CN105985922A - Bacillus aryabhattai J5 and application thereof - Google Patents
Bacillus aryabhattai J5 and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microbial resources, and particularly discloses a strain of Bacillus aryabhattai J5 and application thereof in promoting growth of corn. The Bacillus aryabhattai J5 is collected in CGMCC (China General Microbiological Culture Collection Center) on October 15th, 2015 and is filed with the number of CGMCC NO.11508. The Bacillus aryabhattai J5 has functions of producing auxin, siderophore, ammonia and protease, dissolving phosphorus, resisting to salt, realizing antagonism, and the like. As the strain is capable of growing normally in a culture medium with salt content equal to 11%, the strain has good potential in eliminating soil salinization. As content of siderophore produced by the strain is up to 53.7%, the strain has effective disease-prevention property. By the strain, corn seed germination rate can be increased by 12.5%, and accordingly the strain has good growth promoting effect. By application of microbial inoculum prepared from the strain, corn yield is increased by 37.1%, and hundred-grain weight is increased by 35%. It is possible to develop the strain into microbial inoculum, and accordingly the strain has wide application prospect in terms of improving soil nutrient, eliminating salinization, promoting growth of plants, preventing diseases and the like.
Description
Technical field
The present invention relates to microbial resources field, more particularly, to a strain A Shi bacillus J5 and application thereof.
Background technology
Plant growth-promoting rhizobacteria (Plant growth-promoting rhizobacteria, PGPR) refers to survive in plant
Rhizosphere, root table, and can promote directly or indirectly or the microorganism of coordinate plant growth.Plant growth-promoting rhizobacteria typically has product
Auxin, produces ammonia, produces siderophore, antagonism, phosphorus decomposing, the ability such as salt tolerant or at least have the ability of one of them.Inoculation plant roots
Border Promoting bacteria, can make soil nutrient validation, promotes that root system of plant absorbs soil moisture and nutrient, raw to improve plant
Long.Bacillus produces the gemma having very strong resistance to heat, ionising radiation and some chemicals etc..Therefore, there is root
The gemma bacillus agent of border growth-promoting functions, production technology is simple, and shelf life is long, it is easy to survives in soil environment and surely grows, thus
Improve field crops growth, microbial manure industry shows huge application prospect.
A Shi bacillus (Bacillus aryabhattai) in Shivaji in 2009 etc. first from height above sea level be 27～
Screening in the air of 41km separates, and as a kind of new mushroom-seed culturing resource, has significant rhizosphere growth-promoting functions.Such as, Zuo Shan etc.
The A Shi bacillus B8W22T of screening, has product auxin feature, can significantly improve smoke tree growth.The A Shi of the literary composition screening of Liu
Bacillus has effect of solubilizing phosphate, has facilitation to plant germination and growth.Duan Jiali reports A Shi bacillus B5 pair
Muskmelon seeds plumular axis and radicle growth have facilitation.The A Shi bacillus MASR14 that Aketi etc. screen at Central India
There is product auxin, produce siderophore and produce three kinds of beneficial characteristics of ammonia, facilitation is had to plant growth.Li Zuhong etc. utilize Ah
The antagonistic effect effect preventing and treating black shank of family name bacillus, has significant effect.Sol etc. are in the barren lakeside of Korea S
Screen four strains and there is the A Shi bacillus producing IAA and two kinds of functions of molten phosphorus, promote 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
The parsing of single or two or three kind of function, it is difficult to meet the market demands of Modern microbiological fertilizer functional diversities.
In addition, the soil salinization is the major reason seriously restricting plant growth.According to current UNESCO and
The incomplete statistics of food and agricultural organization, world's salination area is 109hm2, the Second National overall survey of soil result of China shows,
The salinisation gross area is 3.6 × 107 hm2, account for the 4.88% of the available area in the whole nation.Cultivated area salination reaches 9.21 ×
106 hm2, account for the 6.62% of whole nation cultivated area.Forefathers study, and A Shi bacillus has preferable salt tolerant feature, such as Lv Zhao
The A Shi bacillus of the separation screening from the middle level pit mud sample of Luzhou, Sichuan liquor production factory such as brightness, at salt content (NaCl)
Under the conditions of being 0.5%, remarkably promote sorghum saccharatum plant strain growth.A Shi bacillus MDSR 7, the MDSR 11 of the screening such as Aketi,
The salt tolerance of MDSR 14 is between 4～10%.The strain that Du Chuanying screens in the Northeast of China have 6% salt tolerance Ah
Family name bacillus.Liu Xin Linli variable concentrations NaCl have studied the growing state of A Shi bacillus A4, the increment of bacterial strain
First increasing with the increase of NaCl concentration, when the left and right that NaCl concentration is 6%, increment reaches maximum.These progress show
Great potential in terms of soil salinization improvement for the A Shi bacillus, needs to be studied further.
The siderophore of bacterial strain has preferable facilitation equally to plant growth.Existing research shows, at iron deficiency ring
Plant under border can directly utilize the siderophore that microorganism produces, thus adds the absorption to iron for the plant, and beneficially promotion is planted
Thing grows.Siderophore can also promote plant growth by suppressing harmful microorganism, therefore, siderophore can by directly or
Indirectly-acting promotes the growth of plant.The A Shi bacillus WRP5 of the screening such as Sheetal can produce siderophore.Sol etc. screen
Four strain A Shi bacillus all judge that it can produce siderophore by qualitative experiment, but also do not quantitative determine.Sun Lei
It is respectively 11.8% and 9.6% Deng the siderophore content of A Shi bacillus N44 and N23 of screening；Major part research at present is only
Can only cross qualitative judgement A Shi bacillus and produce siderophore, the research of quantitative determination siderophore units activity be relatively fewer, still
Treat to study further.
A Shi bacillus is also limited to laboratory as the dominant strain of a kind of new microbial bacterial agent, current research
With the potted plant experiment stage.Such as, Jian etc. have carried out the research of some physicochemical properties to A Shi bacillus GZ03 in laboratory.
The microcosm soil incubation such as Aketi have studied the growth-promoting effect to crop for the A Shi bacillus.Seulki uses pot experiment,
Have studied A Shi bacillus and promote the effect of soil phophorus validation.Sol etc. are probing into the growth-promoting to plant for the A Shi bacillus
During effect, microcosm experiment is make use of to be verified.Therefore, the field growth-promoting needing to be confirmed further A Shi bacillus is made
With.
Content of the invention
The invention aims to overcome in prior art and there is above-mentioned deficiency, provide a strain to have concurrently efficiently and produce length
The multi-functional compound A Shi bacillus J5 such as element, product ammonia, product siderophore, phosphorus decomposing, salt tolerant, antagonism, this bacterial strain can improve seed
Germination percentage, promotion plant growth and raising plant products etc..
Further object is that offer A Shi bacillus J5 is producing auxin or/and produce ammonia or/and produce iron
Carrier is or/and phosphorus decomposing is or/and salt tolerant is or/and antagonism is or/and promote germination or/and promote the application in plant growth.
To achieve these goals, the present invention is achieved by the following technical programs:
One strain A Shi bacillus (Bacillus aryabhattai) J5, is called for short J5 bacterial strain, and this bacterial strain was October 15 in 2015
Day is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and deposit number is CGMCC
No.11508, Classification And Nomenclature: A Shi bacillus aryabhattai；Preservation address: the Chaoyang District, Beijing City North Star
West Road 1 institute 3, Institute of Microorganism, Academia Sinica.
A Shi bacillus of the present invention (Bacillus aryabhattai) J5 is from south China, Guangzhou, Guangdong
Isolated and purified gained in the vegetable garden soil on agriculture university farm.This bacterial strain has an endogenous spore, belongs to gram-positive bacteria, thalline
For shaft-like, there is motility, aerobic；The bacterium colony cultivated on beef-protein medium 24 hours is circular or irregular shape,
After 48h, all circles of bacterium colony, slightly yellow, and diameter is about 2～3mm, and edge is irregular, flat moistening.Bacterial strain has stronger
Producing IAA, siderophore and dissolving P capacity, being A Shi bacillus through Physiology and biochemistry and Molecular Identification, its Latin is entitled:Bacillus aryabhattai, the 16S rRNA gene sequencing result of bacterial strain is as shown in SEQ ID NO:1.
The method for preserving of J5 bacterium, the composition of its storage medium is beef extract 3.0 g, peptone 10.0 g, sodium chloride 5.0
G, agar 15.0 g, distilled water 1000 mL, or the culture medium becoming according to this proportions, pH value is 7.0～7.2.Bacterium routinely
Plant preservation temperature conservation.
A kind of microbial bacterial agent, comprise A Shi bacillus as above (Bacillus aryabhattai) J5.
Preferably, in described microbial bacterial agent, the concentration of J5 bacterial strain is 1.0～9.0 × 108cfu/ml。
Microbial bacterial agent described above is producing auxin or/and produce ammonia or/and produce siderophore or/and protease is or/and mistake
Hydrogen oxide enzyme is or/and decompose insoluble phosphorus or/and salt tolerant is or/and application in antibacterial.
Microbial bacterial agent described above is promoting germination or/and promote the application in plant growth.
A Shi bacillus as above (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 in antibacterial should
With.
A Shi bacillus as above (Bacillus aryabhattai) J5 promote germination or/and promote
Enter the application in plant growth.
It is highly preferred that described plant is corn.
Compared with prior art, the method have the advantages that
The A Shi bacillus J5 bacterial strain of the present invention under the condition of culture of salt content 11%, well-grown, it is shown that super good is resistance to
Salt, in the A Shi Bacillus strain reported, effect is best；The siderophore experiment of bacterial strain J5 shows that it has bigger
Orange haloing, produces siderophore content and is up to 53.7%, and this is the A Shi bacillus bacterium of reported first height sequestering power siderophore
Strain.Bacterial strain J5 not only achieves good laboratory and pot experiment effect, is investigated bacterial strain J5 answering in maize field
With result shows, corn yield increasing 37.1%, and spike length increases by 10.6%, and fringe slightly improves 7%, and corn total particle number increases by 9%, corn
Fresh weight improves 35.8%, and dry weight improves 44.4%, and 100-grain weight improves 35%.Therefore, the A Shi bacillus J5 tool of the present invention
For product auxin, produce siderophore, produce ammonia, produce protease, salt tolerant, antagonism, the comprehensive function of molten phosphorus, and effect is notable.In addition,
This research also compares the impact that different microbial inoculum method of application carries out potted plant milpa growth to bacterial strain J5, closes for later stage science
Reason fertilizing method provides data supporting.If utilize A Shi bacillus J5 make microbial bacterial agent as cereal crops grow new
Bacteria agent, will have huge market application foreground.
Brief description
Fig. 1 is J5 bacterial strain spore staining microphoto, and in thalline, oval shaped portion is gemma.
Fig. 2 is J5 bacterial strain Gram's staining microphoto, and thalline 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 produces 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 the antagonistic experiment result to banana blight for the J5 bacterial strain.
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 the promotion corn germination test photo of J5 bacterial strain.
Figure 14 is that J5 microbial inoculum promotes corn growth pot experiment photo.
Figure 15 is to promotion corn growth pot experiment photo under J5 microbial inoculum Different Fertilization.
Figure 16 is that J5 microbial inoculum promotes corn growth field plot trial photo.
Detailed description of the invention
Below in conjunction with Figure of description and specific embodiment the present invention made and elaborating further, described embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiment is as without spy
Different explanation, is conventional method；The material that used, reagent etc., if no special instructions, for the reagent commercially obtaining
And material.The separation of embodiment 1 bacterial strain
S1. separate: with the vegetable garden soil on farm, Tianhe District, Guangzhou City, Guangdong Province Agricultural University Of South China leap north for screening soil sample, use
Spread plate accurately weighs screening soil sample 10.0g, puts into and (adds bead) equipped with in the 250mL triangular flask of 90mL sterilized water,
Shaking table vibrates 30min, makes cell fully dispersed, stands 20～30s, takes supernatant and carries out 10 times of dilutions of successively decreasing, uses 104～106
Dilution factor, draws dilution 0.1mL respectively with liquid-transfering gun, is coated on beef extract-peptone flat board, is inverted for 28 DEG C and cultivates 48h.
Select suitable concentration flat board, according to form, size, color, the single bacterium colony of typical case of picking different strains is pure on culture medium
After change, the bacterial strain of screening is carried out inclined-plane preservation, standby.
S2. the screening of auxin IAA is produced: through plate dilution method, according to growing the form of bacterium colony, size, matter on flat board
The difference of the morphological features such as ground, color, is divided into and separates out 45 strain bacteriums.Qualitative and quantitative screening, J5 strain secretes through product IAA
IAA is most, converts IAA and reach 20.1 mg L during 24 h-1Through repeatedly passing on after, produce IAA stable.The J5 bacterium that will filter out
It after strain utilizes plate streaking, is stored in the made inclined-plane of beef-protein medium, be placed in 4 DEG C of refrigerators standby.
Embodiment 2 J5 strain characteristic is identified
Thalli morphology characteristic: gram-positive bacteria, has gemma.Thalline is shaft-like, has motility, obligate aerobic.Spore staining is shone
Piece is shown in Fig. 1.
Colonial morphology characteristic: the bacterium colony being formed after cultivating 24h on beef-protein medium is circular or irregular
Shape, after 48h, all circles of bacterium colony, slightly yellow, and diameter is about 2-3mm, and edge is irregular, flat moistening.
Growth characteristics: at beef extract 3.0 g, peptone 10.0 g, sodium chloride 5.0 g, in the fluid nutrient medium of water 1L,
Rotating speed 113 rpm, temperature 30 DEG C, under conditions of initial ph value is 7.0, cultivate 18h, record viable count for (1.94 ± 0.60) ×
108cfu·mL-1。
Physiology, biochemical characteristic: with reference to the normal experiment method of " primary Jie Shi Bacteria Identification handbook ", this bacterial strain is carried out
Physiological and biochemical test, experimental result is shown in Table 1.
Table 1 bacterial strain J5 physiological and biochemical test
| Test name | Result | Test name | Result |
| Produce IAA test | + | Solve starch test | - |
| Siderophore is tested | + | Catalase | + |
| Indoles | + | Proteasome degradation | + |
| Produce ammonia test | + | Methyl red test | - |
Note: in table "+" it is the positive, "-" is feminine gender.
Molecular biological characteristic: use RNA isolation kit to extract the STb gene of J5 bacterial strain.Use 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
Increase the 16S rDNA of bacterium, obvious band occur near 1000bp, pcr amplification product is reclaimed laggard row sequencing,
The DNA sequence dna input GenBank that will obtain, compares analysis with Blast program to all sequences in database, and result is sent out
Show the 16S rDNA sequence of bacterial strain of the present invention and with A Shi bacillus in GenBank, there is higher homology, its phase
It is 100% (see Fig. 3) like degree.In conjunction with above-mentioned flat-plate bacterial colony feature, physio-biochemical characteristics, the result of 16S rDNA sequence, just
Step identifies that J5 bacterial strain is A Shi bacillus (Bacillus aryabhattai strain), Classification And Nomenclature: A Shi bacillus
Bacillus aryabhattai.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms on October 15th, 2015
Common micro-organisms center (CGMCC), deposit number is CGMCC No.11508, preservation address: BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica.
Embodiment 3 J5 bacterium produces auxin effect
Auxin qualitative determination: J5 bacterium is inoculated in the LB Liquid Culture of 50mL containing L-Trp (100 g/mL)
In base, 30 DEG C, 24h cultivated by 150r/min shaking table.Take 300 μ L cultivate drops on whiteware plate, add 300 μ L simultaneously
Salkowski color solution, if the sterile LB medium of 300 μ L is as negative control, the 10th, the 20th, the 30th, 50(g/mL) auxin
Color solution is as positive control.Observe after whiteware plate is placed under the conditions of room temperature, lucifuge 30 min, the color person of reddening
Expression can produce auxin.Result of the test is shown in Fig. 4.
Auxin quantitative determines: J5 bacterium is inoculated in the 50mL LB Liquid Culture containing L-Trp (100 g/mL)
In base, three repetitions, 30 DEG C, 24h cultivated by 150r/min shaking table.Nutrient solution is centrifuged respectively at 8,000 xg 10min, takes
4mL supernatant adds 4mL Salkowski color solution, mixes, and stands 30min, use uv-spectrophotometric in room temperature, dark
Meter, surveys absorbance under 530nm, does same treatment as comparison with aseptic culture medium simultaneously, use the 0th, the 10th, the 20th, the 30th, 50 (
G/mL) calibration curve made by auxin.J5 bacterium produces long cellulose content and reaches 20.1 mg/L.
Embodiment 4 J5 bacterium Salt resistant test
J5 bacterial strain is scoring to 0%, on the nutrient agar panel of 1%, 3%, 5%, 7%, 9%, 11%, 13%, cultivates 24h, observe bacterial strain
Growing state.Result of the test such as Fig. 5.The salt resistance ability of bacterial strain J5 can reach 11 g 100mL-1.So strong salt tolerance exists
The A Shi bacillus reported at present is the highest.
Embodiment 5 J5 bacterium produces siderophore ability and measures
Qualitative: after bacterial strain being transferred on LB flat board cultivate 24-48 h, use some inocalation method to be connected on the detection of MSA-CAS solid flat
On plate, 3 repetitions of point, cultivate 72 h for 30 DEG C, observe the size of the outer yellow halo of bacterium colony on detection flat board, secrete the thin of siderophore
Bacterium periphery of bacterial colonies there will be obvious orange siderophore haloing.Results strain J5 haloing is relatively big, produces siderophore ability strong.Such as Fig. 6.
Quantitative: by inoculation in limit iron MSA fluid nutrient medium, (150 rpm) 48 h cultivated by 28 DEG C of shaking tables；Bacterium is hanged
Liquid centrifuges 15 min through 10000rpm, takes supernatant, and the volume of bacterium solution and CAS detection liquid 1:1 fully mixes, and after static 1 h, surveys
Light absorption value (As) at fixed 630 nm wavelength, takes distilled water and returns to zero as comparison, with the nonvaccinated MSA liquid training being measured in the same method
The light absorption value of foster base is as reference value (Ar), and calculates siderophore active unit ([(Ar-As)/Ar] × 100).The work of bacterial strain J5
Property is up to 53.7%, and reported first belongs to the A Shi bacillus producing high chelated iron carrier.
Embodiment 6 J5 bacterium produces the effect experimental of water-soluble phosphorus
Triangular flask loads 50mL sterilized indissoluble Phos culture medium (glucose 10.0g, ammonium sulfate 0.5g, sodium chloride
0.3g, potassium chloride 0.3g, ferrous sulfate heptahydrate 0.03g, manganese sulfate 0.03g, yeast extract 0.4g, calcium phosphate 10g, steam
Distilled water 1000mL), the concentration of pH 7.0 ~ 7.5(calcium phosphate is 5.0g L-1), access J5 bacterium and make after bacterium solution at 28 DEG C,
150rpm, 7d cultivated by shaking table.By nutrient solution at 10000 rpm, 4 DEG C of centrifugal 15min, take supernatant molybdenum antimony resistance colorimetric method and measure
Available phosphorus content.Through the cultivation of 5 days, effective amount of phosphorus dissolved was 94.0mg kg-1。
Embodiment 7 J5 bacterium produces ammonia effect test
Being inoculated in J5 bacterium in peptone water medium in right amount, putting incubated at room temperature 3d, add Nessler reagent few drops, observation is
No there is tan precipitate.Result of the test is shown in Fig. 7.Result shows: J5 bacterium has product ammonia function.
Embodiment 8 J5 bacterium produces catalase test
After J5 bacterium is activated 24 h on beef-protein medium, choose appropriate strains tested on slide, drip 3% mistake
Hydrogen oxide, on strains tested, is observed immediately, has a large amount of bubble to produce, and is the positive, otherwise is feminine gender.Right with A15 for the positive
According to result of the test as shown in Figure 8, shows that the catalase test of J5 bacterium is the positive.
Embodiment 9 J5 bacterium produces protease effect test
J5 inoculation to protease is detected culture medium (peptone 10 g, sodium chloride 5 g, calcium chloride 0.1 g, skimmed milk power
10 g, agar 18 g, 115 DEG C of sterilizings 30 min, pH 7.2～7.4.On), cultivate 2 d for 30 DEG C, observe the transparent of periphery of bacterial colonies
Circle size judges the proteolysis ability of bacterial strain, the protein as it is shown in figure 9, compared with the control, on bacterial strain flat board for the result
It is nearly all utilized completely, illustrate that this bacterial strain utilizes the very capable of protein.
The inhibition to pathogen for the embodiment 10 J5 bacterium is tested
By banana blight bacteria(Fusarium oxysporumf.sp.cubense)It is inoculated in nutrient agar to put down
The central authorities of plate, simultaneously in each streak inoculation in the both sides J5 bacterial strain of this flat board, cultivate, observe.Result shows that banana is withered by J5 bacterium
Germ has certain inhibitory action, such as Figure 10.
The impact on corn seed germination for the embodiment 11 J5 bacterial strain
By corn seed by 75% alcohol immersion 1min, then recycle 5% liquor natrii hypochloritis and soak 2min, then rush with sterilized water
Washing 3～4 times, then with aseptic filter paper dry seed, all operations ensures to carry out under aseptic condition as far as possible.The kind handled well
Son is placed in dry aseptic container standby.Two process groups (zymotic fluid of first group of addition J5 bacterial strain, the 2nd group of addition are set
The culture medium of non-inoculating strain is as blank), corn seed is soaked respectively in one or two groups 30min.Training by sterilizing
Support and complete two-layer aseptic filter paper in advance in ware, be placed on the seed of seed soaking in culture dish in (10 seeds of every ware), planting
Cover a metafiltration paper on son, cultivate (30 DEG C).Every day, every ware added 2mL sterilized water, and add water time unification.Each bacterial classification processes and arranges 4
Individual repetition.Measure its root length, bud length and germination percentage (see Table 2).The root length of bacterial strain J5 and bud length and germination percentage are the 5th germinateing
It reaches remarkable result, has been respectively increased 47.2% compared with control treatment, and 4.6%, 12.5%.As shown in figure 13.
The impact on corn seed germination for the table 2 J5 bacterial strain
| Bacterial strain | Root length (cm) | Bud length (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: in table, each numerical value is mean value ± standard error (n=4), represents variance analysis without showing with the same letter person in column data
Writing difference (P ＜ 0.05), following table data processing method is identical.
Embodiment 12 J5 bacterial strain promotes the test of potted plant milpa growth result
Prepared by bacteria suspension: point after J5 actication of culture is accessed LB fluid nutrient medium, and through 24h shaken cultivation, cultivation temperature is 30
DEG C, rotating speed is 150r/min；Bacterium solution is instilled blood counting chamber, counts under an optical microscope, draw somatic cells in bacterium solution
Concentration；Bacterium solution is centrifuged in supercentrifuge, rotating speed 6000r/min, centrifugal 5min；By resuspended for thalline aqua sterilisa；For
The bacterial strain concentration of inoculation romaine lettuce is 3.0 × 108The bacteria suspension of cfu/ml.
Corn nursery: vernalization washed by corn, soaks into preserve moisture with filter paper, is placed in 30 DEG C of incubators, reveals two days later
Bud is sowed to seedbed, paramount about 25 cm of seedling growth, can transplant seedlings, four seedlings of every basin.
Test sets 4 process altogether, and each processes 4 repetitions, and each repeats 4 seedlings.Every basin soil loading amount 10.0 kg(does not executes
Add any chemical fertilizer).It is respectively processed as: do not apply microbial inoculum and be called for short CK；Apply microbial inoculum J5.Bacterium solution viable count reaches 105~6 cfu·mL-1, move
Seedling two days later, is inoculated bacterium solution respectively and is used sterilized water re-suspension liquid 30 mL, CK to do same process through centrifugal.Water every day in right amount, every basin
The water yield identical, sow uniformly, water must not be made to flow out at the bottom of basin in order to avoid fertilizer loss causes error.
The assay method that corn growth is affected by each microbial inoculum: after inoculating 20 days and 40 days, the jade that sampling determination 4 is processed respectively
Rice plant height, the number of blade, chlorophyll, root length, leaf is long, Ye Kuan, and stem is thick, fresh weight and dry weight.(corn ground is to jade for measurement corn plant height
Rice blade smoothes out with the fingers the most advanced and sophisticated height of straight posterior lobe), measurement maize leaf (including all blades growing), chlorophyll (uses chlorophyll meter
Measure the 3rd leaf from bottom to up), leaf length and leaf width (measuring the 3rd leaf from bottom to up with tape measure), stem slightly (uses vernier
Slide calliper rule measure).Stem, leaf, after cleaning milpa, are separated by fresh weight: take the maize seedling of every basin with under ground portion, with ten thousand/
One electronic balance weighs its fresh weight respectively, and then in 105 DEG C of 15 min that complete, 65 DEG C of constant temperature dryings are to constant weight, and with very much
One of scales/electronic balance weighing.Result shows, the growth to milpa for the J5 bacterial strain has facilitation.The growth of milpa
Situation such as Figure 14.
After table 3 inoculates 10 days, impact that potted plant milpa is grown by J5 bacterial strain
After table 3 inoculates 10 days, impact that potted plant milpa is grown by J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (cm) | Leaf length (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: in table 3 to table table 17, each numerical value is the measurement result of each.
After table 4 inoculates 20 days, impact (watering bacterium solution 20 days) that potted plant milpa is grown by J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (cm) | Leaf length (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 inoculates 30 days, impact (watering bacterium solution 30 days) that potted plant milpa is grown by J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (cm) | Leaf length (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 |
When table 6 is gathered in the crops, the fresh weight of milpa compares
| Process | 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 |
When table 7 is gathered in the crops, the dry weight of milpa compares
| Process | 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 |
Embodiment 13 under Different Fertilization mode, influence research that potted plant milpa is grown by J5 bacterial strain
Test sets 4 process altogether, and each processes 4 repetitions, and each repeats 4 seedlings.Every basin soil loading amount 10.0 kg(is according to every thousand
Gram soil applies 50 g N, 10 g P2O5、10 g K2O).It is respectively processed as: do not apply bacterial manure and be called for short CK；Bacterium solution directly edge is planted
Water around strain stem, be called for short to water and execute；Bacterium solution is transplanted plant after soil mix and blend, is called for short mixed executing；By maize seedling
Root portion is transplanted after soaking 10 min in bacterium solution, is called for short and dips in root.Bacterium solution viable count reaches 105-106 cfu·mL-1, transplant seedlings two days
After, inoculating re-suspension liquid 30 mL through centrifugal aqua sterilisa for the bacterium solution respectively, CK does same process.Water every day in right amount, the water of every basin
Amount is identical, sows uniformly, and water must not be made to flow out at the bottom of basin in order to avoid fertilizer loss causes error.
After inoculating 20 days and 40 days, the corn plant height of sampling determination 4 process, the number of blade, chlorophyll, root length respectively, leaf is long,
Ye Kuan, stem is thick, fresh weight and dry weight.Measurement corn plant height (the most advanced and sophisticated height of straight posterior lobe is smoothed out with the fingers on corn ground to maize leaf), measurement
Maize leaf (includes all blades growing), and chlorophyll (measures the 3rd leaf from bottom to up with chlorophyll meter), Ye Changhe
Leaf width (measures the 3rd leaf from bottom to up with tape measure), and stem is thick (measuring with slide measure).Fresh weight: take the maize seedling of every basin,
Milpa is cleaned after drying, stem, leaf is separated with under ground portion, weighs its fresh weight with ten thousand/electronic balance respectively,
Then in 105oC completes 15 min, and 65oC constant temperature drying is to constant weight, and with ten thousand/scales/electronic balance weighing.Result shows,
Use different administration microbial inoculum modes, different to the growth-promoting effect of corn, provide important evidence for applying fertilizers scientifically from now on.
Milpa growing state such as Figure 15.
Table 8 is inoculated 20 days, the impact on the growth of potted plant milpa under Different Fertilization mode of J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (piece) | Leaf length (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 |
| Dip in root | 68.41±2.92ab | 8.68±0.19ab | 8.06±0.13a | 36.67±1.79ab | 2.07±0.07ab |
| Mix and execute | 67.12±1.54ab | 9.21±0.14a | 7.63±0.14b | 37.41±1.06a | 2.28±0.05a |
| Water and execute | 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 the measurement result of each, and be mean value (n=4), the same letter of the data of same column indicate without
Significant difference.
Table 9 is inoculated 40 days, the impact on the growth of potted plant milpa under Different Fertilization mode of J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (piece) | Leaf length (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 |
| Water and execute | 94.89±1.46a | 8.56±0.20ab | 9.81±0.31a | 59.34±1.12a | 2.69±0.11a |
| Mix and execute | 82.89±0.83b | 8.33±0.46ab | 10.44±0.19a | 54.61±1.50a | 2.81±0.11a |
| Dip in root | 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 the measurement result of each, and be mean value (n=4), the same letter of the data of same column indicate without
Significant difference.
When table 10 is gathered in the crops, the comparison to potted plant milpa each position fresh weight under Different Fertilization mode of J5 bacterial strain
| Process | Root (g) | Leaf (g) | Stem (g) |
| CK | 1.64±0.32b | 30.23±5.44a | 36.67±6.43b |
| Water and execute | 2.48±0.33ab | 41.85±0.05a | 52.23±3.06ab |
| Mix and execute | 4.45±1.32a | 40.74±4.16a | 46.53±5.74ab |
| Dip in root | 5.03±0.89a | 41.90±1.24a | 53.63±6.01a |
Note: each numerical value is the measurement result of each, and is mean value (n=4), indicates without significantly with column data same letter
Sex differernce.
When table 11 is gathered in the crops, the comparison to potted plant milpa each position dry weight under Different Fertilization mode of J5 bacterial strain
| Process | Leaf dry weight (g) | Stem weight (g) |
| CK | 8.48±0.73a | 3.30±0.09b |
| Water and execute | 9.88±0.24a | 4.41±0.14a |
| Mix and execute | 8.84±0.91a | 3.96±0.29b |
| Dip in root | 9.71±0.36a | 4.10±0.28a |
Note: each numerical value is the measurement result of each, and is mean value (n=4), indicates without significantly with column data same letter
Sex differernce.
Embodiment 14 J5 bacterial strain improves the research of field corn plant strain growth
Test and devise 3 process altogether: CK process；Microbial inoculum 4 is processed；Microbial inoculum 5 is processed.Each chemical fertilizer processing all applying equivalent.Examination
Testing totally 9 cells, totally 3 row, often arranging 3 cells, each processes 3 repetition, according to arrange the principle of equal different disposal of going together with
The planting location that machine distribution is processed.Each cell maize planting plant 2 row, line-spacing is 50 cm, and colleague's plant longitudinal separation is
20 cm, each cell planted 40 milpas altogether, and each cell includes that left and right protection row occupied area is about 8.2 m2,
Dose applies 1～2 time according to the standard of 7.5 kilograms every mu.
With conventional method measure that different times corn plant height, root length, the number of blade, leaf length, Ye Kuan, stem be thick, fresh weight and dry weight, become
(the yield: for the yield of a test block in single process such as ripe phase spike length, fringe are thick, 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: on sample plant corn fruit choose that the longest position of girth goes out straight
Footpath；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: be
The dry weight of corn fruit on sample plant；100-grain weight: the weight of 100 iblets selected at random on the sample plant of collection
Amount).Economic flow rate sampling method is each cell corn fresh weight.Result shows, the growth to field corn for the administration microbial inoculum J5 has
There is significant facilitation.Current existing report, the research of A Shi bacillus mainly concentrates on laboratory and potted plant rank
Section, this research is confirmed the growth-promoting functions to cereal crops for the A Shi bacillus further by field test.The growth of corn
Situation such as Figure 16.
After table 12 inoculates 30 days, the impact on field corn plant strain growth for the J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (mm) | The number of blade (piece) | Leaf length (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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
After table 13 inoculates 30 days, the impact on field corn plant fresh weight for the J5 bacterial strain
| Process | 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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
After table 14 inoculates 30 days, the impact on field corn plant weights for the J5 bacterial strain
| Process | 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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
After table 15 inoculates 75 days, the impact on field corn plant strain growth index for the J5 bacterial strain
| Process | Plant height (cm) | Stem is thick (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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
After table 16 inoculates 75 days, the impact on field corn plant fresh weight for the J5 bacterium
| Process | 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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
After table 17 inoculates 75 days, the impact on field corn plant weights for the J5 bacterial strain
| Process | 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 mean value (n=4), and the different alphabets of the data of same column are shown with
Significant difference.
Each Indexes Comparison of the yield of corn and fruit when table 18 is gathered in the crops
| Process | 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 mean value, and n=4, is shown with significant difference with the different alphabets of column data.Yield: be single
The total output of a test block in process.Spike length: for the length of corn fruit on sample plant；Fringe is thick: beautiful on sample plant
Rice cracker is real chooses the diameter that the longest position of girth goes out；Total particle number: the result of line number * columns on corn fruit；Fresh weight: be sample
The fresh weight of corn fruit on plant；Dry weight: for the dry weight of corn fruit on sample plant；100-grain weight: on the sample plant of collection
The weight of 100 iblets selected at random.
<110>Agricultural University Of South China
<120>one strain A Shi bacillus J5 and application thereof
<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
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<210> 3
<211> 22
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Claims (8)
1. a strain A Shi bacillus (Bacillus aryabhattai) J5, it is characterised in that described 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 microbial bacterial agent, it is characterised in that comprise A Shi bacillus described in claim 1 (Bacillus aryabhattai) J5.
3. microbial bacterial agent according to claim 2, it is characterised 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 or/and produce ammonia or/and produce siderophore or/and protease or/and
Catalase is or/and decompose insoluble phosphorus or/and salt tolerant is or/and application in antibacterial.
5. microbial bacterial agent described in claim 2 is promoting germination or/and promote the application in plant growth.
6. the A Shi bacillus described in claim 1 (Bacillus aryabhattai) J5 produce auxin or/and produce ammonia
Or/and produce siderophore or/and protease is or/and catalase is or/and decompose insoluble phosphorus or/and salt tolerant is or/and in antibacterial
Application.
7. the A Shi bacillus described in claim 1 (Bacillus aryabhattai) J5 promote germination or/and
Promote the application in plant growth.
8. the application according to claim 5 or 7, described plant is corn.
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