CN103571760A - Separation and purification method of beauveria bassiana - Google Patents
Separation and purification method of beauveria bassiana Download PDFInfo
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Abstract
The invention relates to the technical field of identification, separation and purification application of beauveria bassiana strains, and discloses a separation and purification method of beauveria bassiana. By adopting the separation and purification method, a culture medium is scientifically optimized and improved; simple, quick and effective separation and purification of the beauveria bassiana are achieved by combining improvement of cultivation device coverings on the basis of the culture medium with a scientific formula. The method disclosed by the invention is simple and quick to operate, wide in samples applicable to separation and purification of the beauveria bassiana, low in operation cost and free of pollution on environment; the targets of separation and purification can be well achieved.
Description
Technical field
The present invention relates to the applied technical fields such as the evaluation of muscardine kind, biological control, family's muscardine prevention and control, more specifically, relate to the separation purification method of a kind of muscardine.
Background technology
Muscardine (
beauveria bassiana) be classified as now Chinese caterpillar fungus section (
cordycipitaceae), Cordyceps (
cordyceps) important fungi, the gondola scientist Bassi(1835 as far back as 1816) from Bombyx Batryticatus, separate.
Muscardine can invade 700 various insects of 15 521 genus of Ge Mu149Ge section and mite class (Li Zengzhi. Chinese entomogenous fungi research and application (first roll). Beijing: academic journal press, 1988).Due to muscardine have meet some basic demands of biotechnological formulation, to harmless, the easy cultivation of warm-blooded animal and plant, virulence is strong, pest control is effective and the advantage such as wide spectrum, host is extensive, highly pathogenic and adaptability, its bacterial classification is found broad application in the biological control of agriculture, woods insect, be also simultaneously one of important pathogen of economic insects silkworm etc., affect the healthy and stable development that silkworm and mulberry are produced always.In addition, Bombyx Batryticatus is as the traditional Chinese medicine of China, and application is clinically also quite extensive.Therefore set up separation and purification muscardine method particularly important.
At present, for the separation and purification of muscardine, all there is technology report both at home and abroad.Muscardine is for there being a fungi, can be according to traditional fungi line separation and purification, but the method operation element is loaded down with trivial details, and transfer repeatedly could be separated by muscardine.For improving, researchist has developed the selective medium for muscardine both at home and abroad, Martin (Martin J P. Use of acid for example, rose bengal, and streptomycin in the plate method for estimating soil fungi. Soil Science, 1950) in substratum, add rose-bengal (ose bengale) and oxgall has reached miscellaneous bacteria inhibition to a certain degree; Boerema(Boerema G H etl. Some notable fungus infections II. Verslagen van de Plantenziektenkundige Dienst te Wageningen, 1963) with cherry leach liquor, make selective medium, but undesirable to the inhibition of miscellaneous bacteria; (the Veen K H such as Veen, Ferron P. A selective medium for the isolation of Beauveria tenella and of Metarrhizium anisopliae. Journal of invertebrate pathology, 1966) utilize the MartinShi substratum of improvement successfully separated beauveria bassiana and green muscardine fungus, but muscardine is poor growth on this kind of substratum, be difficult for differentiating; (the Doberski J W such as Doberski, Tribe H T. Isolation of entomogenous fungi from elm bark and soil with reference to ecology of Beauveria bassiana and Metarhizium anisopliae. Transactions of the British Mycological Society, 1980), utilize Viola crystallina and cycloheximide to there is good selection specificity to muscardine; (Beilharz V C.etl. Dodine:a selective agent for certain soil fungi. Transactions of the British Mycological Society such as Beilharz, 1982) design a kind of selective medium that dodine (dodine) is fungistat of take, there is good separating effect; (the Chase A R.etl. Selective isolation of the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae from an artificial potting medium. Florida Entomologist such as Chase, 1986) Optimal Medium condition adds dodine, F-1991 and Viola crystallina muscardine can be carried out from other fungies effective separated in oat medium; Bright (the Shimazu M of Shimadzu, Sato H. Media for selective isolation of an entomogenous fungus, Beauveria bassiana. Applied entomology and zoology, 1996) characteristic of utilizing muscardine to grow within the scope of very wide pH, make the barren alkaline medium of nutrition, obtain good separating effect; The domestic force literary composition of presenting oneself before (a monarch) waits (the force literary composition of presenting oneself before (a monarch), Gao Zongqing, Yao Junmei. cultivate reagent pine cream and the loose cream substratum of fungi. fungus journal, 1985) utilize pine needle extract-loose cream to come the microorganism growth such as anti-bacteria, actinomycetes to promote fungal growth with this, but specificity is inadequate.(the Wang Bin such as Wang Bin, Fan Meizhen, Li Zengzhi. the screening of beauveria bassiana selective medium. Agricultural University Of Anhui's journal, 2000) select 25% Sportak missible oil (containing 25% Prochloraz), 77% Cocide wettable powder (containing 77% copper hydroxide), 5% octicin solution aqua (containing 5% amino acid pattern systemic fungicide) to replace dodine and cycloheximide, but experimental result is undesirable; (the Shi Hongxia such as Shi Hongxia, Hu Minglong, Yan Jun. the screening of beauveria bassiana selective medium and detection application. Zhejiang Forestry Institute journal, 2002) for the muscardine that colonizes in cone flowerfly, select rose-bengal, cycloheximide, paraxin as fungistat, obtain good separation; According to muscardine, there is certain host preference, Cai Guogui (Cai Guogui. just bamboo poison moth Fine Strains of Beauveria bassiana screening and production application research. forest-science, 2003, screening and the application of the high virulence muscardine of cryptomeria caterpillar Bbd3 bacterial strain. the journal .2006 of Nanjing Forestry University) to traditional substratum, add firm bamboo poison moth or cryptomeria caterpillar worm dry powder, can provide obligate nutrition for invading the muscardine of firm bamboo poison moth or cryptomeria caterpillar; Xu Wen wait quietly (Zhang Zhengkun, etc. the new bacterial strain screening of the efficient beauveria bassiana of control of maize snout moth's larva for Xu Wenjing, Yin Wei phoenix. Jilin agricultural sciences, 2011) application adds the PDA substratum of peptone to carry out purifying to invading the muscardine of Pyrausta nubilalis (Hubern).; Wang Tao far waits that (Wang Tao is far away, Xu Wenjing, Zhang Zhengkun, Deng. the screening of beauveria bassiana isolation medium and application. Chinese agronomy circular, 2013) to adding dodine, cephamycin, sulphuric acid kanamycin in PPDA substratum, can effectively suppress miscellaneous bacteria, filter out for detecting the obligate substratum of muscardine at the bacterium colony content in field.
In sum, although prior art has been made a large amount of research for selective medium, only have the expensive dodine of use or cycloheximide relatively good as the separating effect of fungistat, practical application high cost; And cycloheximide is carcinogenic, affect HUMAN HEALTH.The deficiencies in the prior art have limited the practical application of above-mentioned technology, can not solve the common requirements to muscardine separation and purification at present.
Lack at present the demand that a kind of simple and easy, safe, efficient, lower-cost separation and purification muscardine method adapts to practical application.
Summary of the invention
The technical problem to be solved in the present invention is the technical deficiency that overcomes existing muscardine separation purification method, has set up a kind of method of simple and easy, safe, efficient, lower-cost separation and purification muscardine.
Object of the present invention is achieved by the following technical programs:
The separation purification method of a kind of muscardine is provided, the spore liquid of muscardine is evenly coated on selective medium and cultivated, obtain muscardine mycelia and the conidium of separation and purification; The composition of described selective medium is: in the PDA substratum of every 1000 mL sterilising treatment, add following component: 2%～2.5% Deoxycholic Acid sodium solution 20 mL, 0.1%～0.3% rose-bengal 3.3 mL, the above percentage ratio is mass percent concentration, adds 10 before facing use
4u/mL～10
6vetstrep solution 3.3 mL of U/mL.
PDA substratum described in each (15～20 mL) contains 660U Vetstrep, 0.8 % sodium deoxycholate, 0.007 % rose-bengal.
Described PDA substratum consists of: potato 200 g, and glucose 20 g, agar 20 g, tap water 1000 mL, regulate pH value to 6 with 1M NaOH or 1M HCL.
Preferably, after culture medium solidifying, under aseptic technique, with tweezers, aseptic glassine paper is covered on agar plate, the spore liquid of muscardine is evenly coated on aseptic glassine paper.
Described glassine paper is cellulose film material, is a kind of transparent film made from fiber, and culture dish size, is used after sterilizing.
Wherein, the method that the spore liquid of muscardine preparation, coating spore liquid, cultivation and purifying are cultivated can be with reference to the routine of the art.Preferably, when coating spore liquid, the present invention is that the spore liquid preparing is even with aseptic spreading rod coating on substratum, make spore liquid be dispersed in the planar surface of whole substratum, and then use this spreading rod, coating the 2nd or the 3rd flat board, can prevent that bacterium colony is overlapping well continuously, guarantees that substratum is easy to occur single bacterium colony.
More specifically, the separation purification method of muscardine of the present invention comprises the following steps:
S1. prepare spore liquid:
With aseptic inoculation ring from Bombyx Batryticatus or insect body surface picking 2～3 ring conidial powders that infected muscardine in sterilized water, vibration is fully scattered spore;
S2. preparation is dull and stereotyped: the PDA substratum of sterilising treatment is cooled to 50～60 ℃, add sodium deoxycholate, rose-bengal, Vetstrep solution; After culture medium solidifying, under aseptic technique, with tweezers, aseptic glassine paper is covered on agar plate;
S3. the spore liquid of being prepared by S1 is evenly coated flat board;
Get spore liquid prepared by 200 μ L on described flat board, with aseptic spreading rod coating evenly, make spore liquid be dispersed in whole planar surface, and then use this spreading rod, be coated with continuously the 2nd or the 3rd flat board, its object prevents that bacterium colony is overlapping, and this makes substratum be easy to occur single bacterium colony.
S4. cultivate;
It is that 92 ± 5% environment are cultivated 3～7d that the flat-plate inverted that S3 has been coated with to spore liquid is placed in 26 ± 2 ℃, humidity; The colonies typical that select that mycelial growth is vigorous, spore is many is to 1.5mL centrifuge tube, in centrifuge tube, there are the aseptic insect physiological saline of 1mL and appropriate granulated glass sphere, after vibration evenly, get 200 μ L and be coated on the flat board that posts glassine paper, be inverted in 26 ± 2 ℃, humidity is that 92 ± 5 % cultivate 3～7d;
Described aseptic insect physiological saline is that mass percent concentration is 6.5% sodium-chlor (NaCL) aqueous solution, is wherein added with the tween-80 that volume ratio percentage concentration is 0.1 %, in every 1000mL insect physiological saline, adds 1 mL tween-80.
S5. treat that mycelia covers with, glassine paper is taken off, can obtain muscardine mycelia and the conidium of separation and purification.
Further, the present invention also comprised the step that the cause of disease to gathering is processed before S1:
Bombyx Batryticatus 75 %(v/v that natural infection or artificial challenge are obtained) ethanol cotton balls carries out the sterilization of silkworm surface, then it is put into sterile petri dish, in ware, put a cotton balls soaking with sterilized water, be placed in 26 ℃ and cultivate 3～5d, after silkworm surface grows white fine hair mycelia and conidium, carry out again strain separating;
Preferably, described in S2, the compound method of flat board is as follows:
S21. prepare following reagent: Vetstrep concentration is 10
4～10
6u/mL(prepares with sterilized water, without autoclaving); Sodium deoxycholate mass percent concentration is 2%～2.5%, and rose-bengal mass percent concentration is 0.1%～0.3%, and autoclaving is standby;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000 mL poach are rotten (boils 20～30 min, can poke with glass stick), by 4 layers of filtered through gauze, add after 20 g agar and 20 g glucose, supply again moisture to 1000 mL, use 1M NaOH or 1M HCL that the pH of substratum is adjusted to sterilizing 20 min at 6.0,121 ℃, then add ready sodium deoxycholate 20 mL of S21 and 3.3 mL Vetstreps and 3.3 mL rose-bengals.
The separation purification method of muscardine of the present invention is more suitable for from having infected the silkworm separation and purification muscardine of muscardine.
The present invention has following beneficial effect:
The invention provides a kind of selective medium that is applicable to muscardine separation and purification, with sodium deoxycholate, Vetstrep and rose-bengal coupling, obtained good technique effect, the microbiotic of suitable concentration and usage quantity and tensio-active agent are brought into play synergy jointly, the effectively growth of anti-bacteria, mould, do not affect the growth of muscardine, more quickly by muscardine separation and purification out.
The present invention does not use carcinogenic cycloheximide, and having overcome it affects the unfavorable of HUMAN HEALTH, is a kind of safe separation purification method.
Cost of the present invention is lower, according to existing market valency, investigates, and the dodine that prior art adopts is 580～590 yuan/g, and the 7 yuan/g of sodium deoxycholate that the present invention adopts, 3 yuan/g of sulfuric acid Sodium cholic acid, 1.4 yuan/g of rose-bengal, reduces costs greatly.
From substratum, collect separated mycelia and conidium is also very difficult, easily mix other miscellaneous bacteria, pollute.The present invention creatively uses a kind of glassine paper, and the glassine paper using is a kind of regenerated cellulose, and as semi-permeable membranes, its molecular gap exists ventilation property, and muscardine can draw the required nutrition of growth, normal growth from substratum.Treat that muscardine grows to required degree and just glassine paper can be taken off, just can be easily quickly that mycelia and conidium is separated with substratum, in order to muscardine DNA extracting or conidial purifying or other application.And glassine paper raw material comes from natural, under environment, easily decompose, free from environmental pollution.
In sum, separation and purification muscardine method of the present invention is simple and easy, safe, efficient, cost is lower, has good actual application prospect.
Accompanying drawing explanation
Fig. 1 is to add Vetstrep etc. for the substratum of muscardine separation.
The common PDA substratum of Fig. 2 carries out the result of separation and purification to the muscardine of Bombyx Batryticatus.
Fig. 3 substratum posts glassine paper and collects muscardine mycelia and conidium.
Fig. 4 is placed in 26 ℃ of cultivations by Bombyx Batryticatus and treats that silkworm surface grows white fine hair mycelia and conidium after 3～5 days.
Embodiment
Below in conjunction with the drawings and specific embodiments, further set forth the present invention.Unless stated otherwise, the method and apparatus that the present invention adopts is this area ordinary method and equipment, and the reagent of employing unless stated otherwise, is the reagent of the art routine.
Embodiment 1
The cause of disease gathering is processed: the Bombyx Batryticatus of natural infection (also can adopt conventional means artificial challenge's Bombyx Batryticatus) is used to 75 %(v/v) ethanol cotton balls carries out the sterilization of silkworm surface, then it is put into sterile petri dish (ware is put a cotton balls soaking with sterilized water), be placed in 26 ℃ and cultivate 3～5 days (d), after silkworm surface grows white fine hair mycelia and conidium, carry out again strain separating;
S1. prepare spore liquid: with aseptic inoculation ring, from silkworm surface picking 2～3 ring conidial powders, in the triangular flask that fills 50 mL sterilized waters, (include sterile glass beads), with vortex oscillation 5 min, spore is fully scattered;
S2. preparation is dull and stereotyped:
S21. prepare following reagent: Vetstrep concentration is that 1000U/mL(prepares with sterilized water, without autoclaving, adds before use); Sodium deoxycholate mass percent concentration is 2%,, rose-bengal mass percent concentration is 0.1%, autoclaving is standby;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000 mL poach are rotten (boils 20～30 min, can poke with glass stick), by 4 layers of filtered through gauze, add after 20 g agar and 20 g glucose, then supply moisture to 1000 mL, use 1M NaOH or 1M HCL that medium pH is adjusted to sterilizing 20 min at 6.0,121 ℃.
The PDA substratum of sterilising treatment is cooled to 50～60 ℃, add 2 % sodium deoxycholate 20 ml/L; 0.1 % rose-bengal 3.3 mL/L; Vetstrep solution (1000 U/mL) 3.3 mL/L; Separately by the PDA flat board that does not add sodium deoxycholate and Vetstrep in contrast;
S3. be coated with spore liquid: get 200 uL spore liquid and with aseptic spreading rod, be coated with evenly on flat board, make spore liquid be scattered in whole planar surface, and then use this spreading rod, be coated with continuously the 2nd the 3rd flat board, flat-plate inverted is placed in to 26 ℃, and humidity is that 95 % cultivate 3～7d;
S4. the colonies typical that select that mycelial growth is vigorous, spore is many enters in 1.5 mL, to have that 1ml is aseptic to be added with in the insect physiological saline of 0.1 % tween-80 and the centrifuge tube of appropriate granulated glass sphere, and vibration evenly;
S5. get 200 μ L and coat on the glassine paper being attached on antibiotic flat boards such as containing Vetstrep, being inverted in 26 ℃, humidity is to cultivate 3～7d in 95 % environment; Treat that mycelia covers with, glassine paper can be taken off, can obtain muscardine mycelia and the conidium of separation and purification, see shown in accompanying drawing 1 and accompanying drawing 3; The muscardine of common PDA substratum separation is coated on to the colonial morphology on common PDA substratum, sees shown in accompanying drawing 2.Shown in accompanying drawing 1 and accompanying drawing 3, adding on antibiotic substratum, there is not mould and bacterium, bacterium colony is pale pink, is easy to explanation; By ordinary culture medium shown in accompanying drawing 2, had mould and other miscellaneous bacterias when the separated muscardine for the first time.
Embodiment 2
The cause of disease gathering is processed: will infect silkworm chrysalis 75 %(v/v of natural infection or artificial challenge muscardine) ethanol cotton balls carries out surface sterilization, then it is put into sterile petri dish, in ware, put a cotton balls soaking with sterilized water, be placed in 26 ℃ and cultivate 3～5 days (d), after silkworm surface grows white fine hair mycelia and conidium, carry out again strain separating;
S1. prepare spore liquid: with aseptic inoculation ring, from silkworm surface picking 2～3 ring conidial powders, in the triangular flask that fills 50 mL sterilized waters, (include sterile glass beads), with vortex oscillation 5 min, spore is fully scattered;
S2. preparation is dull and stereotyped:
S21. prepare following reagent: Vetstrep concentration is that 10000U/mL(prepares with sterilized water, without autoclaving, adds before use); Sodium deoxycholate mass percent concentration is 2.5%,, rose-bengal mass percent concentration is 0.2%, autoclaving is standby;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000 mL poach are rotten (boils 20～30 min, can poke with glass stick), by 4 layers of filtered through gauze, add after 20 g agar and 20 g glucose, then supply moisture to 1000 mL, use 1M NaOH or 1M HCL that medium pH is adjusted to sterilizing 20 min at 6.0,121 ℃.
The PDA substratum of sterilising treatment is cooled to 50～60 ℃, add 2.5 % sodium deoxycholate 20 ml/L; 0.2 % rose-bengal 3.3 mL/L; Vetstrep solution (10000 U/mL) 3.3 mL/L; Separately by the PDA flat board that does not add sodium deoxycholate and Vetstrep in contrast;
S3. be coated with spore liquid: get 200 uL spore liquid and with aseptic spreading rod, be coated with evenly on flat board, make spore liquid be scattered in whole planar surface, and then use this spreading rod, be coated with continuously the 2nd the 3rd flat board, flat-plate inverted is placed in to 26 ℃, and humidity is that 95 % cultivate 3～7d;
S4. the colonies typical that select that mycelial growth is vigorous, spore is many enters in 1.5 mL, to have that 1ml is aseptic to be added with in the insect physiological saline of 0.1 % tween-80 and the centrifuge tube of appropriate granulated glass sphere, and vibration evenly;
S5. get 200 μ L and coat on the glassine paper being attached on antibiotic flat boards such as containing Vetstrep, being inverted in 26 ℃, humidity is to cultivate 3～7d in 95% environment; Treat that mycelia covers with, glassine paper can be taken off, can obtain muscardine mycelia and the conidium of separation and purification.
Embodiment 3
S1. the biological pesticide that contains beauveria bassiana spore of making for biological control (biological control station, Guangzhou is chosen and provided at random, and those skilled in the art also can adopt the biological pesticide that similarly contains beauveria bassiana spore to test) is diluted to the diluent that mass percent concentration is 1 or 10 %.
S2. preparation is dull and stereotyped;
S21. prepare following reagent: Vetstrep concentration is that 100000U/mL(prepares with sterilized water, without autoclaving, adds before use); Sodium deoxycholate mass percent concentration is 2.2%,, rose-bengal mass percent concentration is 0.25%, autoclaving is standby;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000 mL poach are rotten (boils 20～30 min, can poke with glass stick), by 4 layers of filtered through gauze, add after 20 g agar and 20 g glucose, then supply moisture to 1000 mL, use 1M NaOH or 1M HCL that medium pH is adjusted to sterilizing 20 min at 6.0,121 ℃.
The PDA substratum of sterilising treatment is cooled to 50～60 ℃, add 2.2 % sodium deoxycholate 20 ml/L; 0.25 % rose-bengal 3.3 mL/L; Vetstrep solution (100000 U/mL) 3.3 mL/L; Separately by the PDA flat board that does not add sodium deoxycholate and Vetstrep in contrast;
S3. be coated with spore liquid: get 200 uL biological pesticide diluents even with aseptic spreading rod coating on flat board, make spore liquid be scattered in whole planar surface, and then be coated with continuously the 2nd the 3rd flat board with this spreading rod, and flat-plate inverted is placed in to 26 ℃, humidity is that 95 % cultivate 3～7d;
S4. the colonies typical that select that mycelial growth is vigorous, spore is many enters in 1.5 mL, to have that 1ml is aseptic to be added with in the insect physiological saline of 0.1 % tween-80 and the centrifuge tube of appropriate granulated glass sphere, and vibration evenly;
S5. get 200 μ L and coat on the glassine paper being attached on antibiotic flat boards such as containing Vetstrep, being inverted in 26 ℃, humidity is to cultivate 3～7d in 95% environment; Treat that mycelia covers with, glassine paper can be taken off, can obtain muscardine mycelia and the conidium of separation and purification.
According to existing market valency, investigate, 7 yuan/the g of sodium deoxycholate using in the embodiment of the present invention 1,2,3,3 yuan/g of sulfuric acid Sodium cholic acid, 1.4 yuan/g of rose-bengal, and the dodine that prior art adopts is 580～590 yuan/g, the present invention reduces the cost of separation and purification greatly.
After testing, in the muscardine mycelia that separation and purification of the present invention obtains and conidium, not containing other miscellaneous bacterias (bacterium, mould), muscardine mycelia is dense is spherical or oval with conidium, occurs individually healthy sprout.The present invention collects separated mycelia and conidium is very convenient quick from substratum.
Claims (7)
1. a method for separation and purification muscardine, is characterized in that, is the spore liquid of muscardine is evenly coated on selective medium and cultivated, and obtains muscardine mycelia and the conidium of separation and purification; The composition of described selective medium is: in the PDA of every 1000mL sterilising treatment substratum, add following component: 2%～2.5% Deoxycholic Acid sodium solution 20mL, 0.1%～0.3% rose-bengal 3.3mL, the above percentage ratio is mass percent concentration, adds 10 before facing use
4u/mL～10
6the Vetstrep solution 3.3mL of U/mL.
2. the method for separation and purification muscardine according to claim 1, is characterized in that, described PDA substratum consists of: potato 200g, and glucose 20g, agar 20g, tap water 1000mL, regulates pH value to 6 with 1MNaOH or 1MHCL.
3. the method for separation and purification muscardine according to claim 1, is characterized in that, the spore liquid of muscardine is evenly coated on selective medium, after culture medium solidifying, under aseptic technique, aseptic glassine paper is covered on agar plate.
4. the method for separation and purification muscardine according to claim 1, it is characterized in that, the method that the spore liquid of muscardine is evenly coated on selective medium is that spore liquid is even with aseptic spreading rod coating on substratum, and then is coated with continuously another flat board with this spreading rod.
5. according to the method for separation and purification muscardine described in claim 1 to 4 any one, it is characterized in that, comprise the following steps:
S1. prepare spore liquid:
With aseptic inoculation ring from Bombyx Batryticatus or insect body surface picking 2～3 ring conidial powders that infected muscardine in sterilized water, vibration is fully scattered spore;
S2. preparation is dull and stereotyped: the PDA substratum of sterilising treatment is cooled to 50～60 ℃, add sodium deoxycholate, rose-bengal, Vetstrep solution; After culture medium solidifying, under aseptic technique, with tweezers, aseptic glassine paper is covered on agar plate;
S3. the spore liquid of being prepared by S1 is evenly coated flat board;
S4. cultivate; It is that 92 ± 5% environment are cultivated 3～7d that the flat-plate inverted that S3 has been coated with to spore liquid is placed in 26 ± 2 ℃, humidity; The colonies typical that select that mycelial growth is vigorous, spore is many is to 1.5mL centrifuge tube, in centrifuge tube, there are the aseptic insect physiological saline of 1mL and granulated glass sphere, after vibration evenly, get 200 μ L and be coated on the flat board that posts glassine paper, be inverted in 26 ± 2 ℃, humidity is that 92 ± 5 % cultivate 3～7d;
Described aseptic insect physiological saline is that mass percent concentration is 6.5% sodium chloride aqueous solution, is wherein added with the tween-80 that volume by volume concentration is 0.1 %;
S5. treat that mycelia covers with, glassine paper is taken off, can obtain muscardine mycelia and the conidium of separation and purification.
6. the method for separation and purification muscardine according to claim 5, it is characterized in that, before S1, also comprise the step that the cause of disease to gathering is processed, the ethanol cotton balls that the Bombyx Batryticatus that soon natural infection or artificial challenge obtain is 75% with concentration of volume percent carries out the sterilization of silkworm surface, then put it in sterile petri dish, in ware, put a cotton balls soaking with sterilized water, be placed in 26 ℃ and cultivate 3～5d, after silkworm surface grows white fine hair mycelia and conidium, carry out again strain separating.
7. the method for separation and purification muscardine according to claim 5, is characterized in that, compound method dull and stereotyped described in S2 is as follows:
S21. prepare following reagent: Vetstrep concentration is 10
4～10
6u/mL; Sodium deoxycholate mass percent concentration is 2%～2.5%, and rose-bengal mass percent concentration is 0.1%～0.3%, and autoclaving is standby;
S22. by peeling potatoes, be cut into small pieces, add poach rotten, filter, add after agar and glucose, supply moisture, pH is adjusted to 6.0, adds the ready sodium deoxycholate of S21, Vetstrep and rose-bengal after sterilizing.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104016811A (en) * | 2014-05-09 | 2014-09-03 | 安徽福瑞德生物科技有限公司 | Beauveria bassiana biological compound medicine fertilizer and preparation method thereof |
| CN104611239A (en) * | 2015-02-11 | 2015-05-13 | 广东省微生物研究所 | Culture collection method for fungi mycelium |
| CN105232580A (en) * | 2015-07-28 | 2016-01-13 | 河池学院 | Method of using grasserie silkworms for producing medicinal white muscardine silkworms |
| CN106834123A (en) * | 2016-11-30 | 2017-06-13 | 四川省农业科学院蚕业研究所 | A kind of muscardine separates expanding propagation method |
| CN108715813A (en) * | 2018-05-25 | 2018-10-30 | 福建农林大学 | A method of collecting pure mycelia |
| CN109810907A (en) * | 2019-03-18 | 2019-05-28 | 福建农林大学 | One plant of beauveria bassiana BbJL-01 for having High pathogenicity to Dendrolimus houi linal-instar larvae |
| CN110352920A (en) * | 2019-08-30 | 2019-10-22 | 江苏海洋大学 | A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine |
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| CN114540203A (en) * | 2022-02-22 | 2022-05-27 | 山西农业大学 | Beauveria bassiana with young silkworm infection function, microbial inoculum and application thereof, and preparation method of white muscardine silkworm |
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| CN104016811A (en) * | 2014-05-09 | 2014-09-03 | 安徽福瑞德生物科技有限公司 | Beauveria bassiana biological compound medicine fertilizer and preparation method thereof |
| CN104611239A (en) * | 2015-02-11 | 2015-05-13 | 广东省微生物研究所 | Culture collection method for fungi mycelium |
| CN105232580A (en) * | 2015-07-28 | 2016-01-13 | 河池学院 | Method of using grasserie silkworms for producing medicinal white muscardine silkworms |
| CN106834123A (en) * | 2016-11-30 | 2017-06-13 | 四川省农业科学院蚕业研究所 | A kind of muscardine separates expanding propagation method |
| CN108715813A (en) * | 2018-05-25 | 2018-10-30 | 福建农林大学 | A method of collecting pure mycelia |
| CN109810907A (en) * | 2019-03-18 | 2019-05-28 | 福建农林大学 | One plant of beauveria bassiana BbJL-01 for having High pathogenicity to Dendrolimus houi linal-instar larvae |
| CN110352920A (en) * | 2019-08-30 | 2019-10-22 | 江苏海洋大学 | A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine |
| CN110352920B (en) * | 2019-08-30 | 2021-09-14 | 江苏海洋大学 | Method for artificially culturing white muscardine silkworms based on beauveria bassiana |
| CN113278578A (en) * | 2021-06-15 | 2021-08-20 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| CN114540203A (en) * | 2022-02-22 | 2022-05-27 | 山西农业大学 | Beauveria bassiana with young silkworm infection function, microbial inoculum and application thereof, and preparation method of white muscardine silkworm |
| CN114540203B (en) * | 2022-02-22 | 2023-04-14 | 山西农业大学 | Beauveria bassiana with young silkworm infection function, microbial inoculum and application thereof, and preparation method of white muscardine silkworm |
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