CN105018386A - Bacillus methylotrophicus and application thereof - Google Patents
Bacillus methylotrophicus and application thereof Download PDFInfo
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- CN105018386A CN105018386A CN201510462747.3A CN201510462747A CN105018386A CN 105018386 A CN105018386 A CN 105018386A CN 201510462747 A CN201510462747 A CN 201510462747A CN 105018386 A CN105018386 A CN 105018386A
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Abstract
The invention provides bacillus methylotrophicus with a collection number of CCTCC M2015297. Bacillus methylotrophicus is named bacillus methylotrophicus XK-4. The invention further provides a screening method of bacillus methylotrophicus XK-4, an application of bacillus methylotrophicus XK-4 to inhibition of sclerotinia sclerotiorum, rhizoctonia solani, fusarium solani and fusarium oxysporum and a bacillus methylotrophicus XK-4 agent. Bacillus methylotrophicus XK-4 is a rape rhizosphere microorganism, is safe to human, livestock and crops, is environment-friendly, has a wider antibacterial spectrum, has better inhibitory effects on mycelial growth of common pathogenic bacteria, such as sclerotinia sclerotiorum, rhizoctonia solani, fusarium solani and fusarium oxysporum, has obvious inhibitory effects on mycelial growth of sclerotinia sclerotiorum and causes mycelia deformation. The further prepared agent has obvious control effects on sclerotinia rot of colza, is short in production cycle and has a very extensive application prospect in biological control of plant diseases.
Description
Technical field
The invention belongs to technical field of plant disease biological control, particularly relate to a kind of Methylotrophic genus bacillus and application thereof.
Background technology
Rape is one of important oil crops of China, and its cultivated area and output all occupy the first in the world.By sclerotinite, i.e. Sclerotinia sclerotiorum, infecting the sclerotinia rot of colza caused is a kind of important disease, occupies first of the large disease of China's rape three, and in five continents, nearly more than 100 countries have and to occur and popular.In recent years, along with the popularization of rape no-tillage Direct Hole Sowing, the generation of sclerotinia rot of colza is in ascendant trend year by year, and annual sickness rate in area, the Yangtze valley rises to 15-30%.The generation of sclerotinia rot of colza can have a strong impact on the yield and quality of rape, causes great financial loss, and general time sickness rate is between 10%-40%, and the serious time can up to 80%, the production of serious threat China rape.
At present, the still aphylactic rape variety of the control about sclerotinia rot of colza, although cultural control, as, crop rotation, reduction field humidity, can reduce the generation of this disease to a certain extent, but be usually subject to the restriction of geographical configuration or cropping pattern, be difficult to spread.Although chemical prevention can effectively suppress cause of disease mycelial growth and spore germination rapidly, use chemical agent in a large number for a long time, not only easily make pathogenic bacteria develop immunity to drugs, and cause the Pesticide Residue of ecological environmental pollution and Semen Brassicae campestris.Biological control is to the mankind and environmental friendliness, and be prevent and treat the ideal a kind of prevention and controls of sclerotinia rot of colza, the parent being subject to researcher in recent years gradually looks at.Therefore, the antagonistic microbe finding control sclerotinia rot of colza is significant.
Utilize biocontrol strain controlling plant diseases all to have report at home and abroad, 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 Methylotrophic genus bacillus and patent in existing patent and non-patent literature.Such as, number of patent application is 2014106017754 to disclose " Methylotrophic genus bacillus and the application in control Fusarium graminearum thereof ", utilizes Methylotrophic genus bacillus to prevent and treat Fusarium graminearum and infects the disease caused.Number of patent application is 2014104032209 to disclose " a kind of Methylotrophic genus bacillus (Bacillus methylotrophicus) three torches-04 and application thereof ", utilize Methylotrophic genus bacillus three torch-04 to prevent and treat pepper ralstonia solanacearum, citrus anthracnose, all there is comparatively stable prevention effect.Number of patent application is that 201210498378X discloses " acquisition of a strain ginseng diseases biocontrol strains and application ", utilize the zymocyte liquid of Methylotrophic genus bacillus NJ13 and filtrate thereof to prevent and treat common 7 kinds of diseases such as ginseng gray mold, black spot, all there is good prevention effect.Number of patent application is 2012102472347 to disclose " a kind of Methylotrophic Bacillus strain 4-L-16 of preventing and controlling banana fusarium wilt and application thereof ", utilizes Methylotrophic genus bacillus 4-L-16 to prepare the microbiobacterial agent of preventing and controlling banana fusarium wilt.But Sclerotinia sclerotiorum is not had to the report of the Methylotrophic genus bacillus of inhibition at present.
Summary of the invention
For improving the deficiency of existing Prevention Technique, the object of the present invention is to provide a kind of Methylotrophic genus bacillus and application thereof, this Methylotrophic genus bacillus has significant antagonistic action to multiple pathogenic bacterias such as Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon.
First aspect present invention provides a kind of Methylotrophic genus bacillus, it is preserved in Wuhan University's China typical culture collection center on May 15th, 2015, preserving number is CCTCC M 2015297, called after Methylotrophic genus bacillus (Bacillus methylotrophicus) XK-4.
The bacteria characteristic of Methylotrophic genus bacillus XK-4:
A, morphological specificity: XK-4 is gram-positive microorganism, and in shaft-like, gemma is oval, and the bacterium colony that LB flat board is formed is circular, oyster white, surface irregularity, edge indentation, and protuberance is moistening, glossy.
B, physiological and biochemical property: hydrolyzed starch, peritrichous, oxidase negative, catalase is positive.
The 16S rDNA sequence of Methylotrophic genus bacillus XK-4 detected and Genbank has reported sequence is compared, finally determining that strain X K-4 is Methylotrophic genus bacillus.
Second aspect present invention provides Methylotrophic genus bacillus XK-4 and is suppressing the application in Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon.
Third aspect present invention provides the screening method of Methylotrophic genus bacillus XK-4, and concrete steps comprise:
(1) be formulated as suspension with sterilized water after being cultivated by the sclerotium of sclerotinia rot of colza, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) from single bacterium colony that step (1) is chosen, bacterial strain Sclerotinia sclerotiorum being had to bacteriostatic action is filtered out by dull and stereotyped face-off method.
Fourth aspect present invention provides a kind of Methylotrophic genus bacillus XK-4 microbial inoculum, and described microbial inoculum is that Methylotrophic genus bacillus XK-4 is activated, seed culture, the microbial inoculum that obtains after fermentation.
The invention has the beneficial effects as follows:
(1) Methylotrophic genus bacillus XK-4 provided by the invention is a strain rape rhizospheric microorganism, to people, animal, Crop securify, environmentally friendly.
(2) Methylotrophic genus bacillus XK-4 provided by the invention has wider antimicrobial spectrum, to common pathogenic bacteria, as Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Rhizoctonia solani Kuhn (Rhizoctoniasolani), withered germ of water-melon (Fusarium oxysporum), walnut pine root fungus (Fusarium solani) etc., all there is good inhibition, inhibiting rate reaches 83.8% respectively, 78.6%, 79.2%, 73.9%, to Sclerotinia sclerotiorum mycelial growth, there is obvious restraining effect, cause myceliolysis, fracture, the deformities such as vegetative point expands.
(3) microbial inoculum that Methylotrophic genus bacillus XK-4 provided by the invention is prepared into has significant prevention effect to sclerotinia rot of colza, and with short production cycle, cost is low, uses simple, has very wide application prospect in the biological control of Plant diseases.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of Methylotrophic genus bacillus XK-4 at LB cultured on solid medium;
Fig. 2 is the antagonistic action schematic diagram of Methylotrophic genus bacillus XK-4 to Sclerotinia sclerotiorum;
Fig. 3 is the antagonistic action schematic diagram of Methylotrophic genus bacillus XK-4 to Rhizoctonia solani Kuhn;
Fig. 4 is the antagonistic action schematic diagram of Methylotrophic genus bacillus XK-4 to withered germ of water-melon;
Fig. 5 is the antagonistic action schematic diagram of Methylotrophic genus bacillus XK-4 to walnut pine root fungus;
Fig. 6 is the teratogenesis schematic diagram of Methylotrophic genus bacillus XK-4 to Sclerotinia sclerotiorum mycelial growth, and wherein, A is the control group without Methylotrophic genus bacillus XK-4 process, and B is the experimental group through Methylotrophic genus bacillus XK-4 process.
Embodiment
First aspect present invention provides a kind of Methylotrophic genus bacillus, it is preserved in Wuhan University's China typical culture collection center on May 15th, 2015, preserving number is CCTCC M 2015297, called after Methylotrophic genus bacillus (Bacillus methylotrophicus) XK-4.
Second aspect present invention provides Methylotrophic genus bacillus XK-4 and is suppressing the application in Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon.
Third aspect present invention provides the screening method of Methylotrophic genus bacillus XK-4, and concrete steps comprise:
(1) be formulated as suspension with sterilized water after being cultivated by the sclerotium of sclerotinia rot of colza, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) from single bacterium colony that step (1) is chosen, bacterial strain Sclerotinia sclerotiorum being had to bacteriostatic action is filtered out by dull and stereotyped face-off method.
Preferably, the described sclerotium of step (1) cultivate for after sterilizing at 28-30 DEG C shaking culture 40-50h, add after sterilized water is formulated as suspension and coat LB flat board, at 28-30 DEG C, cultivate 23-26h, picking list bacterium colony; The described dull and stereotyped face-off method concrete steps of step (2) are: Sclerotinia sclerotiorum is seeded in PDA plate center, be that triangular shape symmetry access PDA is dull and stereotyped by step (1) gained list bacterium colony, distance center 1-3cm, cultivate at 26 DEG C, obtain bacteriostatic action bacterial strain, proceeded on LB flat board and cultivate preservation.
Fourth aspect present invention provides a kind of Methylotrophic genus bacillus XK-4 microbial inoculum, and described microbial inoculum is that Methylotrophic genus bacillus XK-4 is activated, seed culture, the microbial inoculum that obtains after fermentation.
Preferably, described activation, for be inoculated in substratum by Methylotrophic genus bacillus XK-4, cultivates 20-24h at 28-30 DEG C.
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 be inoculated in substratum by the Methylotrophic genus bacillus XK-4 after activated, in 28-30 DEG C, shaking culture 20-24h under 155-165r/min.
Preferably, described fermentation be by the Methylotrophic genus bacillus XK-4 after seed culture with 5% inoculative proportion be inoculated in substratum, in 28-30 DEG C, shaking culture 40-44h under 135-145r/min.
Be more preferably, described substratum is LB liquid nutrient medium, and its collocation method is yeast extract 5g, tryptone 10g, sodium-chlor 10g, adjusts pH to 7.0-7.2, autoclaving 20min at 121 DEG C with NaOH.
Preferably, described Methylotrophic genus bacillus XK-4 microbial inoculum is diluted to active constituent content 1.0 × 10
6-1.0 × 10
8use after cfu/mL, for suppressing Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon.
Below in conjunction with embodiment, a kind of Methylotrophic genus bacillus provided by the invention and application thereof are further described.
Embodiment 1
Described in the present embodiment, Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, withered germ of water-melon, walnut pine root fungus are provided by plant pathology research department of agricultural college of Changjiang University, and the Sclerotinia sclerotiorum that alternate manner obtains, Rhizoctonia solani Kuhn, withered germ of water-melon, walnut pine root fungus are also applicable to the present invention.
One, the separation of Methylotrophic genus bacillus XK-4, selection systems
Be separated: from Hubei Jingzhou City periphery rape field, gather the vigorous rapeseed plants of growing way, get the rhizosphere soil sticking to root, be placed in 250mL triangular flask.The sclerotinia rot of colza sclerotium that a upper season of growth collects is loaded 5cm
2size, mesh is in the nylon pouch of 1.5mm2,10 every bag, then Nylon Bag is imbedded above-mentioned filling in the triangular flask of rhizosphere soil.After three weeks, take out the sclerotium in Nylon Bag, be placed in the culture dish of sterilizing, with 75% alcohol-pickled 1min, then soak 30s with 0.1% mercuric chloride, then rinsed with sterile water 4 times, be placed in LB liquid nutrient medium 28-30 DEG C, 160r/min shaking culture 2d, get the bacterium liquid 1mL cultivating gained, 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, altogether picking list bacterium colony 135 strain, transfers and to cultivate on LB flat board, save backup.
The bacterial strain of above-mentioned separation and purification is that triangular shape is symmetrical with the label of sterilizing by screening: Sclerotinia sclerotiorum is placed in PDA culture medium flat plate central authorities accesses PDA flat board, and distance center is about 2cm, cultivates for 26 DEG C and observes the antibacterial situation of opposite culture.Obvious for bacteriostatic action strain X K-4 is proceeded on LB flat board and cultivates, save backup.Methylotrophic genus bacillus XK-4 is shown in accompanying drawing 1 at the colonial morphology of LB cultured on solid medium, and the bacterium colony that Methylotrophic genus bacillus XK-4 is formed on LB flat board is circular, oyster white, surface irregularity, edge indentation, protuberance, moistening, glossy.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 customary physiological biochemical identification to the strain X K-4 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.1; 1429R:5 '-GGTTACCTTGTTACGACTT-3 ', i.e. sequence table SEQ ID No.2.PCR reaction system is 15 μ L reaction systems: DNA profiling 0.6 μ L; H
2o 8.57 μ L; Buffer 1.65 μ L; Mg
2+2.75 μ L; DNTP 0.66 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, is accredited as Methylotrophic genus bacillus according to sequencing result by strain X K-4.
Two, Methylotrophic genus bacillus XK-4 is to the antagonistic action of Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon
Accessed in LB liquid nutrient medium by the most obvious strain X K-4 of bacteriostatic action filtered out, at 28 DEG C, 140r/min shaking culture 36h, makes bacteria suspension.Sclerotinia sclerotiorum is accessed respectively at different PDA culture medium flat plate center, Rhizoctonia solani Kuhn, withered germ of water-melon, the pathogenic fungies such as walnut pine root fungus, being that triangular shape is symmetrical with pipettor by XK-4 bacteria suspension accesses PDA flat board, often some access 10 μ L, distance center is about 2cm, take sterilized water as contrast, cultivate for 26 DEG C and observe the antibacterial situation of opposite culture, colony radius is measured after 4d, the antibacterial situation of Methylotrophic genus bacillus XK-4 to Sclerotinia sclerotiorum is shown in accompanying drawing 2, accompanying drawing 3 is shown in the antibacterial situation of Rhizoctonia solani Kuhn, accompanying drawing 4 is shown in the antibacterial situation of withered germ of water-melon, accompanying drawing 5 is shown in the antibacterial situation of walnut pine root fungus.
The relative bacteriostasis rate adopting following formulae discovery strain X K-4 to grow pathogenic fungi, often processes in triplicate.
Relative inhibition=(contrast colony radius-process colony radius)/contrast colony radius × 100%
Methylotrophic genus bacillus XK-4 all has obvious antagonistic action to several important soil-borne disease fungal pathogenses such as Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, withered germ of water-melon, walnut pine root fungus, the results are shown in Table 1, its relative inhibition is respectively 83.8%, 78.6%, 79.2%, 73.9%.Observe Methylotrophic genus bacillus XK-4 to the restraining effect of Sclerotinia sclerotiorum mycelial growth by microscopy, the results are shown in accompanying drawing 6, find that dissolving appears in the mycelia of strain X K-4 process, fracture, the deformities such as vegetative point expands, and it is normal to contrast mycelial growth, form is even.
Table 1: Methylotrophic genus bacillus XK-4 is to the antagonistic action of several Different Kinds of Pathogens fungi
Three, the preparation of bacillus amyloliquefaciens XK-2 microbial inoculum
The present embodiment completes the preparation of Methylotrophic genus bacillus XK-4 microbial inoculum according to following key step:
(1) described Methylotrophic genus bacillus XK-4 is inoculated in LB solid medium, at 28 DEG C, cultivates 20h, 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 20h, 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, 140r/min shaking culture 40h at 28 DEG C.
By above step i.e. obtained Methylotrophic genus bacillus XK-4 microbial inoculum.
Four, the application of Methylotrophic genus bacillus XK-4 in control sclerotinia rot of colza
(1) prepared Methylotrophic genus bacillus XK-4 microbial inoculum 0.25%Tween20 is diluted to 1.0 × 10
8cfu/mL, OD
600=0.82-0.84, for the in vitro control experiment of sclerotinia rot of colza.
Gather the blade that rape 4-5 leaf phase size is close, leaf position is consistent, with aqua sterilisa cleaning, dry.Arrange 3 process: 1. blade is soaked in 30s in the XK-4 microbial inoculum diluent of above-mentioned preparation, dries, by the hyphal cluster germ inoculated by hypha block of PDA flat board activation in rape leaf front, every blade connects 2 bacterium cakes; 2. blade is soaked in 30s in sterilized water, dries, at front access Sclerotinia sclerotiorum bacterium cake; 3. blade is soaked in 30s in the XK-4 microbial inoculum diluent of above-mentioned preparation, dries.Often process repetition 3 times, blade is placed in 25 DEG C of crispers, and blade base moistening cotton balls parcel, in case dehydration.After 4d, measure the diameter of each process scab.According to the relative prevention effect of formulae discovery.
Relative prevention effect=[(contrast lesion diameter-process lesion diameter)/contrast lesion diameter] × 100%
The in vitro controlling experiment of Methylotrophic genus bacillus XK-4 to sclerotinia rot of colza the results are shown in Table 2, and only the leaf spot lesion of inoculation Sclerotinia sclerotiorum is 2.93cm; Only the blade of inoculation XK-4 microbial inoculum is without scab, shows that it is safe to rapeseed plants; The leaf spot lesion diameter of Simultaneous vaccination sclerotium bacteria and microbial inoculum XK-4 is 0.57cm, and relative prevention effect is 80.5%.
Table 2: Methylotrophic genus bacillus XK-4 is to the in vitro prevention effect of sclerotinia rot of colza
(2) Methylotrophic genus bacillus XK-4 prevents and treats the pot experiment of sclerotinia rot of colza
By prepared XK-4 microbial inoculum preservative fluid, i.e. 0.25%Tween20, is made into diluent 1.0 × 10
8cfu/mL, OD
600=0.82-0.84, for the potted plant controlling experiment of sclerotinia rot of colza.By the sclerotinite inoculated by hypha block of PDA flat board activation in the 250mL triangular flask that 100mL PDB nutrient solution is housed, every bottle graft kind 3-4 block, 25 DEG C, 140r/min cultivates 72h, then pours fermented liquid into tissue mashing machine and makes hyphal suspension, for subsequent use.
Through the Semen Brassicae campestris of warm water soaking, in two No. 10, be seeded in seedling pan, the 4-5 leaf phase to be grown to, for subsequent use.Choose healthy rapeseed plants to transplant, the strain of every basin 5.Arrange following three process: 1. spray inoculation XK-4 microbial inoculum diluent, every strain is about 5mL; 2. spray inoculation sclerotinite hyphal suspension, every strain is about 5mL; 3. first spray inoculation XK-4 microbial inoculum diluent, every strain is about 5mL, spray inoculation sclerotinite hyphal suspension after 48h, and every strain is about 5mL; 4. clear water of spraying is blank.All be disposed after sleeving plastic bag moisturizing 48h, add up sickness rate after 5d, with reference to severity Scaling criterion calculation disease index and prevention effect.
Sclerotinia rot of colza grade scale:
0 grade: anosis;
1 grade: slightly fall ill, onset area accounts for less than 10% of blade table area;
2 grades: slightly fall ill, onset area accounts for the 11%-30% of blade table area;
3 grades: medium morbidity, onset area accounts for the 31%-50% of blade table area;
4 grades: highly fall ill, onset area accounts for the 50%-70% of blade table area;
5 grades: highly fall ill, onset area accounts for more than 70% of blade table area;
Disease index=[∑ (the sick number of sheets at different levels × corresponding sick progression)/(investigating total number of sheets × highest numerical value)] × 100
Relative prevention effect=[(contrast disease index-process contrast disease index)/contrast disease index] × 100%
The indoor pot controlling experiment of Methylotrophic genus bacillus XK-4 to sclerotinia rot of colza the results are shown in Table 3, the sickness rate of contrast is 81.4, and disease index is the rape sickness rate of 66.0, XK-4 microbial inoculum process is 23.1, disease index is 12.0, and its relative prevention effect reaches 81.8%.
Table 3: Methylotrophic genus bacillus XK-4 is to the indoor control effect of sclerotinia rot of colza
(3) Methylotrophic genus bacillus XK-4 prevents and treats the field experiment of sclerotinia rot of colza
By prepared XK-4 microbial inoculum preservative fluid, i.e. 0.25%Tween20, is made into diluent 1.0 × 10
8cfu/mL, OD
600=0.82-0.84, for the daejeon prevention test of sclerotinia rot of colza.
Test arrangement in rape continuous cropping field, the long-term sclerotium disease sickness rate in test site at 30-50%, and along with the continuous cropping time limit increase sickness rate in rising trend.Rape variety in being two No. 10, this kind is the rape variety of open popularization.Rapeseed Field arranges community, 30m
2/ community, sprays above-mentioned XK-4 microbial inoculum diluent in the rape early flowering season, until there is one deck liquid film on rape leaf surface, every strain rape is about sprayed 25mL, arranges clear water control treatment, often processes 3 communities and repeats, each community stochastic distribution.The incidence of 20d " Invest, Then Investigate " sclerotinia rot of colza, takes 5 samplings, often some investigation 20 strains, and record total strain number, diseased plant number and the sick level of every strain, sick level is with reference to following standard, and disease index and relatively prevention effect calculate with described in embodiment 5.
Sclerotinia rot of colza grade scale:
0 grade: anosis;
1 grade: slightly fall ill, onset area accounts for less than 5% of stem surface-area;
2 grades: slightly fall ill, onset area accounts for the 6%-15% of stem surface-area;
3 grades: medium morbidity, onset area accounts for the 16%-30% of stem surface-area;
4 grades: highly fall ill, onset area accounts for the 31%-50% of stem surface-area;
5 grades: highly fall ill, onset area accounts for more than 50% of stem surface-area;
The rape disease index that the daejeon prevention test of Methylotrophic genus bacillus XK-4 to sclerotinia rot of colza the results are shown in Table the process of 4, XK-4 microbial inoculum is 17.1, and the disease index of contrast is 67.9, and its relative prevention effect reaches 74.8%.
Table 4: Methylotrophic genus bacillus XK-4 is to the field control effect of sclerotinia rot of colza
Embodiment 2
Embodiment 2 is substantially the same manner as Example 1, and difference is:
The present embodiment completes the preparation of Methylotrophic genus bacillus XK-4 microbial inoculum according to following key step:
(1) described Methylotrophic genus bacillus XK-4 is inoculated in LB solid medium, at 30 DEG C, cultivates 22h, activated strains; LB solid medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, agar powder 20g, NaOH adjust pH to 7.1,121 DEG C, 20min autoclaving.
(2), in bacterial strain access LB liquid nutrient medium step (1) activated, at 30 DEG C, 155r/min shaking culture 22h, makes seed liquor; LB liquid nutrient medium is prepared: yeast extract 5g, tryptone 10g, sodium-chlor 10g, NaOH adjust pH to 7.1,121 DEG C, 20min autoclaving.
(3) seed fermentation liquid step (2) obtained 5% is inoculated in LB liquid fermentation medium by volume, 140r/min shaking culture 42h at 30 DEG C.
By above step i.e. obtained Methylotrophic genus bacillus XK-4 microbial inoculum, gained Methylotrophic genus bacillus XK-4 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 Methylotrophic genus bacillus XK-4 microbial inoculum according to following key step:
(1) described Methylotrophic genus bacillus XK-4 is inoculated in LB solid medium, at 30 DEG C, cultivates 24h, 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 30 DEG C, 165r/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, 140r/min shaking culture 44h at 30 DEG C.
By above step i.e. obtained Methylotrophic genus bacillus XK-4 microbial inoculum, gained Methylotrophic genus bacillus XK-4 microbial inoculum is diluted to active constituent content 1.0 × 10
6use 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 (9)
1. a Methylotrophic genus bacillus, is characterized in that: its preserving number is CCTCC M 2015297, called after Methylotrophic genus bacillus (Bacillus methylotrophicus) XK-4.
2. Methylotrophic genus bacillus described in claim 1 is suppressing the application in Sclerotinia sclerotiorum, Rhizoctonia solani Kuhn, walnut pine root fungus, withered germ of water-melon.
3. a screening method for Methylotrophic genus bacillus as claimed in claim 1, its step comprises:
(1) be formulated as suspension with sterilized water after being cultivated by the sclerotium of sclerotinia rot of colza, be coated with dull and stereotyped rear cultivation, choose single bacterium colony;
(2) from single bacterium colony that step (1) is chosen, bacterial strain Sclerotinia sclerotiorum being had to bacteriostatic action is filtered out by dull and stereotyped face-off method.
4. the screening method of Methylotrophic genus bacillus as claimed in claim 3, it is characterized in that: the described sclerotium of step (1) cultivate for after sterilizing at 28-30 DEG C shaking culture 40-50h, add after sterilized water is formulated as suspension and coat LB flat board, 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: rape nuclear disk germ silk block is seeded in PDA plate center, be that triangular shape symmetry access PDA is dull and stereotyped by step (1) gained list bacterium colony, distance center 1-3cm, cultivate at 26 DEG C, obtain bacteriostatic action bacterial strain, proceeded on LB flat board and cultivate preservation.
5. a Methylotrophic genus bacillus XK-4 microbial inoculum, is characterized in that: described microbial inoculum is that Methylotrophic genus bacillus XK-4 is activated, seed culture, the microbial inoculum that obtains after fermentation.
6. Methylotrophic genus bacillus XK-4 microbial inoculum as claimed in claim 5, is characterized in that: described activation, for be inoculated in substratum by Methylotrophic genus bacillus XK-4, cultivates 20-24h at 28-30 DEG C.
7. Methylotrophic genus bacillus XK-4 microbial inoculum as claimed in claim 5, it is characterized in that: described seed culture is be inoculated in substratum by the Methylotrophic genus bacillus XK-4 after activated, in 28-30 DEG C, shaking culture 20-24h under 155-165r/min.
8. Methylotrophic genus bacillus XK-4 microbial inoculum as claimed in claim 5, it is characterized in that: described fermentation be by the Methylotrophic genus bacillus XK-4 after seed culture with 5% inoculative proportion be inoculated in substratum, in 28-30 DEG C, shaking culture 40-44h under 135-145r/min.
9. the Methylotrophic genus bacillus XK-4 microbial inoculum as described in any one of claim 5 to 8 claim, is characterized in that: described Methylotrophic genus bacillus XK-4 microbial inoculum is diluted to active constituent content 1.0 × 10
6-1.0 × 10
8use after cfu/mL.
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| CN105670973A (en) * | 2016-03-11 | 2016-06-15 | 山东京青农业科技有限公司 | Bacillus methylotrophicus, bactericide and preparation method and application of bactericide |
| CN107189964A (en) * | 2017-07-06 | 2017-09-22 | 山西大学 | The Bacillus strain and its fermentation medium of antagonism Root rot disease of Astragallus |
| CN107760631A (en) * | 2017-11-24 | 2018-03-06 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its granule and application |
| CN107955616A (en) * | 2017-10-31 | 2018-04-24 | 浦江县合洪园艺研发有限公司 | A kind of preparation method suitable for harmful microorganism soil conditioner |
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| CN113122479A (en) * | 2021-04-28 | 2021-07-16 | 广东省科学院生物工程研究所 | New application of biocontrol bacillus |
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| CN105543132B (en) * | 2015-12-31 | 2019-01-18 | 河南省农业科学院植物保护研究所 | A kind of Methylotrophic bacillus YB-F7 and its application in controlling plant diseases |
| CN105543132A (en) * | 2015-12-31 | 2016-05-04 | 河南省农业科学院植物保护研究所 | Bacillus methylotrophicus YB-F7 and application thereof in preventing plant diseases |
| CN105670973A (en) * | 2016-03-11 | 2016-06-15 | 山东京青农业科技有限公司 | Bacillus methylotrophicus, bactericide and preparation method and application of bactericide |
| CN105670973B (en) * | 2016-03-11 | 2019-04-30 | 山东京青农业科技有限公司 | A kind of application of Methylotrophic bacillus, microbial inoculum, the preparation method of microbial inoculum and microbial inoculum |
| CN107189964B (en) * | 2017-07-06 | 2020-08-04 | 山西大学 | Bacillus strain for antagonizing astragalus root rot and fermentation culture medium thereof |
| CN107189964A (en) * | 2017-07-06 | 2017-09-22 | 山西大学 | The Bacillus strain and its fermentation medium of antagonism Root rot disease of Astragallus |
| CN107955616A (en) * | 2017-10-31 | 2018-04-24 | 浦江县合洪园艺研发有限公司 | A kind of preparation method suitable for harmful microorganism soil conditioner |
| CN108085269A (en) * | 2017-11-23 | 2018-05-29 | 中国农业科学院麻类研究所 | Methylotrophic Bacillus strain IBFCBF-2 and its application |
| CN107760631A (en) * | 2017-11-24 | 2018-03-06 | 四川农业大学 | A kind of Methylotrophic bacillus B18 and its granule and application |
| CN107760631B (en) * | 2017-11-24 | 2021-04-09 | 四川农业大学 | Bacillus methylotrophicus B18, and granules and application thereof |
| CN111269838A (en) * | 2020-04-30 | 2020-06-12 | 福建省南平市农业科学研究所 | Method for inducing and separating antagonistic bacteria in soil by barley grains infected with gibberellic disease |
| CN112442468A (en) * | 2020-12-16 | 2021-03-05 | 四川农业大学 | Bacillus methylotrophicus and application thereof |
| CN112442468B (en) * | 2020-12-16 | 2022-09-06 | 四川农业大学 | Bacillus methylotrophicus and application thereof |
| CN113122479A (en) * | 2021-04-28 | 2021-07-16 | 广东省科学院生物工程研究所 | New application of biocontrol bacillus |
| CN113122479B (en) * | 2021-04-28 | 2023-02-14 | 广东省科学院生物工程研究所 | New application of biocontrol bacillus |
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