CN104286032A - Preparation method of Talaromyces flavus spore powder, Talaromyces flavus wettable pulvis and preparation method of wettable pulvis - Google Patents

Abstract

The invention relates to a yellow basket-shaped fungus wettable powder, which comprises yellow basket-shaped fungus (Talaromyces flavus) conidia powder, a carrier and an auxiliary agent, and the live bacteria content in the yellow basket-shaped fungus wettable powder is 5× l0 ⁸ cfu/g-5×l0 ⁹ cfu/g, the suspension rate of the yellow basket-shaped fungus wettable powder is greater than 75%, the wetting time is less than 60 seconds, and the water content is less than 6%. The present invention optimizes the spore fermentation process of T. yellow spores qw12 strain; uses the spores of T. yellow as active ingredients to create a wettable powder of T. yellow, and provides a production technology and method for the biocontrol preparation of T. yellow A convenient and practical new dosage form. The invention has laid a foundation for the large-scale commercial production of the yellow basket-shaped fungus wettable powder, is conducive to accelerating the promotion and application of the yellow basket-shaped fungus in biological control, and has important theoretical and practical significance.

Description

A preparation method of yellow basket-shaped fungus spore powder, yellow basket-shaped fungus wettable powder and preparation method thereof

technical field

The present invention relates to the technical field of microbial pesticides, in particular to a wettable powder of microbial pesticides and a preparation method thereof, more specifically, the present invention relates to a method for fermenting and preparing spore powder of yellow basket-shaped fungus, and the yellow basket-shaped fungus can be Wet powder and its preparation method.

Background technique

Pesticides play an important role in preventing and controlling plant diseases and insect pests, ensuring stable and increased agricultural production, improving the quality of agricultural products, and improving people's living standards. Chemical pesticides have played an important role in controlling crop diseases and protecting agricultural safety, but they have also caused a series of environmental and social problems, such as the "3R" problem caused by the widespread use of chemical pesticides, etc., seriously affecting the sustainability of modern agriculture. develop.

Microbial pesticides are preparations made from living organisms or metabolites that can prevent and control crop diseases, insects, and weeds. The objects to be controlled are not easy to produce drug resistance, have strong selectivity, are safer for humans, animals, and natural enemies, and reproduce faster. They can be used Agricultural and sideline products, industrial and agricultural wastewater and waste are widely produced, and are the first choice for the production of pollution-free food. At present, there are mainly 4 kinds of insecticidal bacteria that have been developed into products and put into practical use, namely Bacillus sphaericus, Bacillus japonicus, Bacillus thuringiensis and Bacillus lentipathis; more than 100 genera of insecticidal fungi have been recorded, More than 800 species, the most studied are Beauveria bassiana and Metarhizium anisopliae. Trihoderma (Trihoderma) has a good inhibitory effect on plant pathogenic fungi, is the most studied fungicide, and has been developed as a Trichoderma pesticide preparation.

In order to facilitate the storage, transportation and use of microbial pesticides and improve their efficacy, they need to be made into certain formulations. The processing of microbial pesticides is more difficult than that of chemical pesticides. First, the active ingredients of microbial pesticides are generally living organisms, and their hydrophobicity directly affects the physical properties of the preparation, such as dispersibility, suspension, and wettability; second, the interaction between microorganisms and various additives Biocompatibility is generally poor; in addition, organisms are sensitive to humidity, temperature, light and other environments, and the storage stability of preparations is poor, so additives such as protective agents need to be added.

The formulations of microbial pesticides include wettable powders, granules, suspensions, and microgranules. Wettable Powders (WP) have the characteristics of convenient use, storage and transportation, no addition of organic solvents, and relatively easy processing operations. It is the preferred dosage form in the development of preparations. The wettable powder is composed of the original drug, the carrier and the surfactant and other auxiliary agents. The auxiliary agents have an important impact on the pesticide preparation and directly affect the efficacy of the pesticide. Especially in fungal spore preparations, there are higher requirements for adjuvants. In addition to the excellent performance of adjuvants, they must not affect the germination of fungal spores and the growth of hyphae. Therefore, screening for those with excellent biological compatibility with fungi Auxiliaries are particularly important for the development of fungal agents. Screening suitable auxiliaries for specific strains is the key to the production of fungal wettable powders.

Talaromyces flavus has hyperparasitic effects on the sclerotia produced by various pathogenic bacteria such as Sclerotinia sclerotiorum, Sclerotium rolfsii, and Rhizoctonia solani. Verticillum dahliae and Fusarium oxysporum f.sp.vasinfectum have obvious inhibitory effects. The biocontrol mechanism of this fungus is diverse, mainly including hyperparasitism, competition, bacteriolysis, etc. Among them, hyperparasitism is the most important mechanism of action of the yellow basket fungus, which can be induced to produce chitin during the interaction with the host fungus. Glucose oxidase and hydrolase such as carbozyme, cellulase, β-1,3-glucanase, etc. The growth temperature and pH range of yellow basket fungus are wide, and it can survive and reproduce under different ecological conditions in the south and north of my country. It has obvious control effects on important plant fungal diseases such as sclerotinia and verticillium wilt, and is an important The biological control factor has a good application prospect and research value in biological control.

As one of the important bio-control bacteria, Talaromyces flavus has no corresponding preparation research and development in China, which greatly restricts the use and promotion of Talaromyces flavus in biological control. We isolated and screened a yellow basket-shaped fungus qw12 (preservation number: CGMCC 5067, characteristic gene GenBank accession number: JN602366) from the soil. Phytopathogenic fungi have a strong inhibitory effect.

Contents of the invention

Aiming at the defects existing in the prior art, the object of the present invention is to provide a method for fermenting and preparing conidia powder of the yellow basket-shaped fungus, as well as the wettable powder of the yellow basket-shaped fungus and the preparation method thereof.

Technical scheme of the present invention is as follows:

A yellow basket-shaped fungus wettable powder, which includes yellow basket-shaped fungus (Talaromyces flavus) conidia powder, a carrier and an auxiliary agent, and the live bacteria content in the yellow basket-shaped fungus wettable powder is 5×10 ⁸ cfu /g～5×10 ⁹ cfu/g, the suspension rate of the yellow basket-shaped fungus wettable powder is greater than 75%, the wetting time is less than 60 seconds, and the water content is less than 6%.

The weight percentage of the conidia powder of the yellow basket-shaped fungus in the wettable powder is 5-50%. The carrier is selected from one or more of diatomite, bentonite, and kaolin, and the weight percentage of the carrier in the wettable powder is 40% to 93%; the auxiliary agent includes a wetting agent, a dispersant and an ultraviolet ray Protective agent; the wetting agent is selected from one of sodium lignosulfonate and Tween 80, and the weight percentage of the wetting agent in the wettable powder is 1% to 5%; the dispersing agent is polyethylene glycol Alcohol, the percentage by weight of the dispersant in the wettable powder is 0.5% to 2%; the UV protective agent is selected from a kind of dextrin and methylcellulose, and the percentage by weight of the UV protective agent in the wettable powder is 0.5% %~2%.

The above-mentioned yellow basket-shaped bacterium wettable powder, the specific preparation method is as follows:

(1) Take each component in the wettable powder according to the amount;

(2) The carrier is first pulverized with a pulverizer, and passed through a 325-mesh sieve;

(3) The carrier, the wetting agent, the dispersant, the ultraviolet protecting agent and the conidia powder of the yellow basilisk fungus were thoroughly mixed in sequence in proportion to obtain the yellow basilisk fungus wettable powder.

On the basis of the above scheme, the yellow basket-shaped bacterium is yellow basket-shaped fungus qw12.

On the basis of the above scheme, the conidia content of Talaromyces flavus conidia in the Talaromyces flavus powder is 5×10 ⁹ to 1.42×10 ¹¹ /g, and the water content is less than 12%. The live spore rate is greater than 90%.

The yellow basket-shaped fungus conidia powder is after the solid fermentation of the yellow blue-shaped fungus is completed, and the solid culture is dried and crushed and passed through a 200-mesh sieve; or the yellow basket-shaped fungus conidia collected by separating and collecting with a fungal spore separator pink.

A method for fermenting and culturing conidia powder prepared by using the yellow basket bacterium qw12, solid substrate/water=10/4～10/10 in the culture medium, carbon source is 0.5%～4% of the substrate, and ammonium sulfate is the substrate 0.1%~4%, inorganic salt is 0.5‰~5‰ of the matrix.

The solid matrix is bran and corn stalk powder, wherein the weight percentage of bran in the solid matrix is 20% to 100%; the carbon source is selected from one or both of glucose and maltose; the The inorganic salt is selected from one or a combination of MgSO ₄ , ZnSO ₄ , FeSO ₄ , CuSO ₄ .

The fermentation conditions are as follows: the inoculum amount of T. yellow qw12 seeds is 3% to 22%, 12h to 24h light per day, 25 to 30°C, and the fermentation time is 5 to 13 days.

After the fermentation is finished, the solid culture is dried, pulverized, and passed through a 200-mesh sieve; or the yellow basket-shaped fungus conidia powder collected is separated by a fungal spore separator.

On the basis of the above scheme, the seeds of T. yellow spores are a spore suspension of T. yellow at a concentration of 1×10 ⁶ to 1×10 ⁸ /mL or 1×10 ⁶ cfu/mL to 1× 10 ⁹ cfu/mL culture solution of yellow basket bacteria.

The present invention optimizes the spore fermentation process of T. yellow spores qw12 strain; uses the spores of T. yellow as active ingredients to create a wettable powder of T. yellow, and provides a production technology and method for the biocontrol preparation of T. yellow A convenient and practical new dosage form. The invention has laid a foundation for the large-scale commercial production of the yellow basket-shaped fungus wettable powder, is conducive to accelerating the promotion and application of the yellow basket-shaped fungus in biological control, and has important theoretical and practical significance.

Detailed ways

Example 1

Conditions for Solid Fermentation of Basilisk Yellow Fungus qw12

1. Determine the solid fermentation substrate

Set up three single-substance culture media: bran, corn stalk powder, and corn flour; four mixed-substance culture media: bran+corn stalk powder, bran+corn meal, corn stalk powder+corn meal, bran + corn stalk powder + corn flour, the different ingredients in the mixed substance medium are mixed in equal proportions. Ratio of material to water = 1:1, sterilized at 121°C for 30 minutes, inoculated with 1×10 ⁶ spore suspension/mL, inoculation volume 5%; cultured at 28°C for 7 days, turning once a day. Three replicates were set up for each treatment. After the fermentation, take 0.5 g of the solid fermentation culture, add sterile water, stir on a magnetic stirrer for 5 min, and count the number of spores under a microscope with a hemocytometer. The results show that when the medium is a single substrate, the effect of bran is the best, and the sporulation amount reaches 5.66×10 ⁹ /g; in the mixed substrate, the combination of bran and straw powder has the best effect, and the sporulation amount reaches 6.10×10 ⁹ Individuals/g (Table 1); Further research (Table 2) found that the ratio of bran and straw powder at 4:1 was the best, and the sporulation yield could reach 7.80×10 ⁹ cells/g.

Table 1 The influence of different solid media on sporulation

Table 2 Effects of culture media with different proportions of bran and corn stalks on sporulation

2. Effect of material-water ratio on spore production

In the solid fermentation basal medium, the feed-to-water ratio was respectively set to be 10:3, 10:4, 10:5, 10:6, 10:7, 10:8, 10:9, 10:10 to carry out the test, each treatment Set 3 repetitions, count the number of spores and calculate the average sporulation. The test results showed (as in Table 3) that the treatment with a material-to-water ratio of 5:4 had the highest sporulation yield.

Table 3 Effect of material-water ratio on spore production

3. The promotion effect of mineral elements on sporulation

Add 1‰ mineral elements MgSO ₄ , ZnSO ₄ , FeSO ₄ , CuSO ₄ , CaSO ₄ , K ₂ SO ₄ to the solid fermentation basal medium respectively, without adding mineral elements as a control, each treatment is repeated 3 times , Calculate the average sporulation under the microscope. The results are shown in Table 4. The effects of different mineral elements added on the spore production of T. yellow qw12 were different. Adding Cu element had the most significant promotion effect on the spore production of the bacterium, and the spore production was the highest; and in the culture Adding 3‰CuSO ₄ to the base is the best (Table 5).

Table 4 The effect of different sporulation accelerators on sporulation

Table 5 Effect of CuSO ₄ concentration on sporulation

4 The impact of adding carbon source on sporulation

Three carbon sources, sucrose, glucose, and maltose, were added to the solid basal medium at 1% of the mass of the solid matrix; the control was the solid basal medium without additional carbon sources. Compared with the control, the sporulation of the strains added with carbon source increased, and the sporulation of the culture medium added with glucose was the highest, followed by maltose, as shown in Table 6.

In the case of keeping the fermentation conditions unchanged, glucose was added at 0.5%, 1%, 2%, 3%, and 4% of the mass of the solid substrate respectively. As shown in Table 7, the effects of different concentrations of glucose on the production of T. The spore production was different. With the increase of glucose addition, the sporulation production of T. yellow increased sequentially, but decreased when the addition was over 3%, so adding 3% glucose had the best effect.

Table 6 Effect of different carbon sources on sporulation

Table 7 Effect of glucose concentration on sporulation

5. Effect of additional nitrogen source on sporulation

Inorganic nitrogen sources (ammonium nitrate, ammonium sulfate, urea) and organic nitrogen sources (yeast extract, peptone, bean cake powder) were added to the solid basal medium at 1% of the mass of the solid substrate, and the control was a solid basal medium without nitrogen sources , and keep other fermentation conditions unchanged. Among the six additional nitrogen sources, the culture medium added with ammonium sulfate had the highest sporulation yield, and the sporulation yield of other treatments was lower than that of the control strain, as shown in Table 8. Under the condition of keeping the fermentation conditions unchanged, ammonium sulfate was added in the culture medium by 0.1%, 0.5%, 1%, 2%, 3%, 4% of the solid substrate mass, as shown in Table 9, different concentrations of Ammonium sulfate has different effects on sporulation of T. yellow qw12. With the increase of ammonium sulfate addition, the sporulation of T. % Ammonium Sulfate works best.

Table 8 Effect of different nitrogen sources on sporulation

Table 9 The effect of ammonium sulfate concentration on sporulation

6. Influence of inoculum amount on spore production

Inoculate according to 3%, 5%, 10%, 14%, 18%, 22% inoculum respectively. The test results show that the most ideal inoculum in solid fermentation basal medium is 18%, as shown in Table 10.

Table 10 Effect of inoculum amount on spore production

7. Effect of light conditions on sporulation

The inoculated solid fermentation basal medium was cultured under light conditions of 24 hours of light, 12 hours of darkness and 12 hours of light and 24 hours of darkness. The number of spores was counted under a microscope and the average sporulation yield was calculated. Among them, 24h of light can significantly increase the sporulation of the yellow T. yellow qw12, as shown in Table 11.

Table 11 Effect of light on sporulation

8. Media Orthogonal Test Screening

Choose the ratio of bran to corn stalk powder, material-water ratio, ammonium sulfate and inoculum size to do an orthogonal experiment with 4 factors and 3 levels (L9(34)) (Table 12) to optimize the conditions for sporulation.

Table 12 Orthogonal factor test level design

Within the selected level range, the influence of factors A, B, C, and D on sporulation production is in the following order: D﹥B﹥A﹥C, and the optimal combination is A ₃ B ₂ C ₁ D ₃ (Table 13), After further testing, screening and verification, the optimal formula was obtained as follows: the proportion of bran is 100%, the ratio of material to water is 10:7, the inoculum size is 14%, the concentration of ammonium sulfate is 3%, and the final spore amount reaches 8.39×10 ¹⁰ /g .

Table 13 (L ₉ (3 ⁴ )) Orthogonal Design and Results

9. Determination of solid fermentation time of yellow basket-shaped bacteria

Orthogonally optimized fermentation medium was used to ferment and cultivate yellow basket-shaped bacteria, and the growth status of the bacteria was observed every day, and the amount of spore production was detected to determine the optimal fermentation time. As shown in Table 14, the sporulation of the yellow basket-shaped bacteria was the largest on the 10th day.

Table 14 The change of spore production with time

Embodiment 2: the preparation of yellow basket-shaped fungus spore powder

The optimized spore-forming medium of T. yellow was sterilized at 121°C for 20-30 minutes to inoculate 10 ⁶ spore suspensions/mL, and cultured in the dark at 28°C for 10 days. After the fermentation is over, dry the solid medium in an oven at 35°C to dry the water, dry and crush the solid culture and pass it through a 200-mesh sieve; or use a fungal spore separator to separate the spores and pass through a 200-mesh sieve; the collected yellow basket-shaped fungus Conidia powder.

Weigh 0.05g of the spore powder of the yellow basket-shaped fungus and suspend it in 20mL of sterile water, stir it with a magnetic stirrer for 30 minutes, then use a hemocytometer to measure the concentration of the spore suspension, and calculate the spore content of the yellow basket-shaped fungus conidia powder. Repeat 3 times to calculate the average spore content of spore powder.

Weigh 1g of the spore powder of the yellow basket-shaped fungus, bake it at 100°C for 2 hours, let it cool naturally, weigh the spore powder of the yellow basket-shaped fungus again, repeat 3 times, and calculate the average value of the water content.

Determination of viable spore rate: Prepare spore powder with sterile water to make a suspension with a concentration of ¹⁰⁶ /ml, draw 1mL of the suspension into 9mL of germination liquid, place it on a shaker at 28°C, and culture it with shaking at 120r/min for 24h Finally, dilute the culture solution (about 100 conidia in each visual field), record the number of germinated and ungerminated spores in the culture solution with a hemocytometer under a microscope, repeat the sampling 3 times, and calculate the average germination rate of the spores, which is Live spore rate. The spore germination standard is that the germ tube length is greater than or equal to 1/2 of the spore diameter. The formula of the spore germination liquid is 1% tryptone, 2% glucose, 1000 mL of sterile water, and sterilized at 105-115° C. for 15 minutes.

The results show that the solid culture is dried and crushed and passed through a 200-mesh sieve. The spore powder of the yellow basket-shaped fungus contains 5×10 ⁹ to 1.2×10 ¹⁰ spores/g, the water content is 10%, and the viable spore rate is 92.3%. ; Separate the spores with a fungal spore separator and pass through a 200-mesh sieve. The conidia powder of the yellow basket-shaped fungus collected has a spore content of 9.8×10 ⁹ to 1.0×10 ¹¹ /g, a water content of 9.7%, and a viable spore rate It is 95.31%, and can be used for subsequent wettable powder processing.

Embodiment 3: the screening of auxiliary agent

1 Carrier screening

The effect of carrier on the germination of spores of T. jaundice: Kaolin, talcum powder, bentonite, and diatomaceous earth are mixed with PDA medium at a concentration ratio of 5 mg/mL and 10 mg/mL, sterilized, and prepared under sterile conditions flat. Take 0.1mL of 1.0× ¹⁰³ /ml spore suspension of T. yellow qw12 and spread it on a plate containing different carriers. The PDA medium without carrier is used as the control. Each treatment is repeated 4 times and cultured at 28°C After 48 hours, observe and count the number of colonies formed by spore germination on plates containing different carriers.

The influence of the carrier on the mycelial growth of the yellow basket-shaped fungus: put the yellow basket-shaped fungus qw12 bacterial block with a diameter of 0.5cm in the center of the plate containing different carriers made by the above method, and use the carrier-free PDA medium as a control, each treatment Repeat 4 times, culture at 28°C, measure the diameter of the colony with the cross method after 72 hours, and calculate the average daily growth rate of the colony.

Colony growth rate = (final colony diameter - initial colony diameter) / colony growth days

The result shows: different carrier has difference (table 15, table 16) to the daily average growth rate of yellow basket-shaped bacteria and spore germination influence, and diatomaceous earth, bentonite, kaolin can be used as the carrier of yellow basket-shaped bacteria wettable powder, wherein silicon Algae is better.

Table 15 The effect of the carrier on the mycelia growth of T.

Table 16 The effect of the carrier on the germination of the spores of T.

Note: The cfu value of the number of colonies formed by spore germination is represented by the mean ± standard error (SE). The difference of different mass concentrations of the same adjuvant on the formation of colonies by the germination of T. yellow spores is indicated by capital letters, and different capital letters indicate significant differences (P≤0.05). The difference in the number of single-spore colonies formed by the germination of the spores of Taliformum yellow with different additives at the same mass concentration is represented by lowercase letters, and different small letters indicate significant differences (P≤0.05), the same below.

2. Screening of wetting agent

The wetting agents sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium lignosulfonate and Tween 80 were mixed with PDA medium according to the concentration ratio of 500 μg/mL, 1000 μg/mL and 2000 μg/mL respectively Sterilized and plated. Spread and inoculate the spore suspension of T. yellow qw12, take PDA medium without wetting agent as the control, set 4 repetitions for each treatment, culture at 28°C, observe and count after 48 hours in the PDA medium containing different wetting agents The number of colonies formed by spore germination on the plate was used to determine the biocompatibility of the wetting agent. The method of measuring mycelium growth rate is the same as that of the carrier mycelium. The wetting time is measured according to GB/T5451-2001.

Taking biocompatibility and wetting time as the discriminant indicators, sodium lignosulfonate and Tween 80 can be used as wetting agents for the wettable powder of yellow basket fungus; among them, Tween 80 is better, which can promote the growth of mycelium and protect the The spore germination has the least influence and the wetting time is the shortest (Table 17-19).

Table 17 The effect of wetting agent on the mycelia growth of T.

Table 18 The effect of wetting agent on the spore germination of T.

Table 19 Wetting time measurement results

3. Screening of dispersants

Dispersants polyvinyl alcohol and polyethylene glycol were mixed with PDA medium at concentrations of 500 μg/mL, 1000 μg/mL and 2000 μg/mL, respectively, and then sterilized to make plates. The biocompatibility test method is the same as that of wetting agent.

Determination of dispersing power: the above-mentioned dispersant was formulated into mother liquors with concentrations of 500 μg/mL, 1000 μg/mL and 2000 μg/mL, and the spore suspension of the yellow basket-shaped fungus qw12 was prepared so that the concentration was ¹⁰⁷ spores/ml, and 10 mL of the suspension was taken. Add the solution into 90mL mother liquor, shake well, after 0h, 1h, and 4h, take samples from the upper, middle, and lower layers of the suspension, count them under a microscope with a hemocytometer, and use no dispersant solution as a control. And calculate the dispersion index (I) according to the following formula:

$\mathrm{<span\; class="notranslate">\; I</span>}<span\; class="notranslate">\; =</span>{\mathrm{<span\; class="notranslate">\; \&sigma;</span>}}^{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; /</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; a</span>}}$

—sample mean; σ—standard deviation

I=1, the spores are randomly distributed; I>1, the spores are aggregated and distributed, and when I<1, the spores are evenly distributed.

Taking biocompatibility and dispersing power as the discriminant index, polyethylene glycol can promote the growth of mycelium, has the least effect on spore germination, has strong dispersion ability, and is relatively stable, so the wetting agent is determined to be polyethylene glycol.

Table 20 Effects of different dispersants on the mycelia growth of T.

Table 21 The effect of different dispersants on the spore germination of T.

Table 22 Dispersant performance of dispersant on yellow spores

4. Screening of UV protective agent

Ultraviolet protective agents dextrin, humic acid, methylcellulose, xanthan gum, and trehalose were mixed with PDA medium at a concentration ratio of 500 μg/mL, 1000 μg/mL, and 2000 μg/mL, respectively, and then sterilized to make a plate. The biocompatibility test method is the same as that of wetting agent.

Determination of the protective effect of the protective agent: prepare the spore suspension of the yellow basket bacterium qw12, the concentration is 1.0×10 ³ /ml, the suspension is coated on the PDA plate containing the ultraviolet protective agent, at 30W, the light intensity is Under the ultraviolet light of 120lx at 20cm place, open the cover and irradiate the plate for 30s, cultivate the treated plate in the dark at 28°C, observe and count the number of colonies formed by spore germination after 48h. Screen out the UV protective agent with the best protective effect, and then set different concentration gradients to determine the most suitable ratio through the method of spore germination. Taking ultraviolet irradiation but not adding protective agent plate as control 1 (used to calculate the residual surviving spore rate), taking no ultraviolet irradiating and adding protective agent plate as control 2 (used to calculate the theoretical viable spore rate), each treatment Set 4 repetitions, and calculate the protective efficacy index according to the following formula:

Protection efficiency index = (survival rate after irradiation - residual survival rate) / (theoretical survival rate - residual survival rate)

In the three concentrations of methylcellulose medium, the growth rate of the mycelia of the yellow Tarantula qw12 was faster, reaching (1.003±0.017) cm/d at 2000 μg/mL. Dextrin and trehalose can also promote the growth of mycelia of T. yellow qw12, and the difference is not significant compared with the control. Xanthan gum and humic acid have a significant inhibitory effect on mycelium growth. The spores of T. yellow qw12 can germinate well on the medium containing five kinds of ultraviolet protective agents. On the medium containing xanthan gum, dextrin, methylcellulose and humic acid, the germination rate of spores increases with The growth of spores increased, and the germination rate of spores was the highest on the methylcellulose medium containing 500 μg/mL. Xanthan gum, dextrin, trehalose, methyl cellulose had no significant difference with the control, and had better compatibility. There were differences in the protective efficacy indexes of the five UV protectants against the conidia of T. jaundice qw12. The protective efficacy of dextrin and methylcellulose is better, reaching up to 0.3 (Table 23).

Table 23 Protective efficacy of different UV protective agents

Embodiment 4: the quality inspection of the yellow basket-shaped fungus wettable powder

1. Preparation of wettable powder of yellow basket fungus: 5%, 10%, 30%, 50% of spore powder, 3% of wetting agent Tween 80, 1% of dispersing agent polyethylene glycol, ultraviolet protecting agent methyl Cellulose 1%, carrier diatomite to make up 100%; first crush the carrier and solid additives, pass through a 325 mesh sieve; add various solid additives in proportion to diatomite and mix well, then add wetting agent and mix well , and finally add the dry conidia powder of the yellow basket fungus into the mixture and mix evenly to obtain the wettable powder of the yellow basket fungus.

2. Quality inspection

Spore content assay: with embodiment 2.

Determination of water content: weigh the test vessel after drying it in an oven at 120°C for 15 minutes, weigh 5g of yellow basket-shaped bacteria wettable powder, bake it at 120°C for 2 hours, let it cool naturally, and then add the yellow basket-shaped fungus spore powder Weigh the dried container, repeat 3 times, and calculate the average value of water content. Moisture content (%)=[(5-mass after drying)/5]×100

Determination of wetting time Refer to GB/T5451-2001 to measure the time of complete wetting.

Determination of suspension rate Refer to GB/T14825-2006 to determine the suspension rate of spores.

pH measurement: Weigh 2g of wettable powder and dissolve it in 100mL of distilled water, stir rapidly with a magnetic stirrer for 1min, then let it stand for 1min, measure the pH value of the sample with a pH meter, repeat 3 times for each sample, and the results are different each time The absolute value is less than 0.1, and the average value is calculated.

The content of live spores in the wettable powder of Tasmania yellow is 8.4×10 ⁸ cfu/g～9×10 ⁹ cfu/g, the water content is 0.96%～4.89%, the wetting time is 17s～23s, and the suspension rate is 81.73% ~86.46%, pH value is 7.27 ~ 7.49, the above indicators meet the relevant national standards (Table 24).

Table 24 Determination of various indicators of yellow basket-shaped fungus wettable powder

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications are made to the technical solutions described, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

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.