CN103215210A - 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 the microorganism marine alga fertilizer thereof.
Background technology
Soil depletion is caused at the medium-term and long-term chemical fertilizer that uses of leaf tobacco production in China various places, soil compaction, and nutrient imbalance causes the tobacco leaf interior quality to descend the aroma quality variation.Crop rotation year after year in addition, disease and pest increases the weight of, and resistance strengthens, the vega disruption of ecological balance, even had a strong impact on the Sustainable development of leaf tobacco production.The high speed development of agricultural and problems such as the agriculture soil erosion, soil productivity decline, agricultural-food and the agricultural environment pollution that cause, environmental destruction, caused the extensive concern of society, agricultural product quality and safety problem particularly becomes the focus of public attention.For this reason; the alternative agricultural of various ways such as the Organic farming of Chu Xianing, biological husbantry, natural agriculture, the ecological agriculture and sustainable agriculture in succession, by a lot of countries as solving food safety, protect species diversity, carrying out the approach put into practice of a 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 is along with the development of Organic farming, constantly is subject to people's attention and favors.
Microbial fertilizer has original effect on the solution problem that chemical fertilizer brought, and has obtained good result of use, will be the emphasis and the support of China's agrotechnique development.Whether beneficial microorganism kind in the microbial fertilizer, vital movement be vigorous is the key of its validity, and high-activity microorganism and the meta-bolites thereof of seeking antagonism pathogenic bacterium or pathogenic insect prevent and solve research focus and the breach that soil-borne disease is present microbial fertilizer.
Summary of the invention
In order to overcome the deficiency of traditional chemical fertilizer and common micro-organisms fertilizer, the purpose 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 the plant height that utilization of the present invention screens is used to prepare microorganism marine alga fertilizer efficiently.Not only nutrition is complete but also resistance, disease and insect resistance, promotes growth ability outstanding for this microorganism marine alga fertilizer, alternative agricultural chemicals, chemical fertilizer, hormone, and the proterties of improving the soil is improved 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 kind of Lyceum genus bacillus L13, its classification called after Lyceum bacillus siamensis, its deposit number is CGMCC No.7285.
To further improvement in the technical proposal: 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, and life or end are given birth in the gemma.
To further improvement in the technical proposal: described Lyceum genus bacillus L13 has bacteriostatic action to tobacco alternaric bacteria, tobacco mosaic viruses disease, Alternaria alternate, black shank, sharp sickle spore bacterium oronoco bacterium.
The present invention also provides the application of bacterial strain fermentation liquor in the preparation sterilizing agent of described Lyceum genus bacillus L13.
To further improvement in the technical proposal: the leaf-cutting that is used to sterilize of described Lyceum genus bacillus L13 bacterial strain fermentation liquor is cut operating apparatus, viable count in the fermented liquid 〉=200,000,000/mL.
The present invention also provides the application of bacterial strain fermentation liquor in the preparation growth-promoting agent of described Lyceum genus bacillus L13.
To further improvement in the technical proposal: the bacterial strain fermentation liquor of described Lyceum genus bacillus L13 is long to fresh weight, the root of mung bean, plant height has growth-promoting functions, viable count in the fermented liquid 〉=200,000,000/mL.
The present invention also provides the application of bacterial strain fermentation liquor in preparation microorganism marine alga fertilizer of described Lyceum genus bacillus L13.
To further improvement in the technical proposal: Lyceum genus bacillus living bacteria count 〉=200,000,000/g in the described microorganism marine alga fertilizer, organic 〉=35%.
To further improvement in the technical proposal: the culture medium prescription that is used 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, lime 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 tobacco mosaic viruses disease (Tobacco mosaic virus, restraining effect TMV) is more than 85%; Alternaria alternate (Tobacco blown spot) and black shank (Tobacco black shank) also there is good prevention effect, root rot, virus disease are also all had good result, and the fermented liquid of bacterial strain also can play the disinfectant effect to instrument on producing.
The microorganism marine alga fertilizer that the present invention produced is with marine alga activity extract, amino acid, organic humic acid, micronutrient boron, manganese, copper, zinc, prepare living bacteria count 〉=200,000,000/g by a certain percentage, 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, promotes growth ability are very outstanding, the crop yield amplitude reaches more than 25%, alternative agricultural chemicals, chemical fertilizer, hormone, avoid chemical pollution, the proterties of improving the soil, improve product quality, make product reach green or organic standard.
After the specific embodiment of the present invention, other characteristics of the present invention and advantage will become clearer.Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described in further detail.
Embodiment 1
One, Lyceum separation of bacillus screening
Part rhizosphere soil surplus in the of 50 of taking a sample altogether from Yishui, Shandong, Zhucheng cigarette district tobacco-growing soil on July 4th, 2012 is got the 2g sample respectively and is suspended in the 20ml physiological saline, 180rpm, concussion 30min.Getting 100 μ L sample suspension respectively evenly is coated with at nutrient agar plate, be inverted in 30 ℃ of incubators and cultivate 16h, single bacterium colony that picking comes in every shape is inoculated on the nutrient agar plate with method of scoring, obtains pure bacterium by the repeated multiple times line, filters out the pure bacterium of 88 strains altogether.
Obtained strains is inoculated in the 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 is collected supernatant liquor, and the gained supernatant liquor is carried out anti-TMV Determination of biological activity.Select healthy, eugonic 6-7 leaf phase three lives-NN cigarette to inoculate.The TMV tobacco tender leaf of fully morbidity is ground to form homogenate with the sterilization mortar, with phosphoric acid buffer (PH7.0) the 1:40 dilution proportion after the sterilization.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, the blade face of water sprinkling immediately after the inoculation.3-4 blade of every processing inoculation repeats observations behind the 3d 3 times.There is the bacterial strain of antagonistic activity to adopt the same procedure repeated test through preliminary test, every processing 3 strains, three repetitions.
Withered spot inhibiting rate calculation formula:
Withered spot inhibiting rate experimental result shows that bacterial strain L13 is best to the inhibition effect of TMV, and the withered spot inhibiting rate of TMV is reached 94.16%, and biocontrol bacteria has produced in the growth metabolism process has the antibiotics that suppresses virus activity.
Identify that through bacteria molecule genetic classification authentication method bacterial strain L13 is a bacillus, the Lyceum genus bacillus.Lyceum genus bacillus biological characteristics: Gram-negative, the about 3mm of the colony diameter that on plate culture medium, forms, neat in edge, moistening smooth, high projection, sticky, pure white.It is short and small shaft-like that thalli morphology is, and life or end are given birth in the gemma.
Table 1L13 is to the restraining effect of tobacco mosaic viruses
Two, the bacteria molecule genetic classification of Lyceum genus bacillus is identified
At first extracting the DNA of bacterial strain L13, is template with this DNA, and 16S rDNA universal primer is a primer, and 16S rDNA fragment is increased.The fragment of amplification is directly carried out sequencing.The blast program that the result who checks order imports on the NCBI website is compared, the result shows that the 16s rDNA sequence homology degree of the Bacillus siamensis in the bacillus in this bacterial strain 16S rDNA sequence and the GenBank gene pool is the highest, and homology reaches 99%.By DNAMAN6.0 the 16S rDNA sequence of existing bacillus among the Genbank is carried out the genetic evolution analysis, the result shows, bacterial strain L1316S rDNA and Bacillus siamensis homology are the highest, therefore can judge that this bacterial strain is the Lyceum genus bacillus of bacillus.
The bacterial strain L13 that screens is carried out culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms 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, the 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, at the single bacterium colony of distance indicator bacterium piece 2.8cm place inoculation Lyceum genus bacillus, opposite side is not inoculated the 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 to the inhibition activity of tobacco fungal disease
The Lyceum genus bacillus all has good inhibition effect to several fungal diseases that grow tobacco as can be seen from Table 2, and wherein the inhibiting rate to tobacco black shank bacterium, tobacco alternaric bacteria, sharp sickle spore bacterium oronoco has reached 73%, 85% and 69% respectively.The Lyceum genus bacillus has the prevention effect of wide spectrum to the tobacco fungal disease.
Four, Lyceum fermentation of bacillus Optimum of culture medium
Preparation Lyceum genus bacillus seed liquor is inoculated in the fermention medium by 3% inoculative proportion, then 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, lime 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.
The fermentation result: about pH7.0, living bacteria count is 30,000,000,000/mL.
Five, the disinfection of the bacterial strain fermentation liquor of Lyceum genus bacillus L13
The sterilization scissors in 40 times of TMV juice, soak 1min, after the taking-up respectively 1. following～4. soak 30sec in the liquid, on common cigarette N89 of 5～6 leaf phases, cut 3 leaves in top.Every processing 9 strains, 3 repetitions.15d investigates disease index.1. L13 fermented liquid, viable count is more than 200,000,000/mL, 2. fermention medium, 3. 4. clear water does not soak, and directly cuts the NC89 blade.
The disinfection that table 3 Lyceum fermentation of bacillus liquid is cut leaf-cutting
Found out that by table 3 scissors is immersed in the TMV juice, picked viral juice, soak in the L13 bacterial strain fermentation liquor, can kill whole viruses, sickness rate only is 0, therefore, produces and can operating apparatus such as cut with the leaf-cutting of sterilizing of L13 fermented liquid.Also observe the processing that is immersed in fermention medium and the clear water in the test, sickness rate also is lower than the processing of 4. not soaking, and is because diluted the virus concentration that picks on scissors, has reduced invasiveness.
Six, the growth-promoting functions of Lyceum genus bacillus L13 bacterial strain fermentation liquor
Filter paper is pruned suitably size, places culture dish, the L13 fermented liquid is centrifugal remove thalline after, dilute 1 times, 5 times, 25 times, 125 times, 625 times respectively, moistening filter paper.Choose full mellow and full mung bean, ten in each culture dish, and do contrast with sterilising medium, observe guaranteeing that also filter paper is moistening every day.Investigate percentage of germination, investigate fresh weight, long, the plant height of root.
Table 4L13 fermentation liquid irrigating root is to the growth-promoting functions of mung bean
Find out by table 4, after the diluted fermentation liquor treatment of mung bean, its fresh weight, root is long, plant height is handled 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 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 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 microorganism marine alga fertilizer is to the influence of cigarette strain growth
Get common cigarette NC89 of 2-3 leaf phase, transplant, irritate root with the microorganism marine alga fertilizer during transplanting to the small flower of diameter 10cm, every strain 10ml, interval 3d, the continuous irrigation root is 2 times again, and irritating root with fermention medium, sterilized water is contrast, every processing 6 strains, 3 repetitions.After last is irritated root 3d, investigation fresh weight, maximum leaf length and width.
Table 5 microorganism marine alga fertilizer is irritated the growth-promoting functions of root to NC89
Find out by table 5, common cigarette NC89, continuous three filling roots, the microorganism marine alga fertilizer is handled its fresh weight, and maximum leaf length and width all is higher than substratum and clear water is handled, and is 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 handle.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (10)
1. Lyceum genus bacillus L13 is characterized in that: its classification called after Lyceum bacillus siamensis, its deposit number is CGMCC No.7285.
2. a kind of Lyceum genus bacillus L13 according to claim 1 is characterized in that: the Gram-negative of described Lyceum genus bacillus L13; 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, and life or end are given birth in the 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 disease, Alternaria alternate, black shank, sharp sickle spore bacterium 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 preparation sterilizing agent.
5. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 4 in the preparation sterilizing agent, it is characterized in that: the leaf-cutting that is used to sterilize of described Lyceum genus bacillus L13 bacterial strain fermentation liquor is cut operating apparatus, viable count in the 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 preparation growth-promoting agent.
7. the application of the bacterial strain fermentation liquor of a kind of Lyceum genus bacillus L13 according to claim 6 in the preparation growth-promoting agent, it is characterized in that: the bacterial strain fermentation liquor of described Lyceum genus bacillus L13 is long to fresh weight, the root of mung bean, plant height has growth-promoting functions, viable count in the fermented liquid 〉=200,000,000/mL.
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 the 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 that is used 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, lime 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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