CN105985922B - One plant of A Shi bacillus J5 and its application - Google Patents
One plant of A Shi bacillus J5 and its application Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention belongs to microbial resources fields, specifically disclose one plant of A Shi bacillus J5 and its application in terms of corn growth-promoting.A Shi bacillus J5 was preserved in China General Microbiological culture presevation administrative center (CGMCC) on October 15th, 2015, and deposit number is CGMCC No.11508.A Shi bacillus J5 of the invention, which has, produces auxin, produces siderophore, produces ammonia, produces protease, salt tolerant, the functions such as antagonism and phosphorus decomposing；Bacterial strain normal growth under the culture medium condition of salt content 11%, it is shown that preferable to eliminate soil salinization potentiality；It produces siderophore content and reaches 53.7%, there is efficient diseases prevention characteristic.The bacterial strain can improve corn seed germination rate 12.5%, have preferable growth-promoting effect.After the microbial inoculum application of bacterial strain preparation, corn yield improves 37.1%, and 100-grain weight improves 35%.The bacterial strain be expected exploitation be bacteria agent, improve soil nutrient, eliminate salination, promote plant growth and in terms of have a extensive future.
Description
Technical field
The present invention relates to microbial resources fields, more particularly, to one plant of A Shi bacillus J5 and its application.
Background technique
Plant growth-promoting rhizobacteria (Plant growth-promoting rhizobacteria, PGPR), which refers to, survives in plant
Rhizosphere, root table, and can directly or indirectly promote or the microorganism of coordinate plant growth.Plant growth-promoting rhizobacteria generally has production
Auxin produces ammonia, produces siderophore, antagonism, phosphorus decomposing, the abilities such as salt tolerant or the ability at least with one of them.Inoculated plant root
Border Promoting bacteria can make soil nutrient validation, and root system of plant is promoted to absorb soil moisture and nutrient, raw to improve plant
It is long.Bacillus generates the gemma for having very strong resistance to heat, ionising radiation and certain chemicals etc..Therefore, there is root
The gemma bacillus agent of border growth-promoting functions, simple production process, shelf life is long, and being easy to survive in soil environment colonizes, thus
Field crops growth is improved, huge application prospect is shown in microbial manure industry.
A Shi bacillus (Bacillus aryabhattai) in Shivaji in 2009 etc. for the first time from height above sea level be 27~
Separation is screened in the air of 41km, and as a kind of new mushroom-seed culturing resource, there are significant rhizosphere growth-promoting functions.For example, left mountain etc.
The A Shi bacillus B8W22T of screening has and produces auxin feature, can significantly improve smoke tree growth.The A Shi of the text screening of Liu
Bacillus has effect of solubilizing phosphate, has facilitation to plant germination and growth.Duan Jiali reports B5 pairs of A Shi bacillus
Muskmelon seeds plumular axis and radicle growth have facilitation.The A Shi bacillus MASR14 that Aketi etc. is screened in Central India
With producing auxin, producing siderophore and producing three kinds of beneficial characteristics of ammonia, there is facilitation to plant growth.Li Zuhong etc. using Ah
The antagonistic effect effect prevention and treatment tobacco black shank of family name bacillus, has significant effect.Lakeside Sol etc. barren in South Korea
Screening four plants has the A Shi bacillus for producing two kinds of functions of IAA and Soluble phosphorus, promotes the growth of lakeside Italy Siberian cocklebur.This
A little progress illustrate the huge rhizosphere growth-promoting potentiality of A Shi bacillus, but these results are confined to A Shi bacillus more
Single or two or three kind of function parsing, it is difficult to meet the market demands of Modern microbiological fertilizer functional diversities.
In addition, the soil salinization is the serious major reason for restricting plant growth.According to current UNESCO and
The incomplete statistics of food and agricultural organization, world's salination area are 109hm2, the Second National overall survey of soil in China the results show that
The salinisation gross area is 3.6 × 107 hm2, the 4.88% of area can be utilized by accounting for the whole nation.Cultivated area salination reaches 9.21 ×
106 hm2, the 6.62% of Zhan Quanguo cultivated area.Forefathers' research, A Shi bacillus have preferable salt tolerant feature, for example Lv Zhao
The A Shi bacillus of the separation screening from the middle layer pit mud sample of Luzhou, Sichuan liquor production factory such as brightness, at salt content (NaCl)
Under the conditions of 0.5%, sorghum saccharatum plant strain growth is remarkably promoted.A Shi bacillus MDSR 7, the MDSR 11 of the screenings such as Aketi,
The salt tolerance of MDSR 14 is between 4~10%.Du Chuanying China the Northeast screen one plant with 6% salt tolerance Ah
Family name bacillus.Liu Xin Linli has studied the growing state of A Shi bacillus A4, the increment of bacterial strain with various concentration NaCl
First increase with the increase of NaCl concentration, when NaCl concentration is 6% left and right, increment reaches maximum.These progress are shown
Great potential of the A Shi bacillus in terms of soil salinization improvement needs further to be studied.
The siderophore of bacterial strain equally has preferable facilitation to plant growth.Existing research shows that in iron deficiency ring
The siderophore that plant under border can directly be generated using microorganism is conducive to promote to plant to increase absorption of the plant to iron
Object growth.Siderophore can also by inhibiting harmful microorganism promote plant growth, therefore, siderophore can by directly or
Indirectly-acting promotes the growth of plant.The A Shi bacillus WRP5 of the screenings such as Sheetal can produce siderophore.The screening such as Sol
Four plants of A Shi bacillus pass through qualitative experiment and judge that it can produce siderophore, but also do not quantitative determined.Sun Lei
The siderophore content of A Shi the bacillus N44 and N23 of equal screenings are respectively 11.8% and 9.6%；Most of research at present is only
Can qualitative judgement A Shi bacillus only crossed produce siderophore, and the research for quantitative determining siderophore units activity is relatively fewer, still
To further study.
A kind of dominant strain of the A Shi bacillus as new microbial bacterial agent, current research are also limited to laboratory
With the potted plant experiment stage.For example, Jian etc. has carried out the research of some physicochemical properties in laboratory to A Shi bacillus GZ03.
Aketi etc. has studied A Shi bacillus to the growth-promoting effect of crop with microcosm soil incubation.Seulki uses pot experiment,
Have studied the effect that A Shi bacillus promotes soil phophorus validation.Sol etc. is probing into A Shi bacillus to the growth-promoting of plant
When effect, microcosm experiment is utilized and is verified.Therefore, the field growth-promoting for needing further to be confirmed A Shi bacillus is made
With.
Summary of the invention
The purpose of the invention is to overcome that above-mentioned deficiency exists in the prior art, provides one plant and have both efficiently to generate and grow
Multi-functional compound A Shi bacillus J5, the bacterial strains such as element, production ammonia, production siderophore, phosphorus decomposing, salt tolerant, antagonism can improve seed
Germination percentage promotes plant growth and raising plant products etc..
It is another object of the present invention to provide A Shi bacillus J5 to produce auxin or/and produce ammonia or/and production iron
Application in carrier or/and phosphorus decomposing or/and salt tolerant or/and antagonism or/and promotion germination or/and promotion plant growth.
To achieve the goals above, the present invention is achieved by the following technical programs:
One plant of A Shi bacillus (Bacillus aryabhattai) J5, abbreviation J5 bacterial strain, the bacterial strain was in 2015 10
The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), deposit number CGMCC on the 15th
No.11508, classification naming: A Shi bacillus aryabhattai；Preservation address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica.
A Shi bacillus of the present invention (Bacillus aryabhattai) J5 is from Guangzhou, Guangdong south China
Gained is isolated and purified in the vegetable garden soil on agriculture university farm.The bacterial strain has an endogenous spore, belongs to gram-positive bacteria, thallus
Be it is rod-shaped, have motility, it is aerobic；The bacterium colony cultivated on beef-protein medium 24 hours is round or irregular shape,
The all circles of bacterium colony, yellowish after 48h, and diameter is about 2~3mm, and edge is irregular, flat wet.Bacterial strain has stronger
IAA, siderophore and dissolving P capacity are produced, is A Shi bacillus, latin name by Physiology and biochemistry and Molecular Identification are as follows:Bacillus aryabhattai, the 16S rRNA result of gene sequence determination of bacterial strain is as shown in SEQ ID NO:1.
The method for preserving of J5 bacterium, the ingredient of storage medium are 3.0 g of beef extract, 10.0 g of peptone, sodium chloride 5.0
G, 15.0 g of agar, 1000 mL of distilled water, or the culture medium being configured to according to this ratio, pH value are 7.0~7.2.Routinely bacterium
Kind preservation temperature preservation.
A kind of microbial bacterial agent, comprising A Shi bacillus as described above (Bacillus aryabhattai) J5.
Preferably, the concentration of J5 bacterial strain is 1.0~9.0 × 10 in the microbial bacterial agent8cfu/ml。
Microbial bacterial agent as described above is producing auxin or/and is producing ammonia or/and production siderophore or/and protease or/and mistake
Hydrogen oxide enzyme or/and decompose insoluble phosphorus or/and salt tolerant or/and it is antibacterial in application.
Microbial bacterial agent as described above is promoting germination or/and is promoting the application in plant growth.
As described above A Shi bacillus (Bacillus aryabhattai) J5 produce auxin or/and produce ammonia or/
With produce siderophore or/and protease or/and catalase or/and decompose insoluble phosphorus or/and salt tolerant or/and it is antibacterial in answer
With.
As described above A Shi bacillus (Bacillus aryabhattai) J5 promoting germination or/and promotion
Application in plant growth.
It is highly preferred that the plant is corn.
Compared with prior art, the invention has the following advantages:
A Shi bacillus J5 bacterial strain of the invention is under the condition of culture of salt content 11%, well-grown, it is shown that super good
Salt tolerance, effect is best in reported A Shi Bacillus strain；Bacterial strain J5 siderophore experiment display its have compared with
Big orange haloing produces siderophore content and is up to 53.7%, this is the A Shi bacillus for reporting high sequestering power siderophore for the first time
Bacterial strain.Bacterial strain J5 not only achieves good laboratory and pot experiment effect, is investigated bacterial strain J5 in maize field
Using the results show that corn yield increasing 37.1%, spike length increases by 10.6%, and fringe slightly improves 7%, and corn total particle number increases by 9%, corn
Fresh weight improve 35.8%, dry weight improves 44.4%, and 100-grain weight improves 35%.Therefore, A Shi bacillus J5 of the invention
Have production auxin, produce siderophore, produce ammonia, produces protease, salt tolerant, antagonism, the comprehensive function of Soluble phosphorus, and significant effect.Separately
Outside, this research also compares the influence that different microbial inoculum methods of application carry out the growth of potting plant to bacterial strain J5, is later period section
It learns rational application of fertilizer method and data supporting is provided.If microbial bacterial agent is made as cereal crops using A Shi bacillus J5 to grow
Neoformation microbial inoculum, will have huge market application prospect.
Detailed description of the invention
Fig. 1 is J5 bacterial strain spore staining microphoto, and oval shaped portion is gemma in thallus.
Fig. 2 is J5 bacterial strain Gram's staining microphoto, and thallus is dyed to purple.
Fig. 3 is the phylogenetic tree of J5 bacterial strain.
Fig. 4 is the qualitative color comparison diagram that different strains produce IAA.
Fig. 5 is J5 bacterial strain salt tolerance lab diagram.
Fig. 6 is that J5 bacterial strain produces siderophore figure.
Fig. 7 is that J5 bacterial strain produces ammonia experiment picture, and A15 is positive control.
Fig. 8 is that J5 bacterial strain produces catalase result figure, and A15 is positive control.
Fig. 9 is that J5 bacterial strain produces protease experimental result.
Figure 10 is antagonistic experiment result of the J5 bacterial strain to banana blight.
Figure 11 is that J5 bacterial strain utilizes citrate experimental result, and A15 is positive control.
Figure 12 is the indoles experiment photo of J5 bacterial strain, and A15 is positive control.
Figure 13 is that the promotion corn germination of J5 bacterial strain tests photo.
Figure 14 is that J5 microbial inoculum promotes corn growth pot experiment photo.
Figure 15 is under J5 microbial inoculum Different Fertilization to promotion corn growth pot experiment photo.
Figure 16 is that J5 microbial inoculum promotes corn growth field plot trial photo.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.The separation of 1 bacterial strain of embodiment
S1. separate: the vegetable garden soil with the northern farm of Tianhe District, Guangzhou City, Guangdong Province Agricultural University Of South China leap is screening soil sample,
Screening soil sample 10.0g is accurately weighed using spread plate, is put into the 250mL triangular flask equipped with 90mL sterile water and (adds glass
Pearl), shaking table vibrates 30min, keeps cell fully dispersed, stands 20~30s, takes supernatant to carry out 10 times of dilutions of successively decreasing, using 104
~106Dilution draws dilution 0.1mL with liquid-transfering gun respectively, is coated on beef extract-peptone plate, 28 DEG C of inversion cultures
48h.Suitable concentration plate is selected, according to form, size, color, the typical single bacterium colony of picking different strains, in culture medium
On after purification, the bacterial strain of screening is subjected to inclined-plane preservation, it is spare.
S2. it produces the screening of auxin IAA: passing through plate dilution method, according to the form, size, matter for growing bacterium colony on plate
The difference of the morphological features such as ground, color isolates 45 plants of bacteriums.IAA is qualitative and quantitative screening by producing, J5 strain secretes
IAA is most, and conversion IAA reaches 20.1 mgL when 24 h-1, after by repeatedly passing on, produce IAA and stablize.The J5 bacterium that will be filtered out
Strain is stored in the made inclined-plane of beef-protein medium, it is spare to be placed in 4 DEG C of refrigerators using after plate streaking.
The identification of 2 J5 strain characteristic of embodiment
Thalli morphology characteristic: gram-positive bacteria has gemma.Thallus be it is rod-shaped, have motility, it is obligate aerobic.Gemma dye
Color photo is shown in Fig. 1.
Colonial morphology characteristic: it is round or irregular that the bacterium colony formed afterwards for 24 hours is cultivated on beef-protein medium
Shape, all circles of bacterium colony after 48h, yellowish, diameter is about 2-3mm, and edge is irregular, flat wet.
Growth characteristics: in 3.0 g of beef extract, 10.0 g of peptone, 5.0 g of sodium chloride, the fluid nutrient medium of water 1L,
113 rpm of revolving speed, 30 DEG C of temperature, initial ph value be 7.0 under conditions of, cultivate 18h, measure viable count be (1.94 ± 0.60) ×
108cfu·mL-1。
Physiology, biochemical characteristic: with reference to the routine experiment method of " primary Jie Shi Bacteria Identification handbook ", this bacterial strain is carried out
Physiological and biochemical test, experimental result are shown in Table 1.
1 bacterial strain J5 physiological and biochemical test of table
| Test name | As a result | Test name | As a result |
| Produce IAA test | + | Solve starch test | ? |
| Siderophore test | + | Catalase | + |
| Indoles | + | Proteasome degradation | + |
| Produce ammonia test | + | Methyl red test | ? |
Note: "+" is the positive in table, and "-" is feminine gender.
Molecular biological characteristic: the total DNA of J5 bacterial strain is extracted using RNA isolation kit.Using bacterial 16 S rDNA universal primer
27f:(AGA GTT TGA TCC TGG CTC AG) and 1492r:(TAC GGC TAC CTT GTT ACG ACT T), PCR expands
The 16S rDNA for increasing bacterium, occurs apparent band near 1000bp, carries out sequencing after pcr amplification product is recycled,
The DNA sequence dna of acquisition is inputted into GenBank, analysis is compared to all sequences in database with Blast program, is as a result sent out
The 16S rDNA sequence of existing bacterial strain of the present invention and with A Shi bacillus homology with higher, phase in GenBank
It is 100% (see Fig. 3) like degree.In conjunction with above-mentioned flat-plate bacterial colony feature, physio-biochemical characteristics, 16S rDNA sequence as a result, first
Step identifies that J5 bacterial strain is A Shi bacillus (Bacillus aryabhattai strain), classification naming: A Shi bacillus
Bacillus aryabhattai.The bacterial strain is preserved in China Committee for Culture Collection of Microorganisms on October 15th, 2015
Common micro-organisms center (CGMCC), deposit number are CGMCC No.11508, preservation address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica.
3 J5 bacterium of embodiment produces auxin effect
Auxin qualitative determination: J5 bacterium is inoculated in the LB liquid of the 50mL containing L-Trp (100 μ g/mL)
In culture medium, 30 DEG C, 150r/min shaking table culture is for 24 hours.It takes 300 μ L culture drop on whiteware plate, while adding 300
μ L Salkowski color solution, if the sterile LB medium of 300 μ L is as negative control, 10,20,30,50(μ g/mL) growth
The color solution of element is as positive control.By whiteware plate in room temperature, be protected from light under the conditions of place 30 min after observe, color become
Red person indicates that auxin can be produced.Test result is shown in Fig. 4.
Auxin quantitative determination: J5 bacterium is inoculated in the 50mL LB Liquid Culture containing L-Trp (100 μ g/mL)
In base, three repetitions, 30 DEG C, 150r/min shaking table culture is for 24 hours.Culture solution is centrifuged 10min at 8,000 xg respectively, is taken
4mL Salkowski color solution is added in 4mL supernatant, mixes, and stands 30min in room temperature, dark, uses uv-spectrophotometric
Meter, surveys absorbance at 530nm, while aseptic culture medium being used to do same treatment as control, using 0,10,20,30,50 (μ
G/mL auxin) makees standard curve.J5 bacterium generates long cellulose content and reaches 20.1 mg/L.
4 J5 bacterium Salt resistant test of embodiment
J5 bacterial strain is crossed to 0%, 1%, 3%, 5%, 7%, 9%, 11%, on 13% nutrient agar panel, culture for 24 hours, observation
The growing state of bacterial strain.Test result such as Fig. 5.The salt resistance ability of bacterial strain J5 can reach 11 g100mL-1.So strong salt tolerant
Property is highest in A Shi bacillus reported at present.
5 J5 bacterium of embodiment produces the measurement of siderophore ability
It is qualitative: bacterial strain to be transferred to after cultivating 24-48 h on LB plate, connect using an inocalation method and examined in MSA-CAS solid
On master plate, 3 repetitions, 30 DEG C of 72 h of culture are put, observation detects the size of the outer yellow halo of bacterium colony on plate, secretes siderophore
Surrounding bacterial colonies will appear apparent orange siderophore haloing.Results strain J5 haloing is larger, and it is strong to produce siderophore ability.Such as
Fig. 6.
It is quantitative: by strain inoculated in limit iron MSA fluid nutrient medium, 28 DEG C of 48 h of shaking table culture (150 rpm)；Bacterium is outstanding
Liquid is centrifuged 15 min through 10000rpm, takes supernatant, and the volume that bacterium solution and CAS detect liquid 1:1 mixes well, and after static 1 h, surveys
Light absorption value (As) at fixed 630 nm wavelength takes distilled water as control zeroing, with the nonvaccinated MSA liquid training being measured in the same method
The light absorption value of base is supported as reference value (Ar), and calculates siderophore active unit ([(Ar-As)/Ar] × 100).The work of bacterial strain J5
Property up to 53.7%, report belongs to the A Shi bacillus for producing high chelating siderophore for the first time.
The effect experiment of 6 J5 bacterium of embodiment production water-soluble phosphorus
The sterilized indissoluble Phos culture medium of 50mL (glucose 10.0g, ammonium sulfate 0.5g, chlorination are packed into triangular flask
Sodium 0.3g, potassium chloride 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate 0.03g, yeast extract 0.4g, calcium phosphate 10g,
Distilled water 1000mL), the concentration of pH 7.0 ~ 7.5(calcium phosphate is 5.0gL-1), access J5 bacterium is made after bacterium solution at 28 DEG C,
150rpm, shaking table culture 7d.Supernatant is taken to be measured with molybdenum antimony resistance colorimetric method in 10000 rpm, 4 DEG C of centrifugation 15min culture solution
Available phosphorus content.By culture in 5 days, effective amount of phosphorus dissolved was 94.0mg ﹒ kg-1。
7 J5 bacterium of embodiment produces ammonia effect test
J5 bacterium is inoculated in peptone water medium in right amount, sets incubated at room temperature 3d, is added few drops of Nessler reagent, is seen
It examines and whether tan precipitate occurs.Test result is shown in Fig. 7.The result shows that: J5 bacterium has the function of to produce ammonia.
8 J5 bacterium of embodiment produces catalase test
After J5 bacterium is activated 24 h on beef-protein medium, appropriate strains tested is chosen on glass slide, is added dropwise
3% hydrogen peroxide is observed immediately on strains tested, has a large amount of bubbles to generate, as positive, otherwise is feminine gender.It is sun with A15
Property control, test result is as shown in figure 8, show the catalase test of J5 bacterium for the positive.
9 J5 bacterium of embodiment is laid eggs the test of white enzyme effect
J5 strain inoculated to protease is detected into culture medium (10 g of peptone, 5 g of sodium chloride, 0.1 g of calcium chloride, degreasing
10 g of milk powder, agar 18 g, 115 DEG C of sterilizings 30 min, pH 7.2~7.4.) on, 30 DEG C of 2 d of culture observe periphery of bacterial colonies
Transparent circle size judges the protein hydrolysis ability of bacterial strain, as a result as shown in figure 9, compared with the control, the egg in bacterial strain flat board
White matter is nearly all utilized completely, illustrates the bacterial strain using the very capable of protein.
10 J5 bacterium of embodiment tests the inhibitory effect of pathogen
By banana blight bacteria(Fusarium oxysporumf.sp.cubense)It is flat to be inoculated in nutrient agar
The center of plate, while in each streak inoculation J5 bacterial strain in the two sides of the plate, it cultivates, observation.The result shows that J5 bacterium is withered to banana
Germ has certain inhibiting effect, such as Figure 10.
Influence of the 11 J5 bacterial strain of embodiment to corn seed germination
75% ethyl alcohol of corn seed is impregnated into 1min, then recycles 5% liquor natrii hypochloritis to impregnate 2min, then with sterile
Water rinse 3~4 times, then use sterile filter paper dry seed, it is all operation as far as possible guarantee aseptic condition under carry out.It handles well
Seed be placed on it is spare in dry sterile container.Be arranged two processing groups (fermentation liquid of first group of addition J5 bacterial strain, the 2nd group
The culture medium of non-inoculating strain is added as blank control), corn seed is impregnated into 30min in one or two groups respectively.It will sterilizing
Culture dish in complete two layers of aseptic filter paper in advance, the seed of seed soaking is placed in culture dish (10 seeds of every ware),
One layer of filter paper is covered on seed, is cultivated (30 DEG C).Daily every ware adds 2mL sterile water, adds water time unification.Each strain processing is set
Set 4 repetitions.Measure its root long, bud length and germination percentage (see Table 2).The root long and bud of bacterial strain J5 be long and germination percentage is the of germination
Reach remarkable result within five days, is respectively increased 47.2%, 4.6%, 12.5% compared with control treatment.As shown in figure 13.
Influence of the 2 J5 bacterial strain of table to corn seed germination
| Bacterial strain | Root long (cm) | Bud is long (cm) | Germination percentage (%) |
| CK | 7.40±1.26 b | 5.43±0.14 a | 72.5±0.025 a |
| J5 | 10.89±1.51 a | 5.68± 0.09 a | 85±0.029 a |
Note: each numerical value is average value ± standard error (n=4) in table, indicates variance analysis with the same letter person in column data
Without significant difference (P < 0.05), following table data processing method is identical.
The test of 12 J5 bacterial strain of embodiment promotion potting plant growth result
Bacteria suspension preparation: by point access LB liquid medium after J5 actication of culture, by shaken cultivation for 24 hours, cultivation temperature
It is 30 DEG C, revolving speed 150r/min；Bacterium solution is instilled into blood counting chamber, is counted under an optical microscope, obtains thallus in bacterium solution
Cell concentration；Bacterium solution is centrifuged, revolving speed 6000r/min in supercentrifuge, is centrifuged 5min；Thallus is resuspended with aqua sterilisa；
Bacterial strain concentration for being inoculated with romaine lettuce is 3.0 × 108The bacteria suspension of cfu/ml.
Corn nursery: corn washes vernalization, soaks into preservation moisture with filter paper, is placed in 30 DEG C of incubators, reveals two days later
Bud is sowed to seedbed, and seedling grows supreme about 25 cm, can be transplanted seedlings, four seedlings of every basin.
Test sets 4 processing, 4 repetitions of each processing, 4 seedlings of each repetition altogether.Every 10.0 kg(of basin soil loading amount is not applied
Add any chemical fertilizer).Each processing are as follows: do not apply microbial inoculum abbreviation CK；Apply microbial inoculum J5.Bacterium solution viable count is up to 105~6 cfu·mL-1, move
Seedling two days later, is inoculated with bacterium solution through centrifugation respectively and does same processing with 30 mL of sterile water re-suspension liquid, CK.Appropriate amount of water, every basin are poured daily
Water want identical, sow uniformly, not make water outflow basin bottom in order to avoid fertilizer loss causes error.
The measuring method that each microbial inoculum influences corn growth: after inoculation 20 days and 40 days, the jade of the difference processing of sampling determination 4
Rice plant height, the number of blade, chlorophyll, root long, leaf is long, leaf width, stem thickness, fresh weight and dry weight.Measure corn plant height (corn ground to jade
Rice blade smoothes out with the fingers the height at straight rear blade tip end), it measures maize leaf (including all blades grown), chlorophyll (uses chlorophyll meter
Measure third piece leaf from bottom to up), leaf length and leaf width (measuring third piece leaf from bottom to up with tape measure), stem thickness (uses vernier
Slide calliper rule measurement).Fresh weight: taking the maize seedling of every basin, and after plant is cleaned, stem, leaf and under ground portion are separated, with ten thousand/
One electronic balance weighs its fresh weight respectively, then in 105 DEG C of 15 min of water-removing, 65 DEG C of constant temperature dryings to constant weight, and with very much
One of electronic balance weighing.The result shows that J5 bacterial strain has facilitation to the growth of plant.The growth of plant
Situation such as Figure 14.
After table 3 is inoculated with 10 days, influence that J5 bacterial strain grows potting plant
After table 3 is inoculated with 10 days, influence that J5 bacterial strain grows potting plant
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (cm) | Leaf is long (cm) | Leaf width (cm) | Chlorophyll |
| CK | 50.91±3.97b | 7.59±0.16b | 7.75±0.19b | 35.99±0.95b | 2.10±0.07b | 26.69±1.72b |
| J5 | 62.51±5.32a | 9.80±0.27a | 8.81±0.10a | 43.36±1.45a | 2.61±0.04a | 30.87±0.98a |
Note: each numerical value into table table 17 of table 3 is each measurement result.
After table 4 is inoculated with 20 days, influence (pouring bacterium solution 20 days) that J5 bacterial strain grows potting plant
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (cm) | Leaf is long (cm) | Leaf width (cm) |
| CK | 74.00±1.18b | 11.66±0.31b | 9.81±0.19b | 43.50±0.56b | 3.05±0.06b |
| J5 | 80.19±1.53a | 13.49±0.24a | 11.31±0.12a | 49.84±0.72a | 3.59±0.12a |
After table 5 is inoculated with 30 days, influence (pouring bacterium solution 30 days) that J5 bacterial strain grows potting plant
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (cm) | Leaf is long (cm) | Leaf width (cm) | Chlorophyll |
| CK | 101.68±1.61a | 10.04±0.33b | 14.44±0.27b | 63.91±0.73a | 4.53±0.09a | 19.76±0.37a |
| J5 | 105.57±1.74a | 10.97±0.19a | 15.38±0.20a | 63.19±0.82a | 4.71±0.11a | 20.21±0.60a |
The fresh weight of plant compares when table 6 harvests
| Processing | Leaf (g) | Stem (g) | Root (g) |
| CK | 78.78±1.68a | 127.87±4.70b | 10.74±0.64b |
| J5 | 83.66±2.48a | 141.22±0.87ab | 14.89±2.14ab |
The dry weight of plant compares when table 7 harvests
| Processing | Leaf (g) | Stem (g) | Root (g) |
| CK | 19.60±1.02a | 20.88±0.35b | 7.99±0.28a |
| J5 | 22.52±0.80a | 22.33±0.68a | 7.79±0.32a |
For embodiment 13 under Different Fertilization mode, J5 bacterial strain studies the influence that potting plant grows
Test sets 4 processing, 4 repetitions of each processing, 4 seedlings of each repetition altogether.Every 10.0 kg(of basin soil loading amount according to
Every kilogram of soil applies 50 g N, 10 g P2O5、10 g K2O).Each processing are as follows: do not apply bacterial manure abbreviation CK；Bacterium solution is direct
It pours around plants stems, referred to as pours and apply；Bacterium solution and soil mixing are moved back into plant strain, it is referred to as mixed to apply；By corn
The root portion of seedling is transplanted after impregnating 10 min in bacterium solution, abbreviation root dipping.Bacterium solution viable count is up to 105-106 cfu·mL-1, transplant seedlings
Two days later, it is inoculated with bacterium solution respectively and does same processing through re-suspension liquid 30 mL, CK of centrifugation aqua sterilisa.Appropriate amount of water, every basin are poured daily
Water want identical, sow uniformly, not make water outflow basin bottom in order to avoid fertilizer loss causes error.
After inoculation 20 days and 40 days, the corn plant height that sampling determination 4 is handled respectively, the number of blade, chlorophyll, root long, leaf is long,
Leaf width, stem thickness, fresh weight and dry weight.Measurement corn plant height (height that straight rear blade tip end is smoothed out with the fingers on corn ground to maize leaf), measurement
Maize leaf (including all blades grown), chlorophyll (measure third piece leaf from bottom to up with chlorophyll meter), Ye Changhe
Leaf width (measures third piece leaf from bottom to up with tape measure), and stem thickness (is measured) with vernier caliper.Fresh weight: taking the maize seedling of every basin,
Plant is cleaned after drying, stem, leaf and under ground portion is separated, weigh its fresh weight respectively with a ten thousandth electronic balance,
Then in 105oC finish 15 min, 65oC constant temperature drying is weighed to constant weight, and with a ten thousandth electronic balance.The result shows that
It is different to the growth-promoting effect of corn using different application microbial inoculum modes, important evidence is provided to apply fertilizers scientifically from now on.
Plant growing state such as Figure 15.
Table 8 is inoculated with 20 days, influence of the J5 bacterial strain under Different Fertilization mode to the growth of potting plant
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (piece) | Leaf is long (cm) | Leaf width (cm) |
| Ck | 60.85±3.04b | 7.02±0.28c | 7.44±0.12b | 30.66±2.61b | 1.82±0.12b |
| Root dipping | 68.41±2.92ab | 8.68±0.19ab | 8.06±0.13a | 36.67±1.79ab | 2.07±0.07ab |
| It is mixed to apply | 67.12±1.54ab | 9.21±0.14a | 7.63±0.14b | 37.41±1.06a | 2.28±0.05a |
| It pours and applies | 72.24±5.35a | 8.32±0.22ab | 8.19±0.24a | 34.70±2.07ab | 1.83±0.17b |
Note: each numerical value is each measurement result, and is average value (n=4), the same word matrix of the data of same column
Show that there was no significant difference.
Table 9 is inoculated with 40 days, influence of the J5 bacterial strain under Different Fertilization mode to the growth of potting plant
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (piece) | Leaf is long (cm) | Leaf width (cm) |
| CK | 86.28±3.81b | 7.46±0.52b | 8.88±0.39b | 55.18±2.41a | 2.31±0.10b |
| It pours and applies | 94.89±1.46a | 8.56±0.20ab | 9.81±0.31a | 59.34±1.12a | 2.69±0.11a |
| It is mixed to apply | 82.89±0.83b | 8.33±0.46ab | 10.44±0.19a | 54.61±1.50a | 2.81±0.11a |
| Root dipping | 86.93±1.96b | 8.75±0.28a | 10.13±0.22a | 56.66±2.02a | 2.93±0.15a |
Note: each numerical value is each measurement result, and is average value (n=4), the same word matrix of the data of same column
Show that there was no significant difference.
When table 10 harvests, comparison of the J5 bacterial strain under Different Fertilization mode to each position fresh weight of potting plant
| Processing | Root (g) | Leaf (g) | Stem (g) |
| CK | 1.64±0.32b | 30.23±5.44a | 36.67±6.43b |
| It pours and applies | 2.48±0.33ab | 41.85±0.05a | 52.23±3.06ab |
| It is mixed to apply | 4.45±1.32a | 40.74±4.16a | 46.53±5.74ab |
| Root dipping | 5.03±0.89a | 41.90±1.24a | 53.63±6.01a |
Note: each numerical value is each measurement result, and be average value (n=4), with column data same letter indicate without
Significant difference.
When table 11 harvests, comparison of the J5 bacterial strain under Different Fertilization mode to each position dry weight of potting plant
| Processing | Leaf dry weight (g) | Stem weight (g) |
| CK | 8.48±0.73a | 3.30±0.09b |
| It pours and applies | 9.88±0.24a | 4.41±0.14a |
| It is mixed to apply | 8.84±0.91a | 3.96±0.29b |
| Root dipping | 9.71±0.36a | 4.10±0.28a |
Note: each numerical value is each measurement result, and be average value (n=4), with column data same letter indicate without
Significant difference.
The research of 14 J5 bacterial strain of embodiment raising field corn plant strain growth
Test devises 3 processing: CK processing altogether；The processing of microbial inoculum 4；The processing of microbial inoculum 5.Each processing applies the change of equivalent
Fertilizer.Test totally 9 cells, totally 3 row, 3 cells of every row, 3 repetitions of each processing are gone together the original of equal different disposal according to same row
Then it is randomly assigned a planting location for processing.Each 2 row of cell maize planting plant, line-spacing be 50 cm, colleague plant before and after away from
From for 20 cm, each cell has planted 40 plants altogether, and each cell includes that left and right protection row occupied area is about 8.2
m2, dose is according to 7.5 kilograms of standard applications per acre 1~2 time.
Different times corn plant height, root long, the number of blade, leaf length, leaf width, stem thickness, fresh weight and dry weight are measured with conventional method, at
Ripe phase spike length, fringe be thick, (the yield: for the yield of a test block in individually handling such as grain number per spike, 100-grain weight and economic flow rate；
Spike length: for the length of corn fruit on sample plant；Fringe is thick: corn fruit selection perimeter longest position goes out straight on sample plant
Diameter；Total particle number: the result of line number * columns on corn fruit；Fresh weight: for the fresh weight of corn fruit on sample plant；Dry weight: for
The dry weight of corn fruit on sample plant；100-grain weight: the weight for 100 niblets selected at random on the sample plant of acquisition
Amount).Economic flow rate sampling method is each cell corn fresh weight.The result shows that application microbial inoculum J5 has the growth of field corn
There is significant facilitation.The research of current existing report, A Shi bacillus mainly concentrates on laboratory and potting rank
Section, this research further confirm A Shi bacillus to the growth-promoting functions of cereal crops by field trial.The growth of corn
Situation such as Figure 16.
After table 12 is inoculated with 30 days, influence of the J5 bacterial strain to field corn plant strain growth
| Processing | Plant height (cm) | Stem thickness (mm) | The number of blade (piece) | Leaf is long (cm) | Leaf width (cm) |
| CK | 120.77±1.37b | 16.31±0.45b | 10.00±0.38b | 63.89±0.68a | 6.51±0.11b |
| J5 | 146.67±0.80a | 20.20±1.42a | 11.89±0.29a | 63.34±1.12a | 7.40±0.15a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
After table 13 is inoculated with 30 days, influence of the J5 bacterial strain to field corn plant fresh weight
| Processing | Root (g) | Stem (kg) | Leaf (kg) |
| CK | 7.59±0.59b | 0.12±0.01b | 0.06±0.006b |
| J5 | 22.65±0.62a | 0.24±0.23a | 0.11±0.003a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
After table 14 is inoculated with 30 days, influence of the J5 bacterial strain to field corn plant weights
| Processing | Root (g) | Stem (g) | Leaf (g) |
| CK | 5.69±1.05b | 14.28±0.72b | 13.99±0.77b |
| J5 | 13.84±1.49a | 28.61±0.18a | 27.04±0.19a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
After table 15 is inoculated with 75 days, influence of the J5 bacterial strain to field corn plant strain growth index
| Processing | Plant height (cm) | Stem thickness (mm) |
| CK | 153.77±4.29b | 15.49±1.08b |
| J5 | 177.60±4.29a | 20.25±1.13a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
After table 16 is inoculated with 75 days, influence of the J5 bacterium to field corn plant fresh weight
| Processing | Root (g) | Stem (kg) | Leaf (g) |
| CK | 12.73±0.71b | 0.09±0.01b | 32.59±1.40b |
| J5 | 18.27±1.51a | 0.15±0.02a | 48.52±3.56a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
After table 17 is inoculated with 75 days, influence of the J5 bacterial strain to field corn plant weights
| Processing | Root (g) | Stem (g) | Leaf (g) |
| CK | 10.39±0.47a | 23.21±1.04a | 19.09±1.03b |
| J5 | 12.40±1.66a | 32.09±4.90a | 27.82±1.62a |
Note: each numerical value is the measured value of a plant, and is average value (n=4), the different alphabets of the data of same column
It is shown with significant difference.
The yield of corn and each Indexes Comparison of fruit when table 18 harvests
| Processing | Yield kg | Spike length cm | The thick mm of fringe | Total grain number | Fresh weight g | Dry weight g | 100-grain weight g |
| CK | 5.87±0.40b | 17.08±0.04b | 45.74±1.13a | 397.22±14.59a | 163.31±4.27b | 49.22±3.31b | 10.71±0.90b |
| J5 | 8.05±0.75a | 18.89±0.15a | 48.93±1.04a | 432.89±12.92a | 221.74±9.86a | 71.08±6.42a | 14.46±0.08a |
Note: each numerical value is average value, and n=4, the different alphabets with column data are shown with significant difference.Yield: for
The total output of a test block in single processing.Spike length: for the length of corn fruit on sample plant；Fringe is thick: sample plant
Upper corn fruit chooses the diameter that perimeter longest position goes out；Total particle number: the result of line number * columns on corn fruit；Fresh weight: for
The fresh weight of corn fruit on sample plant；Dry weight: for the dry weight of corn fruit on sample plant；100-grain weight: the sample of acquisition is planted
The weight for 100 niblets selected at random in strain.
<110>Agricultural University Of South China
<120>one plants of A Shi bacillus J5 and its applications
<130> 0
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1451
<212> 16S rDNA
<213>A Shi bacillus (Bacillus aryabhattai)
<400>1
gtcgagcgaa ctgattagaa gcttgcttct atgacgttag cggcggacgg gtgagtaaca 60
cgtgggcaac ctgcctgtaa gactgggata acttcgggaa accgaagcta ataccggata 120
ggatcttctc cttcatggga gatgattgaa agatggtttc ggctatcact tacagatggg 180
cccgcggtgc attagctagt tggtgaggta acggctcacc aaggcaacga tgcatagccg 240
acctgagagg gtgatcggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300
agcagtaggg aatcttccgc aatggacgaa agtctgacgg agcaacgccg cgtgagtgat 360
gaaggctttc gggtcgtaaa actctgttgt tagggaagaa caagtacaag agtaactgct 420
tgtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt atccggaatt attgggcgta aagcgcgcgc aggcggtttc 540
ttaagtctga tgtgaaagcc cacggctcaa ccgtggaggg tcattggaaa ctggggaact 600
tgagtgcaga agagaaaagc ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660
ggaacaccag tggcgaaggc ggctttttgg tctgtaactg acgctgaggc gcgaaagcgt 720
ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780
ttagagggtt tccgcccttt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840
acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaactct 960
agagatagag cgttcccctt cgggggacag agtgacaggt ggtgcatggt tgtcgtcagc 1020
tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080
agcatttagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140
acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggatggtaca 1200
aagggctgca agaccgcgag gtcaagccaa tcccataaaa ccattctcag ttcggattgt 1260
aggctgcaac tcgcctacat gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320
gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380
cgaagtcggt ggagtaaccg taaggagcta gc 1412
<210> 2
<211> 20
<212> DNA
<213>universal primer F27
<400>2
agagtttgat cctggctcag 20
<210> 3
<211> 22
<212> DNA
<213>universal primer R1492
<400>3
tacggctacc ttgttacgac tt 22
Claims (8)
1. one plant of A Shi bacillus (Bacillus aryabhattai) J5, which is characterized in that the bacterial strain was in 2015 10
The moon is preserved in China General Microbiological culture presevation administrative center (CGMCC) on the 15th, and deposit number is CGMCC No.11508.
2. a kind of microbial bacterial agent, which is characterized in that include A Shi bacillus (Bacillus described in claim 1
Aryabhattai) J5.
3. microbial bacterial agent according to claim 2, which is characterized in that the concentration of J5 bacterial strain is 1.0~9.0 × 108cfu/
ml。
4. microbial bacterial agent described in claim 2 produce auxin, produce ammonia, produce siderophore, produce protease, produce catalase,
Decompose the application in insoluble phosphorus, salt tolerant or inhibition banana blight bacteria.
5. microbial bacterial agent described in claim 2 is promoting germination or/and is promoting the application in plant growth.
6. A Shi bacillus (Bacillus aryabhattai) J5 described in claim 1 is producing auxin, is producing ammonia, production iron
Carrier produces protease, produces catalase, decomposing the application in insoluble phosphorus, salt tolerant or inhibition banana blight bacteria.
7. A Shi bacillus (Bacillus aryabhattai) J5 described in claim 1 promote germination or/and
Promote the application in plant growth.
8. the application according to claim 5 or 7, the plant is corn.
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