CN103571760B - 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 muscardine Species estimation, 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) is classified as Chinese caterpillar fungus section (Cordycipitaceae) now, the important fungi of Cordyceps (Cordyceps), as far back as 1816, gondola scientist Bassi (1835) was separated from Bombyx Batryticatus.
Muscardine can invade 15 Ge Mu149Ge section 521 belong to 700 various insects and mite class (Li Zengzhi. Chinese entomogenous fungi research and apply (first roll). Beijing: academic journal press, 1988).Due to muscardine have meet biotechnological formulation some basic demands, namely to warm-blooded animal and non-phytotoxic, easily cultivate, 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, also be one of important pathogen of economic insects silkworm etc. simultaneously, affect the healthy and stable development of Sericultural production always.In addition, Bombyx Batryticatus is as the traditional Chinese medicine of China, and application is clinically also quite extensive.Therefore separation and purification muscardine method is set up particularly important.
At present, for the separation and purification of muscardine, technology is all had to report both at home and abroad.Muscardine, can traditionally fungi line separation and purification for there being a fungi, but the method operation element is loaded down with trivial details, transfer and repeatedly muscardine could be separated.For improving, domestic and international researchist develops the selective medium for muscardine, such as Martin (Martin J P.Use of acid, rosebengal, and streptomycin in the plate method for estimating soil fungi.Soil Science, 1950) rose-bengal (ose bengale) is added in the medium and oxgall reaches miscellaneous bacteria suppression to a certain degree; Boerema (Boerema G H etl.Some notable fungus infections II.Verslagen van de PlantenziektenkundigeDienst te Wageningen, 1963) selective medium is made with cherry leach liquor, 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 tenellaand of Metarrhizium anisopliae.Journal of invertebrate pathology, 1966) the MartinShi substratum of improvement is utilized successfully to be separated beauveria bassiana and green muscardine fungus, but muscardine is poor growth on this kind of substratum, not easily differentiates; (the Doberski J W such as Doberski, Tribe H T.Isolation of entomogenous fungi from elm bark and soilwith reference to ecology of Beauveria bassiana and Metarhizium anisopliae.Transactions of theBritish Mycological Society, 1980), utilize Viola crystallina and cycloheximide can have muscardine and select specificity preferably; (Beilharz V C.etl.Dodine:a selective agent for certain soil fungi.Transactionsof the British Mycological Society such as Beilharz, 1982) devise the selective medium that one is fungistat with dodine (dodine), there is good separating effect; (the Chase A R.etl.Selective isolation of theentomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae from an artificial pottingmedium.Florida Entomologist such as Chase, 1986) Optimal Medium condition, in oat medium, add dodine, F-1991 and Viola crystallina effectively can be separated muscardine from other fungies; Shimadzu light (Shimazu M, Sato H.Mediafor selective isolation of an entomogenous fungus, Beauveria bassiana.Applied entomology andzoology, 1996) characteristic that muscardine can grow within the scope of very wide pH is utilized, make the alkaline medium that nutrition is barren, obtain good separating effect; Domestic force literary composition of presenting oneself before (a monarch) waits that (force is presented oneself before (a monarch) literary composition, Gao Zongqing, Yao Junmei. cultivate the reagent pine cream of fungi and loose cream substratum. fungus journal, 1985) utilize pine needle extract-loose cream to carry out the microorganism growth such as anti-bacteria, actinomycetes with this to promote fungal growth, 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 College, 2002) for the muscardine colonizing in cone flowerfly, select rose-bengal, cycloheximide, paraxin as fungistat, be separated preferably; Certain host speciality is there is according to muscardine, Cai Guogui (Cai Guogui. just bamboo poison moth Fine Strains of Beauveria bassiana screening and production application research. forest-science, 2003, the screening of Dendrolimus houi high virulence muscardine Bbd3 bacterial strain and application. Nanjing Forestry University journal .2006) add firm bamboo poison moth or Dendrolimus houi worm dry powder to conventional medium, obligate nutrition can be provided for the muscardine invading firm bamboo poison moth or Dendrolimus houi; Xu Wen waits quietly (Xu Wenjing, Yin Wei phoenix, Zhang Zhengkun, etc. the efficient beauveria bassiana new strains screening of. control of maize snout moth's larva. Jilin agricultural sciences, 2011) and the application PDA substratum that adds peptone carries out purifying to the muscardine invading Pyrausta nubilalis (Hubern).; Wang Tao far wait (Wang Tao is far away, Xu Wenjing, Zhang Zhengkun, Deng. the screening of beauveria bassiana isolation medium and application. Chinese agronomy circular, 2013) in PDA substratum, adding dodine, cephamycin, sulphuric acid kanamycin effectively can suppress miscellaneous bacteria, filtering out for detecting the obligate substratum of muscardine at the bacterium colony content in field.
In sum, although prior art has made large quantifier elimination for selective medium, only have use expensive dodine 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 limit the practical application of above-mentioned technology, can not solve the common requirements at present to muscardine separation and purification.
A kind of simple and easy, safe, efficient, lower-cost separation and purification muscardine method of current shortage adapts to the demand of practical application.
Summary of the invention
The technical problem to be solved in the present invention is the technical deficiency overcoming existing muscardine separation purification method, establishes 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 spread evenly across on selective medium and cultivates, obtain muscardine mycelia and the conidium of separation and purification; The composition of described selective medium is: in the PDA substratum of every 1000mL sterilising treatment, add following component: the Deoxycholic Acid sodium solution 20mL of 2% ~ 2.5%, the rose-bengal 3.3mL of 0.1% ~ 0.3%, the above percentage ratio is mass percent concentration, adds 10 before facing use
4u/mL ~ 10
6the Vetstrep solution 3.3mL of U/mL.
Described in each, PDA substratum (15 ~ 20mL) is containing 660U Vetstrep, 0.8% sodium deoxycholate, 0.007% rose-bengal.
Described PDA substratum consists of: potato 200g, glucose 20g, agar 20g, tap water 1000mL, by 1M NaOH or 1M HCL adjust ph to 6.
Preferably, after culture medium solidifying, with tweezers, sterile glass paper is covered on agar plate under aseptic technique, the spore liquid of muscardine is spread evenly across on sterile glass paper.
Described glassine paper is cellulose film material, and be the transparent film that a kind of fiber is made, culture dish size, uses after sterilizing.
Wherein, the method that the spore liquid preparation of muscardine, coating spore liquid, cultivation and purifying are cultivated can with reference to the routine of the art.Preferably, when being coated with spore liquid, the present invention is that the spore liquid prepared is even with aseptic spreading rod coating on substratum, spore liquid is made to be dispersed in the planar surface of whole substratum, and then use this spreading rod, continuous coating the 2nd or the 3rd flat board, can prevent bacterium colony overlapping well, ensure 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. spore liquid is prepared:
With aseptic inoculation ring from Bombyx Batryticatus or infected muscardine insect body surface picking 2 ~ 3 ring conidial powder in sterilized water, vibration makes spore fully scatter;
S2. prepare flat board: cool the PDA substratum of sterilising treatment to 50 ~ 60 DEG C, add sodium deoxycholate, rose-bengal, Vetstrep solution; After culture medium solidifying, with tweezers, sterile glass paper is covered on agar plate under aseptic technique;
S3. spore liquid prepared by S1 is spread evenly across 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, continuous coating the 2nd or the 3rd flat board, its object prevents bacterium colony overlapping, and this makes substratum be easy to occur single bacterium colony.
S4. cultivate;
Flat-plate inverted S3 being coated with spore liquid is placed in 26 ± 2 DEG C, humidity is that 92 ± 5% environment cultivate 3 ~ 7d; Select mycelial growth is vigorous, spore is many colonies typical in 1.5mL centrifuge tube, 1mL aseptic insect physiological saline and appropriate granulated glass sphere is had in centrifuge tube, get 200 μ L after shaken well to be coated on and to post on the flat board of glassine paper, be inverted in 26 ± 2 DEG C, humidity is 92 ± 5% cultivate 3 ~ 7d;
Described aseptic insect physiological saline is 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%, namely adds 1mL tween-80 in every 1000mL insect physiological saline.
S5. treat that mycelia is covered with, and takes off glassine paper, muscardine mycelia and the conidium of separation and purification can be obtained.
Further, the present invention also comprised the step processed the cause of disease gathered before S1:
Bombyx Batryticatus natural infection or artificial challenge obtained 75% (v/v) ethanol cotton balls carries out the sterilization of silkworm surface, then it is put into sterile petri dish, a cotton balls soaked with sterilized water is put in ware, be placed in 26 DEG C and cultivate 3 ~ 5d, grow after white fluff mycelia and conidium until silkworm surface and carry out strain separating again;
Preferably, dull and stereotyped described in S2 compound method is as follows:
S21. following reagent is prepared: Vetstrep concentration is 10
4~ 10
6u/mL (with sterilized water preparation, without the need to autoclaving); Sodium deoxycholate mass percent concentration is 2% ~ 2.5%, and rose-bengal mass percent concentration is 0.1% ~ 0.3%, and autoclaving is for subsequent use;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000mL poach is rotten (boils 20 ~ 30min, can poke with glass stick), by 4 layers of filtered through gauze, after adding 20g agar and 20g glucose, supply moisture again to 1000mL, use 1M NaOH or 1M HCL the pH of substratum to be adjusted to 6.0, sterilizing 20min at 121 DEG C, then add the ready sodium deoxycholate 20mL of S21 and 3.3mL Vetstrep and 3.3mL rose-bengal.
The separation purification method of muscardine of the present invention is more suitable for the silkworm separation and purification muscardine from having infected muscardine.
The present invention has following beneficial effect:
The invention provides a kind of selective medium being applicable to muscardine separation and purification, good technique effect is obtained with sodium deoxycholate, Vetstrep and rose-bengal coupling, microbiotic and the tensio-active agent of suitable concentration and usage quantity play synergy jointly, can the growth of effectively 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 overcoming it affects the unfavorable of HUMAN HEALTH, is a kind of safe separation purification method.
Cost of the present invention is lower, investigates according to existing market valency, and the dodine that prior art adopts is 580 ~ 590 yuan/g, and the sodium deoxycholate 7 yuan/g that the present invention adopts, sulfuric acid Sodium cholic acid 3 yuan/g, rose-bengal 1.4 yuan/g, reduce costs greatly.
From substratum collect be separated mycelia and conidium also very difficult, easily mix other miscellaneous bacteria, pollute.The present invention creatively uses a kind of glassine paper, and the glassine paper used is a kind of regenerated cellulose, and as semi-permeable membranes, its molecular gap exists ventilation property, and muscardine can draw the nutrition needed for growing, normal growth from substratum.Treat that muscardine grows to required degree and just glassine paper can be taken off, just can easily quickly mycelia and conidium be separated with substratum, in order to muscardine DNA extracting or conidial purifying or other application.And glassine paper raw material comes from natural, easily decomposes at ambient, 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
The substratum that Fig. 1 is separated for muscardine to add Vetstrep etc.
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.
Bombyx Batryticatus is placed in 26 DEG C of cultivations and treats that silkworm surface grows white fluff mycelia and conidium after 3 ~ 5 days by Fig. 4.
Embodiment
The present invention is set forth further below in conjunction with the drawings and specific embodiments.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 gathered is processed: the Bombyx Batryticatus (also can adopt the Bombyx Batryticatus of conventional means artificial challenge) of natural infection 75% (v/v) ethanol cotton balls is carried out the sterilization of silkworm surface, then it is put into sterile petri dish (ware puts a cotton balls soaked with sterilized water), be placed in 26 DEG C and cultivate 3 ~ 5 days (d), grow after white fluff mycelia and conidium until silkworm surface and carry out strain separating again;
S1. spore liquid is prepared: in the triangular flask filling 50mL sterilized water, (include sterile glass beads) from silkworm surface picking 2 ~ 3 ring conidial powder with aseptic inoculation ring, use vortex oscillation 5min, spore is fully scattered;
S2. flat board is prepared:
S21. following reagent is prepared: Vetstrep concentration is 1000U/mL (with sterilized water preparation, without the need to autoclaving, adding before use); Sodium deoxycholate mass percent concentration is 2%, rose-bengal mass percent concentration is 0.1%, and autoclaving is for subsequent use;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000mL poach is rotten (boils 20 ~ 30min, can poke with glass stick), by 4 layers of filtered through gauze, after adding 20g agar and 20g glucose, then supply moisture to 1000mL, 1M NaOH or 1M HCL is used medium pH to be adjusted to 6.0, sterilizing 20min at 121 DEG C.
The PDA substratum of sterilising treatment is cooled to 50 ~ 60 DEG C, adds 2% sodium deoxycholate 20ml/L; 0.1% rose-bengal 3.3mL/L; Vetstrep solution (1000U/mL) 3.3mL/L; Separately will not add the PDA flat board of sodium deoxycholate and Vetstrep in contrast;
S3. spore liquid is coated with: get 200uL spore liquid and be coated with evenly with aseptic spreading rod on flat board, make spore liquid be scattered in whole planar surface, and then use this spreading rod, continuously coating the 2nd the 3rd flat board, flat-plate inverted is placed in 26 DEG C, humidity is 95% cultivation 3 ~ 7d;
S4. select mycelial growth is vigorous, spore is many colonies typical and enter in 1.5mL to have that 1ml is aseptic is added with in the insect physiological saline of 0.1% tween-80 and the centrifuge tube of appropriate granulated glass sphere, shaken well;
S5. get 200 μ L to coat and be attached to containing on the glassine paper on the antibiotic flat boards such as Vetstrep, be inverted in 26 DEG C, humidity is cultivate 3 ~ 7d in 95% environment; Treat that mycelia is covered with, glassine paper can be taken off, muscardine mycelia and the conidium of separation and purification can be obtained, see shown in accompanying drawing 1 and accompanying drawing 3; The muscardine that common PDA substratum is separated is coated on the colonial morphology on common PDA substratum, sees shown in accompanying drawing 2.Shown in accompanying drawing 1 and accompanying drawing 3, on the antibiotic substratum of interpolation, do not occur mould and bacterium, bacterium colony is pale pink, is easy to explanation; Mould and other miscellaneous bacterias is had when first time is separated muscardine by ordinary culture medium shown in accompanying drawing 2.
Embodiment 2
The cause of disease gathered is processed: silkworm chrysalis 75% (v/v) ethanol cotton balls having infected natural infection or artificial challenge muscardine is carried out surface sterilization, then it is put into sterile petri dish, a cotton balls soaked with sterilized water is put in ware, be placed in 26 DEG C and cultivate 3 ~ 5 days (d), grow after white fluff mycelia and conidium until silkworm surface and carry out strain separating again;
S1. spore liquid is prepared: in the triangular flask filling 50mL sterilized water, (include sterile glass beads) from silkworm surface picking 2 ~ 3 ring conidial powder with aseptic inoculation ring, use vortex oscillation 5min, spore is fully scattered;
S2. flat board is prepared:
S21. following reagent is prepared: Vetstrep concentration is 10000U/mL (with sterilized water preparation, without the need to autoclaving, adding before use); Sodium deoxycholate mass percent concentration is 2.5%, rose-bengal mass percent concentration is 0.2%, and autoclaving is for subsequent use;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000mL poach is rotten (boils 20 ~ 30min, can poke with glass stick), by 4 layers of filtered through gauze, after adding 20g agar and 20g glucose, then supply moisture to 1000mL, 1M NaOH or 1M HCL is used medium pH to be adjusted to 6.0, sterilizing 20min at 121 DEG C.
The PDA substratum of sterilising treatment is cooled to 50 ~ 60 DEG C, adds 2.5% sodium deoxycholate 20ml/L; 0.2% rose-bengal 3.3mL/L; Vetstrep solution (10000U/mL) 3.3mL/L; Separately will not add the PDA flat board of sodium deoxycholate and Vetstrep in contrast;
S3. spore liquid is coated with: get 200uL spore liquid and be coated with evenly with aseptic spreading rod on flat board, make spore liquid be scattered in whole planar surface, and then use this spreading rod, continuously coating the 2nd the 3rd flat board, flat-plate inverted is placed in 26 DEG C, humidity is 95% cultivation 3 ~ 7d;
S4. select mycelial growth is vigorous, spore is many colonies typical and enter in 1.5mL to have that 1ml is aseptic is added with in the insect physiological saline of 0.1% tween-80 and the centrifuge tube of appropriate granulated glass sphere, shaken well;
S5. get 200 μ L to coat and be attached to containing on the glassine paper on the antibiotic flat boards such as Vetstrep, be inverted in 26 DEG C, humidity is cultivate 3 ~ 7d in 95% environment; Treat that mycelia is covered with, glassine paper can be taken off, muscardine mycelia and the conidium of separation and purification can be obtained.
Embodiment 3
S1. be diluted to being used for the biological pesticide (biological control station, Guangzhou random selecting provides, and those skilled in the art also can adopt the similar biological pesticide containing beauveria bassiana spore to test) containing beauveria bassiana spore that biological control makes the diluent that mass percent concentration is 1 or 10%.
S2. flat board is prepared;
S21. following reagent is prepared: Vetstrep concentration is 100000U/mL (with sterilized water preparation, without the need to autoclaving, adding before use); Sodium deoxycholate mass percent concentration is 2.2%, rose-bengal mass percent concentration is 0.25%, and autoclaving is for subsequent use;
S22. by 200g peeling potatoes, be cut into small pieces, add that 1000mL poach is rotten (boils 20 ~ 30min, can poke with glass stick), by 4 layers of filtered through gauze, after adding 20g agar and 20g glucose, then supply moisture to 1000mL, 1M NaOH or 1M HCL is used medium pH to be adjusted to 6.0, sterilizing 20min at 121 DEG C.
The PDA substratum of sterilising treatment is cooled to 50 ~ 60 DEG C, adds 2.2% sodium deoxycholate 20ml/L; 0.25% rose-bengal 3.3mL/L; Vetstrep solution (100000U/mL) 3.3mL/L; Separately will not add the PDA flat board of sodium deoxycholate and Vetstrep in contrast;
S3. spore liquid is coated with: get 200uL biological pesticide diluent even with aseptic spreading rod coating on flat board, spore liquid is made to be scattered in whole planar surface, and then being coated with the 2nd the 3rd flat board continuously with this spreading rod, flat-plate inverted is placed in 26 DEG C, humidity is 95% cultivation 3 ~ 7d;
S4. select mycelial growth is vigorous, spore is many colonies typical and enter in 1.5mL to have that 1ml is aseptic is added with in the insect physiological saline of 0.1% tween-80 and the centrifuge tube of appropriate granulated glass sphere, shaken well;
S5. get 200 μ L to coat and be attached to containing on the glassine paper on the antibiotic flat boards such as Vetstrep, be inverted in 26 DEG C, humidity is cultivate 3 ~ 7d in 95% environment; Treat that mycelia is covered with, glassine paper can be taken off, muscardine mycelia and the conidium of separation and purification can be obtained.
Investigate according to existing market valency, the sodium deoxycholate 7 yuan/g used in the embodiment of the present invention 1,2,3, sulfuric acid Sodium cholic acid 3 yuan/g, rose-bengal 1.4 yuan/g, 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, do not contain other miscellaneous bacterias (bacterium, mould) in the muscardine mycelia that separation and purification of the present invention obtains and conidium, muscardine mycelia is dense is spherical or oval with conidium, indivedual sprout occurring health.The present invention from substratum collect be separated mycelia and conidium fast very convenient.
Claims (7)
1. a method for separation and purification muscardine, is characterized in that, is the spore liquid of muscardine to be spread evenly across on selective medium to cultivate, and obtains muscardine mycelia and the conidium of separation and purification; The composition of described selective medium is: in the PDA substratum of every 1000mL sterilising treatment, add following component: the Deoxycholic Acid sodium solution 20mL of 2% ~ 2.5%, the rose-bengal 3.3mL of 0.1% ~ 0.3%, the above percentage ratio is mass percent concentration, adds 10 before facing use
4u/mL ~ 10
5the Vetstrep solution 3.3mL of U/mL.
2. the method for separation and purification muscardine according to claim 1, it is characterized in that, described PDA substratum consists of: potato 200g, glucose 20g, agar 20g, tap water 1000mL, by 1MNaOH or 1MHCL adjust ph to 6.
3. the method for separation and purification muscardine according to claim 1, is characterized in that, being spread evenly across on selective medium by the spore liquid of muscardine, after culture medium solidifying, being covered on agar plate by sterile glass paper under aseptic technique.
4. the method for separation and purification muscardine according to claim 1, it is characterized in that, be that spore liquid is even with aseptic spreading rod coating on substratum by the method that the spore liquid of muscardine is spread evenly across on selective medium, and then be coated with another flat board continuously with this spreading rod.
5. the method for separation and purification muscardine according to any one of Claims 1-4, is characterized in that, comprise the following steps:
S1. spore liquid is prepared:
With aseptic inoculation ring from Bombyx Batryticatus or infected muscardine insect body surface picking 2 ~ 3 ring conidial powder in sterilized water, vibration makes spore fully scatter;
S2. prepare flat board: cool the PDA substratum of sterilising treatment to 50 ~ 60 DEG C, add sodium deoxycholate, rose-bengal, Vetstrep solution; After culture medium solidifying, with tweezers, sterile glass paper is covered on agar plate under aseptic technique;
S3. spore liquid prepared by S1 step is spread evenly across flat board;
S4. cultivate; The flat-plate inverted being coated with spore liquid that S3 step is obtained be placed in 26 ± 2 DEG C, humidity be 92 ± 5% environment cultivate 3 ~ 7d; Select mycelial growth is vigorous, spore is many colonies typical in 1.5mL centrifuge tube, the aseptic insect physiological saline of 1mL and granulated glass sphere is had in centrifuge tube, get 200 μ L after shaken well to be coated on and to post on the flat board of glassine paper, be inverted in 26 ± 2 DEG C, humidity is 92 ± 5% cultivate 3 ~ 7d;
Described aseptic insect physiological saline is 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 is covered with, and takes off glassine paper, muscardine mycelia and the conidium of separation and purification can be obtained.
6. the method for separation and purification muscardine according to claim 5, it is characterized in that, the step that the Bombyx Batryticatus gathered is processed also was comprised before S1 step, the Bombyx Batryticatus concentration of volume percent obtained by natural infection or artificial challenge be 75% ethanol cotton balls carry out the sterilization of silkworm surface, then put it in sterile petri dish, a cotton balls soaked with sterilized water is put in ware, be placed in 26 DEG C and cultivate 3 ~ 5d, grow after white fluff mycelia and conidium until silkworm surface and carry out strain separating again.
7. the method for separation and purification muscardine according to claim 5, is characterized in that, compound method dull and stereotyped described in S2 step is as follows:
S21. following reagent is prepared: Vetstrep concentration is 10
4~ 10
5u/mL; Sodium deoxycholate mass percent concentration is 2% ~ 2.5%, and rose-bengal mass percent concentration is 0.1% ~ 0.3%, and autoclaving is for subsequent use;
S22. by peeling potatoes, be cut into small pieces, add water well-done, filter, after adding agar and glucose, supply moisture, pH is adjusted to 6.0, adds the ready sodium deoxycholate of S21 step, Vetstrep and rose-bengal after sterilizing.
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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 |
| CN109810907B (en) * | 2019-03-18 | 2020-07-31 | 福建农林大学 | Beauveria bassiana BbJ L-01 with strong pathogenicity on terminal-age larvae of cryptomeria fortunei caterpillars |
| 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 |
| CN113278578B (en) * | 2021-06-15 | 2022-09-16 | 广西大学 | Transplanting method of rice blast bacterium conidia |
| 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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