CN104286032A - Preparation method of Talaromyces flavus spore powder, Talaromyces flavus wettable pulvis and preparation method of wettable pulvis - Google Patents
Preparation method of Talaromyces flavus spore powder, Talaromyces flavus wettable pulvis and preparation method of wettable pulvis Download PDFInfo
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Abstract
The invention relates to a Talaromyces flavus wettable pulvis. The Talaromyces flavus wettable pulvis includes Talaromyces flavus conidiospore powder, a carrier and an assistant, the content of living bacteria in the Talaromyces flavus wettable pulvis is 5*10<8>-5*l0<9>cfu/g/g, the suspension rate of the Talaromyces flavus wettable pulvis is greater than 75% wettable powder, the wetting time is less than 60s, and the water content is less than 6%. In the invention, the spore fermentation technology of Talaromyces flavus aw12 strains is optimized; and the Talaromyces flavus wettable pulvis is created by using Talaromyces flavus spores as an active component. The invention provides a production method of a Talaromyces flavus biocontrol preparation, and a new convenient and practical dosage form. The Talaromyces flavus spore powder preparation method lays a foundation for the large scale commercialization production of the Talaromyces flavus wettable pulvis, is in favor accelerating the application process of Talaromyces flavus in the biological control, and is of theoretic and practical significance.
Description
Technical field
The present invention relates to microbial pesticide technical field, in particular to a kind of microbial pesticide wetting powder and preparation method thereof, more particularly, the present invention relates to a kind of yellow basket bacterium conidial powder fermentation and preparation method, and this yellow basket bacterium wetting powder and preparation method thereof.
Background technology
Agricultural chemicals, at control plant pest, ensures that agriculture stable yields and volume increase, the quality of raising agricultural product, the living standard of improving the people play an important role.Chemical pesticide has played important effect at control corps diseases and protecting agriculture secure context; but result also in a series of environment and social concern; as " 3R " problem that extensive use chemical pesticide occurs etc., have a strong impact on the sustainable development of modern agriculture.
Microbial pesticide is the preparation can preventing and treating crop disease, worm and weeds made with biological living or metabolite, the object of control is not easy to develop immunity to drugs, selectivity is strong, to people and animals and natural enemy safer, breeding is very fast, agricultural byproducts, industrial and agricultural wastewater and discarded object etc. can be utilized to carry out widespread production, is the first-selected medicament producing pollution-free food.At present, having developed becomes product, drops into the actual insecticidal bacteria used and mainly contain 4 kinds, and namely Bacillus sphaericus, Bacillus popilliae, bacillus thuringiensis are eased up disease bacilli; The disinsection fungal recorded have 100 multiple genus, more than 800 plant, most study be white muscardine fungi and green muscardine fungus.Trichoderma (Trihoderma) has good inhibitory action to plant pathogenic fungi, is the fungi bactericide of most study, has been developed as Trichoderma pesticidal preparations.
For the ease of the preservation of microbial pesticide, transport and use, improve drug effect, the preparation of certain formulation need be made into.Microbial pesticide processing is than chemical pesticide difficulty, and first the active ingredient of microbial pesticide is generally the organism lived, and its hydrophobicity directly has influence on the physical properties such as the dispersiveness of preparation, suspension and wetability; Secondly the biocompatibility of microorganism and each analog assistant is general all poor; Organism is more responsive to environment such as humidity, temperature and light photographs in addition, preparation stored poor stability, needs to add the auxiliary agents such as protectant.
The formulation of microbial pesticide contains wetting powder, granule, suspending agent, fine granule etc.Wetting powder (Wettable Powders, WP) possesses use, storage and convenient transportation, the feature such as do not add organic solvent and process operation is relatively easy, is the first-selected formulations of people in formulation development.Wetting powder is made up of auxiliary agents such as former medicine, carrier and surfactants, and auxiliary agent has material impact to pesticidal preparations, directly has influence on pesticide efficacy.Especially in fungal spore preparation, to auxiliary agent, there is higher requirement, except the function admirable requiring auxiliary agent, also must can not affect the sprouting of fungal spore and the growth of mycelia, therefore screen the auxiliary agent with fungi with excellent biocompatibility particularly important to the development of microbial inoculum, the auxiliary agent be suitable for for specific bacterial strain screening is the key that fungi wetting powder is produced.
Yellow basket bacterium (Talaromyces flavus) has hyperparasitism to the sclerotium that the multiple pathogen such as sclerotinite (Sclerotinia sclerotiorum), Sclerotium rolfsii (Sclerotium rolfsii), Rhizoctonia solani Kuhn (Rhizoctonia solani) produces, and has obvious inhibitory action in addition to verticillium dahliae (Verticillum dahliae) and Fusarium oxysporum (Fusarium oxysporum f.sp.vasinfectum) etc.This bacterium Biocontrol Mechanism is various, mainly comprises hyperparasitism, Competition, bacteriolysis etc.; Wherein hyperparasitism is yellow basket bacterium is most important mechanism of action, in doing mutually with host fungi, can be induced to produce multiple glucose oxidase and the hydrolases such as chitinase, cellulase, β-1,3-dextranase.Yellow basket bacteria growing temperature and pH's is in extensive range, can survive under the ecological condition that China south and north is different and breed, obvious preventive and therapeutic effect is had to the important fungal diseases of plants such as stalk break and verticillium wilt, be a kind of important bio-control factors, there is good biological and ecological methods to prevent plant disease, pests, and erosion application prospect and researching value.
Yellow basket bacterium (Talaromyces flavus), as one of important biocontrol microorganisms, domesticly at present there is no the corresponding formulation development research of this bacterium, greatly constrains the using and promoting in biological control of yellow basket bacterium.We from soil separation screening to the yellow basket bacterium qw12 of a strain, (deposit number is: CGMCC 5067, the characterizing gene GenBank number of logging in: JN602366), it has very strong inhibitory action to plant pathogenic fungis such as willow rotten fungus, Rhizoctonia solani Kuhn and Rhizoctonia solani Kahn.
Summary of the invention
For the defect existed in prior art, the object of the present invention is to provide a kind of yellow basket bacterium conidial powder fermentation and preparation method, and this yellow basket bacterium wetting powder and preparation method thereof.
Technical scheme of the present invention is as follows:
A kind of yellow basket bacterium wetting powder, it comprises yellow basket bacterium (Talaromyces flavus) conidial powder, carrier and auxiliary agent, and the viable bacteria content in the basket bacterium wetting powder of described yellow is 5 × l0
8cfu/g ~ 5 × l0
9cfu/g, the suspensibility of the basket bacterium wetting powder of described yellow is greater than 75%, and wetting time is less than 60 seconds, and water content is less than 6%.
The percentage by weight of the basket bacterium conidial powder of described yellow in wetting powder is 5-50%.Described carrier be selected from diatomite, bentonite, kaolin one or more, the percentage by weight of carrier in wetting powder is 40% ~ 93%; Described auxiliary agent comprises wetting agent, dispersant and uv-protector; Described wetting agent is selected from the one in sodium lignin sulfonate, Tween 80, and the percentage by weight of wetting agent in wetting powder is 1% ~ 5%; Described dispersant is polyethylene glycol, and the percentage by weight of dispersant in wetting powder is 0.5% ~ 2%; Described uv-protector is selected from the one of dextrin and methylcellulose, and the percentage by weight of uv-protector in wetting powder is 0.5% ~ 2%.
The basket bacterium wetting powder of yellow as above, concrete preparation method is as follows:
(1) each component in wetting powder is taken according to quantity;
(2) carrier is first pulverized with cracker, cross 325 mesh sieves;
(3) successively basket to carrier, wetting agent, dispersant, uv-protector and yellow bacterium conidial powder is fully mixed in proportion, namely obtain yellow basket bacterium wetting powder.
On the basis of such scheme, the basket bacterium of described yellow is yellow basket bacterium qw12.
On the basis of such scheme, in the basket bacterium of described yellow (Talaromyces flavus) conidial powder, yellow basket bacterium conidium content is 5 × 10
9~ 1.42 × 10
11individual/g, water content is less than 12%, and spore rate of living is greater than 90%.
Described yellow basket bacterium conidial powder is after the solid fermentation of yellow blue shape bacterium terminates, and solid culture is dry, pulverizing is rear crosses 200 mesh sieves; Or with the basket bacterium conidial powder of the yellow of fungal spore separator separated and collected.
Utilize yellow basket bacterium qw12 to prepare a fermentation culture method for conidial powder, solid matrix/water=10/4 ~ 10/10 in medium, carbon source is 0.5% ~ 4% of matrix, and ammonium sulfate is 0.1% ~ 4% of matrix, and mineral salt are 0.5 ‰ ~ 5 ‰ of matrix.
Described solid matrix is wheat bran and corn stalk powder, and wherein the percentage by weight of wheat bran in solid matrix is 20% ~ 100%; Described carbon source be selected from glucose, maltose one or both; Described mineral salt are selected from MgSO
4, ZnSO
4, FeSO
4, CuSO
4in one or both combination.
Fermentation condition is the inoculum concentration of yellow basket bacterium qw12 seed is 3% ~ 22%, 12h ~ 24h illumination every day, and 25 ~ 30 DEG C, fermentation time is 5 ~ 13d,
After fermentation ends, solid culture is dry, pulverizing is rear crosses 200 mesh sieves; Or with the basket bacterium conidial powder of the yellow of fungal spore separator separated and collected.
On the basis of such scheme, basket bacterial classification of described yellow is concentration 1 × 10
6individual/mL ~ 1 × 10
8the basket bacterium spore suspension of yellow of individual/mL or 1 × 10
6cfu/mL ~ 1 × 10
9the basket bacteria culture fluid of yellow of cfu/mL.
Present invention optimizes the spore zymotechnique of yellow basket bacterium qw12 bacterial strain; With the basket bacterium spore of yellow for active component, formulate out yellow basket bacterium wetting powder, provide the production technology of a kind of yellow basket bacterium biological prevention and control agent and convenient and practical novel form.This invention is that yellow basket bacterium wettable powder large-scale commercial production is laid a good foundation, and is conducive to accelerating yellow basket bacterium and applies process in biological control, have most important theories and practice significance.
Embodiment
Embodiment 1
Yellow basket bacterium qw12 solid fermentation condition
1, solid fermentation matrix is determined
The medium of 3 kinds of one matters is set respectively: wheat bran, corn stalk powder, corn flour; The medium of 4 kinds of compounding substances: wheat bran+corn stalk powder, wheat bran+corn flour, corn stalk powder+corn flour, wheat bran+corn stalk powder+corn flour, the heterogeneity equal proportion mixing in compounding substances medium.Material-water ratio=1:1,121 DEG C of sterilizing 30min, inoculation 1 × 10
6the spore suspension of individual/mL, inoculum concentration 5%; Cultivate 7d for 28 DEG C, turn over bent 1 time every day.Three repetitions are established in each process.Get 0.5g solid fermentation culture after fermentation ends, add sterile water, magnetic stirring apparatus stirs 5min, add up the number of spore with blood counting chamber under the microscope.Result shows: when medium is single-matrix, and wheat bran effect is best, and sporulation quantity reaches 5.66 × 10
9individual/g; In mixed-matrix, wheat bran and powder of straw combined effect the best, sporulation quantity reaches 6.10 × 10
9individual/g (table 1); Further research finds (table 2), and it is best that wheat bran and powder of straw press 4:1 proportioning effect, and sporulation quantity can reach 7.80 × 10
9individual/g.
The different solid culture medium of table 1 is on the impact of sporulation quantity
The different ratio medium of table 2 wheat bran and maize straw affects sporulation quantity
2, material-water ratio is on the impact of sporulation quantity
In solid fermentation basal medium, set material-water ratio to test as 10:3,10:4,10:5,10:6,10:7,10:8,10:9,10:10 respectively, 3 repetitions are established in each process, and counting spore count also calculates average sporulation quantity.Result of the test shows (as table 3), is that the process sporulation quantity of 5:4 is the highest with material-water ratio.
Table 3 material-water ratio is on the impact of sporulation quantity
3, mineral element is to the facilitation of producing spore
The mineral element MgSO of solid matrix 1 ‰ is added respectively in solid fermentation basal medium
4, ZnSO
4, FeSO
4, CuSO
4, CaSO
4, K
2sO
4if do not add mineral element in contrast, often kind processes repetition 3 times, calculates average sporulation quantity under microscope.Result is as shown in table 4, and the impact of different mineral elements on the sporulation quantity of yellow basket bacterium qw12 of interpolation is different, and add Cu element and produce the facilitation of spore significantly to this bacterium, sporulation quantity is the highest; And add 3 ‰ CuSO in the medium
4for best (table 5).
Table 4 difference produces spore promoter to the impact of sporulation quantity
Table 5 CuSO
4concentration is on the impact of sporulation quantity
4 add the impact of carbon source on sporulation quantity
In solid-based basal culture medium, press solid matrix quality 1% adds additional carbon sucrose, glucose, maltose 3 kinds of carbon sources; Contrast is the solid-based basal culture medium of not additional carbon.Compared with the control, the sporulation quantity of additional carbon bacterial strain increases to some extent, and the sporulation quantity adding the medium of glucose is the highest, is secondly maltose, as shown in table 6.
When keeping fermentation condition constant, press 0.5% of solid matrix quality, 1%, 2%, 3%, 4% respectively and add glucose, as shown in table 7, the glucose of variable concentrations is different on the impact of yellow basket bacterium qw12 sporulation quantity, the sporulation quantity of the yellow basket bacterium of the increase along with glucose addition raises successively, and addition more than 3% time sporulation quantity reduce, therefore to add the best results of 3% glucose.
Table 6 different carbon source is on the impact of sporulation quantity
Table 7 concentration of glucose is on the impact of sporulation quantity
5, additional nitrogenous source is on the impact of sporulation quantity
Press 1% of solid matrix quality at solid-based basal culture medium and add inorganic nitrogen-sourced (ammonium nitrate, ammonium sulfate, urea) and organic nitrogen source (yeast extract, peptone, beancake powder), contrast is the solid-based basal culture medium of not additional nitrogenous source, and keeps other fermentation condition constant.The highest with the sporulation quantity of the medium adding ammonium sulfate in six kinds of additional nitrogenous sources, the sporulation quantity of other process is lower than the sporulation quantity of control strain, as shown in table 8.When keeping fermentation condition constant, press solid matrix quality in the medium respectively 0.1%, 0.5%, 1%, 2%, 3%, 4% adds ammonium sulfate, as shown in table 9, the ammonium sulfate of variable concentrations is different on the impact of yellow basket bacterium qw12 sporulation quantity, the sporulation quantity of the yellow basket bacterium of the increase along with ammonium sulfate addition raises successively, and addition more than 3% time sporulation quantity reduce, therefore to add the best results of 3% ammonium sulfate.
Table 8 different nitrogen sources is on the impact of sporulation quantity
Table 9 ammonium sulfate concentrations is on the impact of sporulation quantity
6, inoculum concentration is on the impact of sporulation quantity
Inoculate by 3%, 5%, 10%, 14%, 18%, 22% inoculum concentration respectively, result of the test shows, ideal when in solid fermentation basal medium, inoculum concentration is 18%, as shown in table 10.
Table 10 inoculum concentration affects sporulation quantity
7, illumination condition is on the impact of sporulation quantity
Postvaccinal solid fermentation basal medium is placed in respectively illumination condition is 24h illumination, cultivates under dark and 12 h light of 12h, 24h dark condition, counted under microscope spore count also calculates average sporulation quantity.Wherein 24h illumination can significantly improve the sporulation quantity of yellow basket bacterium qw12, as shown in table 11.
Table 11 illumination affects sporulation quantity
8, medium orthogonal experiment screening
Select wheat bran and corn stalk powder ratio, material-water ratio, ammonium sulfate and inoculum concentration to do the orthogonal experiment (table 12) of 4 factor 3 levels (L9 (34)), optimize and produce spore condition of culture.
Table 12 quadrature factor test level designs
In selected horizontal extent, factor A, B, C, D affect size to sporulation quantity and are followed successively by: D ﹥ B ﹥ A ﹥ C, optimum combination is A
3b
2c
1d
3(table 13), through further experiment sieving and checking, to obtain optimum formula be wheat bran ratio is 100%, and material-water ratio is 10:7, and inoculum concentration is 14%, and ammonium sulfate concentrations is 3%, and final spore amount reaches 8.39 × 10
10individual/g.
Table 13 (L
9(3
4)) orthogonal design and result
9, the yellow basket bacteria solid fermentation time is determined
Adopt the yellow basket bacterium of the fermentation medium fermented and cultured after orthogonal optimization, observe the upgrowth situation of bacterium every day, and detect sporulation quantity, determine best fermentation time.Fruit is as shown in table 14, and the sporulation quantity of the 10th day yellow basket bacterium is maximum.
Table 14 sporulation quantity situation over time
Embodiment 2: the preparation of yellow basket bacterium conidial powder
The basket bacterium of the yellow optimized produces spore medium sterilizing 20-30min at 121 DEG C and inoculates 10
6the spore suspension of individual/mL, 28 DEG C of dark culturing 10d.After fermentation ends, solid culture medium is dried moisture in 35 DEG C of baking ovens, after solid culture drying, pulverizing, cross 200 mesh sieves; Or be separated spore with fungal spore separator, cross 200 mesh sieves; The basket bacterium conidial powder of yellow collected.
Taking the yellow basket bacterium conidial powder of 0.05g is suspended from 20mL sterile water, after magnetic stirrer 30min, measures spore suspension concentration with blood counting chamber, calculates the spore content of yellow basket bacterium conidial powder.Repeat for 3 times, calculate the spore content that conidial powder is average.
Take the yellow basket bacterium conidial powder of 1g after 100 DEG C of baking 2h, make it naturally cool, again basket for yellow bacterium conidial powder is weighed, repeat 3 times, calculate water content mean value.
Spore rate of living measures: conidial powder sterile water being mixed with concentration is 10
6the suspension of individual/ml, draw in this suspension of 1mL access 9mL germination fluid, be placed in 28 DEG C of shaking tables, after 120r/min shaken cultivation 24h, dilution culture fluid (about the conidium of 100, each visual field), under the microscope with sprouting in blood counting chamber record culture fluid and the spore count do not sprouted, repeated sampling 3 times, calculate the average germination rate of spore, be spore rate alive.Spore germination standard is that germ tube length is more than or equal to 1/2 of spore diameter.Spore germination formula of liquid is 1% tryptone, 2% glucose, sterile water 1000mL, 105 ~ 115 DEG C of sterilizing 15min.
Result shows, solid culture is dry, pulverizing is rear crosses 200 mesh sieves, and yellow basket bacterium conidial powder spore content is 5 × 10
9~ 1.2 × 10
10individual/g, water content is 10%, and spore rate of living is 92.3%; Be separated spore with fungal spore separator, cross 200 mesh sieves, the yellow basket bacterium conidial powder spore content of collection is 9.8 × 10
9~ 1.0 × 10
11individual/g, water content is 9.7%, and spore rate of living is 95.31%, can be used for the processing of follow-up wetting powder.
Embodiment 3: the screening of auxiliary agent
The screening of 1 carrier
Carrier is on the impact of the basket bacterium spore germination of yellow: sterilizing after kaolin, talcum powder, bentonite, diatomite mix with PDA medium according to the concentration ratio of 5mg/mL, 10mg/mL, and aseptically makes flat board.Get 1.0 × 10
3the yellow basket bacterium qw12 spore suspension 0.1mL of individual/ml is in being coated with containing on the flat board of different carriers, DNAcarrier free PDA medium is contrast, each process arranges 4 repetitions, cultivates, observe and count at the clump count formed containing spore germination on the flat board of different carriers after 48h at 28 DEG C.
Carrier is on the impact of yellow basket bacterium mycelial growth: the center yellow for diameter 0.5cm basket bacterium qw12 bacterium block being placed in the flat board containing different carriers that said method makes, with DNAcarrier free PDA medium for contrast, each process repeats for 4 times, cultivate at 28 DEG C, measure colony diameter by right-angled intersection method after 72h, calculate the average daily growth rate of bacterium colony.
Colony growth speed=(final colony diameter-originally colony diameter)/colony growth number of days
Result shows: different carriers there are differences (table 15, table 16) the average daily growth rate of the basket bacterium of yellow and Spore Germination, and diatomite, bentonite, kaolin can be used as the carrier of yellow basket bacterium wetting powder, and wherein diatomite is more excellent.
Table 15 carrier is on the impact of the basket bacterium mycelial growth of yellow
Table 16 carrier is on the impact of the basket bacterium spore germination of yellow
Note: the clump count cfu value that spore germination is formed is represented by mean+/-standard error (SE).Same auxiliary agent different quality concentration represents with capitalization the difference that the basket bacterium spore germination of yellow forms bacterium colony, and capitalization difference represents significant difference (P≤0.05).The different auxiliary agent of same mass concentration represents with lowercase the difference that the basket bacterium spore germination of yellow forms monospore clump count, and minuscule difference represents significant difference (P≤0.05), lower same.
2, the screening of wetting agent
Sterilizing after wetting agent lauryl sodium sulfate, neopelex, sodium lignin sulfonate and Tween 80 mix with PDA medium according to the concentration ratio of 500 μ g/mL, 1000 μ g/mL, 2000 μ g/mL respectively, makes flat board.The spore suspension of the yellow basket bacterium qw12 of coating inoculation, with the PDA medium without wetting agent for contrast, each process arranges 4 repetitions, cultivates at 28 DEG C, observe after 48h and count at the clump count formed containing spore germination on the flat board of different wetting agent, measuring the biocompatibility of wetting agent.The assay method of mycelial growth rate measures with carrier mycelia.Wetting time measures according to GB/T5451-2001.
With biocompatibility and wetting time for discriminant criterion, sodium lignin sulfonate, Tween 80 can be used as the wetting agent of yellow basket bacterium wetting powder; Wherein Tween 80 is more excellent, can promote mycelial growth, minimum to Spore Germination, wetting time is the shortest (table 17-19).
Table 17 wetting agent is on the impact of the basket bacterium mycelial growth of yellow
Table 18 wetting agent is on the impact of the basket bacterium spore germination of yellow
Table 19 wetting time measurement result
3, the screening of dispersant
Sterilizing after polyethylene of dispersing agent alcohol, polyethylene glycol mix with PDA medium according to the concentration ratio of 500 μ g/mL, 1000 μ g/mL, 2000 μ g/mL respectively, makes flat board.The same wetting agent of biocompatibility assay method.
The mensuration of dispersion force: the mother liquor above-mentioned dispersant being mixed with 500 μ g/mL, 1000 μ g/mL, 2000 μ g/mL concentration, prepares yellow basket bacterium qw12 spore suspension, makes its concentration be l0
7individual/ml, get this suspension of 10mL and join in 90mL mother liquor, fully shake up, the upper, middle and lower layer from suspension after 0h, 1h, 4h samples respectively, counts under the microscope with blood counting chamber, not add dispersant solution in contrast.And calculate dispersion index (I) according to the following formula:
-average of samples; σ-standard deviation
I=1, spore random distribution; I>1, spore Assembled distribution, works as I<1, and spore is uniformly distributed.
With biocompatibility and dispersion force for discriminant criterion, polyethylene glycol can promote mycelial growth, dispersibility minimum to Spore Germination more by force, relatively stable, determine that wetting agent is polyethylene glycol.
The different dispersant of table 20 is on the impact of the basket bacterium mycelial growth of yellow
The different dispersant of table 21 is on the impact of the basket bacterium spore germination of yellow
Table 22 dispersant is to the dispersive property of the basket bacterium spore of yellow
4, the screening of uv-protector
Sterilizing after uv-protector dextrin, humic acid, methylcellulose, xanthans, trehalose mix with PDA medium according to the concentration ratio of 500 μ g/mL, 1000 μ g/mL, 2000 μ g/mL respectively, makes flat board.The same wetting agent of biocompatibility assay method.
The mensuration of protectant protection usefulness: the spore suspension configuring yellow basket bacterium qw12, concentration is 1.0 × 10
3individual/ml; this suspension is coated on the PDA flat board containing uv-protector; under 30W, light intensity are the uviol lamp of 120lx, 20cm place uncaps and irradiates after dull and stereotyped 30s, the flat board of this process of dark culturing at 28 DEG C, observes and count the clump count that spore germination formed after 48h.Filter out the uv-protector of protection usefulness the best, more different concentration gradients is set, by the method for spore germination, determine most suitable ratio.To carry out ultraviolet irradiation but not add protectant flat board for contrasting 1 (being used for calculating Residue viability); not dull and stereotyped for contrasting 2 (be used for the theory of computation live spore rate) with protectant not carry out ultraviolet irradiation yet; each process arranges 4 repetitions, and calculates protection efficiency index according to the following formula:
Protection efficiency index=(spore rate-Residue viability of living after irradiating)/(theoretical spore rate-Residue viability of living)
In the methylcellulose medium of 3 kinds of concentration, the mycelial growth rate of yellow basket bacterium qw12 is very fast, reaches (1.003 ± 0.017) cm/d during 2000 μ g/mL.Dextrin and trehalose also can promote yellow basket bacterium qw12 mycelial growth, and with to compare difference remarkable, xanthans and humic acid have significant inhibitory action to mycelial growth.The spore of yellow basket bacterium qw12 can better sprouted containing on five kinds of uv-protector medium; containing on xanthans, dextrin, methylcellulose, humic acid medium; the germination rate of spore increases along with the increase of concentration, the highest containing the germination rate of spore on the methylcellulose medium of 500 μ g/mL.Xanthans, dextrin, trehalose, methylcellulose and contrast difference are not remarkable, have good compatibility.5 kinds of uv-protectors there are differences between yellow basket bacterium qw12 conidial protection efficiency index.The protection usefulness of dextrin and methylcellulose is better, is up to 0.3 (table 23).
The protection usefulness of the different uv-protector of table 23
Embodiment 4: the quality testing of yellow basket bacterium wetting powder
1, the preparation of yellow basket bacterium wetting powder: conidial powder 5%, 10%, 30%, 50%, wetting agent Tween 80 is 3%, dispersant polyethylene glycol 1%, uv-protector methylcellulose 1%, and carrier diatomaceous earth supplies 100%; Carrier and solid additive are first pulverized, crosses 325 mesh sieves; Various solid additive joins in diatomite in proportion and mixes, and then adds wetting agent mixing, is finally joined in mixture by basket for the yellow of drying bacterium conidial powder and mix, namely obtain yellow basket bacterium wetting powder.
2, quality testing
Spore content measures: with embodiment 2.
The mensuration of water content: the vessel of test are weighed after 120 DEG C of baking ovens dry 15min, take the yellow basket bacterium wetting powder of 5g after 120 DEG C of baking 2h, make it naturally cool, then the vessel of basket for yellow bacterium conidial powder and oven dry are weighed, repeat 3 times, calculate water content mean value.Water content (%)=[(quality after 5-oven dry)/5] × 100
Wetting time measures the time measuring complete wetting with reference to GB/T5451-2001.
Suspensibility measures and measures spore suspension rate with reference to GB/T14825-2006.
PH measures: take 2g wetting powder and be dissolved in 100mL distilled water, stir 1min rapidly with magnetic stirring apparatus, and then standing 1min, the pH value of working sample is carried out with pH meter, each sample repeats 3 times, and each result difference absolute value is less than 0.1, and calculating mean value.
Spore content of living in yellow basket bacterium wetting powder is 8.4 × 10
8cfu/g ~ 9 × 10
9cfu/g, water content is 0.96% ~ 4.89%, and wetting time is 17s ~ 23s, and suspensibility is 81.73% ~ 86.46%, and pH value is 7.27 ~ 7.49, and above index meets the relevant criterion (table 24) of country.
The indices of the yellow basket bacterium wetting powder of table 24 measures
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (6)
1. the basket bacterium wetting powder of yellow, is characterized in that, it comprises yellow basket bacterium (Talaromyces flavus) conidial powder, carrier and auxiliary agent, and the viable bacteria content in the basket bacterium wetting powder of described yellow is 5 × l0
8cfu/g ~ 5 × l0
9cfu/g, the suspensibility of the basket bacterium wetting powder of described yellow is greater than 75%, and wetting time is less than 60 seconds, and water content is less than 6%.
The percentage by weight of the basket bacterium conidial powder of described yellow in wetting powder is 5 ~ 50%.Described carrier be selected from diatomite, bentonite, kaolin one or more, the percentage by weight of carrier in wetting powder is 40% ~ 93%; Described auxiliary agent comprises wetting agent, dispersant and uv-protector; Described wetting agent is selected from the one in sodium lignin sulfonate, Tween 80, and the percentage by weight of wetting agent in wetting powder is 1% ~ 5%; Described dispersant is polyethylene glycol, and the percentage by weight of dispersant in wetting powder is 0.5% ~ 2%; Described uv-protector is selected from the one in dextrin and methylcellulose, and the percentage by weight of uv-protector in wetting powder is 0.5% ~ 2%.
2. the basket bacterium wetting powder of yellow according to claim 1, is characterized in that concrete preparation method is as follows:
(1) each component in wetting powder is taken according to quantity;
(2) carrier and solid additive are pulverized, cross 325 mesh sieves;
(3) various solid additive joins in diatomite in proportion and mixes, and then adds wetting agent mixing, is finally joined in mixture by basket for the yellow of drying bacterium conidial powder and mix, namely obtain yellow basket bacterium wetting powder.
3. the basket bacterium wetting powder of the yellow described in claim 1 or 2, it is characterized in that the basket bacterium of described yellow is yellow basket bacterium qw12, its preserving number is CGMCC 5067.
4. the basket bacterium wetting powder of yellow according to claim 3, is characterized in that, in the basket bacterium of described yellow (Talaromyces flavus) conidial powder, yellow basket bacterium conidium content is 5 × 10
9~ 1.42 × 10
11individual/g, water content is less than 12%, and spore rate of living is greater than 90%.
Described yellow basket bacterium conidial powder is after the solid fermentation of yellow blue shape bacterium terminates, and solid culture is dry, pulverizing is rear crosses 200 mesh sieves; Or with the basket bacterium conidial powder of the yellow of fungal spore separator separated and collected.
5. one kind utilizes yellow basket bacterium qw12 to prepare the fermentation culture method of conidial powder, it is characterized in that solid matrix/water=10/4 ~ 10/10 in medium, carbon source is 0.5% ~ 4% of matrix, and ammonium sulfate is 0.1% ~ 4% of matrix, and mineral salt are 0.5 ‰ ~ 5 ‰ of matrix.
Described solid matrix is wheat bran and corn stalk powder, and wherein the percentage by weight of wheat bran in solid matrix is 20% ~ 100%; Described carbon source be selected from glucose, maltose one or both; Described mineral salt are selected from MgSO
4, ZnSO
4, FeSO
4, CuSO
4in one or both combination.
Fermentation condition is the inoculum concentration of yellow basket bacterium qw12 seed is 3%-22%, 12h ~ 24h illumination every day, and 25 ~ 30 DEG C, fermentation time is 5 ~ 13d,
After fermentation ends, solid culture is dry, pulverizing is rear crosses 200 mesh sieves; Or with the basket bacterium conidial powder of the yellow of fungal spore separator separated and collected.
6. the yellow basket bacterium qw12 of utilization according to claim 5 prepares the fermentation culture method of conidial powder, it is characterized in that basket bacterial classification of described yellow is concentration 1 × 10
6individual/mL ~ 1 × 10
8the basket bacterium spore suspension of yellow of individual/mL or 1 × 10
6cfu/mL ~ 1 × 10
9the basket bacteria culture fluid of yellow of cfu/mL.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106550763A (en) * | 2015-09-28 | 2017-04-05 | 浙江泛亚生物医药股份有限公司 | A kind of artificial culture method of Periostracum cicadae spore powder |
| CN109593658A (en) * | 2018-12-27 | 2019-04-09 | 河南科技大学 | A kind of endogenetic fungus ZXL-SZY-R-9 and its application with antibiosis |
| CN112042472A (en) * | 2020-09-28 | 2020-12-08 | 上海市农业科学院 | Culture medium for slowing down aging of hypsizigus marmoreus plate culture |
| CN113151001A (en) * | 2021-02-02 | 2021-07-23 | 华中农业大学 | Talaromyces flavus strain TF-04 and application thereof |
| CN113717860A (en) * | 2021-07-07 | 2021-11-30 | 昆明理工大学 | Application of Talaromyces flavidus in conversion of panax notoginseng saponins into low-polarity ginsenoside |
| CN116235865A (en) * | 2019-06-27 | 2023-06-09 | 日本史迪士生物科学株式会社 | Plant disease control agent and plant disease control method |
| CN116458357A (en) * | 2023-05-31 | 2023-07-21 | 甘肃省农业科学院林果花卉研究所 | Method for promoting growth of fruit trees by rhizosphere soil microorganisms |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286383A (en) * | 2011-09-06 | 2011-12-21 | 青岛农业大学 | Talaromyces flavus and application of same in prevention of plant pathogens |
| CN102533608A (en) * | 2012-01-19 | 2012-07-04 | 高丙利 | Bacillus subtilis, preparation, preparation method of preparation, and application of bacillus subtilis and preparation |
| WO2013010322A8 (en) * | 2011-07-20 | 2013-03-21 | 上海泽元海洋生物技术有限公司 | Preparation method of paenibacillus polymyxa wettable powder |
| WO2014079724A1 (en) * | 2012-11-22 | 2014-05-30 | Basf Se | Pesticidal mixtures |
| CN103834580A (en) * | 2014-03-12 | 2014-06-04 | 中国农业科学院棉花研究所 | Endophytic fungi CEF-642 of cotton and application thereof |
-
2014
- 2014-09-04 CN CN201410449924.XA patent/CN104286032B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013010322A8 (en) * | 2011-07-20 | 2013-03-21 | 上海泽元海洋生物技术有限公司 | Preparation method of paenibacillus polymyxa wettable powder |
| CN102286383A (en) * | 2011-09-06 | 2011-12-21 | 青岛农业大学 | Talaromyces flavus and application of same in prevention of plant pathogens |
| CN102533608A (en) * | 2012-01-19 | 2012-07-04 | 高丙利 | Bacillus subtilis, preparation, preparation method of preparation, and application of bacillus subtilis and preparation |
| WO2014079724A1 (en) * | 2012-11-22 | 2014-05-30 | Basf Se | Pesticidal mixtures |
| CN103834580A (en) * | 2014-03-12 | 2014-06-04 | 中国农业科学院棉花研究所 | Endophytic fungi CEF-642 of cotton and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| D. R. FRAVEL等: "In situ Evidence for the role of Glucose oxidase in the biocontrol of verticillium wilt by Talaromyces flavus", 《BIOCONTROL SCIENCE AND TECHNOLOGY》 * |
| 彭志荣等: "生防菌株MT-06对黄瓜白粉病的防治及其定殖力测定", 《浙江农业学报》 * |
| 洪永聪: "生防菌株TL2的菌剂研制及大田防病试验", 《中国农学通报》 * |
| 邵龙等: "10%粉红粘帚菌可湿性粉剂的研制", 《安徽农业科学》 * |
Cited By (12)
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|---|---|---|---|---|
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| EP3991559A4 (en) * | 2019-06-27 | 2023-07-26 | Sds Biotech K.K. | Plant disease control agent and plant disease control method |
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| CN113151001B (en) * | 2021-02-02 | 2022-05-27 | 华中农业大学 | Talaromyces flavus strain TF-04 and application thereof |
| CN113717860A (en) * | 2021-07-07 | 2021-11-30 | 昆明理工大学 | Application of Talaromyces flavidus in conversion of panax notoginseng saponins into low-polarity ginsenoside |
| CN113717860B (en) * | 2021-07-07 | 2023-05-16 | 昆明理工大学 | Application of Huang Lanzhuang bacteria in conversion of total saponins of Notoginseng radix into small-polarity ginsenoside |
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