CN106342898A - Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof - Google Patents
Compound microorganism preparation against sclerotinia rot of colza and preparing method thereof Download PDFInfo
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Abstract
The invention discloses a compound microorganism preparation against sclerotinia rot of colza and preparing method thereof. The compound microorganism preparation comprises fermentation broth of streptomyces resistomycificus, fermentation broth of no-branch streptomyces, fermentation broth of bacillus of tequila, fermentation broth of bacillus sim and fermentation broth of stroma trichoderma; the bacterial species in the compound microorganism preparation against sclerotinia rot of colza and preparing method thereof can comprehensively make use of antibiosis, competition, hyperparasitism and bacteriolysis between microorganisms or bacterial species and induce raps to resist diseases by secondary metabolite, generate a plurality of antibiotics such as resistomycin, acetomycin and macrolide bacteria, and generate ultra-high effects of inhibiting pathogen of sclerotinia rot of colza; the 5 bacterial strains used in the compound microorganism preparation against sclerotinia rot of colza and preparing method thereof are drawn by being separated from soil, thereby, with effects of root trace plant growth promotion, which breed and grow on the surface of crops, in the plants or soil while secreting some secondary metabolite to plants in a targeted way, enhancing plants' nutrient absorption and stimulating plants to grow in effects of fertilizer.
Description
Technical field
The invention belongs to Strategies of Agricultural Bio-control and microorganism formulation applied technical field are and in particular to a kind of prevent and treat Sclerotina Sclerotiorum in Winter Rape
Complex microorganism preparations of core disease and preparation method thereof.
Background technology
Brassica campestris L is the important oil crop of China, is a kind of worldwide weight by the microbial sclerotinia rot of colza of nuclear disk
Want disease, occur universal in China and endanger serious, general time sickness rate is 10%-30%, serious time or serious plot
Sickness rate reaches more than 80%, leads to Severe Reduction and quality decline.
Because sclerotinite has: (1) can obstinate in soil survive, and accumulates easily in soil；(2) host's model
Enclose extremely wide；(3) sclerotium can sprout formation mycelium when condition is suitable, mycelium or infect seedling stage crop or using in soil
Nutrition grown, and form more sclerotium, a sclerotium can produce a lot of apotheciums, in spring field sclerotial germination
The features such as persistent period is up to 50d, therefore takes on producing and promotes anti-(resistance to) crop varieties, cultural control and chemical prevention
Measure.Promoting disease-resistant variety is one of the most cost-effective measure in control of plant disease, but Brassica campestris L is to sclerotiniose not table
Now vertical Hangzhoupro property, there is presently no effective disease-resistant variety and comes out.Therefore, for a long time, flower is mainly taken in the preventing and treating to this disease
Phase sprays chemical agent to overground part portion, but prevention effect unstable.In addition, for a long time in a large number using pesticide, exist tight
The leftover problem of weight: first, occur in that drug-fast strain in sclerotiorum population, such as Pan Yilou (1998) reports sclerotiorum population
In oneself occurs in that the bacterial strain high anti-to carbendazim；Second, pollute environment and the human health that causes harm.
In view of the foregoing, since the eighties in 20th century, people have gradually invested Biological control sight. at present, Sclerotina Sclerotiorum in Winter Rape
The Biological control of core disease controls the research of sclerotinite more with living microorganism or its metabolite, the result of study table that oneself has
Bright, many soil, Ye Wei, rhizosphere and interior raw microorganism even include some phytopathy originals sclerotinite is all had a certain degree of
Inhibitory action.It is reported that now oneself know have the above funguses of kind more than 30, antibacterial or other microorganism sclerotinite is had parasitism or
Antagonism, oneself finds that the microorganism to sclerotinite with antagonism includes: penicillium sp, bird's -nest fungus, Pseudomonas alba, bud pole
Bacterium, Mucor, Verticillium, Radix Crotalariae szemoensis top spore, mould, the attached coccus of Paecilomyces lilacinus, purple of monospore branch etc..Hyperparasites include: shield shell is mould,
Trichoderma spp., viscous broom are mould, food sclerotium very spore is mould and Talaromyces flavus etc..
Most researchs to this biological control of diseases are concentrated mainly on Trichoderma spp., glue some funguses of the genus such as broom is mould, shield shell is mould
Species, such as Luo Kuan etc. have studied the parasitical fungi on Sclerotinia sclerotiorum sclerotium in southern cole soil, find that trichoderma is excellent
Gesture population, and the bacterial strain such as Trichoderma viride tv36 has more than 90% parasitism to rot indoors to sclerotium.Liao Xiaolan etc. reports wood
Removing mildew imposes on Rice-rape fields, and field can mitigate disease 50% about.Also have been reported that and process, with viscous broom is mould, the sclerotium number producing after soil
Fewer than natural soils, and determine Trichoderma spp., viscous broom is mould, shield shell is mould and 2 reaping hook bacteria strains are satisfied to nuclear fungal hyphae body, ascus
Son generation and the impact of sclerotium generation quantity, discovery shield shell is mould, G virens, Trichoderma harzianum are entirely capable of controlling sclerotinite to produce
Apothecium, and mould it is played of pink withered broom slightly acts on.
Additionally, bright with table of field test results in ware, antibacterial also has certain inhibitory action and prevents to sclerotinia rot of colza
Control effect.Li Guoqing is separated to 31 plants to Sclerotina Sclerotiorum in Winter Rape from seven kinds of aboveground vegetation part health tissues surfaces such as sclerotium and Brassica campestris L of rotting
Core pathogenic bacteria has the bacterial isolateses of inhibitory action.Liao Xiaolan separates from petal and flower pesticide and screens in Brassica campestris L initial bloom stage and full-bloom stage
There is the antagonistic bacterium of inhibitory action to 6 plants to sclerotinite, be initially identified as bacillus cereuss.Chen Shiyun etc. is separated to one from soil
Bacillus amyloliquefaciens, can suppress rape endophytic bacterial Sclerotia forming.Wu Jiansheng etc. screens 1 plant and has very strong solution to oxalic acid
The bacterial strain of cytotoxic activity, can prevent and treat sclerotinia rot of colza.
In sum, report a lot for the research of sclerotinia rot of colza Biological control, but from the point of view of present Research still
There are some problems.As, in the big biocontrol microorganisms reported, most of research contents is only limitted to test in ware, live body life
Survey and a small amount of cell diseases prevention test, result of study remains in laboratory stage, is seldom related to it extensively should
Report for field large area disease control；Single bacterial strain is mainly for the research preventing and treating this disease using biocontrol bacteria
Controlling experiment, but, due to multiformity and the synchronous evolution characteristic of pathogen, existing single antagonistic strain is due to its effect side
Formula is single, prevention effect is unstable, produce and using being also unable to reach highly desirable prevention effect, therefore, biocontrol microorganisms a lot
Biological function needs to rely on the synergism between two plants or more than two plants of antibacterial, and competence exertion goes out more preferable effect.
Content of the invention
The first object of the present invention is to provide a kind of complex microorganism preparations of preventing and treating sclerotinia rot of colza.This composite microbial
The proportioning raw materials of thing preparation are: X-340. streptomycete fermentation liquid 20-40 part, not branch chain mold fermentation liquid 10-30 part, Te Jila bud
Spore bacillus fermentation liquid 20-40 part, Lyceum fermentation of bacillus liquid 10-30 part, Stroma Trichoderma spp. fermentation liquid 20-40 part.Described micro-
X-340. streptomycete in biological preparation, not branch chain mycete, Te Jila bacillus cereuss, Lyceum bacillus cereuss, 5 kinds of Stroma Trichoderma spp. etc.
Bacterium is the energy symbiotic co-existence that screening obtains from more than 400 plants of microbial strains, can produce multiple antibiotics such as X-340., acetomycin
With macrolide rhzomorph, can be to sclerotinia rot of colza pathogen -- sclerotinite produce superpower inhibition, thus reaching superpower preventing and treating
The effect of sclerotinia rot of colza；On the other hand, the strain of the present invention can comprehensively utilize between microbial species or plant in antibiosis, competing
Strive, superparasitism, bacteriolysiss, and by secondary metabolite induce Brassica campestris L produce disease resistance, strengthen its protection effect；Again
Secondary, this 5 plants of bacterial strains used in the present invention are all can be directly isolated to obtain from soil, all have root mark growth-promoting functions, can
In crop surface, plant inside or soil while flourish, and orient the plant rhizosphere some secondary metabolites of secretion,
Can improve plant to the absorption of nutrient, stimulate plant strain growth to play the effect of fertilizer；
The second object of the present invention is to provide a kind of preparation method of the complex microorganism preparations of preventing and treating sclerotinia rot of colza, the party
Method step is simple it is ensured that the medicine fertilizer efficiency fruit of microorganism formulation is prominent.
In order to achieve the above object, the preparation method of the complex microorganism preparations of this preventing and treating sclerotinia rot of colza, including following
Step:
Step one: the preparation of X-340. streptomycete fermentation liquid
Take out X-340. streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as X-340. streptomycete seed liquor；
Fermentation: by the X-340. streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l refuse mould
In the 1000l fermentation tank of plain streptomycete fermentation culture medium, 28-32 DEG C of temperature control, first 24 hours, ventilation was per minute empty for 200l
Gas, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, opens stirring 200r/min, cultivates 40-48 hour,
Treat that thalline content reaches 60g/l, you can stop tank, that is, obtain X-340. streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7
2～7 4；
Wherein, described X-340. streptomycete fermentation culture medium: rapeseed cake powder 3%, corn starch 1%, stone powder 1%, soy molasses 2%,
Dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 points
Clock.
Step 2: the not preparation of branch chain mold fermentation liquid
Take out not branch chain mold species preservation pipe, draw flat board with Gause I solid medium and recovered, cultivate 7 days for 30 DEG C.
Under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, in incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powders and can use 500 milliliters of physiological saline solution to wash
De-, regulations spore concentration is 0.1 hundred million cfu/ml, as not branch chain mycete seed liquor；
Fermentation: the not branch chain mycete seed liquor of above-mentioned preparation is seeded to the not branch chain of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of mold fermentation culture medium, 28-30 DEG C of temperature control, first 18 hours, ventilation be per minute for 300l air,
18-32 hour, ventilation is 500l, and after 32 hours, ventilation is 700l, opens stirring 200r/min, cultivates 40-48 hour, treat
Thalline content reaches 80g/l, you can stop tank, that is, obtain not branch chain mold fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7
2～7 4；
Wherein, described not branch chain mold fermentation culture medium: Semen Glycines powder 3%, cottonseed meal powder 1%, corn starch 2%, stone powder 1%, cane suger
Honey 2%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of Te Jila fermentation of bacillus liquid
Take out Te Jila bacillus cereuss preservation pipe, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with 300l Te Jila fermentation of bacillus culture medium and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 26-36 hour, treats that total spore content is not less than 4,000,000,000 cfu/ml, you can as Te Jila fermentation of bacillus liquid；
Wherein, described lb solid medium: yeast extract 5.0 g, peptone 10.0, nacl10.0 g, agar 20 g, water
1000ml, ph7.2；
Wherein, described Te Jila fermentation of bacillus culture medium: glucose 5g/l, tapioca 25 g/l, peanut meal powder 30
G/l, Dried Corn Steep Liquor Powder 5 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, Calcium Carbonate 5 g/
L, ph7.0.The condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 4, the preparation of Lyceum fermentation of bacillus liquid
Take out Lyceum bacillus cereuss preservation pipe, draw flat board respectively with nutrient broth solid medium and recovered, 30 DEG C of cultures 48
Hour.Under flat board, picking single bacterium colony is seeded to equipped with nutrient broth solid medium, and in 30 DEG C of incubators, culture 48 is little
When, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with the culture of 300l Lyceum fermentation of bacillus
In the 500l fermentation tank of base, open stirring 120r/min, ventilation is 200l/min within first 8 hours, after 8 hours, ventilation is 300l/
Min, 30 DEG C of culture 24-36 hours, treat that total spore content is not less than 8,000,000,000 cfu/ml, you can as Lyceum fermentation of bacillus
Liquid；
Wherein, described nutrient broth solid medium: peptone 10.0 g/l, Carnis Bovis seu Bubali cream 3.0 g/l, sodium chloride 5.0 g/l,
Agar 20 g/l, ph 7.2 ± 0.2；
Wherein, described Lyceum fermentation of bacillus culture medium: glucose 8g/l, Semen Maydis powder 20 g/l, rapeseed cake powder 30 g/l, fish
Powder 10 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.2 g/l, Calcium Carbonate 5 g/l, ph7.0.Respectively
The condition of medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 5, the preparation of Stroma Trichoderma spp. fermentation liquid
Take out Stroma Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as Stroma Trichoderma spp. seed liquor；
Fermentation: the Stroma Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l Stroma Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, 30 DEG C of culture 32-48 hours, treats that thalline content reaches 100g/l, that is,
Tank can be stopped, you can as Stroma Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described Stroma Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes.
Step 6, mixed fermentation liquid
The bacterium solution of first five step preparation is pressed X-340. streptomycete fermentation liquid 20-40 part, not branch chain mold fermentation liquid 10-30
Part, Te Jila fermentation of bacillus liquid 20-40 part, Lyceum fermentation of bacillus liquid 10-30 part, Stroma Trichoderma spp. fermentation liquid 20-40
Part, mix, that is, obtain preventing and treating the complex microorganism preparations of sclerotinia rot of colza.
Wherein, this complex microorganism preparations is respectively to spray in Brassica campestris L initial bloom stage and full-bloom stage in the method for preventing and treating sclerotinia rot of colza
Apply once, each foliage-spray consumption is 1-2l/ mu, the multiple of its dilution is 100-200 times.
The present invention has the advantage that and beneficial effect:, the X-340. streptomycete that comprises in the present invention, not branch chain mycete,
Te Jila bacillus cereuss, Lyceum bacillus cereuss, 5 kinds of bacterium such as Stroma Trichoderma spp. are screened from more than 400 plants of microbial strains and obtain
Can symbiotic co-existence, can be in the root of plant, stem, in leaf and soil can growth and breeding, multiple antibiotics such as X-340. can be produced,
Acetomycin and macrolide rhzomorph；To suppression, sclerotinia rot of colza pathogen -- sclerotinite has especially powerful effect for they, thus
Reach the effect of superpower preventing and treating sclerotinia rot of colza, it is high to the drug effect of sclerotinia rot of colza, average preventive effect 90. more than 83%, and
With strong points；, the present invention strain can comprehensively utilize microbial species between or plant in antibiosis, competition, superparasitism, bacteriolyze
Effect, or induce Brassica campestris L to produce disease resistance by secondary metabolite, strengthen its protection effect；, used in the present invention
This 5 plants of bacterial strains be all can be directly isolated to obtain from soil, all there are root mark growth-promoting functions, can be in crop surface, plant
In portion or soil while flourish, and orient the plant rhizosphere some secondary metabolites of secretion, it is possible to increase plant is to foster
Point absorption, stimulate plant strain growth to play the effect of fertilizer；(4), complex microorganism preparations of the present invention, to people, animal safety, belong to
Environmentally friendly, it is not likely to produce Drug resistance using the inventive method preventing and treating sclerotinia rot of colza, preparation method of the present invention is stable, strain
Many, with respect to single Antagonistic Fungi, the antagonistic substance of generation is extensive, effect is significant, and low cost, use are simple.
Specific embodiment
Embodiment 1
A kind of complex microorganism preparations of preventing and treating sclerotinia rot of colza are it is characterised in that the proportioning raw materials of this microorganism formulation are: refuse
30 parts of mycin streptomycete fermentation liquid, not 20 parts of branch chain mold fermentation liquid, 30 parts of Te Jila fermentation of bacillus liquid, Lyceum spore bar
20 parts of fermented liquid, 30 parts of Stroma Trichoderma spp. fermentation liquid.
The preparation method of the complex microorganism preparations of this preventing and treating sclerotinia rot of colza, comprises the following steps:
Step one: the preparation of X-340. streptomycete fermentation liquid
Take out X-340. streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god.Under flat board, the streak inoculation of picking single bacterium colony, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as X-340. streptomycete seed liquor；
Fermentation: by the X-340. streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l refuse mould
In the 1000l fermentation tank of plain streptomycete fermentation culture medium, 28-32 DEG C of temperature control, first 24 hours, ventilation was per minute empty for 200l
Gas, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, opens stirring 200r/min, cultivates 44 hours, inspection
Surveying its thalline content is 65g/l, stops tank, that is, obtains X-340. streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7
2～7 4；
Wherein, described X-340. streptomycete fermentation culture medium: rapeseed cake powder 3%, corn starch 1%, stone powder 1%, soy molasses 2%,
Dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 points
Clock.
Step 2: the not preparation of branch chain mold fermentation liquid
Take out not branch chain mold species preservation pipe, draw flat board with Gause I solid medium and recovered, cultivate 7 days for 30 DEG C.
Under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, in incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powders and can use 500 milliliters of physiological saline solution to wash
De-, regulations spore concentration is 0.1 hundred million cfu/ml, as not branch chain mycete seed liquor；
Fermentation: the not branch chain mycete seed liquor of above-mentioned preparation is seeded to the not branch chain of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of mold fermentation culture medium, 28-30 DEG C of temperature control, first 18 hours, ventilation be per minute for 300l air,
18-32 hour, ventilation is 500l, and after 32 hours, ventilation is 700l, opens stirring 200r/min, cultivates 42 hours, detect it
Thalline content is 82g/l, stops tank, that is, obtains not branch chain mold fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 05
Gram, magnesium sulfate: 05 grams, sodium chloride: 05 grams, ferrous sulfate: 0 01 grams, agar: 20 grams, distilled water supplies 1000ml, ph7
2～7 4；
Wherein, described not branch chain mold fermentation culture medium: Semen Glycines powder 3%, cottonseed meal powder 1%, corn starch 2%, stone powder 1%, cane suger
Honey 2%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes.
Step 3, the preparation of Te Jila fermentation of bacillus liquid
Take out Te Jila bacillus cereuss preservation pipe, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C.
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with 300l Te Jila fermentation of bacillus culture medium and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 32 hours, detects that its total spore content is 4,200,000,000 cfu/ml, you can as Te Jila fermentation of bacillus liquid；
Wherein, described lb solid medium: yeast extract 5.0 g, peptone 10.0, nacl10.0 g, agar 20 g, water
1000ml, ph7.2；
Wherein, described Te Jila fermentation of bacillus culture medium: glucose 5g/l, tapioca 25 g/l, peanut meal powder 30
G/l, Dried Corn Steep Liquor Powder 5 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, Calcium Carbonate 5 g/
L, ph7.0.The condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 4, the preparation of Lyceum fermentation of bacillus liquid
Take out Lyceum bacillus cereuss preservation pipe, draw flat board respectively with nutrient broth solid medium and recovered, 30 DEG C of cultures 48
Hour.Under flat board, picking single bacterium colony is seeded to equipped with nutrient broth solid medium, and in 30 DEG C of incubators, culture 48 is little
When, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with the culture of 300l Lyceum fermentation of bacillus
In the 500l fermentation tank of base, open stirring 120r/min, ventilation is 200l/min within first 8 hours, after 8 hours, ventilation is 300l/
Min, cultivates 28 hours for 30 DEG C, detects that its total spore content is 8,300,000,000 cfu/ml, you can as Lyceum fermentation of bacillus liquid；
Wherein, described nutrient broth solid medium: peptone 10.0 g/l, Carnis Bovis seu Bubali cream 3.0 g/l, sodium chloride 5.0 g/l,
Agar 20 g/l, ph 7.2 ± 0.2；
Wherein, described Lyceum fermentation of bacillus culture medium: glucose 8g/l, Semen Maydis powder 20 g/l, rapeseed cake powder 30 g/l, fish
Powder 10 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.2 g/l, Calcium Carbonate 5 g/l, ph7.0.Respectively
The condition of medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes.
Step 5, the preparation of Stroma Trichoderma spp. fermentation liquid
Take out Stroma Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days for 30 DEG C.Under flat board
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as Stroma Trichoderma spp. seed liquor；
Fermentation: the Stroma Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l Stroma Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, cultivates 39 hours for 30 DEG C, detects that its thalline content is 108g/l, stops
Tank, you can as Stroma Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described Stroma Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes.
Step 6, mixed fermentation liquid
The bacterium solution of first five step preparation is pressed 30 parts of X-340. streptomycete fermentation liquid, not 20 parts of branch chain mold fermentation liquid, Te Ji
Draw 30 parts of fermentation of bacillus liquid, 20 parts of Lyceum fermentation of bacillus liquid, 30 parts of Stroma Trichoderma spp. fermentation liquid, mix, that is, prevented
Control the complex microorganism preparations of sclerotinia rot of colza.
The detection test of embodiment 2 complex microorganism preparations bacteriostatic activity
Microorganism formulation prepared by embodiment 1 dilutes 100 times and 200 times, detects two kinds of microorganism systems using flat board face-off method
The impact to Sclerotinia sclerotiorum mycelial growth for the agent.Place Oxford cup, wherein Deca microorganism formulation dilution in pda flat board central authorities
The each 100ul of liquid, surrounding symmetric position places the Sclerotinia sclerotiorum bacteria cake (a diameter of 5mm) of culture 2d.Place with Deca clear water
As comparison, each processes three repetitions of setting to reason.After 25 DEG C of constant temperature culture 5d, observe microorganism formulation to mycelial growth
Suppression situation, measures antibacterial circle diameter.
Result of the test: after complex microorganism preparations of the present invention are diluted 200 times, Sclerotinia sclerotiorum can be significantly inhibited
Growth, antibacterial circle diameter is up to more than 20mm.After complex microorganism preparations of the present invention are diluted 100 times, oil can be significantly inhibited
The growth of dish hyphal cluster germ, antibacterial circle diameter is up to more than 25mm.
Embodiment 3 complex microorganism preparations of the present invention prevent and treat the field test of sclerotinia rot of colza
Test is located at Hunan Province Longhui County beachhead town great Di village Planting household, for many years sclerotinia rot of colza occurs through investigating this plot,
And
Before spraying, investigation has found apothecium on ridge.Setting initial bloom stage sprays once, full-bloom stage sprays once and just
Florescence
Respectively spray 3 different process with full-bloom stage, embodiment 1 is prepared by the initial bloom stage of Brassica campestris L in mid-March, 2015
Complex microorganism preparations of the present invention dilute 200 times and uniformly spray on Brassica campestris L flower and blade, consumption complex microorganism preparations
1l/ mu, after l0d, full-bloom stage sprays second, each cell 66.7m2, and each processes 3 repetitions, can to spray carbendazim 50%
500 times of diluents of WP are as comparison.In the initial stage of results investigation plant incidence, 5 points of samplings, record morbidity strain number
And disease severity, calculate prevention effect；
Disease index=[σ (diseased plant numbers at different levels × represent numerical value)/(the representative numerical value of investigation total strain number × morbidity heavy duty)) ×
100
Prevention effect (%)=[(matched group disease index one treatment group disease index)/matched group disease index] × 100
Result of the test such as table 1, after the complex microorganism preparations of the present invention are diluted 200 times, respectively sprays in initial bloom stage and full-bloom stage
Shi Yi
Preferably, prevention effect reaches 90. 83% for secondary prevention effect, sprays prevention effect once and full-bloom stage spray in initial bloom stage
Shi Yi
Secondary prevention effect has also reached more than 75.35%.
The field control effect to sclerotinia rot of colza for table 1 complex microorganism preparations
| Process | Diseased plant rate (%) | Disease index | Prevention effect (%) | Yield (kg) | Rate of growth (%) |
| Initial bloom stage is once | 2.09 | 1.03 | 85.24 | 26.8 | 26.42 |
| Full-bloom stage is once | 2.96 | 1.72 | 75.35 | 24.6 | 16.04 |
| Just spend+full-bloom stage | 1.78 | 0.64 | 90.83 | 28.4 | 33.96 |
| Matched group | 9.67 | 6.98 | 21.2 |
Claims (3)
1. a kind of complex microorganism preparations of preventing and treating sclerotinia rot of colza are it is characterised in that the proportioning raw materials of this microorganism formulation are:
X-340. streptomycete fermentation liquid 20-40 part, not branch chain mold fermentation liquid 10-30 part, Te Jila fermentation of bacillus liquid 20-40
Part, Lyceum fermentation of bacillus liquid 10-30 part, Stroma Trichoderma spp. fermentation liquid 20-40 part.
2. a kind of complex microorganism preparations of preventing and treating sclerotinia rot of colza according to claim 1 are it is characterised in that it is prepared
Method, comprises the following steps:
Step one, the preparation of X-340. streptomycete fermentation liquid
Take out X-340. streptomyces species preservation pipe, draw flat board with Gause I solid medium and recovered, 30 DEG C of cultures 7
My god, picking single bacterium colony streak inoculation under flat board, equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, is being cultivated
Cultivate 6-8 days for 30 DEG C in case, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution that a large amount of spore powders can use 500 milliliters
Eluting, regulation spore concentration is 0.1 hundred million cfu/ml, as X-340. streptomycete seed liquor；
Fermentation: by the X-340. streptomycete seed liquor of above-mentioned preparation with 1% inoculum concentration be seeded to equipped with sterilizing 600l refuse mould
In the 1000l fermentation tank of plain streptomycete fermentation culture medium, 28-32 DEG C of temperature control, open and stir 200r/min, first 24 hours, ventilation
For per minute for 200l air, 24-36 hour, ventilation is 400l, and after 36 hours, ventilation is 600l, and 40-48 is little for culture
When, treat that thalline content reaches 60g/l, you can stop tank, that is, obtain X-340. streptomycete fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 0.5
Gram, magnesium sulfate: 0.5 gram, sodium chloride: 0.5 gram, ferrous sulfate: 0.01 gram, agar: 20 grams, 1000ml supplied by distilled water,
Ph7.2～7.4；
Wherein, described X-340. streptomycete fermentation culture medium: rapeseed cake powder 3%, corn starch 1%, stone powder 1%, soy molasses 2%,
Dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.05%, the condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 points
Clock；
Step 2: the not preparation of branch chain mold fermentation liquid
Take out not branch chain mold species preservation pipe, draw flat board with Gause I solid medium and recovered, cultivate 7 days for 30 DEG C,
Under flat board, the streak inoculation of picking single bacterium colony is equipped with the Fructus Solani melongenae bottle of 150 milliliters of Gause I solid mediums, in incubator
In 30 DEG C cultivate 6-8 days, treat that lawn covers with Fructus Solani melongenae bottle, produce a large amount of spore powders and can use 500 milliliters of physiological saline solution to wash
De-, regulations spore concentration is 0.1 hundred million cfu/ml, as not branch chain mycete seed liquor；
Fermentation: the not branch chain mycete seed liquor of above-mentioned preparation is seeded to the not branch chain of the 600l equipped with sterilizing with 1% inoculum concentration
In the 1000l fermentation tank of mold fermentation culture medium, 28-30 DEG C of temperature control, open stirring 200r/min, first 18 hours, ventilation was every
Minute is 300l air, 18-32 hour, and ventilation is 500l, and after 32 hours, ventilation is 700l, cultivates 40-48 hour, treats
Thalline content reaches 80g/l, you can stop tank, that is, obtain not branch chain mold fermentation liquid；
Wherein, described Gause I solid medium: potassium nitrate: 1 gram, soluble starch: 20 grams, dipotassium hydrogen phosphate: 0.5
Gram, magnesium sulfate: 0.5 gram, sodium chloride: 0.5 gram, ferrous sulfate: 0.01 gram, agar: 20 grams, 1000ml supplied by distilled water,
Ph7.2～7.4；
Wherein, described not branch chain mold fermentation culture medium: Semen Glycines powder 3%, cottonseed meal powder 1%, corn starch 2%, stone powder 1%, cane suger
Honey 2%, manganese sulfate 0.002%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-
0.15mpa, 121 DEG C sterilize 30 minutes；
Step 3, the preparation of Te Jila fermentation of bacillus liquid
Take out Te Jila bacillus cereuss preservation pipe, draw flat board respectively with lb solid medium and recovered, cultivate 48 hours for 30 DEG C,
Under flat board, picking single bacterium colony is seeded to equipped with lb solid medium, cultivates 48 hours, use 3000ml in 30 DEG C of incubators
Sterilized water, by the lawn eluting in three Fructus Solani melongenae bottles, is seeded to the 500l equipped with 300l Te Jila fermentation of bacillus culture medium and sends out
In fermentation tank, open stirring 120r/min, ventilation is 200l/min within first 12 hours, after 12 hours, ventilation is 320l/min, 30 DEG C
Culture 26-36 hour, treats that total spore content is not less than 4,000,000,000 cfu/ml, you can as Te Jila fermentation of bacillus liquid；
Wherein, described lb solid medium: yeast extract 5.0 g, peptone 10.0, nacl10.0 g, agar 20 g, water
1000ml, ph7.2；
Wherein, described Te Jila fermentation of bacillus culture medium: glucose 5g/l, tapioca 25 g/l, peanut meal powder 30
G/l, Dried Corn Steep Liquor Powder 5 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.5 g/l, Calcium Carbonate 5 g/
L, ph7.0, the condition of each medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes；
Step 4, the preparation of Lyceum fermentation of bacillus liquid
Take out Lyceum bacillus cereuss preservation pipe, draw flat board respectively with nutrient broth solid medium and recovered, 30 DEG C of cultures 48
Hour, under flat board, picking single bacterium colony is seeded to equipped with nutrient broth solid medium, and in 30 DEG C of incubators, culture 48 is little
When, with 3000ml sterilized water by the lawn eluting in three Fructus Solani melongenae bottles, it is seeded to equipped with the culture of 300l Lyceum fermentation of bacillus
In the 500l fermentation tank of base, open stirring 120r/min, ventilation is 200l/min within first 8 hours, after 8 hours, ventilation is 300l/
Min, 30 DEG C of culture 24-36 hours, treat that total spore content is not less than 8,000,000,000 cfu/ml, you can as Lyceum fermentation of bacillus
Liquid；
Wherein, described nutrient broth solid medium: peptone 10.0 g/l, Carnis Bovis seu Bubali cream 3.0 g/l, sodium chloride 5.0 g/l,
Agar 20 g/l, ph 7.2 ± 0.2；
Wherein, described Lyceum fermentation of bacillus culture medium: glucose 8g/l, Semen Maydis powder 20 g/l, rapeseed cake powder 30 g/l, fish
Powder 10 g/l, magnesium sulfate 0.4 g/l, manganese sulfate 0.5 g/l, potassium dihydrogen phosphate 0.2 g/l, Calcium Carbonate 5 g/l, ph7.0, respectively
The condition of medium sterilization is: 0.10-0.15mpa, and 121 DEG C sterilize 30 minutes；
Step 5, the preparation of Stroma Trichoderma spp. fermentation liquid
Take out Stroma Trichoderma spp. strain preservation pipe, draw flat board with pda solid medium and recovered, cultivate 7 days, under flat board for 30 DEG C
Picking single bacterium colony streak inoculation, equipped with the Fructus Solani melongenae bottle of 150 milliliters of pda solid mediums, cultivates 6-8 for 30 DEG C in incubator
My god, treat that lawn covers with Fructus Solani melongenae bottle, produce the physiological saline solution eluting that a large amount of spore powders can use 500 milliliters, adjust spore dense
Spend for 0.05 hundred million cfu/ml, as Stroma Trichoderma spp. seed liquor；
Fermentation: the Stroma Trichoderma spp. seed liquor of above-mentioned preparation is seeded to equipped with 300l Stroma Trichoderma spp. fermentation culture with 1% inoculum concentration
In the 500l fermentation tank of base, open stirring 200r/min, ventilation is 120l/min within first 8 hours, and after 8-24 hour, ventilation is
240l/min, after 24 hours, ventilation is 360l/min, 30 DEG C of culture 32-48 hours, treats that thalline content reaches 100g/l, that is,
Tank can be stopped, you can as Stroma Trichoderma spp. fermentation liquid；
Wherein, described pda solid medium: Rhizoma Solani tuber osi 200g, sucrose 20g, water 1000ml, agar 20g；
Wherein, described Stroma Trichoderma spp. fermentation medium: rapeseed cake 2%, cottonseed meal 2%, corn starch 2%, stone powder 1%, soy molasses 3%,
Manganese sulfate 0.001%, dipotassium hydrogen phosphate 0.04%, magnesium sulfate 0.02%, the condition of each medium sterilization is: 0.10-0.15mpa,
121 DEG C sterilize 30 minutes；
Step 6, mixed fermentation liquid
The fermentation liquid of first five step preparation is pressed X-340. streptomycete fermentation liquid 20-40 part, not branch chain mold fermentation liquid 10-30
Part, Te Jila fermentation of bacillus liquid 20-40 part, Lyceum fermentation of bacillus liquid 10-30 part, Stroma Trichoderma spp. fermentation liquid 20-40
Part, mix, that is, obtain preventing and treating the complex microorganism preparations of sclerotinia rot of colza.
3. a kind of complex microorganism preparations of preventing and treating sclerotinia rot of colza according to claim 1 are it is characterised in that this is compound
Microorganism formulation is respectively to spray once in Brassica campestris L initial bloom stage and full-bloom stage in the method for preventing and treating sclerotinia rot of colza, each foliage-spray
Consumption is 1-2l complex microorganism preparations/mu, and the multiple of its dilution is 100-200 times.
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| CN113207515A (en) * | 2021-05-18 | 2021-08-06 | 上海交通大学 | Prediction and prevention method for lentil sclerotinia rot |
| CN115161212A (en) * | 2021-04-02 | 2022-10-11 | 重庆市畜牧科学院 | Bacillus tequilensis strain |
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