CN104651294A - Fermentation medium suitable for metarhizium anisopliae spore production as well as preparation method and using method thereof - Google Patents
Fermentation medium suitable for metarhizium anisopliae spore production as well as preparation method and using method thereof Download PDFInfo
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Abstract
The invention discloses a fermentation medium suitable for metarhizium anisopliae spore production as well as a preparation method and a using method thereof, relating to a culture medium. The culture medium comprises a seed culture medium and a solid fermentation medium, wherein the seed culture medium comprises a solid seed culture medium and a liquid seed culture medium; the solid seed culture medium contains 1000mL of potato juice, glucose, KH2PO4, MgSO4, agar and the balance of water; the liquid seed culture medium contains peptone, cane sugar, KH2PO4, MgSO4, NaCl and the balance of water; the solid fermentation medium comprises the following components: wheat bran, corn grits and distilled water; the mass ratio of the wheat bran to the corn grits is 1:1; and the mass of the distilled water is 55-60 percent of the total mass of the wheat bran and the corn grits. The fermentation medium suitable for metarhizium anisopliae spore production disclosed by the invention is suitable for growth of metarhizium anisopliae, and more than 1*10<9> spores can be produced by each gram of fermentation raw material; and the germination rate of the prepared spore powder can be 95 percent.
Description
Technical field
The present invention relates to a kind of substratum, suitable Metarhizium anisopliae produces fermention medium and the preparation and application thereof of spore specifically.
Background technology
Green muscardine fungus (
metarhiziumsp.) be the entomogenous fungi of wide spectrum, to invade after insect body wall can in body cavity amount reproduction, cause insect death.Because green muscardine fungus is strong to the virulence of insect, to people, animal, crop toxicological harmless, use safety, and there is longer preventive effect, therefore green muscardine fungus has good application prospect as biotic pesticide.But owing to there is various problem in fermentation process, formulation development and environmental constraints etc., the application of green muscardine fungus on biological control of insect pests had once been very limited, from Mehhnhob in 1879 first amount reproduction green muscardine fungus prevent and treat Austrian chafer (
anisopliae austriacaherbst), since, the application of green muscardine fungus on biological control of insect pests just progressively launches.According to bottle stalk and conidial shape and arrangement mode, Metarhizium comprises seven kinds or mutation: white green muscardine fungus (
m. album), Metarhizium anisopliae var. Anisopliae (
m. anisopliaevar.
anisopliae), the large spore mutation of Metarhizium anisopliae (
m. anisopliaevar.
majus), dimorphism spore green muscardine fungus (
m. bifomisporae), the yellowish green mutation of yellowish green green muscardine fungus (
m. flavoviridevar.
flavoviride), the little spore mutation of yellowish green green muscardine fungus (
m. flavoviridevar.
minus) and Metarhizium taii (
m. taii).At present, Metarhizium anisopliae is applied more on biological control of insect pests.
Because fungus insecticide has contact, popular, the environmentally safe and feature such as not easily to develop immunity to drugs, application green muscardine fungus pest control has obtained certain effect.Except the Austrian chafer of control, application green muscardine fungus control subterranean pest-insect and African locust have also obtained very great achievement.The Metarrhizium anisopliae preparation of current domestic registered registration can be used to control locust, blattaria, there are scale insect, trialeurodes vaporariorum, termite, mosquito, blattaria (U.S.) abroad, grub (Germany), sugarcane froghopper, trialeurodes vaporariorum (Brazil), sugarcane chafer, locust, grasshopper (Australia) etc.Along with the progress of the deep and technology studied, believe that the range of application of green muscardine fungus in pest control can be more and more wider.
Up to the present, the main formulation of fungus insecticide has pulvis, wettable powder, finish, dry mycelia, microcapsule, granule and leafing agent etc.The green muscardine fungus formulation of large-area applications mainly pulvis, dry mycelia and finish.Due to biocontrol fungi the most effectively infect body be bombys batryticatus body surface produce the raw conidium of gas, so in above-mentioned preparation except dry mycelia and leafing agent, all the other several preparations be all with the spore powder of green muscardine fungus be former medicine develop.The fermentative production of muscardine spore powder has consequence in the development of green muscardine fungus biotic pesticide.
Summary of the invention
The present invention seeks to the deficiency for solving the problems of the technologies described above, the fermention medium providing suitable Metarhizium anisopliae to produce spore and preparation and application thereof, it has starting material and is easy to obtain, preparation method is simply easy to grasp, the Metarhizium anisopliae cultivated, sporulation quantity is high, and every gram of fermentation raw material (namely grow on solid fermentation substratum and grow the Metarhizium anisopliae of spore and the mixture of solid fermentation substratum) can produce 1 × 10
9with last spore; The spore powder spore rate alive of preparation is high, and germination rate can reach about 95%.
The present invention for solving the problems of the technologies described above adopted technical scheme is: provide suitable Metarhizium anisopliae to produce the fermention medium of spore, comprise seed culture medium and solid fermentation substratum.
Wherein said seed culture medium comprises solid seed culture medium and liquid seed culture medium; Containing mass concentration in often liter of solid seed culture medium is 20% potato juice 1000mL, glucose 20g, KH
2pO
43g, MgSO
41.5g and agar 20g, surplus is water; In often liter of liquid seed culture medium, containing peptone 10 g, sucrose 10 g, K
2hPO
40.3 g, MgSO
40.3 g and NaCl 0.3 g, surplus is water;
The composition of described solid fermentation substratum is: wheat bran, hominy grits and distilled water; Wherein the mass ratio of wheat bran and hominy grits is 1:1, and the quality of distilled water is wheat bran and hominy grits total mass 55-60%.The preparation method of described solid fermentation substratum is: get wheat bran and hominy grits that mass ratio is 1:1, mix, put into triangular flask, add the distilled water of wheat bran and hominy grits total mass 55-60%, stir with glass stick, beyond the Great Wall tampon, 121 DEG C of steam sterilizing 45min.
Above-described suitable Metarhizium anisopliae produces the using method of the fermention medium of spore, comprises the following steps:
Step one, Metarhizium anisopliae is seeded on solid seed culture medium, be placed in 25 DEG C of constant incubators and cultivate (after a large amount of olive-green spore to be generated) after 7 ~ 12d, Bechtop is the spore that 0.2% tween-80 washes on lower solid seed culture medium with the quality volume of sterilizing, that transfers to sterilizing is placed with in the triangular flask of liquid seed culture medium and granulated glass sphere, shake ten minutes, spore is uniformly dispersed, then be placed in 150rpm on 25 DEG C of constant-temperature tables and shake cultivation 2 ~ 3d, gained fermented liquid is liquid seeds;
Step 2, on Bechtop, draw the liquid seeds of 5 mL with liquid-transfering gun, on solid fermentation substratum after access sterilizing, stir with glass stick above spirit lamp flame, be placed in 25 DEG C of constant incubators to carry out cultivation and terminate after 10 days to cultivate, namely cultivate and produce the Metarhizium anisopliae of spore; Calculate sporogenic amount, prepare Metarhizium anisopliae spore powder, and measure germination rate.
The described step preparing Metarhizium anisopliae spore powder is: after being ground in mortar by the solid fermentation substratum after fermentation, first filter by 20 mesh sieve, filter by 60,100 and 60 mesh sieve successively again, to remove fermentation material and impurity as far as possible, finally filter by 200 mesh sieve, collect the spore that Metarhizium anisopliae produces; Collected spore is put into baking oven dry, temperature is set to 25 DEG C, takes out, namely prepare Metarhizium anisopliae spore powder after dry 24 hours; The germination rate of prepared Metarhizium anisopliae spore powder is more than 95%.
Beneficial effect is:
1, the suitable Metarhizium anisopliae prepared by the present invention produces the fermention medium of spore, and seed culture medium and solid medium organically combine, and appropriately, be suitable for the growth of Metarhizium anisopliae, sporulation quantity is high, and every gram of fermentation raw material can produce 1 × 10 for composition and proportioning
9with last spore; The spore powder spore rate alive of preparation is high, and germination rate can reach about 95%; And hominy grits and wheat bran are all common raw materials, be easy to obtain, with low cost, preparation method is simple and easy, is easy to grasp, solves the cost existed in prior art high, the problem that sporulation quantity is low.The fermention medium that suitable Metarhizium anisopliae of the present invention produces spore has good application prospect in the biological control of crop pests.
2, fermentation culture method of the present invention, adopts liquid-solid two fermentation methods to cultivate Metarhizium anisopliae, promotes that it produces spore; Short, the production efficiency of the method raw material cheap and easy to get, production cycle improve, antipollution degree is high; The hominy grits wherein adopted in solid fermentation substratum is little compared with Semen Maydis powder density, permeability might as well, the nutritive ingredient such as rich in proteins the same as Semen Maydis powder, starch, vitamin H again, so the interpolation of hominy grits is more conducive to for mycelium provides sufficient nutrient than Semen Maydis powder.
Accompanying drawing explanation
Fig. 1 is the experimental result comparison diagram of experiment two;
Number in the figure 1-9 represents the Metarhizium anisopliae that the formula 1-9 in experiment two turns out respectively;
Fig. 2 is the experimental result comparison diagram of experiment three;
Number in the figure 1-15 represents the Metarhizium anisopliae that the formula 1-15 in experiment three turns out respectively.
Embodiment
Suitable Metarhizium anisopliae produces the fermention medium of spore, and comprise seed culture medium and solid fermentation substratum, wherein said seed culture medium comprises solid seed culture medium and liquid seed culture medium; Containing mass concentration in often liter of solid seed culture medium is 20% potato juice 1000mL, glucose 20g, KH
2pO
43g, MgSO
41.5g and agar 20g, surplus is water; In often liter of liquid seed culture medium, containing peptone 10 g, sucrose 10 g, K
2hPO
40.3 g, MgSO
40.3 g and NaCl 0.3 g, surplus is water;
The composition of described solid fermentation substratum is: wheat bran, hominy grits and distilled water; Wherein the mass ratio of wheat bran and hominy grits is 1:1, and the quality of distilled water is wheat bran and hominy grits total mass 55-60%.The preparation method of described solid fermentation substratum is: get wheat bran and hominy grits that mass ratio is 1:1, mix, put into triangular flask, add the distilled water of wheat bran and hominy grits total mass 55-60%, stir with glass stick, beyond the Great Wall tampon, 121 DEG C of steam sterilizing 45min.
Above-described suitable Metarhizium anisopliae produces the using method of the fermention medium of spore, comprises the following steps:
Step one, Metarhizium anisopliae is seeded on solid seed culture medium, be placed in 25 DEG C of constant incubators and cultivate (after a large amount of olive-green spore to be generated) after 7 ~ 12d, Bechtop washes the spore on lower solid seed culture medium with the tween-80 that the quality volume of sterilizing is 0.2%, that transfers to sterilizing is placed with in the triangular flask of liquid seed culture medium and granulated glass sphere, shake ten minutes, spore is uniformly dispersed; Then be placed in 150rpm on 25 DEG C of constant-temperature tables and shake cultivation 2 ~ 3d, gained fermented liquid is liquid seeds;
Step 2, on Bechtop, draw the liquid seeds of 5 mL with liquid-transfering gun, on solid fermentation substratum after access sterilizing, stir with glass stick above spirit lamp flame, be placed in 25 DEG C of constant incubators to carry out cultivation and terminate after 10 days to cultivate, namely cultivate and produce the Metarhizium anisopliae of spore;
Step 3, calculate Metarhizium anisopliae sporogenic amount: accurately taking quality is about m(1g) fermentation, put into the little triangular flask filling a little distilled water, with glass stick, mycelia broken up, through three layers of filtered through gauze, with the mycelia of angled spoon pressure dissipating bind block during filtration, through repeatedly repeatedly breaing up mycelia, washing, filtration, spore is washed out completely, collect filtrate, add granulated glass sphere, 120rpm vibrates 2h, be settled to proper volume V, suitable dilution, with blood counting chamber counting, is designated as N; With reference to the sporulation quantity of formulae discovery certainweight fermentation raw material.
The calculation formula of sporulation quantity is as follows:
Wherein, S---sporulation quantity (the dry substratum of individual spore/g);
N---blood counting chamber count value (individual spore/mL);
D---the extension rate before spore suspension counting;
The volume (mL) of V---spore suspension
W---the total mass (g) of culture and bottle (containing bottle stopper) during sampling;
Wf---the quality (g) of culture vessel triangular flask and tampon;
(W-Wf)---the quality (g) of the wild Oryza species that fermented;
The quality (g) of m---sample;
M0---the dry weight (g) of substratum batching before fermentation.
The preparation of step 4, muscardine spore powder: after the solid fermentation substratum after fermentation is ground in mortar, first filter by 20 mesh sieve, filter by 60,100 and 60 mesh sieve successively again, to remove fermentation material and impurity as far as possible, finally filter by 200 mesh sieve, collect the spore that Metarhizium anisopliae produces; Collected spore is put into baking oven dry, temperature is set to 25 DEG C, takes out, namely prepare Metarhizium anisopliae spore powder after dry 24 hours; The muscardine spore produced by solid fermentation substratum is through grinding, sieving, and spore is separated with fermentation material, and 100g fermentation material obtains 4.7850g spore powder after drying;
The mensuration of step 5, spore powder vitality: take out a small amount of spore powder with 0.2% tween-80 solution be made into certain density spore suspension, drawing 100 μ L coats on SDAY substratum, cultivate in 25 DEG C of incubators and sprout, timing from spread plate, calculates the spore germination rate of the 36th hour.The germination rate of spore powder is 95.79%.
Described SDAY substratum, adds yeast extract paste 5g, glucose 40.0g, peptone 10.0, agar 20.0g, pH value 6.0 ± 0.1 in often liter of SDAY substratum, surplus is water.
relevant comparative tests as follows:
Test in June, one, 2014, in order to carry out the field test of green muscardine fungus control Asiatic migrotory locust at white crane town, Mengjin County, Luoyang City Yellow River's beach, the solid fermentation of green muscardine fungus is carried out in indoor, collects muscardine spore powder, prepares muscardine spore powder finish.Have employed three kinds of substratum to cultivate; The first is the fermention medium adopting the present invention to be suitable for Metarhizium anisopliae product spore, and adopts methods involving of the present invention to cultivate.The second is that (general rice soaks 30 min to the rice medium that green muscardine fungus solid fermentation is conventional, 0.5% (v/w) salad oil is admixed) after draining, the third is the substratum (proportions is 3:3:4, and amount of water is 60% of solid fermentation material dry weight) be made into for fermentation material with wheat bran, hominy grits, Semen Maydis powder.After adding liquid strain, ferment 12 d, prepares muscardine spore powder, and the spore powder output that result shows to adopt the solid fermentation in invention to prepare is maximum, and sporulation quantity is 1.8206 × 10
9the dry substratum of individual spore/g, be that the spore powder of solid fermentation medium preparing is taken second place with rice, sporulation quantity is 1.1572 × 10
9the dry substratum of individual spore/g, and the spore powder output be mixed with wheat bran, hominy grits, Semen Maydis powder is minimum, sporulation quantity is 0.5608 × 10
9the dry substratum of spore/g.The spore powder prepared of three kinds of fermentation materials is living spore rate and in the virulence of Asiatic migrotory locust, no significant difference, further indicate the present invention, the suitable Metarhizium anisopliae solid fermentation substratum produced in the fermention medium of spore is suitable for Metarhizium anisopliae and produces spore.
Experiment two, with wood chip for fixing composition, compare the wheat bran of different ratios, hominy grits, Semen Maydis powder to the impact of the product spore of Metarhizium anisopliae; Wood chip is fermentation raw material conventional in Edible Fungi, and cheap, be easy to buy.This experiment is frozen composition with wood chip, convert the solid fermentation substratum of other composition contrasts; Wherein each formula is as follows:
Formula 1:30% wood chip+70% hominy grits;
Formula 2:40% wood chip+60% hominy grits;
Formula 3:50% wood chip+50% hominy grits;
Formula 4:30% wood chip+70% Semen Maydis powder;
Formula 5:40% wood chip+60% Semen Maydis powder;
Formula 6:50% wood chip+50% Semen Maydis powder;
Formula 7:30% wood chip+70% wheat bran;
Formula 8:40% wood chip+60% wheat bran;
Formula 9:50% wood chip+50% wheat bran.
Experimental result is for mycelial growth after the fermentation of: different ingredients substratum and produce spore character description and sporulation quantity as shown in table 1 and Fig. 1.In Fig. 1, the form contrast of the Metarhizium anisopliae that the solid fermentation substratum prepared according to formula 1-9 is cultivated can be learnt, respectively the fill a prescription sporogenic amount of Metarhizium anisopliae and form thereof of cultivating; From table 1 and Fig. 1, when wood chip is frozen composition, each formula all can produce spore; Along with wood chip proportion in formula reduces and other composition increases, sporulation quantity starts to increase, and wood chip is described and is not suitable for the product spore of Metarhizium anisopliae; Comparatively speaking, when taking wood chip as frozen composition, hominy grits, Semen Maydis powder are more conducive to the product spore of Metarhizium anisopliae compared with wheat bran.
Sporulation quantity when table 1 take wood chip as frozen composition under different ingredients and mycelium growth and sporulation character description
Experiment three, be frozen composition with wheat bran, contrast the material of other different components and ratio to the impact of the product spore of Metarhizium anisopliae; Concrete formula is as follows:
Formula 1:100% wheat bran;
Formula 2:90% wheat bran+10% Semen Maydis powder;
Formula 3:80% wheat bran+20% Semen Maydis powder;
Formula 4:70% wheat bran+30% Semen Maydis powder;
Formula 5:60% wheat bran+40% Semen Maydis powder;
Formula 6:50% wheat bran+50% Semen Maydis powder;
Formula 7:90% wheat bran+10% hominy grits;
Formula 8:80% wheat bran+20% hominy grits;
Formula 9:70% wheat bran+30% hominy grits;
Formula 10:60% wheat bran+40% hominy grits;
Formula 11:50% wheat bran+50% hominy grits;
Formula 12:80% wheat bran+10% Semen Maydis powder+10% bean powder;
Formula 13:80% wheat bran+10% hominy grits+10% bean powder;
Formula 14:80% wheat bran+20% bean powder;
Formula 15:80% wheat bran+20 wood chip.
Experimental result: different ingredients substratum ferments rear mycelial growth and product spore character description and sporulation quantity as shown in table 2 and Fig. 2.In Fig. 2, the form contrast of the Metarhizium anisopliae that the solid fermentation substratum prepared according to formula 1-15 is cultivated can be learnt, the sporogenic amount of Metarhizium anisopliae of cultivating according to the solid fermentation substratum prepared by each formula and form thereof; From table 2 and Fig. 2,50% wheat bran sporulation quantity can reach maximum with being combined in of 50% hominy grits, and when being fixing recipe ingredient with wheat bran, adding of hominy grits is more conducive to producing spore than Semen Maydis powder, bean powder, wood chip.
Sporulation quantity when table 2 take wheat bran as frozen composition under different ingredients and mycelium growth and sporulation character description
Experiment four, be frozen composition with hominy grits, compare the wheat bran of different ratios, hominy grits, Semen Maydis powder to the impact of the product spore of Metarhizium anisopliae; Each formula is as follows:
Formula 1:50% hominy grits+50% worm corpse;
Formula 2:50% hominy grits+50% Semen Maydis powder;
Formula 3:50% hominy grits+50% bean powder;
Formula 4:100% hominy grits;
Formula 5:50% hominy grits+50% wood chip;
Formula 6:50% hominy grits+50% cotton seed hulls;
Formula 7:50% hominy grits+50% corn cob;
Formula 8:50% hominy grits+50% wheat bran.
Experimental result: different ingredients substratum fermentation after mycelial growth and produce spore character description and sporulation quantity as shown in table 3.As shown in Table 3,50% hominy grits reaches maximum with being combined on sporulation quantity of 50% wheat bran.
Sporulation quantity when table 3 take hominy grits as frozen composition under different ingredients and mycelium growth and sporulation character description
Claims (4)
1. suitable Metarhizium anisopliae produces the fermention medium of spore, it is characterized in that: comprise seed culture medium and solid fermentation substratum;
Described seed culture medium comprises solid seed culture medium and liquid seed culture medium; Containing mass concentration in often liter of solid seed culture medium is 20% potato juice 1000mL, glucose 20g, KH
2pO
43g, MgSO
41.5g and agar 20g, surplus is water; In often liter of liquid seed culture medium, containing 10 g peptones, 10 g sucrose, 0.3 g K
2hPO
4, 0.3 g MgSO
4with 0.3 g NaCl, surplus is water;
The composition of described solid fermentation substratum is: wheat bran, hominy grits and distilled water; Wherein the mass ratio of wheat bran and hominy grits is 1:1, and the quality of distilled water is wheat bran and hominy grits total mass 55 ~ 60%.
2. suitable Metarhizium anisopliae as claimed in claim 1 produces the preparation method of the fermention medium of spore, it is characterized in that: the preparation method of described solid fermentation substratum is: get wheat bran and hominy grits that mass ratio is 1:1, mix, put into triangular flask, add the distilled water of wheat bran and hominy grits total mass 55 ~ 60%, stir with glass stick, beyond the Great Wall tampon, 121 DEG C of steam sterilizing 45min, namely prepare solid fermentation substratum.
3. suitable Metarhizium anisopliae as claimed in claim 1 produces the using method of the fermention medium of spore, it is characterized in that: comprise the following steps:
Step one, Metarhizium anisopliae is seeded on solid seed culture medium, after being placed in 25 DEG C of constant incubators cultivation 7 ~ 12d, Bechtop is the spore that 0.2% tween-80 washes on lower solid seed culture medium with the quality volume of sterilizing, that transfers to sterilizing is placed with in the triangular flask of liquid seed culture medium and granulated glass sphere, shake ten minutes, spore is uniformly dispersed; Then be placed in 150rpm on 25 DEG C of constant-temperature tables and shake cultivation 2 ~ 3d, gained fermented liquid is liquid seeds;
Step 2, on Bechtop, draw the liquid seeds of 5 mL with liquid-transfering gun, on solid fermentation substratum after access sterilizing, stir with glass stick above spirit lamp flame, be placed in 25 DEG C of constant incubators carry out cultivation terminate after 10 days cultivate, namely turn out the Metarhizium anisopliae growing spore.
4. suitable Metarhizium anisopliae as claimed in claim 3 produces the using method of the fermention medium of spore, it is characterized in that: the step adopting the Metarhizium anisopliae of turning out to prepare Metarhizium anisopliae spore powder is: after the solid fermentation substratum after fermentation is ground in mortar, first filter by 20 mesh sieve, filter by 60 orders, 100 orders and 60 mesh sieve successively again, finally filter by 200 mesh sieve, collect the spore that Metarhizium anisopliae produces; Collected spore is put into baking oven dry, at temperature 25 DEG C, dry 24h takes out, and namely prepares Metarhizium anisopliae spore powder.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911111A (en) * | 2015-06-24 | 2015-09-16 | 南京林业大学 | Metarhizium anisopliae solid culture method |
| CN104911111B (en) * | 2015-06-24 | 2018-11-23 | 南京林业大学 | A kind of Metarhizium anisopliae solid culture method |
| CN107446955A (en) * | 2017-08-25 | 2017-12-08 | 陕西省微生物研究所 | Pest-resistant soil conditioner and its production method using traditional Chinese medicine waste as matrix |
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| CN114456814A (en) * | 2022-01-17 | 2022-05-10 | 聊城金余肥业有限公司 | Low-density water-retention insect-resistant saline-alkali soil conditioner and preparation method thereof |
| CN114507096A (en) * | 2022-01-17 | 2022-05-17 | 聊城金余肥业有限公司 | Low-density vegetable organic bacterial fertilizer and preparation method thereof |
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