CN101914468A - Nitrogen-fixing bacillus megaterium strain DL7 and application thereof - Google Patents
Nitrogen-fixing bacillus megaterium strain DL7 and application thereof Download PDFInfo
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- CN101914468A CN101914468A CN2010102273768A CN201010227376A CN101914468A CN 101914468 A CN101914468 A CN 101914468A CN 2010102273768 A CN2010102273768 A CN 2010102273768A CN 201010227376 A CN201010227376 A CN 201010227376A CN 101914468 A CN101914468 A CN 101914468A
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Abstract
The invention relates to a nitrogen-fixing bacillus megaterium strain DL7 and application thereof. The strain is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms in June 7, 2010 and has a preserving number as CGMCC NO.3914. The strain is a new nitrogen-fixing strain obtained by using the conventional microorganism separation and purification method for Chinese podocarpus root nodules. In the strain, the nitrogenase activity is 1.5632nmol.g<-1>.h<-1>; after an eucalyptus seedling is planted, the seedling height increases 40.78-56.22 percent, the ground diameter increases 39.41-46.43 percent, the average biomass increases 72.11-158.76 percent, the chlorophyll content increases 12.97-20.98 percent, and the nitrate reductase activity increases 33.82-35.22 percent. The strain can be applied to a biological bacterial fertilizer for promoting eucalyptus seedling growth.
Description
Technical field
The present invention relates to biological technical field, particularly strain fixed nitrogen bacillus megaterium strain (Bacillusmegaterium) DL7 and an application thereof.
Background technology
The whole world has 5,000 ten thousand hm approximately at present
2Tropical artificial forest, and annual 3000000 hm that increase
2About (Binkley et al., 2004).Wherein, eucalyptus (Eucalyptus sp.) accounts for about 40% of the tropical artificial forest total area, and timber yield accounts for more than 50%, is just bringing into play more and more important effect.The eucalyptus plantation area of China has reached 1,700,000 hm
2, eucalyptus has become one of China's three big reproducting tree species, and afforestation area is only second to India and Brazil, occupies the third place in the world.The life of eucalyptus speed, high yield, high-quality, optimum hybridization family mean annual precipitation can reach 69.8m
3/ hm
2, optimum clone mean annual precipitation reaches 64.8m
3/ hm
2The development of eucalyptus industry has driven the development of transportation, papermaking, regenerated fiber, wood-based panel industry etc., provides a large amount of labour employment chances for society simultaneously, for social stability plays an important role.
Also there are a lot of problems in eucalyptus plantation, for example: (1) productivity problem.Eucalyptus plantation 2,3,4 generation timber reserves has descended 20.9%, 38.7% and 50.5% than the 1st generation respectively; Biomass has descended 19.6%, 26.7% and 44.6% respectively.As seen, the soil fertility decline problem of eucalyptus plantation is quite serious.Many eucalyptus woodss only need 4～5 years felling cycle.This means that the nutrient that is pulled away is more and more.Except excessive results, cultivate whole ground and unreasonable fertilising entirely and also can cause the decline of eucalyptus plantation soil fertility.(2) moisture problem.Since the eighties in 20th century, whether eucalyptus plantation consumes the focus that more soil moisture becomes concern than other seeds artificial forest.The comparative observation ground water table finds that the dark 9～11m of water level, bare area are that 1～4m, mixed forest are 3～4m under the eucalyptus forest land.
Eucalyptus is not have the fatten oneself non-leguminous plant of ability of fixed nitrogen, and the fast growing and high yield of eucalyptus plantation relies on fully and uses N, P, K composite fertilizer at present, causes soil trace element to lack, and soil fertility reduces, and productivity occurs and descends; Use chemical fertilizer in a large number, cause soil compaction, Soil structure is destroyed, forest land water retention capacity difference and the moisture problem that occurs.This is the biggest obstacle that realizes the eucalyptus Sustainable development.Yet by the artificial inoculation vinelandii, set up eucalyptus rhizosphere association nitrogen fixation system, make originally can not symbiotic nitrogen fixation eucalyptus obtain nitrogen fixing capacity, airborne molecular nitrogen is become the ammonia-state nitrogen that can absorb for eucalyptus, promote the eucalyptus growth.This is for barren eucalyptus forest land amelioration day by day, and soil fertility keeps significant and application prospect.
Summary of the invention
The purpose of this invention is to provide through screening, have efficient nitrogen fixation activity, the eucalyptus nursery stock had strain fixed nitrogen bacillus megaterium strain (Bacillus megaterium) DL7 of obvious promotion growth effect.
Another object of the present invention provides cultural method and the application in the bio-bacterial manure of preparation promotion eucalyptus seedling growth of strain fixed nitrogen bacillus megaterium strain (Bacillus megaterium) DL7.
The inventor obtains endogenetic bacteria from the separation and purification of non-leguminous plant Podocarpus macrophyllus root nodule on research basis extensively and profoundly, its biological characteristics, physio-biochemical characteristics, fixed nitrogen dross ability, inoculation nursery stock are promoted systematic testing and detections such as growth.
The cultural method of one strain fixed nitrogen bacillus megaterium strain (Bacillus megaterium) DL7 comprises the steps:
Gather the radicula that root nodule is arranged from the Chinese podocarpus lateral root, after rinsing well with tap water, the root nodule surface was carried out disinfection 1 minute, sterilized 5～6 minutes with 0.1% acid chlorization mercury again with 75% ethanol.Place the mortar of the bacterium of going out to grind gently in root nodule.Taking juice is inoculated in method of scoring and adds on the Congo red YMA medium, cultivated 2～3 days for 28 ℃, select and do not inhale Congo red bacterium colony, repeat the line screening, then according to the difference of bacterium colony size, shape, surface luster, protuberance degree, transparency, edge shape and colony colour, select single bacterium colony, obtain required purifying bacterial strain completely through light microscopy.
Described YMA medium composition and content are: dipotassium hydrogen phosphate 0.5g, sal epsom 0.5g, sodium-chlor 0.1g, N.F,USP MANNITOL 10.0g, yeast extract paste 0.4g, agar powder 15g, water 1000ml, natural pH6.8,121 ℃ of steam sterilizings 20 minutes.
The characteristic of nitrogen-fixing bacillus megaterium strain DL 7
1. morphological character
On YMA medium, the bacterium colony of bacterial strain is circle, oyster white, product mucus, neat in edge.Amphitrichous, movable.
2. cultural characters
The optimum growing condition of this bacterium is: pH=7.2～7.4,28 ℃ of temperature.Can utilize carbon source and nitrogenous source widely.
3. physiological property
Negative through gramstaining reaction microscopy, be direct rod shape.
4. functional performance
Nitrogen-fixing bacillus megaterium strain DL 7 16S rDNA PCR sequence is measured, sequencing result is shown in sequence table, the 16S rDNA sequence of this bacterial strain is carried out BLAST on the net relatively at the U.S. state-run bioinformation center NCBI, find with Genbank in the highest correlated series of homology, determine that this bacterial strain is bacillus megaterium (Bacillus megaterium).Detecting this bacterial strain with the acetylene reduction method has nitrogenase activity, and its enzymic activity is 1.5632nmolg
-1H
-1Be inoculated into the eucalyptus nursery stock and can promote seedling growth, wherein height of seedling increases by 40.78～56.22%, and leading thread increases by 39.41～46.43%, and biomass of individual tree increases by 72.11～158.76%, chlorophyll content increases by 12.97～20.98%, and nitrate reductase activity increases by 33.82～35.22%.
Fixed nitrogen bacillus megaterium of the present invention strain (Bacillus megaterium) DL7 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 7th, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.3914.
Embodiment
Below by specific embodiment technical scheme of the present invention is described further.
Embodiment 1
The cultural method of fixed nitrogen bacillus megaterium of the present invention strain (Bacillus megaterium) DL7 comprises the steps:
1, the separation of nitrogen-fixing bacillus megaterium strain DL 7, purifying
Gather the radicula that root nodule is arranged from the Chinese podocarpus lateral root, after rinsing well with tap water, the root nodule surface was carried out disinfection 1 minute, sterilized 5～6 minutes with 0.1% mercuric chloride again with 75% ethanol.Place the mortar of the bacterium of going out to grind together with radicula in root nodule, add a small amount of sterilized water, add a cover after grinding well and leave standstill, clarification for a moment.Get supernatant liquor and be inoculated in method of scoring and add on the Congo red YMA medium, cultivated 2～3 days for 28 ℃, select and do not inhale Congo red bacterium colony, repeat the screening and culturing of ruling, and then select the bacterium colony of circle, oyster white, product mucus, neat in edge.The microscopic examination amphitrichous, movable, no gemma.Obtain purifying bacterial classification completely.Described YMA medium composition and content are: dipotassium hydrogen phosphate 0.5g, sal epsom 0.5g, sodium-chlor 0.1g, N.F,USP MANNITOL 10.0g, yeast extract paste 0.4g, agar powder 15g, water 1000ml, natural pH6.8,121 ℃ of steam sterilizings 20 minutes.
2, nitrogen-fixing bacillus megaterium strain DL 7 tieback test
Test with the composition and the content of Jenson liquid nutrient medium is: secondary calcium phosphate 1.0g, dipotassium hydrogen phosphate 0.2g, sal epsom 0.2g, sodium-chlor 0.2g, iron trichloride 0.1g, 1 liter in water.Every liter adds 1ml trace element mother liquor, and described micro-mother liquor content is as follows: boron 0.05%, manganese 0.05%, zinc 0.005%, molybdenum 0.005%, copper 0.002%.
Select full Chinese podocarpus seed, in 75% alcohol, handled 1 minute earlier, wash 5～6 times, used 0.1% mercuric chloride surface sterilization then 6 minutes, wash 6～7 times, in sterilized water, soak seed and spend the night.The seed of imbibition is moved in the aseptic nutrient agar 25 ℃ of constant temperature culture vernalization.The Chinese podocarpus seedling that radicle is grown to 4～5cm is transferred to and fills the sealing in the triangular flask of Jensen nutrient solution, and every bottle adds 2ml bacterium liquid, places the illumination cultivation chamber to cultivate, not add bacterium liquid as blank.Contrast is handled with inoculation and is respectively established 20 repetitions.
When the Chinese podocarpus seedling that connects bacterium is cultured to 6 months, adopt Japan to produce the GA-9A gas chromatograph, measure the nitrogenase activity of seedlings root with the acetylene reduction method, recording its enzymic activity is 1.5632nmolg
-1H
-1
Embodiment 2
With soaking of the test of root method inoculation nitrogen-fixing bacillus megaterium strain DL 7 to the influence of eucalyptus nursery stock
This test is carried out being positioned at nursery, forest farm, state-run peak, Guangxi, Nanning suburb.
No. 9 (GLGU9) clonal tissue cultures of wide woods Eucalyptus urophylla-grandis bottle seedling that test is produced from forest farm, state-run peak, Guangxi group training factory with nursery stock.After tissue-culture container seedling shifted out bottle, with tap water substratum is cleaned up earlier,, filter behind the thimerosal of dried nursery stock body surface standby again with the thiophanate methyl sterilization.
Soak root method inoculation: the seedling of above processing before being transplanted to nutrient cup, was soaked the nursery stock root 20～30 minutes with nitrogen-fixing bacillus megaterium strain DL 7 bacterium liquid, and field planting is inoculated nursery stock 5000 strains altogether to nutrient cup then, and nutrient cup soil is yellow soil.
Nursery stock method for culturing seedlings management routinely.Measure the average plant height of nursery stock, leading thread, biomass, chlorophyll content and blade nitrate reductase activity after three months.The results are shown in Table 1.
Table 1 soaks the influence of root method inoculation nitrogen-fixing bacillus megaterium strain DL 7 to the eucalyptus nursery stock
As seen from Table 1, the inoculation nitrogen-fixing bacillus megaterium strain DL 7 can obviously increase plant height, leading thread and the biomass of individual tree of eucalyptus nursery stock, has promoted the growth of nursery stock, can also improve the chlorophyll content and the nitrate reductase activity of nursery stock blade.The results of analysis of variance shows, the height of seedling difference between processing reaches utmost point conspicuous level (F=7.5120**＞F0.01=5.06); Leading thread difference reaches conspicuous level (F=5.0060*＞F0.05=3.11); Biomass difference reaches utmost point conspicuous level (F=15.3470**＞F0.01=5.06); Nitrate reductase activity difference reaches utmost point conspicuous level (F=122.9640**＞F0.01=5.06).
Embodiment 3
With of the test of spray method inoculation nitrogen-fixing bacillus megaterium strain DL 7 to the influence of eucalyptus nursery stock.
This test is carried out being positioned at Vegetable Research Institute nursery, Nanning, Nanning suburb.
No. 9 (GLGU9) clonal tissue cultures of wide woods Eucalyptus urophylla-grandis bottle seedling that test is produced from forest farm, state-run peak, Guangxi group training factory with nursery stock.After tissue-culture container seedling shifted out bottle, with tap water substratum is cleaned up earlier,, filter behind the thimerosal of dried nursery stock body surface standby again with the thiophanate methyl sterilization.
The spray inoculation method: after Nursery stock transplanting is gone into nutrient cup, with watering can with nitrogen-fixing bacillus megaterium strain DL 7 bacterium liquid even spraying on the eucalyptus nursery stock of field planting in nutrient cup, inoculate nursery stock 20,000 strains altogether, do not water in 6 hours.Nutrient cup soil is yellow soil.
Nursery stock method for culturing seedlings management routinely.Measure the average plant height of nursery stock, leading thread, biomass, chlorophyll content and blade nitrate reductase activity after three months.The results are shown in Table 2.
Table 2 spray method inoculation nitrogen-fixing bacillus megaterium strain DL 7 is to the influence of eucalyptus nursery stock
As seen from Table 2, inoculate plant height, leading thread and the biomass of individual tree that the nitrogen-fixing bacillus megaterium strain DL 7 bacterial strain also can obviously increase the eucalyptus nursery stock, promoted the growth of nursery stock, can also improve the chlorophyll content and the nitrate reductase activity of nursery stock blade with spray method.The results of analysis of variance shows, the height of seedling difference between processing reaches utmost point conspicuous level (F=6.1890**＞F0.01=5.06); Leading thread difference reaches utmost point conspicuous level (F=5.9330**＞F0.01=5.06); Nitrate reductase activity difference reaches utmost point conspicuous level (F=67.4260**＞F0.01=5.06).
Find out from embodiment 2,3, grow seedlings therebetween eucalyptus that the inoculation nitrogen-fixing bacillus megaterium strain DL 7 can obviously promote the growth of nursery stock, height of seedling, leading thread, biomass, chlorophyll content, nitrate reductase activity all are higher than contrast.Wherein, height of seedling increases by 40.78～56.22%, and leading thread increases by 39.41～46.43%, and biomass of individual tree increases by 72.11～158.76%, and chlorophyll content increases by 12.97～20.98%, and nitrate reductase activity increases by 33.82～35.22%.
Chlorophyll is that plant carries out photosynthetic important pigment, the effect that how much has influence on photosynthesis of plant of plant leaf chlorophyll content, and then have influence on the accumulation of plant materials photosynthate and the nitrogen fixation effect of plant self, important effect is played in plant-growth.Nitrate reductase is first enzyme in the nitrate assimilation, also is rate-limiting enzyme, is in the key position of plant nitrogen metabolism.It utilizes nitrogenous fertilizer relevant with plant absorbing, and plant-growth is had material impact, thereby nitrate reductase activity is taken as plant absorbing and utilizes one of index of nutrition such as nitrogen and growth.As seen nitrogen-fixing bacillus megaterium strain DL 7 is applicable to growing seedlings of eucalyptus nursery stock.
Sequence table
Sequence table
＜110〉Guangxi University
＜120〉nitrogen-fixing bacillus megaterium strain DL 7 and application thereof
<160>1
<170>PatentIn?version?3.3
<210>1
<211>904
<212>DNA
＜213〉fixed nitrogen bacillus megaterium
<400>1
gggccgacgt?cgcatgctcc?cggccgccat?ggcggccgcg?ggaattcgat?ttagggttac 60
cttgttacga?cttcacccca?atcatctgtc?ccaccttagg?cggctagctc?cttacggtta 120
ctccaccgac?ttcgggtgtt?acaaactctc?gtggtgtgac?gggcggtgtg?tacaaggccc 180
gggaacgtat?tcaccgcggc?atgctgatcc?gcgattacta?gcgattccag?cttcatgtag 240
gcgagttgca?gcctacaatc?cgaactgaga?aatggtttta?tgggattggc?ttgacctcgc 300
ggtcttgcag?ccctttgtac?catccattgt?agcacgtgtg?tagcccaggt?cataaggggc 360
atgatgattt?gacgtcatcc?ccaccttcct?ccggtttgtc?accggcagtc?accttagagt 420
gcccaactaa?atgctggcaa?ctaagatcaa?gggttgcact?cgttgcggga?cttaacccaa 480
catctcacga?cacgagctga?cgacaaccat?gcaccacctg?tcactctggt?cccccgaagg 540
ggaacgctct?atctctagag?ttgtcagagg?atgtcaagac?ctggtaaggg?ttcttcgcgt 600
tgcttcgaat?taaaccacat?gctccaccgc?ttgtgcgggc?ccccgtcaat?tccttttgag 660
tttcagtctt?gcgaccgtac?tcccccaggc?ggagtgctta?atgcgttagc?tgcagcacta 720
aagggcggaa?accctctaac?acttagcact?catccgttta?cggcgtggaa?ctaccagggt 780
atcctaatcc?tgtttgctcc?ccacgctttc?gcgccctcag?cgtcagttac?agaccaaaaa 840
gccgccttcg?ccactggggt?tcctccacat?ctctacgcat?tcaccgctac?acgtggaatt 900
ccgc 904
Claims (6)
1. fixed nitrogen bacillus megaterium strain (Bacillus megaterium) DL7, it is characterized in that, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 7th, 2010, preserving number is CGMCC No.3914, the ability that it has fixed nitrogen and promotes the eucalyptus seedling growth.
2. fixed nitrogen bacillus megaterium according to claim 1 strain (Bacillus megaterium) DL7 is characterized in that the nitrogenase activity of described bacterial strain is 1.5632nmolg
-1H
-1
3. fixed nitrogen bacillus megaterium according to claim 1 strain (Bacillus megaterium) DL7 is characterized in that described bacterial strain has the dna sequence dna of sequence table No.1.
4. the cultural method of strain fixed nitrogen bacillus megaterium strain (Bacillus megaterium) DL7 is characterized in that described cultural method comprises the steps:
Gather the radicula that root nodule is arranged from the Chinese podocarpus lateral root, after rinsing well with tap water, the root nodule surface was carried out disinfection 1 minute, sterilized 5～6 minutes with 0.1% acid chlorization mercury again with 75% ethanol.Place the mortar of the bacterium of going out to grind gently in root nodule.Taking juice is inoculated in method of scoring and adds on the Congo red YMA medium, cultivated 2～3 days for 28 ℃, select and do not inhale Congo red bacterium colony, repeat the line screening, then according to the difference of bacterium colony size, shape, surface luster, protuberance degree, transparency, edge shape and colony colour, select single bacterium colony, obtain required purifying bacterial strain completely through light microscopy.
5. the cultural method of fixed nitrogen bacillus megaterium according to claim 4 strain (Bacillus megaterium) DL7, it is characterized in that, described YMA medium composition and content are: dipotassium hydrogen phosphate 0.5g, sal epsom 0.5g, sodium-chlor 0.1g, N.F,USP MANNITOL 10.0g, yeast extract paste 0.4g, agar powder 15g, water 1000ml, nature pH6.8,121 ℃ of steam sterilizings 20 minutes.
6. the application of the described fixed nitrogen bacillus megaterium of claim 1 strain (Bacillus megaterium) DL7 in the bio-bacterial manure of preparation promotion eucalyptus seedling growth.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011002A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus megatherium T317, microbial agent and preparation method of microbial agent |
| CN106011004A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Nitrogen-fixing microorganism G96 as well as bacterium agent preparation method and application thereof |
| CN106011003A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus megatherium T482, microbial agent and preparation method of microbial agent |
| CN106167771A (en) * | 2016-05-20 | 2016-11-30 | 东莞市保得生物工程有限公司 | A kind of bacillus megaterium D122 and microbial inoculum thereof and bacterial preparation process |
| CN107593769A (en) * | 2017-08-24 | 2018-01-19 | 暨南大学 | Applications of the bacillus megaterium YJB3 in plant growth and biological and ecological methods to prevent plant disease, pests, and erosion is promoted |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011002A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus megatherium T317, microbial agent and preparation method of microbial agent |
| CN106011004A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Nitrogen-fixing microorganism G96 as well as bacterium agent preparation method and application thereof |
| CN106011003A (en) * | 2016-05-20 | 2016-10-12 | 东莞市保得生物工程有限公司 | Bacillus megatherium T482, microbial agent and preparation method of microbial agent |
| CN106167771A (en) * | 2016-05-20 | 2016-11-30 | 东莞市保得生物工程有限公司 | A kind of bacillus megaterium D122 and microbial inoculum thereof and bacterial preparation process |
| CN106011003B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus megaterium T482, microbial inoculum thereof and preparation method of microbial inoculum |
| CN106011004B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Nitrogen-fixing microorganism G96, and preparation method and application of microbial inoculum thereof |
| CN106011002B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus megaterium T317, microbial inoculum thereof and preparation method of microbial inoculum |
| CN106167771B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus megaterium D122, microbial inoculum thereof and preparation method of microbial inoculum |
| CN107593769A (en) * | 2017-08-24 | 2018-01-19 | 暨南大学 | Applications of the bacillus megaterium YJB3 in plant growth and biological and ecological methods to prevent plant disease, pests, and erosion is promoted |
| CN107593769B (en) * | 2017-08-24 | 2020-04-14 | 暨南大学 | Application of bacillus megaterium YJB3 in promoting plant growth and biocontrol |
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