CN105018387A - Bacillus subtilis strain and application thereof - Google Patents
Bacillus subtilis strain and application thereof Download PDFInfo
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- CN105018387A CN105018387A CN201510462749.2A CN201510462749A CN105018387A CN 105018387 A CN105018387 A CN 105018387A CN 201510462749 A CN201510462749 A CN 201510462749A CN 105018387 A CN105018387 A CN 105018387A
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Abstract
The invention provides a Bacillus subtilis strain of which the collection number is CCTCC M2015300 and the name is Bacillus subtilis K-2. The invention also provides a screening method of the Bacillus subtilis K-2, application of the Bacillus subtilis K-2 in promoting growth of lawn grass and controlling snow mould leaf blight and dollar spot and a Bacillus subtilis K-2 microbial inoculant. The Bacillus subtilis K-2 has an inhibiting action on growth of the snow mould leaf blight and dollar spot hypha of lawn grass, so that the hypha grow slowly and have the symptom of deformity, thereby performing the disease-resistance functions on the snow mould leaf blight and dollar spot of the lawn grass. The Bacillus subtilis K-2 has the advantages of short production cycle and low cost, is simple to use, and has very wide application prospects in biological control of plant diseases.
Description
Technical field
The invention belongs to technical field of microbiology, particularly relate to one, to turfgrass, there is growth-promoting functions, and to subtilis and the application thereof of avenging mould leaf blight, coin pinta has resistant effect.
Background technology
Turfgrass is one " popular plants landscape " in the last few years, is applicable to beautify the environment, purifies air, conserves water and soil, etc., turf is referred to as in American-European countries, and Japan is referred to as sesame ground, sesame grass.Modern in the world using lawn area coverage as one of important symbol weighing Modernized City Construction, 1969 year's harvest are founded a state lawn, border association.Adopt in gardens and manually sod or the method for grass-seed sowing, cultivate large-scale green ground, be not only the important component part of Scenery of gardens, be also have a rest, the playground of amusement, represent a high-caliber ecological organism.
But Turfgrass Diseases is the principal element hindering current Development of Turf, global grass cultivation is every year because disease causes several hundred million financial losses.Avenging mould leaf blight is by the mould leaf spoting bacteria of snow, i.e. Microdochium nivale, infects a kind of lawn fungal disease caused, with tikka and the withered symptom of leaf the most common, lawn occurs that diameter is less than the circular withered grass spot of 5cm, after expanding, diameter reaches 20cm, survives the winter, propagate with the wind with rainwater with seed, soil and invalid body, when average daily temperature more than 15 DEG C, meet continuous rainy weather, the large happening and prevelence of disease possibility, endangers very serious.Coin pinta is by coin pinta bacterium, i.e. Sclerotinia homoeocarpa, infects a kind of disease caused, and host widely, be injured blade, starts to produce water stain shape chlorisis spot, finally become white scab, can expand and extend to whole blade by individual plant turfgrass; Cheng Ping lawn there is depression, circular withered grass spot, size assigns to 1 yuan of coin from 5, and pathogenic bacteria tides over poor environment on diseased plant He on sick leaf surface, by propagation such as wind, rainwater, instruments, morbidity thermophilic be 15-32 DEG C, from Chun Mo until autumn disease all can occur.
In Turfgrass Diseases control, as golf course disease control, the strategy that people take normally chemical pesticide, the resistance of pathogenic bacteria to chemical agent strengthens year by year, and dosing improves constantly.The long-term a large amount of of chemical agent use, and cause the pollution that natural ecological environment is day by day serious, have run counter to the original intention that turf plant is improved the ecological environment.So; sight is turned to the biological control of Diseases of Turfgrass by people; the beneficial microorganism utilizing nature to exist substitutes chemical bactericide, be a kind of lawn of protecting from the non-chemically environment protection method of disease, great potential, become a focus of current Diseases of Turfgrass study on prevention.
Using microbe controlling plant diseases and Promoting plant growth all have report at home and abroad, and the U.S. has biocontrol fungicide prepared by 4 kinds of genus bacillus and obtains production licence so far; Through retrieval, applicant retrieves some bibliographical informations about subtilis and patent in existing patent and non-patent literature.Such as, number of patent application is 2013100910680 to disclose " strain is for preventing and treating the subtilis of citrus ulcer ", report bacillus subtilis bacterial strain CCCQ080 and have very strong restraining effect to citrus processing, have good control effects to citrus bacterial canker disease.Number of patent application is 2013101243237 to disclose " strain is for preventing and treating the subtilis of black shank ", report bacillus subtilis strain TBSCQ057 and have very strong restraining effect to tobacco black shank bacterium, good control effects is had to black shank, simultaneously, this bacterial strain also has the antagonistic activity of wide spectrum, better to multiple pathogenic fungi fungistatic effects such as tobacco ash arrhizus bacteria, tobacco brown spot pathogen, tobacco sclerotium rolfsii and Colletotricum destructivum bacterium.Number of patent application is 2012104608407, disclose " a kind of plant for prophyiaxis and promoting growth endophytic Bacillus subtilis ", report subtilis Jaased3, to multiple pathogenic fungies such as withered germ of water-melon, tomato early blight bacterium, Lettuce Drop bacterium, Verticillium Wilt Pathogen of Eggplants, there is antagonistic activity; Can enter under lower bacterial concentration in the plant such as watermelon, eggplant body, and can in farm crop Colonization inside plants and conduction; Farm crop are had to the effect of growth promoting effects, controlling disease, especially to soil-borne vascular bundle disease, there is good prevention effect.But at present not to the report avenging mould leaf blight, coin pinta has the subtilis of resistant effect.
Summary of the invention
For improving the deficiency of existing Prevention Technique, the object of the present invention is to provide a kind of subtilis and application thereof, this subtilis has growth-promoting functions to turfgrass, and has resistant effect to the mould leaf blight of snow, coin pinta.
First aspect present invention provides a kind of subtilis, it is preserved in Wuhan University's China typical culture collection center on May 15th, 2015, preserving number is CCTCC M 2015300, called after subtilis (Bacillus subtilis) K-2.
The bacteria characteristic of subtilis K-2:
A, morphological specificity: bacterial strain K-2 is gram-positive microorganism, in rod-short, the blunt circle in two ends, gemma is oval.The bacterium colony that LB flat board is formed is circular, and edge is irregular, and oyster white is to micro-yellow, and surface ruffle is coarse, opaque.
B, physiological and biochemical property: hydrolyzable starch, catalase is positive.
The 16S rDNA sequence of subtilis K-2, as shown in sequence table SEQ ID No.1, and Genbank has reported sequence is compared, and finally determines that bacterial strain K-2 is subtilis.
Second aspect present invention provides subtilis K-2 at promotion Turfgrass Growth, the application of the mould leaf blight of control snow, coin pinta.
Third aspect present invention provides the screening method of a kind of subtilis, and its step comprises:
(1) be that sample sterilized water is formulated as suspension with soil, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) choose single bacterium colony from step (1) bacterial strain filtering out and the mould leaf spoting bacteria of snow and coin pinta bacterium are had to bacteriostatic action by dull and stereotyped face-off method.
Fourth aspect present invention provides a kind of subtilis K-2 microbial inoculum, and described microbial inoculum is that subtilis K-2 is activated, seed culture, the microbial inoculum that obtains after fermentation.
The invention has the beneficial effects as follows: subtilis K-2 of the present invention has growth-promoting effect to turfgrass, to the mould leaf spoting bacteria of snow and coin pinta bacterium mycelial growth inhibited, make germ mycelial growth slow, expand in the middle of occurring, end attenuates, bending, the lopsided symptoms such as collapse, thus resistant effect is played to the mould leaf blight of snow, coin pinta, and it is with short production cycle, with low cost, use simple, in the biological control of Plant diseases, there is boundless application prospect.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of subtilis K-2 at LB cultured on solid medium;
Fig. 2 is the teratogenesis of subtilis K-2 to the mould leaf spoting bacteria mycelial growth of snow, and wherein, A is the control group without subtilis K-2 process, and B is the experimental group through subtilis K-2 process;
Fig. 3 is the teratogenesis of subtilis K-2 to coin pinta bacterium mycelial growth, and wherein, A is the control group without subtilis K-2 process, and B is the experimental group through subtilis K-2 process;
Fig. 4 is the bacteriostatic action schematic diagram of subtilis K-2 to the mould leaf spoting bacteria of snow;
Fig. 5 is the bacteriostatic action schematic diagram of subtilis K-2 to coin pinta bacterium.
Embodiment
First aspect present invention provides a kind of subtilis, and this bacterial strain is preserved in Wuhan University's China typical culture collection center on May 15th, 2015, and preserving number is CCTCC M 2015300, called after subtilis K-2.
Second aspect present invention provides the screening method of a kind of subtilis, and its step comprises:
(1) be that sample sterilized water is formulated as suspension with soil, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) choose single bacterium colony from step (1) bacterial strain filtering out and the mould leaf spoting bacteria of snow and coin pinta bacterium are had to bacteriostatic action by dull and stereotyped face-off method.
Preferably, step (1) described soil is added in sterilized water gets supernatant liquor after shaking culture, dilutes to obtain suspension with sterilized water, coats LB flat board, cultivate 23-26h, picking list bacterium colony at 28-30 DEG C after being diluted by suspension; The described dull and stereotyped face-off method concrete steps of step (2) are: mould for snow leaf spoting bacteria and coin pinta bacterium are seeded in PDA plate center, be that triangular shape symmetry access PDA is dull and stereotyped by step (1) gained bacterium, distance center 1-3cm, cultivate at 28 DEG C, obtain bacteriostatic action bacterial strain, proceeded on LB flat board and cultivate preservation.
Third aspect present invention provides subtilis K-2 microbial inoculum, and described microbial inoculum is that subtilis K-2 bacterial strain is activated, seed culture, the microbial inoculum that obtains after fermentation.
Preferably, described activation be by subtilis K-2 inoculation in substratum, at 28-30 DEG C, cultivate 24-36h.
Be more preferably, described substratum is LB solid medium, and its collocation method is yeast extract 5g, tryptone 10g, sodium-chlor 10g, agar powder 20g, adjusts pH to 7.0-7.2, autoclaving 20min at 121 DEG C with NaOH.
Preferably, described seed culture is by the subtilis K-2 inoculation after activated in substratum, in 28-30 DEG C, shaking culture 23-25h under 155-165r/min.
Preferably, described fermentation be by the subtilis K-2 bacterial strain after seed culture with 5% inoculative proportion be inoculated in substratum, in 28-30 DEG C, shaking culture 36-48h under 155-165r/min.
Described subtilis K-2 microbial inoculum is diluted to active constituent content 1.0 × 10
6-1.0 × 10
8use after cfu/mL.
Below in conjunction with embodiment, a kind of subtilis provided by the invention and application thereof are further described.
Embodiment 1
Turfgrass described in the present embodiment avenges mould leaf spoting bacteria and turfgrass coin pinta bacterium is provided by Canadian Guelph university professor Tom, and the turfgrass obtained by other means avenges mould leaf spoting bacteria and turfgrass coin pinta bacterium is also applicable to the present invention.
One, the acquisition of subtilis K-2 and qualification
Be separated: gather rich soil from periphery vegetable garden, Hubei Jingzhou City, take back laboratory and preserve, for subsequent use.Take pedotheque 10g, add in 90mL aqua sterilisa, 160r/min shaking culture 10min, leave standstill 30min, get supernatant liquor 1mL, be added in the test tube filling 9mL sterilized water, vibration mixing, makes sample suspension.Get 1mL sample suspension to add in the test tube filling 9mL sterilized water and carry out 10 times of serial dilutions successively, get 10 respectively
-5, 10
-6, 10
-7diluent 50 μ L coat LB flat board, be placed in 28 DEG C cultivate 24h.According to features such as form, size, color and lusters, picking list bacterium colony 197 strain altogether, transfer and to cultivate on LB flat board, save backup, subtilis K-2 LB cultured on solid medium colonial morphology as shown in Figure 1, the bacterium colony that subtilis K-2 is formed on LB flat board is circular, edge is irregular, oyster white is to micro-yellow, and surface ruffle is coarse, opaque.
Screening: cultured turfgrass is avenged mould leaf spoting bacteria and turfgrass coin pinta bacterium is inoculated in PDA culture medium flat plate center respectively, being that triangular shape is symmetrical with the toothpick of sterilizing by the bacterium of above-mentioned separation and purification accesses PDA flat board, distance center 2cm, cultivates for 28 DEG C and observes the antibacterial situation of opposite culture.Obvious for bacteriostatic action bacterial strain K-2 is proceeded on LB flat board and cultivates, save backup.By microscopy observation subtilis K-2, turfgrass is avenged to the restraining effect of mould leaf spoting bacteria and turfgrass coin pinta bacterium mycelial growth, exercising result respectively as shown in Figure 2 and Figure 3, the mycelial growth of bacterial strain K-2 process is slow as seen from the figure, centre is expanded, end attenuates, bending, the lopsided symptoms such as collapse, and it is normal to contrast mycelial growth, form is even.The compound method of described PDA substratum is: potato 200g, glucose 20g, and agar 18g supplies distilled water to 1000mL, 121 DEG C of moist heat sterilization 20min.
Qualification: carry out Physiology and biochemistry qualification to the bacterial strain K-2 of screening, be defined as bacillus, the total genomic dna then extracting bacterial strain is its 16S rDNA sequence of template amplification.Forward used and reverse primer are respectively 27F:5 '-AGAGTTTGATCCTGGCTCAG-3 ', i.e. sequence table SEQ ID No.2; 1429R:5 '-GGTTACCTTGTTACGACTT-3 ', i.e. sequence table SEQ ID No.3.PCR reaction system is 15 μ L systems: DNA profiling 0.6 μ L; H
2o 8.57 μ L; Buffer 1.65 μ L; Mg
2+2.75 μ L; DNTP0.66 μ L; 27F:0.33 μ L; 1429R 0.33 μ L; Tsg 0.11 μ L.PCR response procedures is: 94 DEG C of 3min; 94 DEG C of 30s, 50 DEG C of 45s, 72 DEG C of 100s, 35 circulations; 72 DEG C of 7min.Reclaim and purified pcr product, order-checking, bacterial strain K-2, as shown in sequence table SEQ ID No.1, is accredited as subtilis according to sequencing result by its 16S rDNA sequence.
Further, subtilis K-2 avenges mould leaf spoting bacteria, coin pinta bacterium antagonistic action test to turfgrass has been carried out.
Accessed in LB liquid nutrient medium by the most obvious bacterial strain K-2 of bacteriostatic action filtered out, at 28 DEG C, 130r/min shaking culture 26h, makes bacteria suspension.The mould leaf spoting bacteria of snow, coin pinta bacterium is accessed respectively at different PDA plate center, being that triangular shape is symmetrical with pipettor by K-2 bacteria suspension accesses PDA flat board, often some access 10 μ L, distance center is about 2cm, take sterilized water as contrast, cultivate for 28 DEG C and observe the antibacterial situation of opposite culture, measure colony radius after 4d, the antibacterial situation of subtilis K-2 to the mould leaf spoting bacteria of snow is shown in accompanying drawing 4, sees accompanying drawing 5 to the antibacterial situation of coin pinta bacterium.
The relative bacteriostasis rate adopting following formulae discovery bacterial strain K-2 to grow pathogenic fungi, often processes in triplicate.
Relative inhibition=(contrast colony radius-process colony radius)/contrast colony radius × 100%
Subtilis K-2 all has obvious antagonistic action to the mould leaf spoting bacteria of snow, coin pinta bacterium, and the results are shown in Table 1, its relative inhibition is respectively 80.0%, 81.6%.
Table 1: subtilis K-2 is to the antagonistic action of several Different Kinds of Pathogens fungi
Two, the preparation of subtilis K-2 microbial inoculum
The present embodiment completes the preparation of subtilis K-2 microbial inoculum according to following key step:
(1) subtilis K-2 is inoculated in LB solid medium, at 28 DEG C, cultivates 25h, activated strains; LB solid medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, agar powder 20g, NaOH adjust pH to 7.0,121 DEG C, 20min autoclaving.
(2), in bacterial strain access LB liquid nutrient medium step (1) activated, at 28 DEG C, 160r/min shaking culture 24h, makes seed liquor; LB liquid nutrient medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, NaOH adjust pH to 7.0,121 DEG C, 20min autoclaving.
(3) seed fermentation liquid step (2) obtained 5% is inoculated in LB liquid fermentation medium by volume, 160r/min shaking culture 36h at 28 DEG C.
Subtilis K-2 microbial inoculum can be obtained through as above step.
Three, subtilis K-2 is promoting the application in turfgrass creeping bentgrass Penncross growth
Being loaded by sterilized soil is highly 13.5cm, and diameter is in the tapered tube of 3.5cm, from mouth of pipe 1cm, turfgrass creeping bentgrass Penncross seed, to the mouth of pipe, in water saturates pipe subject to sterilization after soil, is evenly sprinkled in pipe by sprinkle aqua sterilisa, often pipe 0.04g, repeats for five times.Every day, often pipe dripped 5mL aqua sterilisa, until seed germination drips a water every other day after showing money or valuables one carries unintentionally, and often pipe 5mL.Dark 8h is kept after every illumination 16h.After one week, the subtilis K-2 microbial inoculum of preparation in embodiment 2 is diluted to 1.0 × 10
8cfu/mL, OD600=0.86-0.88, fill with root inoculation, often pipe 5mL, with LB liquid nutrient medium for blank.Measure height, the DGCI value of turfgrass respectively at 5d, 10d, 15d, 20d after process, and turfgrass is trimmed to and mouth of pipe level, survey its dry weight.
Result is as shown in table 2, and result shows 5d-20d after subtilis K-2 process, can show significant growth-promoting functions to turfgrass creeping bentgrass Penncross, is significantly higher than blank through the turfgrass height of K-2 process, DGCI value and dry weight.
Table 2: subtilis K-2 is to the growth-promoting effect table of turfgrass creeping bentgrass Penncross
In table 2, data are repeat mean value 5 times, according to SAS 9.1 software analysis statistics, remarkable at 5% level difference with letter representation different after column data.DGCI is plant deep green index, can reflect plant-growth health degree, and DGCI value becomes positive correlation with turfgrass health degree.
As seen from the data in Table 2,5d after inoculation, the height of process and contrast turfgrass is respectively 7.1cm, 5.2cm, increases by 26.7%, DGCI value and is respectively 0.699,0.584; Dry weight is respectively 144.2mg, 46.9mg, increases by 67.5%; 10d after inoculation, the height of process and contrast turfgrass is respectively 7.2cm, 5.4cm, and increase by 25.0%, DGCI value and be respectively 0.650,0.581, dry weight is respectively 133.4mg, 45.2mg, increases by 66.1%; 15d after inoculation, the height of process and contrast turfgrass is respectively 5.9cm, 4.2cm, and increase by 28.8%, DGCI value and be respectively 0.555,0.497, dry weight is respectively 108.8mg, 19.7mg, increases to 81.9%; 20d after inoculation, the height of process and contrast turfgrass is respectively 6.2cm, 4.2cm, and increase by 32.3%, DGCI value and be respectively 0.528,0.460, dry weight is respectively 186.1mg, 50.3mg, increases by 73.0%.
Four, subtilis K-2 is promoting the application in turfgrass creeping bentgrass A4 growth
Being loaded by sterilized soil is highly 13.5cm, and diameter is in the tapered tube of 3.5cm, and from mouth of pipe 1cm, turfgrass creeping bentgrass A4 seed, to the mouth of pipe, in water saturates pipe subject to sterilization after soil, is evenly sprinkled in pipe, often pipe 0.04g by sprinkle aqua sterilisa, repeats for five times.Every day, often pipe dripped 5mL aqua sterilisa, until seed germination drips a water every other day after showing money or valuables one carries unintentionally, and often pipe 5mL.Dark 8h is kept after every illumination 16h.After one week, the subtilis K-2 microbial inoculum of preparation is diluted to 1.0 × 10
8cfu/mL, OD600=0.86-0.88, fill with root inoculation, often pipe 5mL, with LB liquid nutrient medium for blank.Measure height, the DGCI value of turfgrass respectively at 5d, 10d, 15d, 20d after process, and turfgrass is trimmed to and mouth of pipe level.
The results are shown in Table 3, result shows, at 10d-20d after subtilis K-2 process, can show significant growth-promoting functions to turfgrass creeping bentgrass A4, is significantly higher than blank through the turfgrass height of K-2 process and DGCI value.
Table 3: subtilis K-2 is to the growth-promoting effect table of turfgrass creeping bentgrass A4
In table 3, data are repeat mean value 5 times, according to SAS 9.1 software analysis statistics, remarkable at 5% level difference with letter representation different after column data.DGCI is plant deep green index, can reflect plant-growth health degree, and DGCI value becomes positive correlation with turfgrass health degree.
As seen from the data in Table 3,5d after inoculation, the height of process and contrast turfgrass is respectively 5.62cm, 4.74cm, increases by 15.7%, DGCI value and is respectively 0.753,0.734; 10d after inoculation, the height of process and contrast turfgrass is respectively 5.16cm, 3.63cm, increases by 29.7%, DGCI value and is respectively 0.598,0.573; 15d after inoculation, the height of process and contrast turfgrass is respectively 6.61cm, 4.76cm, increases by 28.0%, DGCI value and is respectively 0.602,0.515; 20d after inoculation, the height of process and contrast turfgrass is respectively 5.74cm, 4.21cm, increases by 26.7%, DGCI value and is respectively 0.611,0.521.
Five, subtilis K-2 avenges the application in mould leaf blight at control turfgrass
Turfgrass avenges the preparation of the wheat inoculum of mould leaf blight: cleaned by wheat, soaked overnight, loads in 250mL triangular flask, every bottle of about 50g, sterilizing.By in mould for the cultivation snow of one week on PDA flat board leaf spoting bacteria access triangular flask, be placed in 25 DEG C of cultivations, cover with after wheat substratum until mycelia, pour out natural air drying, pulverize, load in aseptic plastic bag, for subsequent use.
Being loaded by sterilized soil is highly 7.0cm, and diameter is in the vial of 2.5cm, and every bottle adds 20g, adds 1mL aqua sterilisa, in water saturates pipe subject to sterilization after soil, is evenly sprinkled in pipe by turfgrass creeping bentgrass A4 seed, often pipe 0.015g, repeats for five times.Dark 8h is kept after every illumination 16h.After one week, the subtilis K-2 microbial inoculum of preparation is diluted to 1.0 × 10
8cfu/mL, OD600=0.86-0.88, fill with root inoculation, every bottle of 1mL, with LB liquid nutrient medium for blank.After one week, above-mentioned turfgrass is avenged mould leaf blight wheat inoculum and be evenly seeded on the turfgrass in bottle, often pipe 0.015g, after inoculation, 5d starts to observe incidence, records the yellowing leaf rate of turfgrass in every pipe.
The results are shown in Table 4, result shows, at 5-20d after subtilis K-2 process, to avenge mould leaf blight can show significant preventive and therapeutic effect to turfgrass, and the turfgrass yellowing leaf rate through the process of K-2 bacterial strain obviously will lower than blank.
Table 4: subtilis K-2 avenges the prevention effect table of mould leaf blight to turfgrass
In table 4, data are repeat mean value 5 times, according to SAS 9.1 software analysis statistics, remarkable at 5% level difference with letter representation different after column data.
From the data of table 4,5d after inoculation, process and contrast turfgrass yellowing leaf rate are respectively 8.0%, 30.0%, reduce 22.0%; 10d after inoculation, the yellowing leaf rate of process and contrast turfgrass is respectively 12.5%, 65.0%, reduces 52.5%; 15d after inoculation, the yellowing leaf rate of process and contrast turfgrass is respectively 15.0%, 72.5%, reduces 57.5%; 20d after inoculation, the yellowing leaf rate of process and contrast turfgrass is respectively 30.0%, 85.0%, reduces 55.0%.
Six, the application of subtilis K-2 in control turfgrass coin pinta
Turfgrass avenges the preparation of the wheat inoculum of mould leaf blight: cleaned by wheat, soaked overnight, loads in 250mL triangular flask, every bottle of about 50g, sterilizing.By in the turfgrass coin pinta bacterium of a week of cultivation on PDA flat board access triangular flask, be placed in 25 DEG C of cultivations, cover with after wheat substratum until mycelia, pour out natural air drying, pulverize, load in aseptic plastic bag, for subsequent use.
Being loaded by sterilized soil is highly 13.5cm, and diameter is 3.5cm, and volume is about in the tapered tube of 180mL, from mouth of pipe 1cm, sprinkle aqua sterilisa to the mouth of pipe, in water saturates pipe subject to sterilization after soil, turfgrass creeping bentgrass A4 seed is evenly sprinkled in pipe, often pipe 0.04g, repeats for five times.Every day, often pipe sprayed 5mL water, until seed germination sprays a water every other day after showing money or valuables one carries unintentionally, and often pipe 5mL.Keep dark 8h after every illumination 16h, after one week, the subtilis K-2 microbial inoculum of preparation in embodiment 2 is diluted to 1.0 × 10
8cfu/mL, OD600=0.86-0.88, fill with root inoculation, often pipe 5mL, with LB liquid nutrient medium for blank.After one week, be evenly seeded on turfgrass by above-mentioned coin pinta bacterium wheat inoculum, often pipe 0.05g, after inoculation, 5d starts to observe incidence, records the yellowing leaf rate of turfgrass in every pipe.
The results are shown in Table 5, result shows, at 5-20d after subtilis K-2 process, can show significant preventive and therapeutic effect, turfgrass yellowing leaf rate and blank significant difference to turfgrass coin pinta.
Table 5: subtilis K-2 is to the prevention effect table of turfgrass coin pinta
In table 5, data are repeat mean value 5 times, according to SAS 9.1 software analysis statistics, remarkable at 5% level difference with letter representation different after column data.
From the data of table 5, after K-2 inoculation 5d, the turfgrass yellowing leaf rate of process and contrast is respectively 16.5%, 55.0%, is reduced to 70.0%; After inoculation 10d, the turfgrass yellowing leaf rate of process and contrast is respectively 17.5%, 65.0%, reduces 73.1%; After inoculation 15d, the turfgrass yellowing leaf rate of process and contrast is respectively 23.3%, 74.8%, reduces 68.9%; After inoculation 20d, the turfgrass yellowing leaf rate of process and contrast is respectively 30.0%, 88.4%, reduces 66.1%.Can find out thus, adopt K-2 bacterial strain process turfgrass in 20d, to have good preventive and therapeutic effect to coin pinta, turfgrass yellowing leaf rate and blank significant difference.
Embodiment 2
Embodiment 2 is substantially the same manner as Example 1, and difference is:
The present embodiment completes the preparation of subtilis K-2 microbial inoculum according to following key step:
(1) subtilis K-2 is inoculated in LB solid medium, at 30 DEG C, cultivates 30h, activated strains; LB solid medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, agar powder 20g, NaOH adjust pH to 7.2,121 DEG C, 20min autoclaving.
(2), in bacterial strain access LB liquid nutrient medium step (1) activated, at 28 DEG C, 160r/min shaking culture 24h, makes seed liquor; LB liquid nutrient medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, NaOH adjust pH to 7.2,121 DEG C, 20min autoclaving.
(3) seed fermentation liquid step (2) obtained 5% is inoculated in LB liquid fermentation medium by volume, 160r/min shaking culture 40h at 28 DEG C.
By above step i.e. obtained subtilis K-2 microbial inoculum, gained subtilis K-2 microbial inoculum is diluted to active constituent content 1.0 × 10
7use after cfu/mL.
Embodiment 3
Embodiment 3 is substantially the same manner as Example 1, and difference is:
The present embodiment completes the preparation of subtilis K-2 microbial inoculum according to following key step:
(1) subtilis K-2 is inoculated in LB solid medium, at 30 DEG C, cultivates 35h, activated strains; LB solid medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, agar powder 20g, NaOH adjust pH to 7.0,121 DEG C, 20min autoclaving.
(2), in bacterial strain access LB liquid nutrient medium step (1) activated, at 28 DEG C, 160r/min shaking culture 24h, makes seed liquor; LB liquid nutrient medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, NaOH adjust pH to 7.0,121 DEG C, 20min autoclaving.
(3) seed fermentation liquid step (2) obtained 5% is inoculated in LB liquid fermentation medium by volume, 160r/min shaking culture 46h at 28 DEG C.
By above step i.e. obtained subtilis K-2 microbial inoculum, gained subtilis K-2 microbial inoculum is diluted to active constituent content 1.0 × 10
7use after cfu/mL.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. a subtilis, is characterized in that: the deposit number of described subtilis is CCTCC M 2015300, called after subtilis (Bacillus subtilis) K-2.
2. subtilis as claimed in claim 1, is characterized in that: the 16S rDNA sequence of described subtilis is as shown in SEQ ID No.1.
3. subtilis described in claim 1 or 2 is promoting the application in Turfgrass Growth, the mould leaf blight of control snow, coin pinta.
4. a screening method for subtilis as claimed in claim 1, its step comprises:
(1) be that sample sterilized water is formulated as suspension with soil, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) from single bacterium colony that step (1) is chosen, the bacterial strain mould leaf spoting bacteria of snow and coin pinta bacterium being had to bacteriostatic action is filtered out by dull and stereotyped face-off method.
5. the screening method of subtilis as claimed in claim 4, it is characterized in that: step (1) described soil is added in sterilized water gets supernatant liquor after shaking culture, suspension is diluted to obtain with sterilized water, LB flat board is coated after being diluted by suspension, 23-26h is cultivated, picking list bacterium colony at 28-30 DEG C; The described dull and stereotyped face-off method concrete steps of step (2) are: mould for snow leaf spoting bacteria and turfgrass coin pinta bacterium are seeded in PDA plate center, be that triangular shape symmetry access PDA is dull and stereotyped by step (1) gained bacterium, distance center 1-3cm, cultivate at 28 DEG C, obtain bacteriostatic action bacterial strain, proceeded on LB flat board and cultivate preservation.
6. a subtilis K-2 microbial inoculum, is characterized in that: described bacillus subtilis microbial agent is that subtilis K-2 is activated, seed culture, the microbial inoculum that obtains after fermentation.
7. subtilis K-2 microbial inoculum as claimed in claim 6, is characterized in that: described activation, for be inoculated in substratum by subtilis K-2, cultivates 24-36h at 28-30 DEG C.
8. subtilis K-2 microbial inoculum as claimed in claim 6, is characterized in that: described seed culture is be inoculated in substratum by the subtilis K-2 after activated, in 28-30 DEG C, shaking culture 23-25h under 155-165r/min.
9. subtilis K-2 microbial inoculum as claimed in claim 6, it is characterized in that: described fermentation be by the subtilis K-2 after seed culture with 5% inoculative proportion be inoculated in substratum, in 28-30 DEG C, shaking culture 36-48h under 155-165r/min.
10. the subtilis K-2 microbial inoculum as described in any one of claim 6 to 9 claim, is characterized in that: described subtilis K-2 microbial inoculum is diluted to active constituent content 1.0 × 10
6-1.0 × 10
8use after cfu/mL.
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| EP4105317A4 (en) * | 2020-02-12 | 2023-08-23 | JAN153 BIOTECH Inc. | Bacillus subtilis jck-1398 strain inducing resistance in various plants, and composition and method for controlling pine wilt disease by using same |
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| EP1404179B1 (en) * | 2001-06-22 | 2008-06-04 | David J. Drahos | Biofungicide |
| US20110230345A1 (en) * | 2010-03-19 | 2011-09-22 | Novozymes Biologicals, Inc. | Bacillus amyloliquefaciens Strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1404179B1 (en) * | 2001-06-22 | 2008-06-04 | David J. Drahos | Biofungicide |
| US20110230345A1 (en) * | 2010-03-19 | 2011-09-22 | Novozymes Biologicals, Inc. | Bacillus amyloliquefaciens Strain |
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| EP4105317A4 (en) * | 2020-02-12 | 2023-08-23 | JAN153 BIOTECH Inc. | Bacillus subtilis jck-1398 strain inducing resistance in various plants, and composition and method for controlling pine wilt disease by using same |
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