalline of leaving away, collects supernatant liquor, and gained supernatant liquor is carried out to anti-TMV Determination of biological activity.Select health, the eugonic 6-7 leaf phase three lives-NN cigarette to inoculate.The TMV tobacco tender leaf of fully morbidity is ground to form to homogenate with sterilizing mortar, with phosphoric acid buffer (PH7.0) the 1:40 dilution proportion after sterilizing.The mixed liquid of Zuo Banye inoculation NB substratum and viral equal-volume compares, and the mixed liquid of right half leaf inoculation fermentation supernatant liquor and viral equal-volume deals with, and after inoculation, water sprays blade face immediately.3-4 blade of every processing inoculation, repeats observations after 3d 3 times.Through preliminary test, have the bacterial strain of antagonistic activity to adopt same procedure repeated test, every processing 3 strains, repeat for three times.
Withered spot inhibiting rate calculation formula:
Withered spot inhibiting rate experimental result shows that bacterial strain L13 is best to the inhibition of TMV, and the withered spot inhibiting rate of TMV is reached to 94.16%, and biocontrol bacteria has produced in growth metabolism process has the antibiotics that suppresses virus activity.
Through bacteria molecule genetic classification authentication method, identify, bacterial strain L13 is bacillus, Lyceum genus bacillus.Lyceum genus bacillus biological characteristics: Gram-negative, the about 3mm of colony diameter forming on plate culture medium, neat in edge, moistening smooth, high projection, sticky, pure white.It is short and small shaft-like that thalli morphology is, raw or end life in gemma.
The restraining effect of table 1L13 to tobacco mosaic viruses
Two, the bacteria molecule genetic classification of Lyceum genus bacillus is identified
First extract the DNA of bacterial strain L13, take this DNA as template, 16S rDNA universal primer is primer, and 16S rDNA fragment is increased.The fragment of amplification is directly carried out to sequencing.Blast program on the result input NCBI website of order-checking is compared, result shows that the 16s rDNA sequence homology degree of the Bacillus siamensis in bacillus in this bacterial strain 16S rDNA sequence and GenBank gene pool is the highest, and homology reaches 99%.By DNAMAN6.0, the 16S rDNA sequence of existing bacillus in Genbank is carried out to phylogenetic analysis, result shows, bacterial strain L1316S rDNA and Bacillus siamensis homology are the highest, therefore can judge the Lyceum genus bacillus that this bacterial strain is bacillus.
The bacterial strain L13 screening is carried out to culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on March 6th, 2013; Deposit number is: Lyceum genus bacillus L13(Bacillus siamensis) be numbered CGMCC No.7285.
Three, Lyceum genus bacillus suppresses active detection to fungal disease
Adopt the bacteriostatic activity of dull and stereotyped face-off method test Lyceum genus bacillus to indicator, the dull and stereotyped central authorities of 9cm PDA inoculation indicator bacterium piece, apart from indicator bacterium piece 2.8cm place, inoculating the single bacterium colony of Lyceum genus bacillus, opposite side is not inoculated Lyceum genus bacillus, for measuring blank indicator radius, 25 ℃ of constant temperature culture 5d, observe indicator mycelial growth situation, measure antibacterial result.
Antibacterial width (mm)=blank side indicator radius-inoculation L13 genus bacillus side indicator radius
Table 2 Lyceum genus bacillus is active to the inhibition of tobacco fungal disease
Lyceum genus bacillus has good restraining effect to several fungal diseases that grow tobacco as can be seen from Table 2, wherein the inhibiting rate of tobacco black shank bacterium, tobacco alternaric bacteria, sharp Fusariumsp oronoco has been reached respectively to 73%, 85% and 69%.Lyceum genus bacillus has the prevention effect of wide spectrum to tobacco fungal disease.
Four, the optimization of Lyceum fermentation of bacillus substratum
Prepare Lyceum genus bacillus seed liquor, then by 3% inoculative proportion, be inoculated in fermention medium, by adjusting fermentative medium formula, pH value, leavening temperature, inoculative proportion optimization of fermentation conditions.
The fermentative medium formula of optimizing is as follows: Semen Maydis powder 6.0-8.0g/L, yeast powder 0.5-2.0g/L, glucose 3.0-5.0g/L, peptone 1.0-3.0g/L, soybean cake powder 10.0-12.0g/L, dipotassium hydrogen phosphate 0.5-1.0g/L, calcium carbonate 3.0-5.0g/L, magnesium sulfate heptahydrate 0.8-1.0g/L, manganous sulfate 0.1-0.5g/L, defoamer 1.5-3.0g/L, pH8.0-8.5.
Fermentation results: pH7.0 left and right, living bacteria count is 30,000,000,000/mL.
Five, the disinfection of the bacterial strain fermentation liquor of Lyceum genus bacillus L13
Sterilizing scissors soaks 1min in 40 times of TMV juice, after taking-up respectively 1. following～4. in liquid, soak 30sec, on 5～6 common cigarette N89 of leaf phase, cut 3, top leaf.Every processing 9 strains, repeat for 3 times.15d investigates disease index.1. L13 fermented liquid, viable count is more than 200,000,000/mL, 2. fermention medium, 3. clear water, does not 4. soak, and directly cuts NC89 blade.
The disinfection that table 3 Lyceum fermentation of bacillus liquid is cut leaf-cutting
By table 3, found out, scissors is immersed in TMV juice, has picked viral juice, then soaks in L13 bacterial strain fermentation liquor, can kill whole virus, and sickness rate is only 0, therefore, produces and can the operating apparatus such as cut with the leaf-cutting of sterilizing of L13 fermented liquid.In test, also observe the processing being immersed in fermention medium and clear water, sickness rate, also lower than the processing of 4. not soaking, is because diluted the virus concentration picking on scissors, has reduced invasiveness.
Six, the growth-promoting functions of Lyceum genus bacillus L13 bacterial strain fermentation liquor
Filter paper is pruned to suitably size, be placed in culture dish, L13 fermented liquid is centrifugal to be gone after thalline, dilutes respectively 1 times, 5 times, 25 times, 125 times, 625 times, moistening filter paper.Choose full mellow and full mung bean, ten, each culture dish, and contrast with sterilising medium, observe every day and guarantee that filter paper is moistening.Investigate percentage of germination, investigate fresh weight, root length, plant height.
The growth-promoting functions of table 4L13 fermentation liquid irrigating root to mung bean
By table 4, found out, after the diluted fermentation liquor treatment of mung bean, its fresh weight, root is long, plant height is processed apparently higher than fermention medium, and the bacterium liquid of not diluted suppresses the growth of mung bean, and percentage of germination only has 50%, and bud length only has 0.5cm.The bacterium liquid that dilutes 5 times has restraining effect to taking root of mung bean, but the increase of plant height is had to promoter action.
Seven, the preparation of Lyceum genus bacillus and marine alga compound manure
Zymocyte liquid and marine alga activity extract, amino acid, organic humic acid, micronutrient boron, manganese, copper, zinc are prepared to a kind of living bacteria count >=200,000,000/g by a certain percentage, the effectively compound microbial marine alga fertilizer of organic >=35%.
Eight, the impact of microorganism marine alga fertilizer on cigarette strain growth
Get common cigarette NC89 of 2-3 leaf phase, transplant to the small flower of diameter 10cm, during transplanting, with microorganism marine alga fertilizer, fill with root, every strain 10ml, interval 3d, then continuous irrigation root 2 times, take fermention medium, sterilized water, to fill with root be contrast, every processing 6 strains, repeat for 3 times.Last is filled with after root 3d, investigation fresh weight, maximum leaf length and width.
Table 5 microorganism marine alga fertilizer is filled with the growth-promoting functions of root to NC89
By table 5, found out, common cigarette NC89, fills with root continuous three times, and microorganism marine alga fertilizer is processed its fresh weight, and maximum leaf length and width is all processed higher than substratum and clear water, extremely remarkable with clear water processing difference.Wherein microorganism marine alga fertilizer, its cigarette strain growth of fermention medium root irrigation are better than clear water and process.
Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, the technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. a Lyceum genus bacillus L13, is characterized in that: its Classification And Nomenclature is Lyceum bacillus siamensis, and its deposit number is CGMCC No.7285.
2. a kind of Lyceum genus bacillus L13 according to claim 1, is characterized in that: described Lyceum genus bacillus L13 Gram-negative; Colony diameter 2-4 mm, neat in edge, moistening smooth, high projection, sticky, be pure white; It is short and small shaft-like that thalli morphology is, raw or end life in gemma.
3. a kind of Lyceum genus bacillus L13 according to claim 1 and 2, is characterized in that: described Lyceum genus bacillus L13 has bacteriostatic action to tobacco alternaric bacteria, tobacco mosaic viruses, tobacco black shank bacterium, sharp Fusariumsp oronoco bacterium.
4. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 3 in the sterilizing agent of preparation kill tobacco mosaic virus TMV.
5. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 4 in preparing sterilizing agent, is characterized in that: the leaf-cutting that is used for sterilizing of described Lyceum genus bacillus L13 bacterial strain fermentation liquor is cut, viable count in fermented liquid >=200,000,000/mL.
6. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 3 in the growth-promoting agent of preparation promotion mung bean fresh weight, root length, plant height.
7. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 6 in preparing growth-promoting agent, it is characterized in that: the bacterial strain fermentation liquor of described Lyceum genus bacillus L13 has growth-promoting functions, viable count in fermented liquid >=200,000,000/mL to the fresh weight of mung bean, root length, plant height.
8. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 3 in preparation microorganism marine alga fertilizer.
9. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 8 in preparation microorganism marine alga fertilizer, is characterized in that: Lyceum genus bacillus living bacteria count >=200,000,000/g in described microorganism marine alga fertilizer, organic >=35%.
10. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 9 in preparation microorganism marine alga fertilizer, it is characterized in that: the culture medium prescription for strain fermentation is as follows: Semen Maydis powder 6.0-8.0 g/L, yeast powder 0.5-2.0 g/L, glucose 3.0-5.0 g/L, peptone 1.0-3.0 g/L, soybean cake powder 10.0-12.0 g/L, dipotassium hydrogen phosphate 0.5-1.0 g/L, calcium carbonate 3.0-5.0 g/L, magnesium sulfate heptahydrate 0.8-1.0 g/L, manganous sulfate 0.1-0.5 g/L, defoamer 1.5-3.0 g/L, pH 8.0-8.5.
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| CN106282067B (en) * | 2016-08-24 | 2019-10-11 | 李丽丽 | Multifunctional agricultural complex micro organism fungicide and probiotics and application |
| CN106342898A (en) * | 2016-08-29 | 2017-01-25 | 佛山市艳晖生物科技有限公司 | Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof |
| CN108018233B (en) * | 2017-12-12 | 2021-09-21 | 广州融新科技发展有限公司 | Siamese bacillus and preparation and application of microbial agent thereof |
| CN108004145B (en) * | 2018-01-24 | 2021-03-02 | 吉林省农业科学院 | Black fungus wall breaking method |
| CN108849986B (en) * | 2018-07-09 | 2020-09-15 | 江苏师范大学 | Application of bacillus simmer in preventing and treating white rot |
| CN110964654A (en) * | 2018-09-29 | 2020-04-07 | 福建省农业科学院农业生物资源研究所 | Bacillus antagonistic to fusarium wilt and application thereof |
| CN109619283B (en) * | 2019-01-04 | 2022-10-04 | 大连民族大学 | Method for improving fermentation efficiency of soybean meal |
| CN109730198B (en) * | 2019-01-04 | 2022-10-04 | 大连民族大学 | Method for improving soybean meal fermentation efficiency by using response surface experiment |
| CN110564651B (en) * | 2019-09-30 | 2021-03-09 | 中国农业科学院烟草研究所 | Bacillus siamensis and application thereof |
| CN110651891B (en) * | 2019-11-05 | 2023-03-31 | 大连民族大学 | Probiotic preparation for improving growth performance, immunity and nutritional quality of turbot |
| CN111040961B (en) * | 2019-11-26 | 2021-09-03 | 广东省石油与精细化工研究院 | Bacillus siamensis for degrading efficient cypermethrin and application thereof |
| CN111034441B (en) * | 2019-12-27 | 2022-03-01 | 沈阳农业大学 | Irrigation method for continuously repairing microbial flora in continuous cropping soil of melon crops |
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