CN102027998B - Bacillus spore and trichoderma chlamydospore compound preparation and preparation method and application thereof - Google Patents
Bacillus spore and trichoderma chlamydospore compound preparation and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a compound preparation for controlling plant diseases. The compound preparation consists of a bacillus spore and a trichoderma chlamydospore. The invention also provides a method for preparing the compound preparation and application of the compound preparation.
Description
Technical field
The application relates to biological technical field, especially agricultural biological technical field.Particularly, the application relates to bacillus brood cell and Trichoderma chlamydospore compound formulation, its preparation method and application.
Background technology
Bacillus subtilis (Bacillus subtilis) is a kind of mesophilous aerobic sporiferous bacillus, and its physiological characteristic is rich and varied, widely distributed, very easily separated cultivation.This bacterium is widespread in nature, nontoxic to people and animals, free from environmental pollution, can produce multiple antibiotic and enzyme, has broad spectrum antibiotic activity and extremely strong anti-adversity ability.Bacillus subtilis not only can be in the external environments such as soil, plant rhizosphere, body surface extensively exists, and common endophytic bacterium simultaneously or in plant corpus, especially in root, the stem of plant.This bacterium shows good disease-controlling effect on the crops such as paddy rice, capsicum, cotton, wheat, cucumber, soybean, corn at present.The most outstanding feature of bacillus subtilis is that growth is fast, nutrition is simple, can produce the contrary gemma of heat-resistant, this be not only conducive to biocontrol fungicide production, formulation and the survival in environment, surely grow and breeding, and mass production processes is simple, cost is also lower, uses conveniently, and the storage life is long, be a kind of desirable Biocontrol microorganism, have broad application prospects.
Bacillus subtilis, by surely growing in plant rhizosphere, body surface or body, suppresses pathogen growth with pathogen competitive plant nutrition, secretion antibacterial material around, and inducing plant system of defense is resisted pathogen invasion simultaneously, thereby reaches the object of biological and ecological methods to prevent plant disease, pests, and erosion.Main controlling object major part is the caused plant disease of filamentous fungi, as rice sheath blight disease (Stagonospora curtisii), cladosporium leaf and fruit mould of tomato (Cladosporium fulvum), root rot (Fusariumgraminerarum), Apple Mould Core (Alternaria alternata), cotton seedling blight (Rhizoctonia solani), cotton wilt (Fusarium oxysporum f.sp.vasinfectum) etc.Bacillus subtilis Biocontrol Mechanism mainly comprises Competition, antagonism and 3 aspects of induction of resistance effect.In addition, the antibacterial material with biological and ecological methods to prevent plant disease, pests, and erosion activity that producing bacillus subtilis is raw, comprises the multiple compounds such as lipopeptid class, peptide class, protide, phospholipid, polyenoid class, amino acids and nucleic acid, and they are inhibited to fungi, bacterium, virus etc.
Although bacillus subtilis has been obtained certain achievement in the biological control of plant disease as biological bactericide, see on the whole and also have many problems (R.campbell, 1993).
First, as biological bactericide, bacillus subtilis is in field effect performance unstable, this may be owing to conventionally adopting the indoor preservation bacterial strain in many generations, there is larger difference with wild strain activity, and the micro-ecological environment of indoor pot experiment and field complexity is far different, cause indoor inhibition zone measurement result and field result difference larger.Secondly, its toxicity of microorganism formulation and safety be it is generally acknowledged and chemical agent, tend to nontoxic and noresidue, but some microorganism formulations also may cause pathogen resistance, and particularly those suppress the bacterial strain of pathogen with antibiotics secretion.In forbidding list is listed with preparation the bacillus subtilis poultry that produces antibiotic Bacitracin Zinc in Europe, and reason is easily to produce drug resistance and antibiotic residue.To the research of microbial pesticide safety also seldom, majority does not cause enough attention in China.Finally, as microbial pesticide, its cost accounting generally will be higher than chemical agent, and is mainly protectant, after plant morbidity, uses, and effect is poor, and some is even invalid, and this is the disadvantage that many microbial pesticides all exist.
For solving the problem of above-mentioned existence, strengthen the biocontrol effect of biocontrol microorganisms, and improve them to adaptability of environmental condition etc., domestic and international many laboratories are utilizing cell engineering and technique for gene engineering to carry out the genetic improvement of Biocontrol microorganism, build new and effective biocontrol microorganisms (Wang Jinsheng, 2002).As (1999) such as Chen Zhongyi by plasmid vector by Bt gene Introduced into Rice Biocontrol Bacillus B916, laboratory test shows that recombinant bacterial strain has good disinsection prophylaxis effect and good genetic stability.But due to many antagonistic genes particularly antibiotic synthetic gene by baroque gene cluster, form, genetic manipulation is more difficult.Therefore, relevant technologies also needs further research.
Existing more than 70 year of the mould history for controlling plant diseases of wood, its biocontrol effect gains public acceptance, is the widely used biocontrol microorganisms in the whole world.Trichoderma is in Deuteromycotina, Hyphomycetes, hyphomycetales, Moniliaceae, sticky spore mushroom, is that a class is prevalent in the fungi in soil, it is the important group of edaphon, it can parasitize in plant residue and animal wastes, from plant rhizosphere, blade and seed, bulb surface, also often can be separated to, and wood is mould can be parasitized on the pathogen that multiple soil passes plant.Since the eighties, along with molecular biological fast development, the mould research of wood mainly concentrates on how to improve its biological preventive effect aspect, as means such as the expression by reinforcement chitinase, glucanase gene, Interspecific fusions.It is just flocculence or fine and close clump pencil that Trichoderma PDA cultivates bacterium colony, and color is that white is to canescence, without solid shape.After conidium maturation, bacterium colony to edge, graduates into green in various degree in central authorities, and only a few is white powdery.Conidiophore bears from the side shoot of mycelia, upright branch, and sprig is often to life, and does not expand on top, upper raw spore ball estranged.Conidium is spherical, light or colourless.Most of wood is mould not strict to the demand of nutrition, and they can grow under various Carbon and nitrogen sources, and some harmful or lasting harmful environmental contaminants can transform and degrade simultaneously; Can directly utilize derivative, the organic acid of various monose, monose; There are the various polysaccharide of significant degraded (cellulose, hemicellulose) and relevant polysaccharide (chitin); Some agricultural chemicals that also can transform and degrade, as: malathion, dalapon, pcnb etc.The wooden mould complicated and simple nitrogen that utilizes, all fine utilizations of energy of casamino acids mixed liquor, asparatate, alanine, glutamic acid are conducive to wooden some enzymes of mould generation, as cellulase, lactase etc. under high nitrogen condition.Wood is mould is aerobic bacteria, and applicable oxygen is pressed with growth and the Sporulation that is beneficial to mycelia; Best pH is 4.0-6.5; Growth temperature is because of kind difference, and great majority are very rapid about 25 ℃ growths, and the pH value of self-control environment, make its growth conditions of environmental adaptation.
The antagonism scope of Trichoderma has broad spectrum activity, correlative study demonstration, and Trichoderma at least belongs to 29 kinds of disease funguses to 18 antagonism.The parasitic phytopathogen of Trichoderma energy is that antagonism object comprises Rhizoctonia (Rhizotonoa), pyrenomycetes (Sclerotium), Sclerotinia (Sclerotinia), Helminthosporium (Helminth osporium), Fusarium (Fusarium), hair disc spore belongs to (Colletotrichum, Verticillium (Verticillium), Venturia (Venturia), inner seat shell belongs to (Endothia), pythium (Pythium), Phytophthora (Phytophthora), between seat shell belong to (Diaporthe) and Fusicladium (Fusicladium) etc.
Wooden removing mildew is in the market all conidium preparation and mycelia type preparation, is mostly by solid fermentation.Solid state fermentation is not only current fungal culture and is obtained a large amount of conidial best method by aerial hyphae, and its distinctive advantage has made it be widely used and study.In addition, the conidium quality that the conidium that solid fermentation produces produces than liquid fermentation is good, and resistance to drying power is strong, and the time-to-live is long.And while applying in field, product by solid-state fermentation can directly be used, without making preparation, post processing is simple, and cost is low.But its production and apply smallly, traces it to its cause, and a difficult problem for most critical does not have to solve: the shelf life of product is short.The preparation that the conidium of generally take is active ingredient, the time that guarantees viable bacteria content can be over 3 months, the mould survival volume of wood that later stage connects in preparation is difficult to reach requirement, the stability of its preventive effect effectively do not guaranteed, other control measures such as substituted chemistry control completely still at present in control of plant disease.
Summary of the invention
For addressing the above problem, the invention provides following technical scheme.
First aspect present invention provides a kind of compound formulation for controlling plant diseases, and described compound formulation comprises bacillus brood cell and Trichoderma chlamydospore.
At one, preferably in embodiment, described compound formulation is wetting powder, and described compound formulation comprises carrier and wetting dispersing agent, and described carrier is selected from White Carbon black or diatomite, and described wetting dispersing agent is selected from alkylnaphthalene sulfonate.In a better embodiment, the content of described carrier is 5-30% (W/W), the content of described wetting dispersing agent is 0.5-5% (W/W), and the weight of zymotic fluid of take is benchmark (before the wetting powder of take is dry, the weight of zymotic fluid is benchmark).At another, preferably in embodiment, bacillus brood cell's content described in described compound formulation is 10
8-10
10individual cell/gram (preparation), the chlamydosporic content of described Trichoderma is 10
8-10
9individual spore alive/gram (preparation).
The present invention provides a kind of method of preparing above-mentioned compound formulation on the other hand, and the method comprises the following steps:
(1) provide containing bacillus brood cell's zymotic fluid and containing the chlamydosporic zymotic fluid of Trichoderma;
(2) will then mix with the zymotic fluid containing bacillus brood cell containing the chlamydosporic zymotic fluid homogenate of Trichoderma, obtain described compound formulation.
At one preferably in embodiment, obtaining with following methods containing the chlamydosporic zymotic fluid of Trichoderma in described step (1):
In fermentation medium, 25～33 ℃ of temperature, seed inoculum concentration 5～10%, seed concentration 10
5-10
6individual/mL, ventilation ratio 1: 2 to 2: 1, under tank pressure 0.03-0.05MPa, fermentation 84-96 hour, obtains containing the chlamydosporic zymotic fluid of Trichoderma, and the composition of wherein said fermentation medium is: wheat bran 0.4-0.6%, (NH
4)
2sO
40.1-0.3%, KH
2pO
40.1-0.2%, CaCO
30.1-0.2%, glucose 1-3%, the soybean oil of 0.2-0.5%, pH5.5-6.0.
At another preferably in embodiment, described compound formulation is wetting powder, it is by the compound formulation of step (2) gained is further dried and is made, and also comprise the step that is mixed into carrier and wetting dispersing agent in described step (2), described carrier is selected from White Carbon black powder or diatomite, and described wetting dispersing agent is selected from alkylnaphthalene sulfonate.In better embodiment, the content of described carrier is 5-30% (W/W), and the content of described wetting dispersing agent is 0.5-5% (W/W), and the weight of zymotic fluid of take is benchmark.
The present invention relates to the application of the above-mentioned compound formulation of the present invention in controlling plant diseases on the other hand.In embodiment preferably, described plant disease is selected from watermelon blight and climing rot.
Compound formulation of the present invention can be used for preventing and treating various plants soil-borne disease, and meanwhile, this microbial inoculum can Promoting plant growth, improves utilization rate of fertilizer, and inducing plant produces the ability of degeneration-resistant border and disease resistance.The chlamydospore preparation of producing by this invention has stronger vitality and anti-adversity ability than with the conidium preparation of produced in conventional processes, and action function is more, is more suitable in production application.
Embodiment
First aspect present invention provides a kind of compound formulation for controlling plant diseases, and described compound formulation comprises bacillus brood cell and Trichoderma chlamydospore.Described bacillus is preferably bacillus subtilis.
In a preferred embodiment, bacillus brood cell's content described in described compound formulation is 10
8-10
10individual cell/restraint agent, the chlamydosporic content of described Trichoderma is 10
8-10
10individual spore/restraint agent alive.
Compound formulation of the present invention can adopt all kinds of formulations of pesticide well known to those skilled in the art, it is including, but not limited to pulvis, wetting powder, missible oil, granule, colloidal suspending agent, aqueous solvent, aerosol, tablet, fumicants, fumigant, mist of oil agent, sustained release agent, microcapsule formulations, suspended emulsion, solid missible oil, microemulsion and the pulvis etc. that can flow, wherein preferably wetting powder, microcapsule formulation, suspended emulsion, solid missible oil, microemulsion and the pulvis that can flow.Because wetting powder and microcapsule formulations safety easy to use, continuation are strong, be conducive to the long-acting of biopesticide, and the auxiliary agent using is harmless, does not contain organic solvent, environmentally friendly, so be highly preferred.
In compound formulation of the present invention (as wetting powder), also can comprise other carrier and auxiliary agent etc.In a preferred embodiment, compound formulation of the present invention is wetting powder, and this wetting powder comprises carrier and wetting dispersing agent.The carrier of inertia is usually used in processing the solid dosage formss such as pulvis, wetting powder, granule, and its effect is that dilution active ingredient is convenient to processing, improves physicochemical character easy to use.As the inert material of preparations carrier, can affect to a certain extent the stability of preparation miospore.The survival of preparation miospore shows as the metabolism of certain level.Even metabolic activity is very faint under specific storage requirement, but the metabolic by-product accumulating can produce inhibitory action to spore self.
In the present invention, preferred inert carrier is that described carrier is selected from White Carbon black powder or diatomite.
The surface tension that wetting dispersing agent can reduce water makes active ingredient very soon by the material of water-wet.Processing wetting powder, suspending agent, dry suspending agent, water-dispersible granules etc., be watered to be made into when suspension sprays the formulation of use and add wetting agent, makes the very fast wetted and dispersion suspension in water-fast powder surface in water.Medicine liquid spray is easy to spread to target body surface, expands area coverage.Conventional wetting agent has that Chinese honey locust, tea are withered, lignosulfonates, alkylbenzenesulfonate, pull open powder, multiple nonionic surface active agent etc.In the present invention, preferred wetting dispersing agent is alkylnaphthalene sulfonate.
The content of described carrier and wetting dispersing agent is easy to be determined according to limited experiment by those skilled in the art.At one, preferably in embodiment, the content of described carrier is 5-30% (W/W), and the content of described wetting dispersing agent is 0.5-5% (W/W), and the weight of zymotic fluid of take is benchmark.
In addition, also can add other and have active component in compound formulation of the present invention, this can be selected by routine test according to specific purposes by those skilled in the art and determine.
Preparation method
The present invention provides a kind of method of preparing compound formulation of the present invention on the other hand, and the method comprises the following steps:
(1) provide containing bacillus brood cell's zymotic fluid and containing the chlamydosporic zymotic fluid of Trichoderma;
(2) will then mix with the zymotic fluid containing bacillus brood cell containing the chlamydosporic zymotic fluid homogenate of Trichoderma, obtain described compound formulation.
Contain bacillus brood cell's zymotic fluid and can by routine test, be determined as the case may be by those skilled in the art containing the consumption (or its mixed proportion) of the chlamydosporic zymotic fluid of Trichoderma, conventionally preferably wishing that the brood cell's of bacillus described in compound formulation content is 10
8-10
10individual cell/gram, the chlamydosporic content of described Trichoderma is 10
8-10
10individual spore alive/gram.In described step (2), the object of described homogenate step is to make mycelium fully broken, specifically can adopt various Mechanical Crushing method well known to those skilled in the art.
In a preferred embodiment, described compound formulation is wetting powder.For the ease of long-term storage, after zymotic fluid can being mixed according to certain ratio with carrier, wetting dispersing agent or other auxiliary agent, obtain liquid-solid mixture, then adopt modes such as press spray, centrifugal spraying to be dried, thereby make described wetting powder.Therefore, in this embodiment, be also included in the step that is mixed into carrier and wetting dispersing agent in described mixed liquor.The interpolation order of described carrier and wetting dispersing agent has no particular limits, they one of or both can first partly or entirely add containing being mixed to get mixed liquor with the homogenate containing after the chlamydosporic zymotic fluid homogenate of Trichoderma again after bacillus brood cell's zymotic fluid, also can first partly or entirely add with homogenate containing after the chlamydosporic zymotic fluid homogenate of Trichoderma after be mixed to get mixed liquor with the zymotic fluid containing bacillus brood cell again, or can after being mixed with the zymotic fluid that contains bacillus brood cell, the homogenate containing after the chlamydosporic zymotic fluid homogenate of Trichoderma add again part or all of carrier and/or wetting dispersing agent.In a preferred embodiment, described carrier should be selected from White Carbon black powder or diatomite, and described wetting dispersing agent should be selected from alkylnaphthalene sulfonate.In a more preferred embodiment, the addition of described carrier, in zymotic fluid gross weight (being the weight of the zymotic fluid mixture of step (2)), the wetting dispersing agent of the carrier of 5-30% (preferably 10-20%) and 0.5-5% (preferably 1-2%).
The tolerance that how to improve the spore in existing preparation is also the importance that solves problems of the prior art.The mould chlamydospore of wood is wooden mould opposing poor environment and the existence structure of dormancy.Chlamydospore, is generally under bad condition, and the protoplasm in hyphal cell shrinks, and cell wall is thickeied and formed.In siphon, a part for mycelia is concentrated and is stored nutriment, produces heavy wall simultaneously, and two ends form the barrier film of sealing and cut off with other cell of mycelia, form chlamydospore; Older mycelia position at septate hypha often can form chlamydospore.Once chlamydospore runs into adapt circumstance condition and just sprouts generation mycelia.So, utilize the biological prevention and control agent that chlamydospore is main body just may there is the advantages such as storage tolerance and shelf life length length, can meet the requirement as biopesticide.Yet, because chlamydospore is harsher to artificial fermentation's fostering requirement condition, so find suitable fermentation culture conditions, in the hope of obtaining the chlamydospore of high-biomass, become problem demanding prompt solution.
In the present invention, also provide a kind of method that obtains Trichoderma chlamydospore or contain Trichoderma chlamydospore zymotic fluid, the method is included in fermentation medium, 25～33 ℃ of temperature, seed inoculum concentration 5～10%, seed concentration 10
5-10
6individual/mL, ventilation ratio 1: 2 to 2: 1, under tank pressure 0.03-0.05MPa, carry out liquid fermentation 84-96 hour to Trichoderma, thereby obtain, is rich in the chlamydosporic zymotic fluid of Trichoderma.The composition of fermentation medium used is: wheat bran 0.4-0.6%, (NH
4)
2sO
40.1-0.3%, KH
2pO
40.1-0.2%, CaCO
30.1-0.2%, glucose 1-3%, the soybean oil of 0.2-0.5%, pH5.5-6.0.
In a more preferred embodiment, the method is included in fermentation medium, temperature 28-30 ℃, seed inoculum concentration 6-9%, seed concentration 10
5-10
6individual/mL, ventilation ratio 1: 2 to 2: 1, under tank pressure 0.03-0.05MPa, carry out liquid fermentation 96 hours to Trichoderma, thereby obtain, is rich in the chlamydosporic zymotic fluid of Trichoderma.The composition of fermentation medium used is: wheat bran 0.5%, (NH
4)
2sO
40.2%, KH
2pO
40.1%, CaCO
30.1%, glucose 2%, the soybean oil of 0.2-0.3%, pH5.5-6.0.
The application of controlling plant diseases
Compound formulation provided by the invention can be for preventing and treating various plant diseases effectively.At one preferably in embodiment, compound formulation of the present invention can be for preventing and treating and comprise Rhizoctonia (Rhizocionia) effectively, shell ball spore belongs to (Macrophomina), Fusarium (Fusarium), Sclerotinia (sclerotium), pythium (Phythium), Phytophthora (Phytophthora), pyrenomycetes (Sclerotium), Helminthosporium (Helminth osporium), hair disc spore belongs to (Colletotrichum, Verticillium (Verticillium), Venturia (Venturia), inner seat shell belongs to (Endothia), between seat shell belong to plant disease or the soil biography plant disease that (Diaporthe) and Fusicladium (Fusicladium) etc. cause.In addition, compound formulation of the present invention can also be prevented and treated the powdery mildews of the plants such as strawberry and gray mold etc., can also control the rear various rot of adopting of melon, fruit.
More particularly, compound formulation of the present invention can be for preventing and treating following plant disease: rice sheath blight disease, false smut; Pseudo-ginseng, tobacco, flowers, wheat, the soil-borne disease of Chinese cabbage etc.; Powdery mildew, graywall, the epidemic diseases such as anthracnose; Wheat sharp eyespot; Rice blast, black spot of cabbage; Powdery mildew of cucumber, powdery mildew of strawberry and gray mold; Damping off (as tomato, cucumber, eggplant, green pepper, watermelon, muskmelon, muskmelon), root rot, damping off, the diseases such as fusarium wilt.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.All technical schemes of the present invention (comprising technical scheme preferably, or best) all can be combined and form new technical scheme.All these technical schemes are all considered as within the scope of the application's literature record.Unless otherwise described, enforcement of the present invention is by the routine techniques adopting known to those skilled in the art.Or the specification that can provide according to reagent manufacturer carries out.
Embodiment 1: the 500L tank liquid fermentation of wooden mould T4 produces chlamydosporic research
To the mould T4 of wood, the generation of the chlamydospore in 500L fermentation tank is studied the present embodiment.Bacterial strain uses therefor is Trichoderma viride (T.viride) T4, separated acquisition from plant rhizosphere soil, bio-reactor engineering National Key Laboratory of Bing Kecong East China University of Science buys, separately can be referring to document (Xia Siqin, Wang Wei, chemistry and biotechnology, " solid fermentation process of Trichoderma viride T4 and the research of preparation stability thereof ", 2008, Vol.25, No.12,52-56 page).
In fermentation medium, temperature 28-30 ℃, seed inoculum concentration 6-9%, seed concentration 10
5-10
6individual/mL, ventilation ratio 1: 2 to 2: 1, under tank pressure 0.03-0.05MPa, carry out liquid fermentation 96 hours to Trichoderma, thereby obtain, is rich in the chlamydosporic zymotic fluid of Trichoderma.The composition of fermentation medium used is: wheat bran 0.5%, (NH
4)
2sO
40.2%, KH
2pO
40.1%, CaCO
30.1%, the soybean oil of glucose 2%, 0.2%, pH5.5-6.0.
Found that, wooden mould T4 carries out liquid fermentation in 500L fermentation tank has good chlamydospore to form ability.In 500L fermentation tank, front 24h forms mycelia in a large number, without chlamydospore, forms, and started to form, but quantity seldom to 36h chlamydospore, and at this moment wooden mould mycelia forms abundant, denser; Along with fermentation time extends, chlamydospore forms gradually, to 72h chlamydospore amount, reaches 1.5 * 10
7individual/mL, 72h～84h chlamydospore increases very fast, to 84h chlamydospore existing 3.16 * 10
7individual/mL, slightly rises to 96h chlamydospore amount, and 3.24 * 10
7individual/mL, in the obviously visible zymotic fluid of naked eyes, hypha material attenuates, and zymotic fluid is thinning, stops fermentation.From seed liquor, to fermentation, stop finishing to need the 6d time, to compare required time relative less with domestic and international research, and chlamydospore output also reaches higher level.
Embodiment 2: vegetable oil is on the chlamydosporic impact of the mould T4 bacterium of wood
The zymotechnique that repeats embodiment 1 just adds the soybean oil of four kinds of concentration in medium, then measures the chlamydospore output of wooden mould T4.
Found that, compare with not adding soybean oil, add soybean oil can obviously promote the chlamydosporic generation of wooden mould T4, along with the increase of soybean oil concentration, the chlamydospore of generation is increased gradually, and under the concentration of 5g/L, chlamydospore number is maximum, reaches 3.3 * 10
7individual/mL, the chlamydospore amount producing under the concentration of 2g/L has also reached 3.28 * 10
7individual/mL, almost approaches the output of 5g/L.Under the concentration of 5g/L, find simultaneously, during microscopy counting, find to still have large oil droplet to exist in zymotic fluid, the concentration excess of 5g/L is described, can not be by the mould whole absorptions of wood, therefore, the interpolation of the soybean oil of 2-3g/L (being 0.2%-0.3%) is beneficial to the chlamydosporic generation of wooden mould T4 most.
Embodiment 3: the preparation of wooden mould wetting powder
Described in embodiment 1, utilize 500L fermentation tank to cultivate and obtain chlamydospore zymotic fluid, condition: 30 ℃, 180r/min, 2.1L/min, after inoculum concentration is 6%, 96 hour a large amount of formation of chlamydospore, fermentation ends.By after zymotic fluid high-speed homogenization (5000r/min homogenate 3 minutes), carrier and 1% (W/W) (in the zymotic fluid gross weight) wetting dispersing agent that adds 10% (W/W), makes wetting powder after dry with spraying dry (condition: 155 ℃ of inlet temperatures, outlet temperature 105-110 ℃, charging rate 4.5L/h).It is detected by wetting powder standard.In table 1, listed the impact of different carriers (precipitated calcium carbonate (1250 order), kaolin (1250 order), white carbon (500 order), talcum powder (800 order), diatomite (500 order), bentonite (325 order)) on the mould vigor of wood.
Table 1
| For examination carrier | Germination rate after 2d (%) 12h | Germination rate after 60d (%) 24h | Bacterium colony daily growth amount (mm/d) | Bacterium colony daily growth amount (adjusting pH) (mm/d) |
| PDA contrast | 99.24±0.80A | 98.36±0.58A | 32.5±0.71 | 32.5±0.71 |
| Kaolin | 86.28±0.32D | 68.72±0.20D | 30.3±0.58 | 31.5±0.71 |
| White carbon | 96.93±0.76B | 91.79±0.32B | 31.5±0.71 | 31.6±0.58 |
| Talcum powder | 88.52±0.65C | 85.22±0.64C | 27.5±0.71 | 29.3±0.58 |
| Diatomite | 81.17±0.53E | 38.48±0.37F | 32.3±0.58 | 32.5±0.71 |
| Bentonite | 62.45±0.56G | 22.41±0.38G | 22.6±0.58 | 20.3±0.58 |
| Precipitated calcium carbonate | 76.34±0.81F | 46.26±0.41E | 7.3±0.58 | 7.5±0.71 |
As can be seen from Table 1, after conidial powder developing agent storage a period of time of carrier, there is variation in the germination rate of spore, and the spore germination rate that white carbon and talcum powder are carrier changes relatively little; No matter bentonite is initial or spore germination rate is all minimum after 60d.In addition, also find that the sprout time of spore all extends to some extent after a period of time generally.From the various bacterium colony daily growth amount treadmill tests for examination carrier, can find out that the pH value of various carriers itself is little on the impact of the mould growth of wood, can get rid of the interference of pH value, also can find out precipitated calcium carbonate can severe inhibition the mould mycelial growth of wood, white carbon, diatomite and kaolin are on the mould mycelial growth of wood almost without impact, and talcum powder and bentonite can suppress the growth of Trichoderma silk to a certain extent.From above-mentioned research, also can find out that identical carrier has influence in various degree to the mould spore of wood and mycelia.
That the germination rate of different disposal of take changes is low, mycelial growth rate is soon for screening foundation.Through comprehensive, analyze, the carrier that can be used as wooden mould wetting powder processing use in test sample has white carbon and talcum powder, kaolin etc.Wherein white carbon water absorbing properties is large, density is lighter, is more preferred Trichoderma chlamydospore zymotic fluid carrier.
Following table 2 has been listed 5 kinds of wetting dispersing agents: EFW (alkylnaphthalene sulfonate, Nanjing Jie Run Science and Technology Ltd.), Tea Saponin (Ningbo associating Bioisystech Co., Ltd), Morwet D-425 (polycondensation naphthalene sulfonate, Nanjing Jie Run Science and Technology Ltd.), T-shaped lignin (Tumen, Jilin dan Xian advance welfare Chemical Co., Ltd.), D110 (polycondensation naphthalene sulfonate, Nanjing Jie Run Science and Technology Ltd.), MT-150 (naphthalene sulfonic acid-formaldehyde condensation product sodium salt, Japanese Kao crystal soda Co., Ltd.) is on take the wetability of the pulvis that White Carbon black is carrier and dispersed impact.
Table 2
Result shows, along with the amount of wetting dispersing agent increases, the wetting time of pulvis reduces, and suspensibility rises to some extent.The principle shorter according to wetting time, wetability better and suspensibility is higher, dispersiveness is better, thinks that EFW is best wetting dispersing agent, 1% and 3% addition, and its wetability, dispersiveness have all met national standard.The chlamydospore mould due to wood is larger, has sedimentation in various degree in suspension, and for increasing its suspending power, the concentration of interpolation 3% is more preferred.
Utilize drying process with atomizing to prepare Trichoderma chlamydospore wetting powder, selecting white carbon is absorption carrier, and addition is 10%; EFW is wetting dispersing agent, and addition is 3%, and after being dried, to it, the standard according to wetting powder detects, and result is as shown in table 3 below.
Table 3
| Project | Measured value | National standard |
| Spore content (individual/g) alive | 1.4×10 8 | ---- |
| Moisture content (%) | 4.6 | 5 |
| PH value | 6.95 | 6.0～7.5 |
| Suspensibility (%) | 48.3 | 34 |
| Wetting time (s) | 55 | 180 |
| Fineness (sieving by 44um) (%) | 97.25 | 95 |
In addition, in the storage of 6 months by a definite date, at 4 ℃, the germination rate of wooden mould wetting powder is still retained in more than 90%, confirm that deepfreeze is conducive to maintain spore vigor, under room temperature condition (20 ℃～25 ℃), the spore rate alive of preparation is with the prolongation slow decreasing of period of storage, and the spore rate of living after 6 months is still up to 62%.Be significantly higher than conidial granula subtilis (30%).
Embodiment 4: the development of fermentation of bacillus subtilis process optimization and wetting powder
Material and instrument
Bacterial classification:
Bacillus subtillis B99-2 (separated acquisition from plant rhizosphere soil, bio-reactor engineering National Key Laboratory of Ke Cong East China University of Science buys).
Medium:
LB plating medium: potato 200g, peeling, section, boils 30min, filters, and adds 20g glucose in filtrate, is settled to 1000mL, agar 20g, pH nature.
SDAY seed culture medium: glucose 20g, peptone 10g, yeast extract 5g, water 1000ml, pH7.0.
Reagent
Carbon source: glucose, soluble starch, maltose, sucrose, corn flour, glycerine, brown sugar, lactose, wheat bran, glutinous rice flour, sorghum flour, wheat flour
Nitrogenous source: peptone, yeast extract, dusty yeast, peanut powder, beancake powder, analysis for soybean powder, casein, urea, ammonium sulfate, potassium nitrate
Mineral salt: MgSO
47H
2o, MnSO
4h
2o, KH
2pO
4, K
2hPO
4
Carrier: precipitated calcium carbonate (1250 order), kaolin (1250 order), white carbon (500 order), talcum powder (800 order), diatomite (800 order), bentonite (325 order).
Auxiliary agent: D110 (polycondensation naphthalene sulfonate); T-shaped sodium lignin sulfonate; CMC (sodium carboxymethylcellulose); PVA (polyvinyl alcohol); PEG 8000; Gum Arabic; Lauryl sodium sulfate (SDS); EFW (alkylnaphthalene sulfonate); MorwetD-425 (polycondensation naphthalene sulfonate); Neopelex; Tween-60; Tea Saponin, My100
Protectant: ascorbic acid (VC), carboxymethyl cellulose (CMC), dextrin, SDS.
Stabilizing agent: calcium carbonate, potassium phosphate, dipotassium hydrogen phosphate, carboxymethyl cellulose
Key instrument equipment: biochemical cultivation case, drying box, three order biomicroscopes, micro-wave oven, thermostat water bath, precision acidity meter, constant temperature blender with magnetic force, micromill.
Method and result
With initial fermentation medium: glucose, peptone, MgSO
47H
2o, MnSO
4h
2o, KH
2pO
4, K
2hPO
4for minimal medium, select respectively industrial conventional and 11 kinds of cheap carbon sources: soluble starch, maltose, sucrose, corn flour, glycerine, brown sugar, lactose, wheat bran, glutinous rice flour, sorghum flour, wheat flour, as carbon source, are replaced the glucose in initial fermentation medium.Form altogether 12 groups, as the medium of different carbon sources.With the medium without carbon source, contrast, each processes 3 repetitions, every 6 hours sampling and measuring thalline biomasss and gemma rate, to determine the impact of different carbon source medium on thalline biomass.Result shows, from thalline biomass (CFU), in these 12 kinds of carbon sources, bacillus subtilis B99-2 the most easily utilizes corn flour, wheat flour.From a large amount of time forming of gemma, the time that wheat bran forms gemma in a large number early, but wheat bran biomass is lower; Although wheat flour biomass is higher, the time that forms gemma is slower, therefore selects corn flour as the carbon source of medium.
The carbon source that previous step is filtered out is replaced the carbon source of initial fermentation medium, select respectively industrial conventional and 9 kinds of cheap nitrogenous sources: yeast extract, dusty yeast, peanut powder, beancake powder, analysis for soybean powder, casein, urea, ammonium sulfate, potassium nitrate, as carbon source, are replaced the peptone in initial fermentation medium.Form altogether 10 groups, as the medium of different nitrogen sources.With the medium without nitrogenous source, contrast, each processes 3 repetitions, every 6 hours sampling and measuring thalline biomasss and gemma rate, to determine the impact of different nitrogen sources source medium on thalline biomass.From thalline biomass (CFU), in these 10 kinds of nitrogenous sources, bacillus subtilis B99-2 the most easily utilizes beancake powder, analysis for soybean powder.Again from a large amount of time forming of gemma, the time that analysis for soybean powder forms gemma in a large number, early and that beancake powder forms the time of gemma was slower, therefore selects analysis for soybean powder as the carbon source of medium.Consider corn flour, analysis for soybean powder is a kind of slow effect carbon source, imitates late nitrogenous source, as adds a small amount of quick-acting carbon source, and quick-acting nitrogenous sources can promote the early growth of thalline, therefore culture medium prescription reserve part glucose adds KNO
3.
Will be by carbon source, in the medium of nitrogenous source screening test screening, add respectively glucose 10g, KNO
310g, glucose 10g and KNO
310g is as quick-acting carbon sources and nitrogenous source, analyze respectively CFU value and the gemma rate situation of change of B99-2 in these three kinds of medium, with quick-acting carbon sources, quick-acting nitrogenous sources all un-added medium contrast, every 6h measure different time CFU amount and gemma rate, to determine that B99-2 is at the growing states quick-acting and medium that effect carbon nitrogen source combines late.
The development of wetting powder
Carrier in wetting powder is very large to the performance impact of preparation.Selection does not have impact or the very little carrier of impact to thalli growth, according to the factor of the aspects such as the adsorption capacity of carrier, mobility, price, determines optimum carrier.Pesticide original medicine is liquid preparation, select the filler that oil absorbency is high, if the former medicine of solid, selects the filler of general oil absorbency, and the sample that selected filler is made is loose mobile dry powder body.
Select white carbon, kaolin, diatomite, bentonite, talcum powder, six kinds of carriers of fine particle calcium carbonate are done filler, contrast, relatively adsorption capacity, price and the impact on gemma content of different carriers with blank.
Table 4
As shown in table 5, white carbon and diatomite performance are more or less the same, but pesticide original medicine is liquid preparation, select the filler that adsorption rate is high, and white carbon absorption property is larger and mobility is better than diatomite.Therefore the sample of selecting white carbon to make as filler is loose easily mobile.
Wetting powder Quality Index Analysis is measured
By the wetting powder of previous step test system, according to < < State Standard of the People's Republic of China > >, measure wettability determination GB 5451-2001; Suspensibility is measured GB/T 14825-1993; Fineness is measured GB/T16150-1995; Determination of moisture GB/T 1600-2001; PH measures GB/T 1601-1993; Accelerated storage test GB/T19136-2003.Other indexs are tested according to pertinent literature method.
Table 5
| Project | State quota value | Practical measurement value |
| Active constituent content (cfu/g) | 1.8×10 9Individual cell/gram | |
| PH scope | 5-7 | 6.8 |
| Moisture (%) | 4 | 3.7 |
| Suspensibility (%) | 70 | 88 |
| Wetting time (s) | 180 | 65 |
| Fineness (%) | 98 | 99 |
| Heat storage test (resolution ratio < 5%) | Qualified | |
| Cold storage stability | Qualified | Qualified |
| Room temperature | Qualified |
Embodiment 5: the preparation of bacillus brood cell and Trichoderma chlamydospore compound formulation
(1) adopt the brood cell of the method cultivation bacillus of liquid state fermentation
Bacterial strain uses therefor: Bacillus subtillis B99-2 (separated acquisition from plant rhizosphere soil, bio-reactor engineering National Key Laboratory of Ke Cong East China University of Science buys).
Fermentation medium: wheat flour 2%, soybean meal 2%, MgSO47H
2o 0.5%, MnSO4H
2o 0.0003%, KH
2pO
40.05%, K
2hPO
40.2%, pH6.5-7.5.
Condition of culture: 25～35 ℃ of temperature, seed inoculum concentration 5～10%, seed concentration 1 * 10
6individual/mL, ventilation ratio 1: 1, tank pressure 0.04MPa, fermentation time is 28～36 hours.
(2) adopt the method for liquid state fermentation to cultivate the mould chlamydospore of wood
Bacterial strain uses therefor: Trichoderma viride (T.viride) T4, separated acquisition from plant rhizosphere soil, bio-reactor engineering National Key Laboratory of Bing Kecong East China University of Science buys, separately can be referring to document (Xia Siqin, Wang Wei, chemistry and biotechnology, " solid fermentation process of Trichoderma viride T4 and the research of preparation stability thereof ", 2008, Vol.25, No.12,52-56 page).
Fermentation medium: wheat bran 0.5%, (NH
4)
2sO
40.2%, KH
2pO
40.1%, CaCO
30.1%, the soybean oil of glucose 2%, 0.2%, pH5.5-6.0.
Condition of culture: 25～33 ℃ of temperature, seed inoculum concentration 5～10%, seed concentration 1 * 10
6individual/mL, ventilation ratio 1: 1, tank pressure 0.04MPa, fermentation time 96 hours.
(3) fermentation liquor treatment
By after the resulting Trichoderma chlamydospore of above-mentioned steps (2) zymotic fluid high-speed homogenization (5000r/min 3 minutes), mix with brood cell's zymotic fluid 1: 1 (V/V) of the resulting bacillus of above-mentioned steps (2).The carrier white carbon powder (500 order) that adds 10% (W/W), and 1% (W/W) wetting dispersing agent alkylnaphthalene sulfonate, fully mix.
(4) dry
The zymotic fluid mixing that step (3) is obtained is sprayed dry, and concrete technology is: 130～170 ℃ of inlet temperatures, outlet temperature 80-120 ℃, make compound wetting powder after being dried.Through dilution-plate method, record, total content can reach 2 * 10
9individual cell/gram wetting powder.
Embodiment 6: biological and ecological methods to prevent plant disease, pests, and erosion test
Materials and methods
Reagent agent: bacillus, the single preparation of Trichoderma and compound wetting powder (embodiment makes); Contrast medicament is 50% carbendazol wettable powder.
For studying thing: Hui Nan town, Nanhui District of Shanghai greenhouse watermelon, kind 8424.
Experimental scheme and application process
Wetting powder processing is carried out in test in the booth of continuous cropping; With 50% carbendazol wettable powder, do medicament contrast, with clear water, make blank.Random district group is arranged, and repeats 4 times, and 2 row (24 strain) are processed in each community.
When administration time is whole ground of watermelon field and after surely growing one week, twice dispenser, executed and filled with March 1 root, 500 times (10 of wettable powder dilution agent respectively at spilling January 30
5individual/mL), carbendazim dilution is 1000 times.Every strain watermelon is executed 100mL, and clear water is all filled with in contrast.
State of an illness investigation
Preharvest period investigate respectively fusarium wilt, climing rot incidence, calculate relative control effect.Fusarium wilt is counted diseased plant rate and control efficiency in morbidity strain.Climing rot is in the climing portion of stem disease index morbidity severity.
The climing part grade standard of climing rot stem:
0 grade-anosis;
There is fusiformis or circular scab in I level-Jie portion;
The skin breakage of II level-scab has amber coloring agent material to overflow, and has the stem of 1/3 length withered;
The skin breakage of III level-scab has amber coloring agent material to overflow, and has the stem of 1/2 length withered;
The skin breakage of IV level-scab has amber coloring agent material to overflow, and has the stem of 2/3 length withered; V level-complete stool is withered.
Disease index=[∑ (diseased plant numbers at different levels * this grade of typical value) ÷ (investigating total strain number * highest typical value)] * 100.
Preventive effect (%)=[(check plot disease refers to-prevent and treat that district's disease refers to) ÷ check plot disease refers to] * 100.
Results and analysis
Preparation is to greenhouse watermelon fusarium wilt control efficiency
With preparation, carry out result of the test demonstration, watermelon growing phase fusarium wilt is had to obvious control efficiency, compound wetting powder preventive effect reaches 84.02%, all better than the control efficiency of independent wooden removing mildew and independent bacillus subtilis formulation.In process of the test, find that the watermelon plant growing way that initial stage preparation is processed is better than control treatment.
The protection effect of table 6 preparation to watermelon blight
| Chemicals treatment | Investigation strain number | Diseased plant number | Diseased plant rate (%) | Relative control effect (%) |
| The mould wetting powder of wood | 94 | 8 | 8.51 | 77.14 |
| Bacillus subtilis wetting powder | 96 | 16 | 16.67 | 55.22 |
| Brood cell and chlamydospore compound formulation | 95 | 5 | 5.26 | 85.87 |
| 1000 times of liquid of carbendazim | 95 | 17 | 17.89 | 51.95 |
| Clear water contrast | 94 | 35 | 37.23 | -- |
Preparation is to the climing rot control efficiency of greenhouse watermelon
The investigation of disease is found, the disease index of clear water contrast is significantly higher than the processing of four kinds of medicaments, use the disease index of brood cell and chlamydospore compound formulation minimum, be only 9.36%, wood removing mildew is 16.29%, bacillus subtilis formulation is 14.22%, and the disease index that bacterium spirit is processed also has 54.73%.Visible compound Wettable Powder is the highest to the preventive effect of climing rot, is 86.67%, is significantly higher than and uses the mould and independent bacillus subtilis formulation of wood separately.
The protection effect of table 7 preparation to the climing rot of watermelon
| Chemicals treatment | Investigation strain number | Disease index (%) | Relative control effect (%) |
| The mould wetting powder of wood | 94 | 16.29 | 78.33 |
| Bacillus subtilis wetting powder | 95 | 14.22 | 81.08 |
| Brood cell and chlamydospore compound formulation | 92 | 9.36 | 87.55 |
| 1000 times of liquid of carbendazim | 95 | 54.73 | 27.18 |
| Clear water contrast | 94 | 75.16 | -- |
From booth test, can find out that compound wetting powder all has good preventive effect to watermelon blight and climing rot, is better than fusarium wilt to the control of climing rot.Carbendazim has certain preventive effect to fusarium wilt, but lower to the preventive effect of climing rot.In experiment, also find, the watermelon seedlings growing way of processing through preparation is obviously healthy and strong, well developed root system, and leaf look dark green, has the effect of obvious promotion watermelon plant strain growth.
Test booth is 2 years melon second crop soils, and upper one year, diseased plant rate can reach more than 50% sometimes, and output is lower.By result of the test, can be found out: can several Wettable Powder all have good preventive effect to the control of watermelon blight and climing rot disease, apparently higher than chemical agent; Preparation has obvious growth promoting function to watermelon seedlings.
Watermelon blight and climing rot are serious plant disease comparatively common in cultivating watermelon, fusarium wilt is the soil-borne disease that a kind of canonical system infects, in watermelon continuous crop situation, and susceptible disease more, the general incidence of disease of second crop soil is in 30% left and right, when serious up to more than 80%.Climing rot is commonly called as " dead arm ", and at China's growth of watermelon, all there is generation in district.According to reports, the climing rot diseased plant rate of watermelon is generally 15%～25%, when serious, up to 60%～80%, can make melon patch occur a large amount of dead rattans when disease is popular, and the underproduction is more than 30%.The output that has had a strong impact on watermelon and the quality of disease.
This result of the test shows, bacillus, the compound chlamydospore wetting powder of Trichoderma all have good inhibitory action to watermelon blight and climing rot.In addition, compare with the result of alone bacillus subtilis wetting powder and alone Trichoderma chlamydospore wetting powder, adopt the result of bacillus of the present invention, the compound chlamydospore wetting powder of Trichoderma to be significantly improved.
In addition, find that the control efficiency of chlamydospore wetting powder is better than conidium granula subtilis, reason may be to belong to chlamydospore survival ability and colonization ability in soil to be better than conidium, generally at least can survive 20 months; Even same bacterial strain, Spore Types is different, not of uniform size, also different to Soil fungistasis sensitivity, and individual less spore is more responsive to the mycostasis of soil.
Although the invention describes concrete example, having is a bit significantly to those skilled in the art, can the present invention be made various changes and be changed under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.All publications, patent and the patent application of quoting herein all included in for referencial use herein.
Claims (6)
1. the compound formulation for controlling plant diseases, it is characterized in that, described compound formulation comprises bacillus brood cell and Trichoderma chlamydospore, described compound formulation is wetting powder, described compound formulation comprises carrier and wetting dispersing agent, described carrier is selected from White Carbon black or diatomite, and described wetting dispersing agent is selected from alkylnaphthalene sulfonate, and bacillus brood cell's content described in described compound formulation is 10
8-10
10individual cell/gram, the chlamydosporic content of described Trichoderma is 10
8-10
9individual spore alive/gram.
2. compound formulation as claimed in claim 1, is characterized in that, described wetting powder makes in order to below method:
(1) provide containing bacillus brood cell's zymotic fluid and containing the chlamydosporic zymotic fluid of Trichoderma;
(2) will then mix with the zymotic fluid containing bacillus brood cell containing the chlamydosporic zymotic fluid homogenate of Trichoderma, obtain compound formulation;
The compound formulation of step (2) gained is further dried and makes described wetting powder,
And also comprise the step that is mixed into carrier and wetting dispersing agent in described step (2), the content of described carrier is 5-30% (W/W), the content of described wetting dispersing agent is 0.5-5% (W/W), and before the wetting powder of take is dry, the weight of zymotic fluid is benchmark.
3. a method of preparing compound formulation described in claim 1, the method comprises the following steps:
(1) provide containing bacillus brood cell's zymotic fluid and containing the chlamydosporic zymotic fluid of Trichoderma;
(2) will then mix with the zymotic fluid containing bacillus brood cell containing the chlamydosporic zymotic fluid homogenate of Trichoderma, obtain described compound formulation,
Described compound formulation is wetting powder, it is by the compound formulation of step (2) gained is further dried and is made, and also comprise the step that is mixed into carrier and wetting dispersing agent in described step (2), described carrier is selected from White Carbon black powder or diatomite, and described wetting dispersing agent is selected from alkylnaphthalene sulfonate.
4. method as claimed in claim 3, is characterized in that, obtaining with following methods containing the chlamydosporic zymotic fluid of Trichoderma in described step (1):
In fermentation medium, 25～33 ℃ of temperature, seed inoculum concentration 5～10%, seed concentration 10
5-10
6individual/mL, ventilation ratio 1:2 to 2:1, under tank pressure 0.03-0.05MPa, fermentation 84-96 hour, obtains containing the chlamydosporic zymotic fluid of Trichoderma, and the composition of wherein said fermentation medium is: wheat bran 0.4-0.6%, (NH
4)
2sO
40.1-0.3%, KH
2pO
40.1-0.2%, CaCO
30.1-0.2%, glucose 1-3%, the soybean oil of 0.2-0.5%, pH5.5-6.0.
5. method as claimed in claim 3, is characterized in that, the content of described carrier is 5-30% (W/W), and the content of described wetting dispersing agent is 0.5-5% (W/W), and the weight of zymotic fluid of take is benchmark.
6. the application of compound formulation claimed in claim 1 in controlling plant diseases, described plant disease is selected from watermelon blight and climing rot.
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| CN102499265B (en) * | 2011-11-10 | 2013-08-28 | 宁夏农林科学院 | Trichoderma hamatum biological agent for controlling soil borne disease for greenhouse vegetable cultivation |
| CN102718577B (en) * | 2012-06-15 | 2013-10-30 | 山东光大肥业科技有限公司 | Special organic culturing substrate capable of preventing and treating diseases for peppers and preparation method of special organic culturing substrate |
| CN102870822B (en) * | 2012-10-29 | 2015-01-14 | 黄永 | Aqueous dispersible granules of trichoderma asperellum chlamydospores biopesticide and preparation of aqueous dispersible granules |
| CN103704273B (en) * | 2014-01-13 | 2015-08-12 | 湖南农业大学 | Raw Brevibacillus brevis wetting powder in a kind of |
| CN104322564B (en) * | 2014-10-11 | 2017-01-11 | 河北百奥生物制品有限公司 | Trichoderma chlamydospore wettable powder composition and preparing method thereof |
| CN104872200A (en) * | 2015-04-21 | 2015-09-02 | 苏州市新泾村农业基地专业合作社 | Biological pesticide suitable for strawberry powdery mildew and preparation method of biological pesticide |
| CN105165824A (en) * | 2015-09-21 | 2015-12-23 | 南京农业大学 | Solid carrier or carrier liquid for producing agricultural microbial preparation |
| SG11202010778TA (en) * | 2018-05-08 | 2020-11-27 | Locus Agriculture Ip Co Llc | Microbe-based products for enhancing plant root and immune health |
| CN110907600A (en) * | 2019-11-22 | 2020-03-24 | 云南省烟草公司临沧市公司 | Method for measuring tobacco planting soil micro-ecological environment |
| CN111088191B (en) * | 2020-01-08 | 2021-04-02 | 华东理工大学 | Bacillus and trichoderma combined culture method and application |
| CN113604394B (en) * | 2021-08-19 | 2024-04-09 | 山东京青农业科技有限公司 | Bacillus subtilis water dispersible granule and preparation method and application thereof |
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| CN1778886A (en) * | 2004-11-17 | 2006-05-31 | 杨谦 | Production of insect-proof sick wooden mold and its use |
| CN101124915A (en) * | 2007-10-12 | 2008-02-20 | 沈阳农业大学 | Biocontrol bacteria compound agent for preventing and controlling crop soil-infectious diseases and preparation method |
| CN101352177A (en) * | 2008-06-30 | 2009-01-28 | 康坦生物技术（山东）有限公司 | Compound successive crop-resistance micro-ecological formulation special for cotton and special bacterial strain and use thereof |
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| CN101124915A (en) * | 2007-10-12 | 2008-02-20 | 沈阳农业大学 | Biocontrol bacteria compound agent for preventing and controlling crop soil-infectious diseases and preparation method |
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