CN104593302A - Preparation method of new bacterial strain t16 of nitrogen-fixing bacterium for astragalus membranaceus and fermentation liquor - Google Patents
Preparation method of new bacterial strain t16 of nitrogen-fixing bacterium for astragalus membranaceus and fermentation liquor Download PDFInfo
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Abstract
The invention discloses a preparation method of a new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus, which is collected in China General Microbiological Culture Collection Centre on September 18, 2014, with a collection number of CGMCC No. 9576. The invention describes the morphological characteristics and the physiological and biochemical indexes of the new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus, a 16S rDNA sequence determination result and the like in details. A nitrogen-fixing test indicates that the fermentation liquor of the new bacterial strain t16 of nitrogen-fixing bacterium (Rhizobium sp.) for astragalus membranaceus has a good nitrogen-fixing effect on astragalus membranaceus, and can effectively provide a natural nitrogen source need for the growth of astragalus membranaceus, thus providing a corresponding nutrition source for the natural quality of astragalus membranaceus.
Description
Technical field
What the present invention relates to plant nitrogen-fixing bacteria utilizes field, specifically relates to the preparation method of a kind of genuine traditional Chinese medicine Radix Astragali vinelandii t16 and fermented liquid.
Background technology
In recent years, grown worldwide industry too relies on and uses in a large number fertilizer waste that chemical fertilizer causes, soil and groundwater pollutes and disruption of ecological balance has caused the extensive concern of countries in the world.Reducing the use of chemical fertilizer, improving utilization rate of nitrogen fertilizer is the major issue being badly in need of during current agricultural is produced solving.The biological nitrogen fixation giving full play to nitrogen-fixing bacteria reduces the most effective approach of nitrogenous fertilizer usage quantity, and research, the development and utilization of biological nitrogen fixation resource occupy critical role in China's agricultural sustainable development.Biological nitrogen fixation is that occurring in nature is only second to photosynthetic complex biological reactive system, and the plant for the whole world provides the nitrogen of about 75%.Biological nitrogen fixation increasing soil fertility, strengthen in plant disease-resistant ability etc. and also play extremely important effect.
Nitrogen-fixing bacteria can be divided into self vinelandii, combination azotobacter and symbiotic nitrogen-fixing bacteria three class according to its life habit.Self vinelandii refers under free living state can the bacterium of fixed nitrogen, and its kind is few, and distribution is very wide, as azotobacter chroococcum and Bai Yelin kirschner vinelandii.Combination azotobacter refers to and is colonizated in plant root table and nearly root soil, the intercellular substance that can invade epidermis and exodermis had, and by root exudates existence, procreation, has the nitrogen-fixing bacteria of close relationship with root system of plant, as ditch millet vinelandii and product fat azospirillum.The bacterium of symbiotic nitrogen-fixing bacteria only has root nodule bacterium one class, and it is extensively present in soil, contaminates leguminous plants root and forms root nodule, the nitrogen in air can be converted into ammonia and be supplied to plant.In above-mentioned three class vinelandii, self vinelandii due to its nitrogen-fixing efficiency low, and fixed nitrogen is special, is not suitable for agriculturally widespread use, so combination azotobacter and symbiotic nitrogen-fixing bacteria are mainly applied in current agriculture aspect.
The Radix Astragali (
astragalus membranaceusbunge) be pulse family Astragalus per nnial herb, have another name called continuous stilbene.The ground such as the Inner Mongol, Shanxi, Gansu, Heilungkiang are originated in China.The Radix Astragali has enhancing body immunologic function, protects the liver, diuresis, anti-ageing, anti-stress, step-down and anti-microbial effect more widely, as the history of medicinal existing more than 2000 year.Owing to excavating in a large number for a long time, the quantity of the wild Radix Astragali sharply reduced in recent years, had the danger being tending towards dying out.In astragalus cultivation process, during every means of agricultural production drops into, especially nitrogenous fertilizer proportion is very large for chemical fertilizer, and its impact assembled the output of the Radix Astragali and activeconstituents is also the most remarkable.And the Radix Astragali is as leguminous plants, in its rhizosphere soil, also symbiosis has a large amount of vinelandii, plays important nitrogen fixation.Hunyuan County, Shanxi is the place of production of the Chinese medicinal materials Radix Astragali.Have benefited from local water and soil, Astragaloside IV that is produced from Huiyuan, polysaccharide equal size be domestic first of, and in 2011 the granted national geography mark certification mark " positive northern stilbene ".According to this geographical indication product of the useful composition of authentic medical and country of origin of the genuine Radix Astragali in Huiyuan, with its Proterozoic soil root nodule bacterium for Research foundation, the nitrogen-fixing rhizobia of the genuine Radix Astragali is separated, screening and identification, and its culture condition and Physiology and biochemistry and information biology aspect are studied.In order to utilize biological nitrogen fixation to realize on the Radix Astragali is produced, joint fertilizer subtracts consumption, the benign cycle of the reinforcement astragalus cultivation industry ecosystem provides scientific basic and technical support in this research.
Summary of the invention
The object of this invention is to provide the preparation method of a strain genuine Radix Astragali vinelandii new strains t16 and fermented liquid, this fermented liquid has obviously nitrogenase activity.
The object of the invention is to be achieved through the following technical solutions.
The genuine Radix Astragali vinelandii of one strain (
rhizobiumsp.) new strains t16 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2014, and preserving number is CGMCC No. 9676, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Described genuine Radix Astragali vinelandii (
rhizobiumsp.) new strains t16 be from the Radix Astragali soil for growth in area, Huiyuan, Shanxi Province be the root nodule bacteria that material separation obtains.
By genuine Radix Astragali vinelandii on Ashby nitrogen-free agar, the bacterium colony of new strains t16 is rounded on YMA flat board, white, translucent, moistening (see figure 1).Gramstaining, display new strains t16 is gram negative bacillus (see figure 2), and Victoria Green WPB dyeing is negative, not containing brood cell's (see figure 3) in prompting new strains t16 cell.According to " common bacteria system identification handbook ", Physiology and biochemistry qualification is carried out to new strains t16, the results are shown in Table 1.
The physiological and biochemical property of table 1 new strains t16 of the present invention
Note: "+" expression is positive; "-" represents the reaction that is negative.
Extract the genomic dna of Radix Astragali vinelandii new strains t16, utilize PCR to react its 16S rRNA partial sequence of amplification, obtain the DNA fragmentation (Fig. 4) that is about about 1500bp.Obtaining sequence length after order-checking is that 1309bp(is in table 2), sequencing result uses BLAST software to carry out sequence analysis in GeneBank database, and result shows, new strains t16 with
rhizobium fabae16S rRNA gene homology be 100%(KF465964.1), combining form observe and physiological and biochemical test, new strains t16 is defined as root nodule bacterium (
rhizobiumsp.).
The 16S sequencing result of table 2 new strains t16 of the present invention
The preparation method of genuine Radix Astragali vinelandii new strains t16 fermented liquid, comprises the steps:
(1) seed liquor preparation is from test tube slant inoculating needle picking new strains t16 on slant medium (add 1.5% agar by YMA liquid nutrient medium and make cultivation inclined-plane), constant temperature 28 DEG C is inoculated in the triangular flask of the LB nutrient solution that 100mL is housed after cultivating 24h, 180r/min shaking culture 12h, obtained seed liquor;
(2) fermented liquid prepare seed liquor prepared by (1) by volume 1% ratio access fermention medium, culture condition is: 28 DEG C, initial pH is 6.8 ~ 7.0, shaking speed 180-200r/min concussion cultivates 30h, obtained fermented liquid.
Described fermention medium is grouped into by the one-tenth of following masses volume ratio: 14 parts, N.F,USP MANNITOL, yeast powder 5 parts, MgSO
47H
2o 0.1 part, K
2hPO
40.3-0.4 part, NaCl 0.3-0.4 part, CaCl
20.01 part, H
2o 1000 parts, the medium pH 6.8 ~ 7.0 of joining.
The fermented liquid of the genuine Radix Astragali fixed nitrogen new strains t16 adopting above-mentioned steps to prepare carries out nitrogen fixation effect test
1. test method
Thered is provided by astragalus cultivation base, Hunyuan County, Shanxi for examination astragalus membranaceus seed.Adopt full N differential technique to measure the nitrogen fixing capacity of bacterial strain.Be inoculated in 100ml YMA liquid nutrient medium by genuine Radix Astragali fixed nitrogen new strains t16, after 28 DEG C of shake-flask culture, with blood counting chamber counting, with deionized water dilution, obtaining bacteria containing amount is 10
6ml
-1, 10
7ml
-1, 10
8ml
-1microbial inoculum.The vermiculite (particle diameter is 2mm) of clean sterilizing is put into diameter 8cm, high 10cm plastic tub, cultivates place as without nitrogen.Test points 3 process and 1 contrast totally four groups, often group 5 sample basins, in triplicate, random alignment.The astragalus membranaceus seed of each treatment group steeps 3h by the bacterium immersion of above-mentioned three concentration, and contrast deionized water soaks 3h.Then by planting seed in the plastic tub that vermiculite is housed, and each for above-mentioned microbial inoculum 30ml is poured in corresponding basin, cultivates in Sheng Ke institute of Agricultural University Of Shanxi laboratory.Water every day 30ml, cultivates 30 days results Radix Astragali seedlings, often organize stochastic sampling 100 strain seedling complete stool, metering average height of seedling and the number of blade, clean to be placed in baking box and dry, measure the nitrogen content (Bao Shidan, 2000) of Radix Astragali seedling.
2. test-results
(1) Radix Astragali height of seedling and number of blade test-results are in table 3.
Impact on Radix Astragali seedling after table 3 new strains t16 of the present invention fermentation liquor treatment astragalus membranaceus seed
| Group | Ck | 10 6 | 10 7 | 10 8 |
| Average height of seedling (cm) | 11.8±1.83 | 13.9±0.83 ** | 13.3±0.9 * | 13.1±1.04 * |
| Mean number of sheets (sheet/strain) | 5.7±1.42 | 7.2±0.87 ** | 6.2±1.9 | 6.9±1.51 * |
Note: * to compare P<0.01 with Ck with the Ck P<0.05 * * that compares
Test-results shows, after fermentation liquor treatment astragalus membranaceus seed with Radix Astragali fixed nitrogen new strains t16 of the present invention, significant promoter action is all had to Radix Astragali growth of seedling speed and leaf development speed, height of seedling and the number of blade of cultivating the Radix Astragali after 30d are all significantly higher than contrast (see Fig. 5-8), and wherein bacteria containing amount is 10
6ml
-1effect after this concentration process is better than the effect of two other higher concentration.This also points out, and when with bacterium liquid process seed, be not that concentration is the bigger the better, suitable concentration effect may be better.
(2) Radix Astragali seedling nitrogen content test-results is in table 4.
On the impact (mg/ strain) of the nitrogen content of Radix Astragali seedling after table 4 new strains t16 of the present invention fermentation liquor treatment astragalus membranaceus seed
| Group | Ck | 10 6 | 10 7 | 10 8 |
| Nitrogen content | 57.34±0.4234 | 68.04±0.6986 ** | 65.5±1.6381 ** | 66.65±1.1334 ** |
Note: * * to compare P<0.01 with Ck
Test-results shows, with the fermented liquid bacteria containing amount of Radix Astragali fixed nitrogen new strains t16 of the present invention 10
6ml
-1, 10
7ml
-1, 10
8ml
-1microbial inoculum astragalus membranaceus seed is soaked seed 3 hours after sowing, cultivate Radix Astragali seedling nitrogen content pole after 30 days and be significantly higher than control group, show that t16 has significant nitrogenase activity.The amount of nitrogen fixation of average every strain Radix Astragali seedling is about 8.16-10.7mg.According to reality plantation situation, every mu of ground plantation 15000 strain seedling, the amount of nitrogen fixation of per hectare is 0.01*15000*15 ≈ 2250g, and namely t16 microbial inoculum is 2.25kg/km the fixed nitrogen value of 30 days
2left and right.Quite in Radix Astragali milpa, per hectare can reduce amount of urea 4.9 kilograms (calculating according to urea nitrogen content 46%) every month, and along with the prolongation of Radix Astragali rhizome, root nodule numbers also can be more, fixed nitrogen value also can increase, and this can reduce a very large expenditure for plantation family.
Beneficial effect of the present invention
(1) by genuine Radix Astragali vinelandii of the present invention (
rhizobiumsp.) the fermented liquid dilution that prepared by new strains t16 is 10
6ml
-1after the microbial inoculum process astragalus membranaceus seed of concentration, Radix Astragali height of seedling and the number of blade are significantly higher than control group (P < 0.01), nitrogen-fixing efficiency be equivalent to per hectare monthly amount of nitrogen fixation at more than 2.5kg, show the genuine Radix Astragali vinelandii of the present invention (
rhizobiumsp.) new strains t16 has the nitrogen fixation of highly significant in the process of growth of the Radix Astragali.(2) genuine Radix Astragali vinelandii of the present invention (
rhizobiumsp.) the strain fermentation condition of new strains t16 is convenient, and bacterial classification is easily preserved, and microbial inoculum preparation process is simple, easily realizes suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of new strains t16 of the present invention.
Fig. 2 is the Gram-stained form of new strains t16 of the present invention (100 ×).
Fig. 3 is the form (100 ×) of new strains t16 Victoria Green WPB of the present invention dyeing.
Fig. 4 is the 16S rRNA amplification of new strains t16 of the present invention.
Fig. 5 is the growing state cultivating 30 days Radix Astragali seedling after deionized water process astragalus membranaceus seed.
Fig. 6 is new strains t16 fermented liquid concentration 10 of the present invention
6ml
-1the growing state of 30 days Radix Astragali seedling is cultivated after process astragalus membranaceus seed.
Fig. 7 is new strains t16 fermented liquid concentration 10 of the present invention
7ml
-1the growing state of 30 days Radix Astragali seedling is cultivated after process astragalus membranaceus seed.
Fig. 8 is new strains t16 fermented liquid concentration 10 of the present invention
8ml
-1the growing state of 30 days Radix Astragali seedling is cultivated after process astragalus membranaceus seed.
embodiment 1
The genuine Radix Astragali vinelandii of one strain (
rhizobiumsp.) new strains t16, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2014, Classification And Nomenclature is root nodule bacterium
rhizobiumsp., preserving number is CGMCC No. 9676, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Genuine Radix Astragali vinelandii of the present invention (
rhizobiumsp.) new strains t16 is that material separation obtains from the Radix Astragali soil for growth in area, Huiyuan, Shanxi Province.By new strains t16 on Ashby nitrogen-free agar, the bacterium colony of new strains t16 is rounded on YMA flat board, white, translucent, moistening (see figure 1).Gramstaining, display new strains t16 is gram negative bacillus (see figure 2), and Victoria Green WPB dyeing is negative, not containing brood cell's (see figure 3) in prompting new strains t16 cell.According to " common bacteria system identification handbook ", Physiology and biochemistry qualification is carried out to new strains t16, the results are shown in Table 1.
Extract the genomic dna of Radix Astragali vinelandii new strains t16, utilize PCR to react its 16S rRNA partial sequence of amplification, obtain the DNA fragmentation (Fig. 4) that is about about 1500bp.Obtaining sequence length after order-checking is that 1309bp(is in table 2), sequencing result uses BLAST software to carry out sequence analysis in GeneBank database, and result shows, new strains t16 with
rhizobium fabae16S rRNA gene homology be 100% (KF465964.1), combining form observe and physiological and biochemical test, new strains t16 is defined as
rhizobiumsp..
embodiment 2
The preparation method of genuine Radix Astragali vinelandii new strains t16 fermented liquid of the present invention, comprises the steps:
(1) seed liquor preparation is from test tube slant inoculating needle picking new strains t16 on slant medium (add 1.5% agar by YMA liquid nutrient medium and make cultivation inclined-plane), constant temperature 28 DEG C is inoculated in the triangular flask of the LB nutrient solution that 100mL is housed after cultivating 24h, 180r/min shaking culture 12h, obtained seed liquor;
(2) fermented liquid prepare seed liquor prepared by (1) by volume 1% ratio access fermention medium, culture condition is: 28 DEG C, initial pH is 6.8 ~ 7.0, shaking speed 180-200r/min concussion cultivates 30h, obtained fermented liquid.
Described fermention medium is grouped into by the one-tenth of following masses volume ratio: 14 parts, N.F,USP MANNITOL, yeast powder 5 parts, MgSO
47H
2o 0.1 part, K
2hPO
40.3-0.4 part, NaCl 0.3-0.4 part, CaCl
20.01 part, H
2o 1000 parts, the medium pH 6.8 ~ 7.0 of joining.
By new strains t16 fermented liquid concentration 10 of the present invention
6ml
-1cultivate after 30 days after process astragalus membranaceus seed, Radix Astragali height of seedling and the number of blade are significantly higher than control group (P < 0.01), nitrogen-fixing efficiency be equivalent to per hectare monthly amount of nitrogen fixation at more than 2.5kg.
Claims (5)
1. the genuine Radix Astragali vinelandii of a strain (
rhizobiumsp.) new strains t16 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 18th, 2014, and preserving number is CGMCC No.9676.
2. genuine Radix Astragali vinelandii according to claim 1 (
rhizobiumsp.) new strains t16, is characterized in that, described genuine Radix Astragali vinelandii (
rhizobiumsp.) new strains t16 is separated the bacterium obtained from the soil of Radix Astragali base, Hunyuan County, Datong City, Shanxi Province; By genuine Radix Astragali vinelandii on Ashby nitrogen-free agar, the bacterium colony of new strains t16 is rounded on YMA flat board, white, translucent, moistening; Gramstaining, display new strains t16 is gram negative bacillus; Victoria Green WPB dyeing is negative, not containing brood cell in prompting new strains t16 cell; Carried out Physiology and biochemistry qualification according to " common bacteria system identification handbook " to new strains t16, result is as shown in the table:
The physiological and biochemical property of new strains t16 of the present invention
Note: "+" expression is positive; "-" represents the reaction that is negative
Extract the genomic dna of Radix Astragali vinelandii new strains t16, utilize PCR to react its 16S rRNA partial sequence of amplification, obtain the DNA fragmentation that is about about 1500bp; Obtaining sequence length after order-checking is 1309bp, and sequencing result uses BLAST software to carry out sequence analysis in GeneBank database, and result shows, new strains t16 with
rhizobium fabae16S rRNA gene homology be 100%(KF465964.1), combining form observe and physiological and biochemical test, new strains t16 is defined as
rhizobiumsp..
3. genuine Radix Astragali vinelandii new strains t16 according to claim 1, the preparation method of its fermented liquid comprises the steps:
(1) seed liquor preparation is from test tube slant inoculating needle picking new strains t16 on slant medium (add 1.5% agar by YMA liquid nutrient medium and make cultivation inclined-plane), constant temperature 28 DEG C is inoculated in the triangular flask of the LB nutrient solution that 100mL is housed after cultivating 24h, 180r/min shaking culture 12h, obtained seed liquor;
(2) fermented liquid prepare seed liquor prepared by (1) by volume 1% ratio access fermention medium, culture condition is: 28 DEG C, initial pH is 6.8 ~ 7.0, shaking speed 180-200r/min concussion cultivates 30h, obtained fermented liquid.
4. the preparation method of genuine Radix Astragali vinelandii new strains t16 fermented liquid according to claim 3, it is characterized in that, described fermention medium is grouped into by the one-tenth of following masses volume ratio: 14 parts, N.F,USP MANNITOL, yeast powder 5 parts, MgSO
47H
2o 0.1 part, K
2hPO
40.3-0.4 part, NaCl 0.3-0.4 part, CaCl
20.01 part, H
2o 1000 parts, the medium pH 6.8 ~ 7.0 of joining.
5. the method for the use of genuine Radix Astragali vinelandii new strains t16 according to claim 1 is: will be 10 containing bacteria concentration
6ml
-1fermented liquid seed soaking 3h after sow.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399148A (en) * | 2016-06-30 | 2017-02-15 | 上海交通大学 | Kosakonia radicincitans and application thereof |
| CN112552128A (en) * | 2020-12-17 | 2021-03-26 | 新疆农业大学 | Microbial compound bacterial fertilizer for cultivating selenium-rich astragalus and cultivation method of astragalus |
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| CN102174429A (en) * | 2010-12-30 | 2011-09-07 | 领先生物农业股份有限公司 | Rhizobium astragula capable of effectively fixing nitrogen and culture method and application thereof |
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102174429A (en) * | 2010-12-30 | 2011-09-07 | 领先生物农业股份有限公司 | Rhizobium astragula capable of effectively fixing nitrogen and culture method and application thereof |
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| CHEN W X ET AL: "Rhizobium kuakuii sp.nov.Isolated from the root nodules of Astragalus sinicus", 《INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399148A (en) * | 2016-06-30 | 2017-02-15 | 上海交通大学 | Kosakonia radicincitans and application thereof |
| CN106399148B (en) * | 2016-06-30 | 2019-09-27 | 上海交通大学 | One kind secreting ammonium nitrogen-fixing bacteria and its application |
| CN112552128A (en) * | 2020-12-17 | 2021-03-26 | 新疆农业大学 | Microbial compound bacterial fertilizer for cultivating selenium-rich astragalus and cultivation method of astragalus |
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