CN105018387B - A kind of bacillus subtilis and its application - Google Patents
A kind of bacillus subtilis and its application Download PDFInfo
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Abstract
The invention provides a kind of bacillus subtilis, its preserving number is CCTCC M 2015300, it is named as bacillus subtilis (Bacillus subtilis) K 2, and bacillus subtilis K 2 screening technique and the application in promoting Turfgrass Growth, preventing and treating to avenge mould leaf blight, coin pinta are further provided, and a kind of microbial inoculums of bacillus subtilis K 2.Bacillus subtilis K 2 is inhibited to turfgrass avenge mould leaf spoting bacteria and coin pinta bacterium mycelial growth, make germ mycelial growth slow, there is lopsided symptom, so as to play resistant effect to the mould leaf blight of snow, the coin pinta of turfgrass, it is and with short production cycle, cost is cheap, using simple, has boundless application prospect in the biological control of plant disease.
Description
Technical field
The invention belongs to technical field of microbiology, has growth-promoting functions to turfgrass more particularly to a kind of, and to snow
Mould leaf blight, coin pinta have bacillus subtilis and its application of resistant effect.
Background technology
Turfgrass is one " popular plants landscape " in the last few years, suitable for beautifying the environment, purifying air, keep water
Soil, be referred to as turf etc., American-European countries, Japan with being referred to as sesame, sesame grass.It is modern in the world that lawn area coverage is existing as weighing
One of important symbol of generationization urban construction, 1969 year's harvest found a state border lawn association.Sodded or grass-seed using artificial in gardens
The method of sowing, large-scale green ground is cultivated, is not only the important component of Scenery of gardens, and had a rest, entertain
Playground, represent a high-caliber ecological organism.
However, Turfgrass Diseases are the principal elements for hindering current Development of Turf, global grass cultivation is every year caused by disease
Several hundred million economic losses.It is by avenge mould leaf spoting bacteria, i.e. Microdochium nivale to avenge mould leaf blight, infects caused one
Kind lawn fungal disease, most commonly seen with tikka and the withered symptom of leaf, there is the circular withered grass spot that diameter is less than 5cm in lawn, expands
Diameter reaches 20cm afterwards, is survived the winter with seed, soil and invalid body, is propagated with the wind with rainwater, when more than 15 DEG C of average daily temperature, meets continuous
Rainy weather, the possible big happening and prevelence of disease, harm are very serious.Coin pinta is by coin pinta bacterium, i.e. Sclerotinia
Homoeocarpa, a kind of caused disease is infected, host is very extensive, the aggrieved blade of individual plant turfgrass, starts to produce water stain shape
Chlorisis spot, white scab is eventually become, can expand and extend to whole blade；Occur depression, circular withered grass spot on into level ground lawn,
Size assigns to 1 yuan of coin from 5, and pathogen on diseased plant and tides over poor environment on sick leaf surface, passes through wind, rainwater, instrument etc.
Propagate, the thermophilic of morbidity is 15-32 DEG C, from late spring until autumn disease can all occur.
In Turfgrass Diseases preventing and treating, such as golf course disease control, the strategy that people take is typically chemical pesticide,
Pathogen strengthens the resistance of chemical agent year by year, and dosing improves constantly.Long-term a large amount of uses of chemical agent, are caused certainly
The pollution of right ecological environment getting worse, has run counter to the original intention that turf plant is improved the ecological environment.Then, people turn to sight
The biological control of Diseases of Turfgrass, chemical bactericide is substituted using beneficial microbe existing for nature, be that a kind of protection lawn is exempted from
By the non-chemical environment protection method of disease, great potential, turn into a focus of current Diseases of Turfgrass study on prevention.
Microbe controlling plant diseases and promotion plant growth at home and abroad have been reported that the U.S. is existing 4 kinds so far
Biocontrol agent prepared by bacillus obtains production licence；Through retrieval, applicant is in existing patent and non-patent literature
In retrieve some document reports and patent on bacillus subtilis.For example, number of patent application is 2013100910680 public
Open " one plant of bacillus subtilis for being used to prevent and treat citrus ulcer ", it was recently reported that bacillus subtilis bacterial strain CCCQ080 is to citrus
Ulcer bacteria has very strong inhibitory action, there is good control effect to citrus bacterial canker disease.Number of patent application is
2013101243237 disclose " one plant of bacillus subtilis for being used to prevent and treat tobacco black shank ", it was recently reported that bacillus subtilis
Bacterial strain TBSCQ057 has very strong inhibitory action to tobacco black shank bacterium, there is good control effect to tobacco black shank, meanwhile,
The bacterial strain also has the antagonistic activity of wide spectrum, to tobacco ash arrhizus bacteria, tobacco brown spot pathogen, tobacco sclerotium rolfsii and tobacco anthrax
A variety of disease fungus fungistatic effects such as germ are preferable.Number of patent application is 2012104608407, discloses a kind of " disease prevention growth-promoting
Long plant endogenesis bacillus subtilis ", it was recently reported that bacillus subtilis Jaased3 to withered germ of water-melon, tomato early blight bacterium,
A variety of disease funguses such as Lettuce Drop bacterium, Verticillium Wilt Pathogen of Eggplant have antagonistic activity；It can enter under relatively low bacterial concentration
In the plant body such as watermelon, eggplant, and can be in crops Colonization inside plants and conduction；Have to crops and promote growth, controlling disease
Effect, especially there is good prevention effect to soil-borne vascular bundle disease.But at present not to avenging mould leaf blight, coin pinta
The report of bacillus subtilis with resistant effect.
The content of the invention
To improve the deficiency of existing Prevention Technique, it is an object of the invention to provide a kind of bacillus subtilis and its answer
With the bacillus 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 bacillus subtilis, and it was preserved in Wuhan University on May 15th, 2015
China typical culture collection center, preserving number are CCTCC M 2015300, are named as bacillus subtilis (Bacillus
subtilis)K-2。
Bacillus subtilis K-2 bacteria characteristic：
A, morphological feature：Bacterial strain K-2 is gram-positive bacteria, in rod-short, both ends blunt circle, gemma ellipse.Put down in LB
For the bacterium colony formed on plate into circle, edge is irregular, and for milky to slightly yellow, surface ruffle is coarse, opaque.
B, physiological and biochemical property：Hydrolyzable starch, contact enzyme positive.
Bacillus subtilis K-2 16S rDNA sequences are as shown in sequence table SEQ ID No.1, with having been reported on Genbank
Road sequence is compared, final to determine that bacterial strain K-2 is bacillus subtilis.
Second aspect of the present invention provides bacillus subtilis K-2 and is promoting Turfgrass Growth, and mould leaf blight, coin are avenged in preventing and treating
The application of pinta.
Third aspect present invention provides a kind of screening technique of bacillus subtilis, and its step includes：
(1) suspension is formulated as using soil as sample with sterilized water, is cultivated after applying flat board, choose single bacterium colony；
(2) choose to filter out in single bacterium colony from step (1) with flat board face-off method has to avenge mould leaf spoting bacteria and coin pinta bacterium
The bacterial strain of bacteriostasis.
Fourth aspect present invention provides a kind of bacillus subtilis K-2 microbial inoculums, and the microbial inoculum is bacillus subtilis K-2
The microbial inoculum obtained after activated, seed culture, fermentation.
The beneficial effects of the invention are as follows：The bacillus subtilis K-2 of the present invention has growth-promoting effect to turfgrass, to avenging mould leaf
Rot bacterium and coin pinta bacterium mycelial growth are inhibited, make germ mycelial growth slow, are expanded among appearance, and end becomes
Carefully, bend, the lopsided symptom such as collapse, so as to which to avenging mould leaf blight, coin pinta plays resistant effect, and with short production cycle, cost
It is cheap, using simple, there is boundless application prospect in the biological control of plant disease.
Brief description of the drawings
Fig. 1 is colonial morphologies of the bacillus subtilis K-2 in LB cultured on solid medium；
Fig. 2 is teratogenesis of the bacillus subtilis K-2 to avenge mould leaf spoting bacteria mycelial growth, wherein, A is without withered grass
The control group of bacillus K-2 processing, B are the experimental group handled through bacillus subtilis K-2；
Fig. 3 is teratogenesis of the bacillus subtilis K-2 to coin pinta bacterium mycelial growth, wherein, A is without withered grass gemma
The control group of bacillus K-2 processing, B are the experimental group handled through bacillus subtilis K-2；
Fig. 4 is bacteriostasis schematic diagrames of the bacillus subtilis K-2 to avenge mould leaf spoting bacteria；
Fig. 5 is bacteriostasis schematic diagrames of the bacillus subtilis K-2 to coin pinta bacterium.
Embodiment
First aspect present invention provides a kind of bacillus subtilis, and the bacterial strain was preserved in Wuhan on May 15th, 2015
University's China typical culture collection center, preserving number are CCTCC M 2015300, are named as bacillus subtilis K-2.
Second aspect of the present invention provides a kind of screening technique of bacillus subtilis, and its step includes：
(1) suspension is formulated as using soil as sample with sterilized water, is cultivated after applying flat board, choose single bacterium colony；
(2) choose to filter out in single bacterium colony from step (1) with flat board face-off method has to avenge mould leaf spoting bacteria and coin pinta bacterium
The bacterial strain of bacteriostasis.
Preferably, step (1) described soil, which adds, takes supernatant into sterilized water after shaken cultivation, diluted with sterilized water
Suspension, LB flat boards are coated on after suspension is diluted, 23-26h, picking single bacterium colony are cultivated at 28-30 DEG C；Step (2) is described
Flat board face-off method concretely comprises the following steps：Avenge mould leaf spoting bacteria and coin pinta bacterium are seeded in PDA plate center, by obtained by step (1)
Bacterium symmetrically accesses PDA plate in triangular shape, distance center 1-3cm, is cultivated at 28 DEG C, has obtained bacteriostasis bacterial strain, be transferred to
Cultivate and preserve on LB flat boards.
Third aspect present invention provides bacillus subtilis K-2 microbial inoculums, and the microbial inoculum is bacillus subtilis K-2 bacterial strains
The microbial inoculum obtained after activated, seed culture, fermentation.
Preferably, it is described to activate as bacillus subtilis K-2 inoculations in culture medium, are cultivated at 28-30 DEG C
24-36h。
More preferred, the culture medium is LB solid mediums, and its collocation method is yeast extract 5g, pancreas albumen
Peptone 10g, sodium chloride 10g, agar powder 20g, pH to 7.0-7.2, the autoclaving 20min at 121 DEG C are adjusted with NaOH.
Preferably, the seed culture is the bacillus subtilis K-2 inoculations after will be activated in culture medium, in
28-30 DEG C, shaken cultivation 23-25h under 155-165r/min.
Preferably, the fermentation is to connect the bacillus subtilis K-2 bacterial strains after seed culture with 5% inoculative proportion
Kind is in culture medium, the shaken cultivation 36-48h under 28-30 DEG C, 155-165r/min.
Described bacillus subtilis K-2 microbial inoculums are diluted to active constituent content 1.0 × 106-1.0×108Make after cfu/mL
With.
A kind of bacillus subtilis provided by the invention and its application are further described below in conjunction with embodiment.
Embodiment 1
Turfgrass avenge mould leaf spoting bacteria described in the present embodiment and turfgrass coin pinta bacterium are taught by Canadian Guelph universities Tom
Offer is awarded, the turfgrass avenge mould leaf spoting bacteria and turfgrass coin pinta bacterium obtained by other means is also applied for the present invention.
First, bacillus subtilis K-2 acquisition and identification
Separation：Rich soil is gathered from Hubei Jingzhou City periphery vegetable garden, takes back laboratory preservation, it is standby.Weigh soil-like
Product 10g, add in 90mL aqua sterilisas, 160r/min shaken cultivation 10min, stand 30min, take supernatant 1mL, be added to and fill
In the test tube of 9mL sterilized waters, vibration mixes, and sample suspension is made.Take 1mL sample suspensions to add and fill 9mL sterilized waters
10 times are carried out successively in test tube to be serially diluted, and take 10 respectively-5、10-6、10-7The μ L of dilution 50 be coated on LB flat boards, be placed in 28
DEG C culture 24h.According to the features such as form, size, color and luster, 197 plants of picking single bacterium colony, transfers and is cultivated on LB flat boards altogether, preserves
It is standby, bacillus subtilis K-2 LB cultured on solid medium colonial morphology as shown in figure 1, bacillus subtilis K-2 exists
For the bacterium colony formed on LB flat boards into circle, edge is irregular, and for milky to slightly yellow, surface ruffle is coarse, opaque.
Screening：Cultured turfgrass avenge mould leaf spoting bacteria and turfgrass coin pinta bacterium are inoculated in PDA culture medium respectively
Plate center, the above-mentioned bacterium isolated and purified is symmetrically accessed into PDA plate in triangular shape with the toothpick of sterilizing, distance center 2cm,
28 DEG C of culture observation opposite culture antibacterial situations.The obvious bacterial strain K-2 of bacteriostasis is transferred on LB flat boards and cultivated, is preserved standby
With.Bacillus subtilis K-2 is observed to turfgrass avenge mould leaf spoting bacteria and turfgrass coin pinta bacterium mycelial growth by microscopy
Inhibitory action, respectively as shown in Figure 2 and Figure 3, the mycelial growth of bacterial strain K-2 processing is slow as seen from the figure for exercising result, middle swollen
Greatly, end attenuates, bending, the lopsided symptom such as collapse, and compares that mycelial growth is normal, and form is uniform.The PDA culture medium is matched somebody with somebody
Method processed is：Potato 200g, glucose 20g, agar 18g, distilled water are supplied to 1000mL, 121 DEG C of moist heat sterilization 20min.
Identification：Physiology and biochemistry identification is carried out to the bacterial strain K-2 of screening, is defined as bacillus, then extracts bacterial strain
Total genomic dna is its 16S rDNA sequence of template amplification.Forward and reverse primer used is respectively 27F：5’-
AGAGTTTGATCCTGGCTCAG-3 ', i.e. sequence table SEQ ID No.2；1429R：5 '-GGTTACCTTGTTACGACTT-3 ',
That is sequence table SEQ ID No.3.PCR reaction systems are 15 μ L systems：The μ L of DNA profiling 0.6；H2O 8.57μL；buffer 1.65
μL；Mg2+2.75μL；dNTP 0.66μL；27F：0.33μL；1429R 0.33μL；Tsg 0.11μL.PCR response procedures are：94
℃3min；94 DEG C of 30s, 50 DEG C of 45s, 72 DEG C of 100s, 35 circulations；72℃7min.Simultaneously purified pcr product is reclaimed, is sequenced, its
Bacterial strain K-2 is accredited as bacillus subtilis by 16S rDNA sequences as shown in sequence table SEQ ID No.1, according to sequencing result.
Further, bacillus subtilis K-2 has been carried out to turfgrass avenge mould leaf spoting bacteria, coin pinta bacterium antagonism
Test.
The most obvious bacterial strain K-2 of the bacteriostasis filtered out is accessed in LB fluid nutrient mediums, the 130r/min at 28 DEG C
Shaken cultivation 26h, is made bacteria suspension.Avenge mould leaf spoting bacteria, coin pinta bacterium are respectively connected at different PDA plate centers, uses liquid relief
K-2 bacteria suspensions are symmetrically accessed PDA plate by device in triangular shape, every access 10 μ L, distance center about 2cm, using sterilized water as pair
According to opposite culture antibacterial situations are observed in 28 DEG C of cultures, colony radius are measured after 4d, bacillus subtilis K-2 is to avenging mould leaf blight
The antibacterial situation of bacterium is shown in that accompanying drawing 4, the antibacterial situation to coin pinta bacterium are shown in accompanying drawing 5.
The relative bacteriostasis rate grown using below equation calculating bacterial strain K-2 to disease fungus, is often handled in triplicate.
Relative inhibition=(control colony radius-processing colony radius)/control colony radius × 100%
Bacillus subtilis K-2 is respectively provided with obvious antagonism to avenge mould leaf spoting bacteria, coin pinta bacterium, the results are shown in Table 1,
Its relative inhibition is respectively 80.0%, 81.6%.
Table 1：Antagonisms of the bacillus subtilis K-2 to several Different Kinds of Pathogens fungies
2nd, the preparation of bacillus subtilis K-2 microbial inoculums
The present embodiment completes the preparation of bacillus subtilis K-2 microbial inoculums according to following key step：
(1) bacillus subtilis K-2 is inoculated in LB solid mediums, 25h, activated strains is cultivated at 28 DEG C；LB consolidates
Body culture medium is prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, agar powder 20g, NaOH adjust pH to 7.0,121
DEG C, 20min autoclavings.
(2) bacterial strain by step (1) activation is accessed in LB fluid nutrient mediums, the 160r/min shaken cultivations 24h at 28 DEG C,
Seed liquor is made；LB fluid nutrient mediums are prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, NaOH adjust pH extremely
7.0,121 DEG C, 20min autoclavings.
(3) the seed fermentation liquid for being obtained step (2) 5% is inoculated in LB liquid fermentation mediums by volume, in 28
160r/min shaken cultivations 36h at DEG C.
Bacillus subtilis K-2 microbial inoculums are can be prepared by through as above step.
3rd, applications of the bacillus subtilis K-2 in turfgrass creeping bentgrass Penncross growths are promoted
It is highly 13.5cm that sterilized soil, which is loaded, and in a diameter of 3.5cm conical pipe, from mouth of pipe 1cm, sprinkle aqua sterilisa is extremely
The mouth of pipe, in water saturates pipe subject to sterilization after soil, turfgrass creeping bentgrass Penncross seeds are uniformly sprinkled into pipe, often managed
0.04g, five repetitions.Often 5mL aqua sterilisas are added dropwise in pipe daily, until seed is sprouted after showing money or valuables one carries unintentionally is added dropwise a water every other day, often manage
5mL.Dark 8h is kept after per illumination 16h.After one week, the bacillus subtilis K-2 microbial inoculums prepared in embodiment 2 are diluted to
1.0×108Cfu/mL, OD600=0.86-0.88, pouring root are inoculated with, often pipe 5mL, using LB fluid nutrient mediums as blank control.Point
5d, 10d, 15d, 20d measure height, the DGCI values of turfgrass not after processing, and turfgrass is trimmed to horizontal, the survey with the mouth of pipe
Its dry weight.
As a result it is as shown in table 2, the results showed that the 5d-20d after bacillus subtilis K-2 processing, Jian's stock of being crawled to turfgrass
Clever Penncross can show significant growth-promoting functions, and turfgrass height, DGCI values and the dry weight handled through K-2 is significantly high
In blank control.
Table 2：Growth-promoting effect tables of the bacillus subtilis K-2 to turfgrass creeping bentgrass Penncross
Data are 5 repetition average values in table 2, are counted according to the software analysis of SAS 9.1, the different alphabets with after column data
Show notable in 5% level difference.DGCI is the dark green colour index of plant, can reflect plant growth health degree, DGCI values and lawn
Careless health degree is into positive correlation.
As seen from the data in Table 2, the height of 5d after inoculation, processing and control turfgrass is respectively 7.1cm, 5.2cm, is increased
26.7%, DGCI value are respectively 0.699,0.584；Dry weight is respectively 144.2mg, 46.9mg, increases by 67.5%；10d after inoculation,
The height of processing and control turfgrass is respectively 7.2cm, 5.4cm, and 25.0%, DGCI of increase values are respectively 0.650,0.581, are done
It is respectively 133.4mg, 45.2mg again, increases by 66.1%；15d after inoculation, is handled and the height of control turfgrass is respectively
5.9cm, 4.2cm, 28.8%, DGCI of increase values are respectively 0.555,0.497, and dry weight is respectively 108.8mg, 19.7mg, is increased
For 81.9%；The height of 20d after inoculation, processing and control turfgrass is respectively 6.2cm, 4.2cm, increases by 32.3%, DGCI values
Respectively 0.528,0.460, dry weight is respectively 186.1mg, 50.3mg, increases by 73.0%.
4th, applications of the bacillus subtilis K-2 in turfgrass creeping bentgrass A4 growths are promoted
It is highly 13.5cm that sterilized soil, which is loaded, and in a diameter of 3.5cm conical pipe, from mouth of pipe 1cm, sprinkle aqua sterilisa is extremely
The mouth of pipe, in water saturates pipe subject to sterilization after soil, turfgrass creeping bentgrass A4 seeds are uniformly sprinkled into pipe, often pipe 0.04g, five
Secondary repetition.Often 5mL aqua sterilisas are added dropwise in pipe daily, a water are added dropwise every other day after seed is sprouted and showed money or valuables one carries unintentionally, often pipe 5mL.Per illumination
Dark 8h is kept after 16h.After one week, the bacillus subtilis K-2 microbial inoculums of preparation are diluted to 1.0 × 108Cfu/mL, OD600
=0.86-0.88, pouring root are inoculated with, often pipe 5mL, using LB fluid nutrient mediums as blank control.After processing 5d, 10d, 15d,
20d measures height, the DGCI values of turfgrass, and turfgrass is trimmed to horizontal with the mouth of pipe.
It the results are shown in Table 3, the results showed that the 10d-20d after being handled through bacillus subtilis K-2, to turfgrass creeping bentgrass
A4 can show significant growth-promoting functions, and the turfgrass height and DGCI values handled through K-2 is significantly higher than blank control.
Table 3：Growth-promoting effect tables of the bacillus subtilis K-2 to turfgrass creeping bentgrass A4
Data are 5 repetition average values in table 3, are counted according to the software analysis of SAS 9.1, the different alphabets with after column data
Show notable in 5% level difference.DGCI is the dark green colour index of plant, can reflect plant growth health degree, DGCI values and lawn
Careless health degree is into positive correlation.
As seen from the data in Table 3, the height of 5d after inoculation, processing and control turfgrass is respectively 5.62cm, 4.74cm, is increased
15.7%, DGCI value are respectively 0.753,0.734；10d after inoculation, processing and control turfgrass height be respectively 5.16cm,
3.63cm, 29.7%, DGCI of increase value are respectively 0.598,0.573；The height point of 15d after inoculation, processing and control turfgrass
Not Wei 6.61cm, 4.76cm, increase by 28.0%, DGCI values respectively 0.602,0.515；20d after inoculation, processing and control lawn
The height of grass is respectively 5.74cm, 4.21cm, and 26.7%, DGCI of increase values are respectively 0.611,0.521.
5th, bacillus subtilis K-2 avenges the application in mould leaf blight in preventing and treating turfgrass
Turfgrass avenges the preparation of the wheat inoculum of mould leaf blight：Wheat is cleaned, soaked overnight, load 250mL triangles
In bottle, every bottle of about 50g, sterilizing.The avenge mould leaf spoting bacteria cultivated on PDA plate one week is accessed in triangular flask, is placed in 25 DEG C of trainings
Support, after mycelia covers with wheat culture medium, pour out natural air drying, crush, be fitted into aseptic plastic bag, it is standby.
It is highly 7.0cm that sterilized soil, which is loaded, in a diameter of 2.5cm vial, every bottle plus 20g, adds 1mL sterilizings
Water, in water saturates pipe subject to sterilization after soil, turfgrass creeping bentgrass A4 seeds are uniformly sprinkled into pipe, often pipe 0.015g, five
Secondary repetition.Dark 8h is kept after per illumination 16h.After one week, the bacillus subtilis K-2 microbial inoculums of preparation are diluted to 1.0 ×
108Cfu/mL, OD600=0.86-0.88, pouring root inoculation, every bottle of 1mL, using LB fluid nutrient mediums as blank control.After one week
Afterwards, above-mentioned turfgrass is avenged into mould leaf blight wheat inoculum to be uniformly 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 often pipe.
It the results are shown in Table 4, the results showed that in the 5-20d after bacillus subtilis K-2 processing, avenging mould leaf blight to turfgrass is
Significant preventive and therapeutic effect can be shown, the turfgrass yellowing leaf rate handled through K-2 bacterial strains will substantially be less than blank control.
Table 4：Bacillus subtilis K-2 avenges the prevention effect table of mould leaf blight to turfgrass
Data are 5 repetition average values in table 4, are counted according to the software analysis of SAS 9.1, the different alphabets with after column data
Show notable in 5% level difference.
The 5d after the data of table 4, inoculation, processing and control turfgrass yellowing leaf rate be respectively 8.0%,
30.0%, reduce 22.0%；The yellowing leaf rate of 10d after inoculation, processing and control turfgrass is respectively 12.5%, 65.0%,
Reduce 52.5%；The yellowing leaf rate of 15d after inoculation, processing and control turfgrass is respectively 15.0%, 72.5%, is reduced
57.5%；The yellowing leaf rate of 20d after inoculation, processing and control turfgrass is respectively 30.0%, 85.0%, reduces 55.0%.
6th, applications of the bacillus subtilis K-2 in preventing and treating turfgrass coin pinta
Turfgrass avenges the preparation of the wheat inoculum of mould leaf blight：Wheat is cleaned, soaked overnight, load 250mL triangles
In bottle, every bottle of about 50g, sterilizing.The turfgrass coin pinta bacterium cultivated on PDA plate one week is accessed in triangular flask, is placed in 25 DEG C
Culture, after mycelia covers with wheat culture medium, pours out natural air drying, crushes, is fitted into aseptic plastic bag, standby.
It is 13.5cm that sterilized soil is loaded into height, a diameter of 3.5cm, in volume about 180mL conical pipe, from mouth of pipe 1cm,
Sprinkle aqua sterilisa in water saturates pipe subject to sterilization after soil, turfgrass creeping bentgrass A4 seeds is uniformly sprinkled into pipe to the mouth of pipe,
Often pipe 0.04g, five repetitions.Often pipe sprinkling 5mL water daily, until seed is sprouted after showing money or valuables one carries unintentionally sprays a water every other day, often manage
5mL.Dark 8h is kept after per illumination 16h, after one week, the bacillus subtilis K-2 microbial inoculums prepared in embodiment 2 are diluted to
1.0×108Cfu/mL, OD600=0.86-0.88, pouring root are inoculated with, often pipe 5mL, using LB fluid nutrient mediums as blank control.Again
After one week, above-mentioned coin pinta bacterium wheat inoculum is uniformly seeded on turfgrass, often pipe 0.05g, 5d starts after inoculation
Incidence is observed, records the yellowing leaf rate of turfgrass in often pipe.
It the results are shown in Table 5, the results showed that, can table to turfgrass coin pinta in the 5-20d after bacillus subtilis K-2 processing
Reveal significant preventive and therapeutic effect, turfgrass yellowing leaf rate and blank control significant difference.
Table 5：Prevention effect tables of the bacillus subtilis K-2 to turfgrass coin pinta
Data are 5 repetition average values in table 5, are counted according to the software analysis of SAS 9.1, the different alphabets with after column data
Show notable in 5% level difference.
After the data of table 5, K-2 inoculations 5d, the turfgrass yellowing leaf rate of processing and control is respectively
16.5%th, 55.0%, it is reduced to 70.0%；After being inoculated with 10d, the turfgrass yellowing leaf rate of processing and control is respectively
17.5%th, 65.0%, reduce 73.1%；After being inoculated with 15d, the turfgrass yellowing leaf rate of processing and control is respectively 23.3%,
74.8%, reduce 68.9%；After being inoculated with 20d, the turfgrass yellowing leaf rate of processing and control is respectively 30.0%, 88.4%,
Reduce 66.1%.It will thus be seen that there is good preventive and therapeutic effect in 20d to coin pinta using K-2 bacterial strains processing turfgrass,
Turfgrass yellowing leaf rate and blank control significant difference.
Embodiment 2
Embodiment 2 is substantially the same manner as Example 1, and difference is：
The present embodiment completes the preparation of bacillus subtilis K-2 microbial inoculums according to following key step：
(1) bacillus subtilis K-2 is inoculated in LB solid mediums, 30h, activated strains is cultivated at 30 DEG C；LB consolidates
Body culture medium is prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, agar powder 20g, NaOH adjust pH to 7.2,121
DEG C, 20min autoclavings.
(2) bacterial strain by step (1) activation is accessed in LB fluid nutrient mediums, the 160r/min shaken cultivations 24h at 28 DEG C,
Seed liquor is made；LB fluid nutrient mediums are prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, NaOH adjust pH extremely
7.2,121 DEG C, 20min autoclavings.
(3) the seed fermentation liquid for being obtained step (2) 5% is inoculated in LB liquid fermentation mediums by volume, in 28
160r/min shaken cultivations 40h at DEG C.
Bacillus subtilis K-2 microbial inoculums are made by above step, gained bacillus subtilis K-2 microbial inoculums have been diluted to
Effective component content 1.0 × 107Used 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 bacillus subtilis K-2 microbial inoculums according to following key step：
(1) bacillus subtilis K-2 is inoculated in LB solid mediums, 35h, activated strains is cultivated at 30 DEG C；LB consolidates
Body culture medium is prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, agar powder 20g, NaOH adjust pH to 7.0,121
DEG C, 20min autoclavings.
(2) bacterial strain by step (1) activation is accessed in LB fluid nutrient mediums, the 160r/min shaken cultivations 24h at 28 DEG C,
Seed liquor is made；LB fluid nutrient mediums are prepared：Yeast extract 5g, tryptone 10g, sodium chloride 10g, NaOH adjust pH extremely
7.0,121 DEG C, 20min autoclavings.
(3) the seed fermentation liquid for being obtained step (2) 5% is inoculated in LB liquid fermentation mediums by volume, in 28
160r/min shaken cultivations 46h at DEG C.
Bacillus subtilis K-2 microbial inoculums are made by above step, gained bacillus subtilis K-2 microbial inoculums have been diluted to
Effective component content 1.0 × 107Used after cfu/mL.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (8)
- A kind of 1. bacillus subtilis, it is characterised in that：The deposit number of the bacillus subtilis is CCTCC M 2015300, be named as bacillus subtilis (Bacillus subtilis)K-2。
- 2. bacillus subtilis as claimed in claim 1, it is characterised in that：The 16S rDNA sequences of the bacillus subtilis As shown in SEQ ID No.1.
- 3. the bacillus subtilis of claim 1 or 2 is in promoting Turfgrass Growth, preventing and treating to avenge mould leaf blight, coin pinta Using.
- A kind of 4. bacillus subtilis K-2 microbial inoculums, it is characterised in that：The bacillus subtilis microbial agent is bacillus subtilis K- 2 is activated, seed culture, the microbial inoculum obtained after fermentation, and the deposit number of the bacillus subtilis K-2 is CCTCC M 2015300。
- 5. bacillus subtilis K-2 microbial inoculums as claimed in claim 4, it is characterised in that：The activation is by bacillus subtilis Bacterium K-2 is inoculated in culture medium, and 24-36h is cultivated at 28-30 DEG C.
- 6. bacillus subtilis K-2 microbial inoculums as claimed in claim 4, it is characterised in that：The seed culture is will be activated Bacillus subtilis K-2 afterwards is inoculated in culture medium, the shaken cultivation 23-25h under 28-30 DEG C, 155-165r/min.
- 7. bacillus subtilis K-2 microbial inoculums as claimed in claim 4, it is characterised in that：The fermentation is will be through seed culture Bacillus subtilis K-2 afterwards is inoculated in culture medium with 5% inoculative proportion, is vibrated under 28-30 DEG C, 155-165r/min Cultivate 36-48h.
- 8. the bacillus subtilis K-2 microbial inoculums as described in any one of claim 4 to 7 claim, it is characterised in that：Described Bacillus subtilis K-2 microbial inoculums are diluted to active constituent content 1.0 × 106-1.0×108Used after cfu/mL.
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| Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RBI4;Asaka O 等;《Appl. Environ. Microbiol.》;19961231;第62卷(第11期);4081-4085 * |
| 枯草芽孢杆菌菌剂的开发应用;王星云 等;《广西热带农业》;20071231(第2期);32-35 * |
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