CN101603009A - One strain prevents and treats the mould ZS-1SB of biocontrol microorganisms shield shell and the preparation method and the application of sclerotium disease - Google Patents
One strain prevents and treats the mould ZS-1SB of biocontrol microorganisms shield shell and the preparation method and the application of sclerotium disease Download PDFInfo
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Abstract
The invention belongs to the microbial pesticide technical field, be specifically related to a strain to the sclerotinite sclerotium have strong parasitism, cause rotten active shield shell trichoderma strain branch screening, identify and application in microbiobacterial agent.Described shield shell mould (Coniothyrium minitans) ZS-1SB bacterial strain is deposited in Chinese typical culture collection center, and deposit number is: CCTCC NO:M209114.The invention also discloses the semi-open-type solid fermentation cultural method and the application of shield shell mould (Coniothyriumminitans) ZS-1SB microbial inoculum in the control sclerotinia rot of colza of described microbiobacterial agent.Microbiobacterial agent of the present invention has good biocontrol effect to fungal diseases of plants such as sclerotinia rot of colzas.
Description
Technical field
The present invention relates to the microbial pesticide technical field, be specifically related to cultural method and application that a strain has the shield shell trichoderma strain and the semi-open-type solid fermentation of sclerotium disease biological and ecological methods to prevent plant disease, pests, and erosion potential.
Background technology
Sclerotinite [Sclerotinia sclerotiorum (Lib.) de Bary] can be infected 64 sections, 540 various plants, comprise important vegetable crops such as oil crops that rape, soybean, Sunflower Receptacle etc. are main and lettuce, Chinese cabbage, celery, Radix Dauci Sativae, worldwide cause the sclerotium disease of various plants.Because not having up to now to find does not have disease-resistant variety to utilize to its plantation resource with obvious disease resistance, remains a global difficult problem in the agriculture production by its sclerotium disease that causes.At present adopt chemical pesticides to control more, spray the harm that chemical pesticide " dimetachlone " or " derosal " can be alleviated this disease to a certain extent as the Sheng phase of blooming in rape, obtain comparatively ideal effect.Because sclerotium disease mostly occurs, is popular in middle and later periods of growth of rape, needs specific agricultural chemicals dosage and labour intensity and just can make agricultural chemicals arrive at the position that is injured (stem of rape and branch); And another problem that can not be ignored is the resistance of sclerotinite, and long-term big area uses the agricultural chemicals of single character to have high risk.As far back as 1997, just there is the scholar to find that anti-" derosal " bacterial strain frequency of occurrences of some field piece of area, Tongzhou, Jiangsu Province is up to 100%.What need simultaneously to pay close attention to is food safety and problem of environmental pollution, and food-safety problem is the chip in the international agriculture trade barrier often, take part in international competition, and also must implement environment protective plant protecting.Therefore, pesticide control should be very prudent, need set up the prevention and control technical system of secure persistent in the production in a hurry.Utilizing useful fungi control fungal diseases of plants is the important component part of the prevention and control technical system of secure persistent.
Mould (Coniothyrium minitans) imperfect stage of shield shell belongs to Coelomycetes, is the superparasitism fungi of plant pathogenic fungi sclerotinite, also is the important biocontrol fungi of sclerotium disease, can parasitism and the mycelia and the sclerotium that destroy sclerotinite.The shield shell is mould to be described and name by American scholar Campbell (1947) first, and Whipps and Gerlagh (1992) have given argumentation to its biological characteristics and diseases prevention potential.The mould conidium of shield shell can be added soil to or be sprayed sclerotium disease (Jones et al, 2003 that are used for controlling various crop in crop field top; Li et al, 2006).The applicant finds that in early-stage Study the more mould bacterial strains of shield shell can form pycnidium, produce a large amount of conidiums (Cheng et al, 2003), field experiment demonstration for many years shows to have good protection effect, and the shield shell is mould not to have pathogenecity to plant, and people and animals are not had toxic action.
Similar with other biocontrol fungi, the mould development and application of shield shell is subjected to the restriction of large-scale production, and the mould conidium of High-efficient Production shield shell is the key of commercial applications at present.
Summary of the invention
Purpose screening one of the present invention is selected good strains in the field for seed to select sclerotinite (Sclerotinia sclerotinia) sclerotium disease that causes is had the fungal bacterial strain of remarkable biological control effect; and carry out large-scale production by semi-open-type solid fermentation cultural method easy, easy row, it is developed as the microbiobacterial agent of the biological control of various crop sclerotium disease.
The present invention separates in the growing vegetables vegetable soil of Zhushan County, Hubei Province, screen and obtain a strain has extremely strong parasitization to the sclerotinite sclerotium shield shell trichoderma strain ZS-1SB, the applicant is with its called after shield shell mould (Coniothyriumminitans) ZS-1SB, this bacterial strain has delivered on May 31st, 2009 that the international biological material specimens preservation mechanism that is positioned at Wuhan City, Hubei Province Wuhan University that is used for patented procedure--Chinese typical culture collection center (the English CCTCC of abbreviation), its deposit number is CCTCC NO:M209114.
Embodiment studies show that, the mould ZS-1SB bacterial strain of shield shell of the present invention's screening has stronger antagonistic action to plant pathogenic fungi sclerotinite and plant pathogenetic bacteria rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae), can be used as the microbiobacterial agent that sclerotium disease is had the biological and ecological methods to prevent plant disease, pests, and erosion function.
The mycology feature of the mould ZS-1SB of shield shell:
Conidium chocolate, elliposoidal, air spots, size are 3.5-4.7 μ m * 5.3-8.8 μ m (average 3.9 * 6.4 μ m), get children's's tender (white point) the visible broad conidiogenous cell of in a row arranging of base portion of pycnidium compressing tablet.On the PDA flat board, the mycelia initial stage is colourless, and aerial hyphae is undeveloped, on one side mycelia expands, Yi Bian bacterium colony surface forms white pycnidium, with the prolongation of incubation time, bacterium colony is browning gradually, and the spore device black water droplet that spues promptly overflows conidium.The mould mycelia of shield shell all can grow in 10-28 ℃ of scope, and optimum growth temperature is 20 ℃, and top temperature is 30 ℃, and the shield shell is mould all can grow in the environment of pH2-11, and pH3-4 is most appropriate to its growth.Bacterium colony day expansion 0.7cm on the PDA of pH4 flat board.
The invention still further relates to the large-scaled culture method that the semi-open-type solid fermentation of producing the mould ZS-1SB microbial inoculum of described shield shell is provided.It is material that this method adopts dried rolled oats and rice bran, is fermenting container with the food steamer, has realized the mould large-scale production of shield shell, and its technology is simple and easy to do.
The present invention has following characteristics:
1, obviously antagonism rice leaf spot bacteria, parasitic sclerotinite sclerotium of the shield shell trichoderma strain ZS-1SB of the present invention screening has potential widely exploitation value;
2, the above-mentioned shield shell trichoderma strain of employing can the effectively preventing sclerotinia rot of colza.The rape early flowering season uses once (10
10Spore/mu), the early flowering season uses once (10
10Spore/mu), prevention effect is higher than derosal far away;
3, adopt the cultural method of the mould semi-open-type solid fermentation of above-mentioned shield shell, in the culture material prescription, rolled oats and rice bran material cheapness, be easy to get, solid culture medium is loose, and permeability is good, can make sporulation quantity reach 1.5 * 10
10The dried culture material of spore/g;
4, adopt the cultural method of the mould semi-open-type solid fermentation of above-mentioned shield shell, equipment is simple, only need the culturing room of food steamer, stove and energy temperature control to meet the needs of production, need not specific equipments such as required autoclaving equipment of other solid fermentation methods and solid-state reactor.Therefore greatly reduce production cost, be suitable for the mould large-scale production of shield shell.
5, adopt the cultural method of the mould semi-open-type solid fermentation of above-mentioned shield shell,, can well control the pollution of common assorted bacterium such as rhizopus (Rhizopus sp.), aspergillus tubigensis (Aspergillus sp.) etc. by the measure of 20 ℃ and 16 ℃ temperature-variable fermentations.
Description of drawings
Fig. 1: show the antagonistic action of the mould ZS-1SB bacterial strain of shield shell of the present invention's screening to rice leaf spot bacteria.
Fig. 2: the mould ZS-1SB microbial inoculum of the shield shell semi-open-type solid fermentation situation that is the embodiment of the invention.Fig. 2 A is that the semi-open-type solid culture mode that the present invention designs is illustrated; Fig. 2 B and Fig. 2 C cultivate the final culture that contains the mould ZS-1SB microbial inoculum of shield shell that forms.
Fig. 3: be that the mould ZS-1SB bacterial strain of shield shell is parasitic and cause rotten sclerotinite sclerotium situation.
Embodiment
In the following embodiments, the applicant is in the indoor antagonistic action of shield shell trichoderma strain ZS-1SB to rice leaf spot bacteria of having verified, to this bacterial strain carry out fermentation culture, culture parasitism, the activity etc. that causes rotten sclerotinite sclerotium carries out the system verification test, simultaneously the microbiobacterial agent of preparation is carried out the field controling test of sclerotinia rot of colza, to determine its control action kou to sclerotium disease;
Embodiment 1
1, the separation screening of the mould ZS-1SB bacterial strain of shield shell
Separate and evaluation: from the truck garden soil of Zhushan County, Chinese Hubei Province, separate, screen and obtain a strain has extremely strong parasitization to the sclerotinite sclerotium shield shell trichoderma strain, the applicant is with its called after shield shell mould (Coniothyrium minitans) ZS-1SB, test shows that the isolating ZS-1SB bacterial strain of the present invention has stronger antagonistic action to mycelial growth and the paddy rice main pathogen bacterium (Xanthomonas oryzae pv.oryzae) of sclerotinite (S.sclerotium), can be used as the candidate strain of microbiobacterial agent of the control of sclerotinia rot of colza.
The mould ZS-1SB of shield shell of the present invention separates and sclerotinite sclerotium mass trapping (Adames etc., 1980) and dilution plate method are adopted in screening, and strain identification work is with reference to Wei Jingchao, " fungi identification handbook " Shanghai science tech publishing house, the method for 1979 editions introductions.
2, bacterial strain preservation: the preservation of shield shell trichoderma strain ZS-1SB bacterial strain of the present invention, press the Zhou Deqing chief editor, " microbiology experimental technique handbook, Shanghai science tech publishing house, the method operation of 1986 editions introductions.
3, the preparation of test and control strain:
Present embodiment is a test strain with the mould ZS-1SB bacterial strain of shield shell of the applicant's screening, rice leaf spot bacteria 086 (Cheng Guoying etc. with report, rice leaf spot bacteria monoclonal virulence differentiation research. Plant Pathology, 2002,32:227-234) in contrast.The activation condition of the mould ZS-1SB of shield shell for examination is: activation culture 2d on 20 ℃ of PDA substratum (prescription: potato 200g, glucose 20g, agar 10g replenishes distilled water to 1000ml, transfers pH to 7.0, at 121 ℃ of high pressure steam sterilization 30min) flat board.
The activation condition of rice leaf spot bacteria bacterial strain 086 is: under 28 ℃ rice leaf spot bacteria 086 is seeded to solid potato culture medium and (fills a prescription: potato 300g, Ca (NO
3)
24H
2O 0.5g, Na
2HPO
412H
2O 2.0g, peptone 5.0g, sucrose 15.0g, 17% agar adds water to 1000ml, transfer pH to 6.8-7.0, at 121 ℃ of high pressure steam sterilization 30min, down together) streak culture 2d, picking rice leaf spot bacteria 086 single bacterium colony, be seeded to rice leaf spot bacteria liquid nutrient medium (prescription: potato 300g, Ca (NO
3)
24H
2O 0.5g, Na
2HPO
412H
2O2.0g, peptone 5.0g, sucrose 15.0g adds water to 1000ml, transfers pH to 6.8-7.0, at 121 ℃ of high pressure steam sterilization 30min) in, in 28 ℃, 200r/min shaking culture 36h makes and measures OD
600Value to 2.0.
4, experimental technique:
Get above-mentioned rice leaf spot bacteria 086 bacterium liquid 1.3ml and (make OD
600=2.0) and rice leaf spot bacteria solid medium 60ml (composition is the same) mixing, pour into rapidly in the culture dish of diameter 150mm, get the mould ZS-1SB mycelia of shield shell piece (diameter 5mm) and place culture dish central authorities, observe the antagonistic activity of the mould ZS-1SB of shield shell behind the cultivation 3d down rice leaf spot bacteria in 28 ℃.
Experimental result shows, shows that the mould ZS-1SB of shield shell of the present invention has tangible antagonistic action to rice leaf spot bacteria 086 (see figure 1).
Embodiment 2: the semi-open-type solid fermentation of the mould ZS-1SB microbial inoculum of shield shell is cultivated
1, the mould ZS-1SB spore suspension preparation of shield shell: the mould ZS-1SB bacterial strain of shield shell is placed on PDA substratum (the prescription ginseng together) inclined-plane, under 20 ℃ of conditions, cultivate 7d.Use the aseptic water washing inclined-plane, transferring to conidial suspension is 10
6Spore/ml.
2, the numerous cultivation of the expansion of the mould ZS-1SB of shield shell: is to be inoculated in PDB substratum (prescription: potato 200g at 1: 100 with the conidial suspension of the mould ZS-1SB of shield shell of step 1 with mass ratio, glucose 20g, replenish distilled water to 1000ml, transfer pH to 7.0), described PDB substratum branch is packed in the triangular flask (every bottled 100ml/250ml), at 150r/min, shaking culture 24h under 20 ℃ of conditions, obtain the mould ZS-1SB bacterium of shield shell liquid, standby.
3, the mould ZS-1SB microbial inoculum of shield shell semi-open-type solid fermentation is cultivated: is that 3: 1 mixings add after an amount of water logging rises with dried culture material (water content generally is no more than 8%) rolled oats (being purchased) and rice bran (being purchased) with volume ratio, the internal diameter of packing into is 49mm, highly is the family expenses food steamer of 20.5mm, after adding a cover, very hot oven atmospheric steam sterilization 2h.After the cooling, add sterile purified water and regulate the final water content to 70% of described dried culture material.In culturing room, the mould ZS-1SB bacterium of the described shield shell of step 2 liquid is inoculated in culture material with the ratio that the dried culture material of per kilogram adds 100ml, be tiled in food steamer, the thickness that makes culture material is 5mm, add a cover, place 20 ℃ of culturing room to cultivate 48h, half open wide steamer cover (seeing Fig. 2 A) then, under 16 ℃ of conditions, continue to cultivate 7d.The mould ZS-1SB microbial inoculum of the shield shell of present embodiment contains spore count and can reach 1.5 * 10 after testing
10The dried culture material of spore/g (seeing Fig. 2 B and Fig. 2).
Embodiment 3: the mould ZS-1SB bacterial strain of shield shell parasitism, cause the activity test of rotten sclerotinite sclerotium
1, sclerotinite sclerotium and preparation: with sclerotinite bacterial strain (Sclerotinia sclerotium) Ep-1PNA367 (the Huiquan Liu of report, Yanping Fu, Daohong Jiang, Guoqing Li, Jun Xie, Youliang Peng, Xianhong Yi, and Said A.Ghabrial.A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses.JOURNAL OF VIROLOGY, Feb.2009,1981-1991) in contrast, sclerotinite bacterial strain Ep-1PNA367 is inoculated in the Radix Dauci Sativae substratum (Radix Dauci Sativae is cut into strip, 121 ℃ of sterilization 30min) in the triangular flask, cultivate 60d for 20 ℃ and obtain sophisticated sclerotium.
2, the preparation of the mould ZS-1SB of shield shell: it is an amount of that the fermentation culture of getting ZS-1SB among the embodiment 2 contains the culture of microbial inoculum final period, wash by rubbing with the hands repeatedly after adding tap water, wash out the mould conidium liquid of shield shell, at the centrifugal 5min of 3000r/min, abandon supernatant, with the resuspended conidium of tap water precipitation, and the conidium concentration that is adjusted to the mould ZS-1SB of shield shell is 10
7/ ml.
3, test method: get the river sand that sieves, after tap water cleans, the 2h that sterilizes under 121 ℃ of following high pressure steam in the triangular flask packs into, pour in the culture dish of sterilization, add an amount of sterilized water to husky moist, get the pure culture sclerotinite sclerotium of big or small uniformity then respectively, behind 70% ethanol disinfection, aseptic water washing 3 times is at the conidium liquid (10 of the mould ZS-1SB of shield shell
7/ soak 30min in ml), then this sclerotium is taken out half and immerse in the culture dish that the sterilization fine sand is housed, put 20 in sclerotium in every ware sand table, establish 5 repetitions, cultivate down at 20 ℃.The mould ZS-1SB of record shield shell causes rotten degree to the parasitism of this sclerotinite sclerotium.The rotten grading of sclerotinite sclerotium is as follows: 0 grade, and the mould mycelia of the sclerotium no shield shell in surface; 1 grade, there is the mould mycelia of shield shell on the sclerotium surface, but does not have the pycnidium original hase of white; 2 grades, there is the pycnidium original hase on the sclerotium surface, but the top layer without putrefaction; 3 grades, rot in the sclerotium top layer, inner variable color deliquescing; 4 grades, whole sclerotium erosion.
4, test-results: 100 sclerotinite sclerotium all rot after testing, and rotten degree evaluation is 4 grades, can obviously see the black pycnidium (see figure 3) of the mould ZS-1SB of shield shell on septic sclerotinite sclerotium.
Embodiment 4: the field test of utilizing the mould ZS-1SB microbial inoculum control of shield shell sclerotinia rot of colza
In order to verify the protection effect of the mould ZS-1SB microbial inoculum of shield shell that the present invention produces, the applicant selects the part rape field of Yichang City, Hubei Province, Duchang County, Jiangxi Province and Pengze County, Jiangxi Province to carry out the field controling test of sclerotinia rot of colza.
1, test medicine:
The mould ZS-1SB microbial inoculum of shield shell: it is an amount of to get among the embodiment 2 the final culture that contains the mould ZS-1SB microbial inoculum of shield shell of fermentation, washes by rubbing with the hands repeatedly after adding tap water, and the conidium liquid that washes out at the centrifugal 5min of 3000r/min, is abandoned supernatant.Easy to use in order to produce, the mould ZS-1SB microbial inoculum of shield shell is made spore throw out (10
9/ g), every 10g is a packing bag, vacuum seal is preserved.
The contrast medicament is 40% derosal suspension agent (available from the commodity of plant protection limited liability company of Tianjin Chinese nation).
2, test design: test is established 3 Da Qu altogether and is handled, and handles and does not establish repetition; Totally 51 mu of demonstration areas (every mu is 666 square metres, down together).
Test design is as follows:
Handle 1:40% derosal suspension agent in rape initial bloom stage dispenser 1 time, every mu of usefulness 40% derosal suspension agent 200g converts clear water 30kg spraying, 1 mu of processing area.
Handle 2: the mould ZS-1SB microbial inoculum of shield shell is in rape early flowering season dispenser 1 time, and every mu with 1 bag (10 of the mould ZS-1SB microbial inoculum of shield shell
9Spore/g, 10g altogether) convert clear water 30kg spraying, test area is 20 mu.
Handle 3: the mould ZS-1SB microbial inoculum of shield shell is in rape early flowering season dispenser 1 time, the dispenser 1 time again of rape initial bloom stage, each every mu with 1 bag in mould ZS-1SB preparation of shield shell (10g), convert clear water 30kg spraying, test area is 20 mu.
Contrast: only spray clear water, use clear water 30kg for every mu, spraying, test area is 10 mu.
3, the condition of test site:
(1) experimental field click Bai He village, Yiling District Ya Queling town, Yichang City, Hubei Province, preceding crop is a semilate rice, and soil property is a silty loam, middle fertility;
(2) 5-linked village, lotus pier town, Pengze County, Jiangxi Province, cotton oil crop rotation district, the field sclerotinia rot of colza takes place heavier throughout the year, and fertility is higher, and rape is a culturing and transplanting seedlings before the winter, and irrigation conditions is better;
(3) Duchang County, Jiangxi Province Wang Dun township, cotton oil crop rotation district, the field sclerotinia rot of colza takes place heavier throughout the year, and fertility is higher, and rape is a culturing and transplanting seedlings before the winter, and irrigation conditions is better.
4, Shi Yan rape variety: (this kind is bred by Hua Zhong Agriculture University for local main good quality and high output (two low type) the cabbage type rape hybrid new variety of promoting " assorted No. 6 of China's oil " for formation testing vegetable kind, by Hubei Province's crop varietal approval committee, an open new rape variety of promoting), grow seedlings according to ordinary method and transplant.
5, investigation sampling method: the investigation of the sclerotium disease state of an illness is carried out before rape gathered in the crops for 1 week, and 5 sampling methods of checkerboard type are adopted in every field, investigates 20 strain rapes at every, puts down in writing the stem morbidity disease index and the relative control effect of every processing.
Disease index=∑ [sick progression * this grade diseased plant number]/(investigation sum * the highest sick progression) * 100
Relative control effect (%)=(the contrast sclerotium disease disease index-anti-sclerotium disease disease index of processing)/contrast sclerotium disease disease index
6, interpretation of result:
The mould ZS-1SB microbial inoculum of table 1 shield shell of the present invention is to the prevention effect of sclerotinia rot of colza
Illustrate: the difference that two province's prevention effect occur is the difference of the spontaneous generation degree of field, various places sclerotinia rot of colza
The mould ZS-1SB microbial inoculum of shield shell of the present invention is prevented and treated test-results for each 50 mu on Hubei Province and 3 ground, Jiangxi Province and is shown that the mould ZS-1SB microbial inoculum of shield shell can effectively be controlled the harm of field sclerotinia rot of colza.In rape early flowering season dispenser once (10
10Spore/mu)), prevention effect and chemical pesticide " 40% derosal suspension agent " are suitable, in rape early flowering season dispenser once (10
10Spore/mu), early flowering season dispenser once (10 again
10Spore/mu), its prevention effect is significantly higher than 40% derosal suspension agent.
The above-mentioned rape early flowering season is meant that the rape stem of about 5-10% blooms; The rape initial bloom stage is meant rape after the early flowering season 6 days.
Field test results shows that it is that a strain has the fungal bacterial strain (microbial inoculum) that good biological and ecological methods to prevent plant disease, pests, and erosion is worth that the present invention prepares the mould ZS-1SB microbial inoculum of shield shell, this microbial inoculum has the better prevention effect to the sclerotium disease of sclerotinia rot of colza or other crops, shows that it has the excellent development application prospect.
The main reference document
1.Campbell?W?A.A?new?species?of?Coniothyrium?minitans?parasitic?on?sclerotia.Mycologia，1947，39：190-195
2.Cheng?J?S，Jiang?D?H，Yi?X?H，Fu?Y?P，Li?G?Q，Whipps?J?M.production，survival?and?efficacyof?Coniothyriumminitans?conidia?produced?in?shaken?liquid?culture.FEMS?Microbiology?Letters.2003，127-131
3.Li，G.Q.，Huang，H.C.，Miao，H.J.，Erickson，R.S.，Jiang，D.H.，Xiao，Y.N.，2006.Biologicalcontrol?of?sclerotinia?diseases?of?rapeseed?by?aerial?applications?of?the?mycoparasiteConiothyrium?minitans.Eur.J.Plant?Pathol.114，345-355.
4.Jones，E.E.，Stewart，A.，Whipps，J.M.，2003.Use?of?Coniothyrium?minitans?transformed?withthe?hygromycin?B?resistance?gene?to?study?survival?and?infection?of?Sclerotinia?sclerotiorumsclerotia?in?soil.Mycol.Res.107，267-276.
5.Whipps?J?M，Gerlagh?M.Biology?of?Coniothyrium?minitans?and?its?potential?use?in?diseasebiocontrol.Mycol?Res，1992，11：897-907
6?Adames?P?B.Ayers?W?A.Sporidesmium?sclerotivorum：its?distribution?and?function?in?naturalbiological?control?of?sclerotial?fungi.Phytopathology.1980.71：90-93
Claims (4)
1, the biocontrol microorganisms shield shell of a strain sclerotinia rot of colza mould (Coniothyrium minitans) ZS-1SB is deposited in Chinese typical culture collection center, and deposit number is: CCTCC NO:M209114.
2, a strain is the microbiobacterial agent of shield shell mould (Coniothyrium minitans) the ZS-1SB preparation of CCTCC NO:M209114 by preserving number.
3, the preparation method of the mould microbial inoculum of a kind of shield shell is characterized in that, comprises adopting the semi-open-type solid fermentation to cultivate the mould microbial inoculum of described shield shell, and it also comprises the steps:
(1) preparation of the mould ZS-1SB spore suspension of shield shell: with preserving number is that the mould ZS-1SB bacterial strain of shield shell of CCTCC NO:M209114 places on the PDA medium slant, cultivates 7d under 20 ℃ of conditions; Use the aseptic water washing inclined-plane, adjust conidial suspension to 10
6Spore/ml;
(2) the numerous cultivation of the expansion of the mould ZS-1SB of shield shell: is to be inoculated at 1: 100 in the triangular flask of PDB substratum the conidial suspension of the mould ZS-1SB of shield shell of step (1) with mass ratio, in 150r/min, shaking culture 24h under 20 ℃ of conditions obtains the mould ZS-1SB bacterium of shield shell liquid, and is standby.
(3) the mould semi-open-type solid fermentation of shield shell is cultivated: with dried culture material rolled oats and rice bran is 3: 1 mixings by volume, add after an amount of water logging rises, the food steamer of packing into is added a cover the back in very hot oven atmospheric steam sterilization 2h, after the cooling, the final water content that the adding sterile purified water is regulated described culture material is 70%, in culturing room, the mould ZS-1SB bacterium of the described shield shell of step (2) liquid is inoculated in this culture material with the ratio that the dried culture material of per kilogram adds 100ml, be tiled in food steamer, making the thickness of described culture material is 5mm, adds a cover, place 20 ℃ of culturing room to cultivate 48h, then steamer cover half is opened wide, under 16 ℃ of conditions, continue to cultivate 7d, obtain the mould microbial inoculum of described shield shell;
Wherein:
Described PDA nutrient media components of step (1) and proportioning are: potato 200g, and glucose 20g, agar 10g replenishes distilled water to 1000ml, transfers pH to 7.0, at 121 ℃ of high pressure steam sterilization 30min;
Described PDB nutrient media components of step (2) and proportioning are: potato 200g, glucose 20g replenishes distilled water to 1000ml, transfers pH to 7.0, at 121 ℃ of high pressure steam sterilization 30min.
4, the application of the described shield shell of claim 1 mould (Coniothyrium minitans) ZS-1SB in preparation control sclerotinia rot of colza microbial preparation.
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| CN102234619A (en) * | 2011-06-16 | 2011-11-09 | 四川省农业科学院植物保护研究所 | Biocontrol bacteria coniothyrium minitans Cm2004 strain for preventing and treating sclerotiniose and preparation method and application thereof |
| CN102408996A (en) * | 2010-09-26 | 2012-04-11 | 华中农业大学 | Wettable powder of biocontrol bacteria coniothyrium minitans Chy-1C-1 and application |
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Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE19502065C2 (en) * | 1995-01-14 | 1996-05-02 | Prophyta Biolog Pflanzenschutz | Fungus isolate with fungicidal activity |
| CN1584012A (en) * | 2004-05-28 | 2005-02-23 | 西北农林科技大学 | Membrane culturing method for fibrillar strain solid culturing research |
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