CN104762228B - A kind of bacillus megaterium and application thereof derived from napier grass - Google Patents
A kind of bacillus megaterium and application thereof derived from napier grass Download PDFInfo
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Abstract
The present invention relates to a kind of bacillus megateriums and application thereof derived from napier grass, belong to microbe technical field.The bacterium be napier grass endophyte bacillus megaterium pp02, the entitled bacillus megaterium pp02 that classifies (Bacillus megateriumPp02), on July 4th, 2014 in China Microbiological collection preservation, preserving number CGMCC 9415；The bacterial strain has production siderophore, fixed nitrogen, the characteristic for producing IAA, and IAA yield is and strong in plant root colonization ability up to 10.57 μ g/mL, and salt resistance ability is strong, in 5%NaCl（50g/L）It can be grown in culture medium, be a kind of excellent plant growth-promoting endophyte bacterial strain；Hybrid Chinese pennisetum is infected using the bacterial strain, the salt resistance ability of plant can be significantly improved, promotes growth, plant above ground portion fresh weight, dry weight, plant height dramatically increase；The discovery of the bacterial strain promotes it to have important development prospect in the growth and development of saline and alkaline marginal land plant hybrid Chinese pennisetum microbial manure development.
Description
Technical field
The present invention relates to a kind of bacillus megateriums and application thereof derived from napier grass, belong to microbe technical field.
Background technology
In recent years, with the rise of Biomass Energy Industry, napier grass（Pennisetum purpureumschum.）And it is miscellaneous
The characteristics such as friendship Chinese pennisetum because its biomass is big, power of regeneration is strong, cellulose, hemicellulose level height, and annidation is strong, development
For a kind of novel energy-source plant.China has the suitable of the large area such as Coastal beach, salt-soda soil, wasteland, poor and barren land can marginality
Soil, biomass productive potentialities are more than 200,000,000 tons of standard coals at present.In preferably energy marginal land plantation napier grass, hybrid Chinese pennisetum etc.
Energy grass develops biomass energy, meets the energy development target of China's " not striving ground with grain, do not strive grain with people ".But these
Regional production condition is poor, lacks and irrigates guarantee, perniciousness harm is serious, seriously affects the energy grass such as napier grass, hybrid Chinese pennisetum
Yield of biomass.
Plant growth-promoting endophyte (Plant Growth-Promoting bacteria endophyte, PGPE) refers to one
Class can colonize in plant, and form special symbiosis, and bacterium therefore all helpful to both sides with plant.
Plant growth-promoting endophyte can pass through nitrogen fixation, secretion plant growth regulating substance, synthesis siderophore and inhibition some diseases
The modes such as generation promote the growth of plant, promote the cycle of nutriment in soil in addition, they also have, adjust soil knot
Structure carries out the effects that biological prosthetic to contaminated soil.The excellent efficient plant growth-promoting bacteria (Plant of research and development
Growth-Promoting Rhizobacteria, PGPR) bio-feritlizer, to improve soil in nutriment utilization rate,
The application for reducing pesticide and chemical fertilizer, the growth and development for promoting plant, enhancing plant receive weight to the adaptability and tolerance of adverse circumstance
It acts on.
So the excellent efficient napier grass Promoting bacteria bio-feritlizer of research and development, resists enhancing napier grass, hybrid Chinese pennisetum
Property, promote vine growth and development, it is significant in the marginalitys land yield such as Coastal beach, salt-soda soil to improve it.But at present
Growth promotion endophytic bacterium is mainly from rice, corn, wheat, sugarcane, apple, citrus, high clump blueberry, mulberries, apricot, also
The report of the growth promotion endophyte of napier grass is not derived from（(1) Kloepper, J.W. 1994. Plant growth
promoting bacteria (other systems). In: Okon, J. (Ed.), Azospirillum/Plant
Association. CRC Press, Boca Raton, pp. 137–154.；(2) De Silva, A., Petterson,
K., Rothrock, C., Moore, J. Growth promotion of high bush blue berry by
fungal and bacterial inoculants. 2000. HortScience35, 1228–1230.；(3)
Sudhakar, P., Chattopadhyay, G.N., Gangwar, S.K., Ghosh, J.K. Effect of
foliar application of Azotobacter, Azospirillum and Beijerinckia on leaf
yield and quality of mulberry (Morus alba),2000, J. Agric. Sci. 134, 227–
234.；(4) Esitken, A., Karlidag, H., Ercisli, S., Sahin, F. Effects of foliar
application of Bacillus substilis Osu-142 on the yield, growth and control of
shot-hole disease (Coryneum blight) of apricot, 2002, Gartenbauwissenschaft
67, 139–142；(5) Esitken, A., Karlidag, H., Ercisli, S., Turan, M., Sahin, F.
The effect of spraying a growth promoting bacterium on the yield, growth and
nutrient element composition of leaves of apricot (Prunus armeniaca L. cv.
Hacihaliloglu), 2003, Aus. J. Agric. Res.54, 377–380.）.Huge gemma provided by the invention
Bacillus pp02 is the excellent growth-promoting endophyte bacterial strain screened from napier grass, can more effectively enhance the salt tolerant energy of hybrid Chinese pennisetum
Power significantly improves the yield in hybrid Chinese pennisetum seedling stage, and significant application value is implanted with to its kind in marginal land.
Invention content
The purpose of the present invention is to provide a kind of napier grass bacillus megaterium, which has production siderophore, fixed nitrogen, production
The characteristic of IAA, IAA yield is and strong in plant root colonization ability up to 10.57 μ g/mL, and salt resistance ability is strong, in 5% NaCl
（50g/L）It can be grown in culture medium, be a kind of excellent plant growth-promoting endophyte bacterial strain.It can promote the hybridization wolf tail under salt stress
The growth of careless seedling, it is significant as popularizing planting of the energy grass on saline alkali marginal land to hybrid Chinese pennisetum.
In order to achieve the goal above, technical solution provided by the invention is：
The present invention provides a kind of napier grass endophyte, which is one plant of endophyte being separated to from napier grass plant, through tradition
Method and Molecular Identification show that it belongs to bacillus megaterium, be named as bacillus megaterium pp02 (Bacillus sp.
Pp02), on July 4th, 2014 in China Microbiological collection preservation, preserving number CGMCC 9415.
The napier grass endophyte bacillus megaterium pp02 of the present invention, it is it has the following biological characteristics：
1. colony morphology characteristic：Good in LB cultured on solid medium, bacterium colony is larger and thick, rounded, and white is not
Thoroughly
Bright, surface relatively drying has fold, edge irregular；
2. morphological features：It is rod-shaped, end circle, single or arrangement in pairs, 0.75 ~ 1 2.75 ~ 5 μm of μ m, gram sun
Property；
3. part physiological and biochemical property：It can well be grown on LB culture mediums, 28 DEG C of optimum growth temperature, the most suitable growth pH
7.0；
4. genetics characteristics：According to《Common bacteria system identification handbook》（Eastern show pearl, Cai Miaoying, Science Press,
2001）,
9415 morphological features of bacillus megaterium CGMCC and bacillus of the present invention is closest, logical through 16S rRNA
PCR, which is carried out, with primer 2 7F and 534R expands partial gene sequence（496bp）To National Center for Biotechnology Information
(NCBI) Genbank geneseq databases are submitted, accession number KM886123.The bacillus megaterium CGMCC 9415
16S rDNA sequence analyses show and the most like bacterial strain that is recorded in Genbank international data centersBacillus megateriumThe similitude of DSM319 (NC014103.1) is 99%.Therefore, the bacillus megaterium CGMCC 9415
Bacillus megaterium category should be belonged to（Bacillus sp.）.
The napier grass endophyte bacillus megaterium pp02 of the present invention, is to be separately cultured to obtain as follows from napier grass
's：
（1）It is derived from the napier grass Su Mu 2 in academy of agricultural sciences of Jiangsu Province experimental plot, root is taken to kill material to be tested table through surface sterilization
Face microorganism, adds appropriate amount of quartz sand to grind, and sterile water mixing, 100 μ L is added to be coated on LB solid mediums, 28 DEG C, culture
48h；
（2）Picking single bacterium colony scribing line purifying repeatedly on LB solid mediums, until obtaining pure lines bacterial strain；
（3）Inoculation will be sheerly in LB liquid medium, 28 DEG C of culture 18h take 700 μ L of bacterium solution that mass concentration is added
For 50% 300 μ L of glycerine, mixing is placed on freezen protective in -70 DEG C of refrigerator-freezers；
（4）By the pure lines bacterial strain of gained, after verifying its growth promoting activity by hybrid Chinese pennisetum seedling inoculation test, identification,
And strain is preserved, wherein one plant of endogenetic bacteria for isolating and purifying gained is named as pp02.
It is a further object of the present invention to provide the applications of the napier grass endophyte bacillus megaterium pp02.
The endophyte is used to promote the growth of plant, can verify by the following method：
By hybrid Chinese pennisetum seed disinfection, it is placed in 1/2 MS culture mediums, 25 DEG C, light culture vernalization in 48 hours, after vernalization
Seed be seeded in the basin alms bowl for filling vermiculite, seedling is grown to 10 days or so, with pp02 fermenation raw liquid pouring roots, after infecting 3 days, is started
And 6mg/mL brine was poured every four days, height of seedling is measured after 20 days and claims fresh weight.
The endophyte can be to be verified as follows in plant Colonization inside plants：
Above-mentioned pouring root is taken to infect the root of 3 days hybrid Chinese pennisetum seedlings of bacillus megaterium pp02, through surface sterilization, quartz
Sand is ground, and sterile water mixing, 100 μ L is added to be coated on LB solid mediums, 30 DEG C, cultivates 48h, observes bacterium colony growing state,
And 3 monoclonals of random picking, bacterium is shaken, Molecular Identification is carried out to bacterial strain through 16SrDNA sequencings, with the hybrid Chinese pennisetum not infected
Seedling root is as a contrast.The result shows that bacillus megaterium pp02 successfully infects in hybrid Chinese pennisetum root, on LB solid mediums
Observe that bacterium colony is grown, it is bacillus megaterium pp02 that sequencing result, which also demonstrates it,.
The present invention there is fixed nitrogen, production siderophore, secretion to plant heteroauxin raw bacillus megaterium PP02 in the napier grass
(IAA) etc. growth-promotings characteristic produces the amount of IAA up to 10.57 μ g/mL；It is strong to infect colonization ability, inoculation test proves root irrigation three
It, which can be 4.9cfu/g plant root fresh weights in plant Colonization inside plants, infect efficiency；Salt tolerant
Ability is strong, can be grown in 5%NaCl (50g/L) culture medium.Bacillus megaterium pp02 bacterial strains are to hybrid Chinese pennisetum seedling pouring root
Processing, the relatively nonvaccinated negative control group of plant above ground portion fresh weight quality increase by 74.01%；Dry weight is relatively nonvaccinated
Negative control group increases by 45.70%；The relatively nonvaccinated negative control group of plant height increases 30.92%.This shows huge gemma bar
The higher growth-promoting abilities of bacterium pp02, and it is strong in plant Colonization inside plants ability, and can be conveyed for its body in growing process must
The nutrients such as auxin, nitrogen, the iron wanted enhance to salt stress resistance capacity, the growth and development of growth-promoting plant.
Beneficial effects of the present invention：Compared with prior art, the present invention is special to the bacillus megaterium PP02 of offer
The growth-promoting endophyte bacterial strain that one ground is screened for napier grass, hybrid Chinese pennisetum；With fixed nitrogen, production siderophore ability, it is effectively improved soil
The content of available nitrogen, ferro element in earth；Heteroauxin (IAA) is secreted, yield can enhance plant to salt stress up to 10.57 μ g/mL
Resistance, the growth and development of growth-promoting plant significantly improves the yield of biomass in hybrid Chinese pennisetum seedling stage；The bacterial strain salt resistance ability
By force, strong in plant intramatrical infection colonization ability, it is one plant of excellent growth-promoting endophyte bacterial strain, is hybrid Chinese pennisetum bio-feritlizer
Exploitation is laid a good foundation, and has good application prospect.
Description of the drawings
Fig. 1 is the hybrid Chinese pennisetum seedling of vermiculite potting.Wherein CK indicates to replace bacillus megaterium with sterile water
(Bacillus sp.) pp02 bacterium solutions negative control；
Fig. 2 bacillus megateriums (Bacillus sp.) pp02 is to hybrid Chinese pennisetum seedling plant height under salt stress（㎝）'s
It influences；
Fig. 3 bacillus megateriums (Bacillus sp.) pp02 is to hybrid Chinese pennisetum seedling fresh weight under salt stress（g）Shadow
It rings；
Fig. 4 bacillus megateriums (Bacillus sp.) pp02 is to hybrid Chinese pennisetum seedling stem weight under salt stress（g）Shadow
It rings；
Fig. 5 bacillus megateriums (Bacillus sp.) pp02 growth-promoting characteristics a:Plant auxin secretion IAA b：Production
Siderophore ability c：Nitrogen fixing capacity；
Fig. 6 bacillus megateriums (Bacillus sp.) pp02 colonizes rear root scanning electron microscope (SEM) photograph a:Adjoining tree b：It invades
Contaminate plant；
Fig. 7 differences salinity to bacillus megaterium (Bacillus sp.) pp02 growth influence.
Specific implementation mode
The acquisition to strain of the present invention and purposes further instruction with reference to specific embodiment.
Embodiment 1：
Bacillus megaterium pp02 bacterial strains are separately cultured
Growth-promoting plant endogenesis bacillus megaterium pp02 is separated to from the root of napier grass in present embodiment, napier grass
It is derived from the napier grass Su Mu 2 in academy of agricultural sciences of Jiangsu Province experimental plot, separation method is as follows：
（1）Napier grass root is rinsed well with flowing water, takes 1g as material to be tested, it is 70% wine to use mass concentration successively
Essence 5 minutes, aseptic water washing 3-5 times, the sodium hypochlorite that mass concentration is 1% impregnate 20min, kill the micro- life in material to be tested surface
Object；
（2）In gnotobasis, material to be tested aseptic water washing 3 times takes the last 1 aseptic water washing liquid of 100 μ L to apply
It is distributed on LB solid mediums and cultivates 48h, whether there is or not bacterium colony generations for observation；
（3）It is ground to material to be tested plus appropriate amount of quartz sand, adds 5mL sterile water mixings, 100 μ L is taken to be coated on LB solid cultures
On base (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar powder 15g/L), 28 DEG C, 48h, picking are cultivated
Single bacterium colony scribing line purifying repeatedly on LB solid mediums, until obtaining pure lines bacterial strain；
（4）Inoculation will be sheerly in LB liquid medium（Tryptone 10g/L, yeast extract 5g/L, sodium chloride
In 10g/L), 28 DEG C of culture 18h take 700 μ L of bacterium solution that the 300 μ L of glycerine that mass concentration is 50% are added, and mixing is placed on -70 DEG C
Freezen protective in refrigerator-freezer；The endogenetic bacteria for wherein one plant being isolated and purified gained is named as pp02；
Wherein step 2 purpose is whether verification material to be tested surface microorganism can all kill, if there is bacterium colony generation, demonstrate,proves
Bright material surface microorganism is without all killing, on the contrary then nothing.
The bacterial strain screened through the above method expands 16S rDNA gene orders, production with universal primer 27F and 534R PCR
Wu Song Sangon Biotech (Shanghai) Co., Ltd. is sequenced, and sequencing result is blast points in Genbank international data centers
Analysis,Bacillus megateriumThe similitude of DSM319 (NC014103.1) is 99%.In conjunction with the colony characteristics of the bacterium,
The bacillus megaterium pp02 should belong to bacillus megaterium category（Bacillus sp.）.On July 4th, 2014 in the micro- life of China
Object collection preservation, preserving number CGMCC 9415.
Embodiment 2:
The culture producing method for seed of bacillus megaterium pp02, follows the steps below：
(1) 9415 bacterium solutions of bacillus megaterium CGMCC of -70 DEG C of freezen protectives are taken, streak inoculation is in LB solid mediums
On,
18 ~ 20h is cultivated in 28 DEG C of insulating boxs, picking single bacterium colony is inoculated in the LB liquid medium of 5mL, 28 DEG C of concussion trainings
18 20h are supported, activation bacterium solution is obtained；
(2) 1mL activation bacterium solution is inoculated in the 500mlL triangular flasks equipped with 100mL LB liquid mediums, 28 DEG C,
150
250r/min shaken cultivation 12h 16h obtain the zymotic fluid in exponential phase that OD is about 1；
Embodiment 3:
Experiments of the bacillus megaterium pp02 to Plant growth promotion：
To ensure the homogeneity of experiment material, following experiments of the invention are using hybrid Chinese pennisetum seed as experimental subjects.
（1）The disinfecting process of hybrid Chinese pennisetum seed is：The alcohol 5 minutes for being 70% with mass concentration, aseptic water washing 3-
5 times, the hydrogen peroxide dipping 30min that mass concentration is 10%, aseptic water washing 5 times；
（2）The vernalization of hybrid Chinese pennisetum seed：Seed after disinfection is subsequently placed in 1/2 MS culture mediums, 25 DEG C, light culture
Vernalization in 48 hours；
（3）The transplanting of hybrid Chinese pennisetum young shoot：The cotton seeds of vernalization are seeded in equipped with 50g sterilizing vermiculites（121℃,
2h）Greenhouse basin alms bowl in, per basin 5 seedlings, 5 basins of each processing setting repeat；
（4）Hybrid Chinese pennisetum seedling infects：Seedling is grown to 10 days or so, is invaded with bacillus megaterium pp02 fermenation raw liquid pouring roots
Dye, concrete operations are：50mL bacillus megaterium pp02 fermenation raw liquids are added to every basin hybrid Chinese pennisetum seedling base portion, are opened after 3 days
The processing of beginning salt stress, i.e., pour the NaCl solution of 6 ㎎ of 50mL/mL to every basin hybrid Chinese pennisetum seedling base portion（Sterile water configures）, will be upper
It states sterile water and replaces zymocyte liquid, remaining processing is identical, as blank control.
（5）The culture of hybrid Chinese pennisetum seedling：Above-mentioned experimental group and control group are placed on 28 DEG C, illumination cultivation（Light recycles：
Illumination in 16 hours, 8 hours dark）, intensity of illumination：10000Ix measures height of seedling and claims fresh weight after 20 days.
Test result shows that the bacterial strain has apparent growth promoting function to the hybrid Chinese pennisetum seedling under stress（Fig. 1）, real
The hybrid Chinese pennisetum seedling growth conditions for testing group are significantly better than control group, and experimental group plant height is shone than control increases by 30.92%（Fig. 2）,
Fresh weight and dry weight increase by 74.01% than control（Fig. 3）, 45.7%（Fig. 4）.
Embodiment 4:
The detection of bacillus megaterium pp02 growth-promoting characteristics：
1. the detection of auxin (IAA) ability of secretion
（1）According to bibliography（Sheng XF,Xia JJ, Liang CY, He LY, Qian M.
Characterization of heavy metal-resistant endophytic bacteria from rape
(Brassica napus) roots and their potential in promoting the growth and lead
accumulation of rape, 2008, Envrionmental pollution, 156:1164~1170）Described
Salkowski colorimetric method for determining bacillus megateriums pp02 secretes auxin（IAA）Ability.
By the bacillus megaterium of activation (Bacillus sp.) pp02 bacterium solutions are inoculated in final concentration of 0.5mg/ml's
In SMS culture mediums, 28 DEG C of shaking tables, 180rpm shake cultures 4d.Take 1mL bacteria suspensions (zymotic fluid) that the Salkowski ' of 2ml is added
S color developing agents are sufficiently mixed, and react 20min at room temperature, and it is the positive pink occur, illustrate that this bacterial strain can generate IAA, and
And red deeper illustrate that the ability for producing IAA is stronger.The color solution of 1mL 50mg/L IAA will be added as a contrast.Experiment is set three times
It repeats.
SMS culture medium prescriptions are：Sucrose 10g, (NH4)2SO4 1g、K2HPO4 2g、MgSO4 0.5g, NaCl 0.1g, yeast
Powder 0.5g, CaCO30.5g, it is 7.2 to adjust pH.
Salkowski ' s color developing agents：The FeCl of 250mL deionized waters, the 150mL concentrated sulfuric acids, 7.5mL 0.5mol/L3•H2O。
The results show that bacillus megaterium (Bacillus sp.) pp02 bacteria suspensions dropwise addition Salkowski color solutions
Afterwards, bacterium solution color reddens, show bacillus megaterium (Bacillus sp.) pp02 can secrete auxin (IAA)
（Fig. 5 a）.
（2）Produce heteroauxin（IAA）Quantitative detection
The bacillus megaterium pp02 bacterial strains of activation are cultivated into 4d in King culture mediums, by bacteria suspension at 10000rpm
Centrifuge 10min, take supernatant, each isometric addition 1mLl supernatants in PC color solutions, quiet 30min in the dark, after taking-up
It is measured under OD530nm and uses pure 3-IAA（3-indolyl acetic acid）It makes standard curve and calculates IAA yield, not connect the culture medium of bacterium
It returns to zero as a contrast.Experiment is in triplicate.
King nutrient media components：Peptone 20g, K2HPO4 1.15g MgSO4.7H2O 1.5g, glycerine 15mL, L- color
Propylhomoserin 0.5g.
The composition of PC color solutions：FeCl312g, 7.9M H2SO41L。
The result shows that bacillus megaterium (Bacillus sp.) pp02 secretion heteroauxins (IAA), yield reaches
10.57μg/mL。
2, production siderophore ability detection
According to bibliography（Vendan, Regupathy Thamizh; Yu, Young Joon; Lee, Sun Hee;
Rhee, Young Ha. Isolation and characterization of plant growth promoting
endophytic diazotrophic bacteria from Korean rice cultivars. The Journal of
Microbiology48.5,(Oct2010):559-65.）It is described：After mixing by four kinds of solution, it is flat that siderophore detection is made
Plate.The strain point of activation is connected on siderophore detection culture medium, culture is inverted at 28 DEG C, the color observed on tablet becomes
Change, if periphery of bacterial colonies produces orange circle and turns out and produce siderophore on tablet.
The formula of four kinds of solution is：
Solution one：60.5 mg CAS are dissolved in 50 mL water, 10 mL trivalent ferrous solutions（1mM FeCl3•6H2O, 10 mM
HCl）, the two is uniformly mixed, by 72.9 mg HDTMA（Cetyl trimethylammonium bromide）It is dissolved in 40 mL water, then delays
Slow stirring is added in the above mixed solution, high pressure steam sterilization 20min, cooling at 121 DEG C by above-mentioned navy blue mixed solution
To 50 DEG C.
Solution two：By 0.3 g KH2PO4, 0.5 gNaCl and 1.0 g NH4Cl are dissolved in 750mL water, add
30.24 g PIPES（Two ethanesulfonic acids of piperazine -1,4-）In the above salting liquid, with 50% (w/w) KOH solution adjust pH to
6.8,15g agar powders are added, the high pressure steam sterilization 20min at 121 DEG C is cooled to 50 DEG C.
Solution three：100mL15 × KB culture mediums（Peptone 3.2g, K2HPO4 0.115g, MgSO4·7H20.15 g of O,
Glycerine 1.5g）Contain 2 g glucose, 2 g mannitol, 439 mg MgSO with 70mL4•7H2O、11 mg CaCl2、1.17mg
MnSO4•H2O、1.4mgH3BO3、0.04mgCuSO4•5H2O、1.2mgZnSO4•7H2O、1.0mg Na2MoO4•2H2The solution of O is mixed
It closes, the high pressure steam sterilization 20min at 121 DEG C is cooled to 50 DEG C.
Solution four：The casamino acid solution of 30mL 10%, filtration sterilization.
The results show that bacillus megaterium（Bacillus sp.）Periphery of bacterial colonies on pp02 tablets can generate significantly
Orange circle, it was demonstrated that the bacterium can secrete siderophore, promote absorption (Fig. 5 b) of the plant to iron.
3, nitrogen fixing capacity detects
According to bibliography（Zhang Zhen's powder, the functional diversity of herbage endophytic Bacillus subtilis and 16S rDNA identifications
[D], 2010, Lanzhou, Gansu Agriculture University）It is described, with Ah 's bayesian（Ashby）Cultivate based assays bacillus megaterium
(Bacillus sp.）The nitrogen fixing capacity of pp02.By in activated inoculation to Ah 's shellfish culture medium, 3 weights of each bacterial strain
It is multiple, it is control to be inoculated with sterile water, the constant temperature incubation at 28 DEG C on day 3 and observes growth situation on the 7th day, apparent muddy
Turbid is the positive.
The results show that bacillus megaterium（Bacillus sp.）Pp02 culture tube bacterium solutions are obviously muddy, illustrate huge bud
Spore bacillus（Bacillus sp.）Pp02 has relatively strong nitrogen ability（Fig. 5 c）.
Embodiment 5:
Colonization ability identifications of the bacillus megaterium pp02 in plant
（1）Pouring root described in Example 1 infects the root of 3 days hybrid Chinese pennisetum seedlings of bacillus megaterium pp02, warp
Surface sterilization, quartzite sand grind add sterile water mixing, 100 μ L to be coated on LB solid mediums, 30 DEG C, cultivate 48h, observation
Bacterium colony growing state, and 3 monoclonals of random picking, shake bacterium, Molecular Identification are carried out to bacterial strain through 16SrDNA sequencings, not invade
The hybrid Chinese pennisetum seedling root of dye is as a contrast.
Being inoculated with verification test proves root irrigation three days, the bacillus megaterium pp02 can in plant Colonization inside plants,
Infect efficiency is 4.9cfu/g plant root fresh weights.
（2）Pouring root described in Example 1 infects the root of 3 days hybrid Chinese pennisetum seedlings of bacillus megaterium pp02, washes
Vermiculite is removed, and then -20 DEG C of refrigerator freezings for 24 hours, then are placed in freeze drier dry, root are longitudinally splitted with blade method, scanned
Electronic Speculum observation endophyte colonizes situation, takes pictures.
The result shows that infecting about 7 days, bacillus megaterium pp02 is colonized in the cortex of root on a large scale, and minority colonizes
It has arrived in vascular bundle（Fig. 6）.
Embodiment 6：
The salt resistance ability of bacillus megaterium pp02 is identified
（1）9415 bacterium solutions of bacillus megaterium CGMCC of -70 DEG C of freezen protectives are taken, streak inoculation is in LB solid mediums
On, 18 20h are cultivated in 28 DEG C of insulating boxs, picking single bacterium colony is inoculated in the LB liquid medium of 5mL, 28 DEG C of shake cultures
18 20h obtain activation bacterium solution；
（2）Activated bacterium is seeded in the LB that NaCl concentration is 0%, 0.5%, 1%, 1.5%, 3%, 5%, 7%, 9%, 11% and cultivates
In base, 24 hours light splitting afterwards photometric determination bacterium solution OD values are cultivated（OD600）.
The results show that bacillus megaterium pp02 salt resistance abilities are stronger, for growth when NaCl concentration is below 1.5%
It has little effect, at NaCl concentration 3 ~ 5%, although the growth of pp02 receives part inhibition, but remains to remain normal
Growth metabolism, culture for 24 hours when cell concentration OD up to 2.5 or so（Fig. 7）.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (5)
1. a kind of can promote the bacillus megaterium pp02 (Bacillus that hybrid Chinese pennisetum is grown under condition of salt stress
Megaterium pp02), on July 4th, 2014 in China Microbiological collection preservation, preserving number CGMCC 9415；Institute
It states bacillus megaterium pp02 and derives from napier grass.
2. a kind of bacillus megaterium that can promote hybrid Chinese pennisetum growth under condition of salt stress according to claim 1
Pp02, which is characterized in that the bacillus megaterium pp02 is separately cultured to obtain from napier grass as follows：
（1）It is derived from the napier grass Su Mu 2 in academy of agricultural sciences of Jiangsu Province experimental plot, takes root micro- through surface sterilization killing material to be tested surface
Biology adds appropriate amount of quartz sand to grind, and adds sterile water mixing, is coated on LB solid mediums, 28 DEG C of culture 48h；
（2）Picking single bacterium colony scribing line purifying repeatedly on LB solid mediums, until obtaining pure lines bacterial strain；
（3）Will pure lines inoculation in LB liquid medium, 28 DEG C culture 18h, take bacterium solution be added mass concentration be 50% it is sweet
In oil, mixing is placed on freezen protective in -70 DEG C of refrigerator-freezers；
（4）By the pure lines bacterial strain of gained, after verifying its growth promoting activity by hybrid Chinese pennisetum seedling inoculation test, identification, and protect
Strain is deposited, wherein one plant of endogenetic bacteria for isolating and purifying gained is named as pp02.
3. a kind of bacillus megaterium pp02 described in claim 1 is in the case where promoting condition of salt stress in hybrid Chinese pennisetum growth
Using.
4. a kind of bacillus megaterium that can promote hybrid Chinese pennisetum growth under condition of salt stress according to claim 1
Pp02, which is characterized in that the bacillus megaterium pp02 can be in napier grass or hybrid Chinese pennisetum Colonization inside plants.
5. a kind of bacillus megaterium pp02 that can promote hybrid Chinese pennisetum growth under condition of salt stress described in claim 1,
It is characterized in that, the bacillus megaterium pp02 can secrete auxin heteroauxin, there is nitrogen fixing capacity, and
Siderophore can be produced.
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| CN105018393B (en) * | 2015-08-14 | 2018-02-23 | 山东泰诺药业有限公司 | One plant of bacillus megaterium and its application |
| CN106167770B (en) * | 2016-05-20 | 2020-01-17 | 东莞市保得生物工程有限公司 | Bacillus megaterium B16, microbial inoculum thereof and preparation method of microbial inoculum |
| CN107593769B (en) * | 2017-08-24 | 2020-04-14 | 暨南大学 | Application of bacillus megaterium YJB3 in promoting plant growth and biocontrol |
| CN108102984B (en) * | 2018-01-12 | 2018-10-12 | 江苏省农业科学院 | Raw bacillus megaterium BM18-2 and its application in one plant of somatotrophic hybrid Chinese pennisetum of cadmium |
| CN110358697B (en) * | 2018-04-11 | 2022-01-14 | 中国科学院分子植物科学卓越创新中心 | Bacillus megaterium and application thereof in promoting plant growth |
| CN109234183A (en) * | 2018-05-18 | 2019-01-18 | 苏州农业职业技术学院 | A kind of salt tolerant growth-promoting bacteria strain, screening technique and application |
| CN111088197B (en) * | 2020-01-20 | 2021-07-23 | 华南农业大学 | Provevelis alcaligenes and application thereof in degrading tetracycline and producing auxin |
| CN112159786B (en) * | 2020-11-04 | 2022-03-01 | 河北科技大学 | Cr (VI) reducing strain C6, and culture condition and application thereof |
| CN113875525A (en) * | 2021-10-11 | 2022-01-04 | 江苏沿江地区农业科学研究所 | Planting method for increasing grain number per ear and yield of rice under salt stress |
| CN116018954B (en) * | 2022-10-19 | 2023-11-17 | 江苏省农业科学院 | Improved variety breeding method for forage sorghum-bacillus megaterium symbiota |
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