CN111705004A - Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode - Google Patents
Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode Download PDFInfo
- Publication number
- CN111705004A CN111705004A CN202010724152.1A CN202010724152A CN111705004A CN 111705004 A CN111705004 A CN 111705004A CN 202010724152 A CN202010724152 A CN 202010724152A CN 111705004 A CN111705004 A CN 111705004A
- Authority
- CN
- China
- Prior art keywords
- protoplast
- preparing
- acremonium
- root
- regenerating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing and regenerating a biocontrol fungus Acremonium cladosporium protoplast of a root-knot nematode. Belongs to the technical field of agricultural plant protection. The method comprises the following steps: activating a strain; preparing a spore suspension; preparing a massive mycelium; preparing a protoplast; and (4) regeneration of the protoplast. Compared with the prior art, the invention has the following beneficial effects: the preparation and regeneration method of the root-knot nematode biocontrol fungus Acremonium cladosporium protoplast is simple to operate, good in repeatability and high in protoplast preparation activity, the regeneration rate of the protoplast reaches 17.78%, and a good foundation is laid for the establishment of an A.implicatum genetic transformation system and the research of a biocontrol mechanism of the fungi.
Description
Technical Field
The invention relates to the technical field of agricultural plant protection, in particular to a preparation and regeneration method of a root-knot nematode biocontrol fungus Acremonium cladosporium protoplast.
Background
Meloidogyne spp, a kind of important plant pathogens distributed worldwide, brings huge economic loss to the global agricultural production. The root-knot nematodes are various in species, wide in distribution, strong in pathogenicity, wide in host range and strong in environmental adaptability, and can infect almost all vegetable crops. Among vegetable crops, the solanaceae, cruciferae, cucurbitaceae and the like are the most harmful. The vegetable crops are infected by root-knot nematodes, which can cause the yield to be reduced and the quality to be reduced. In addition, root-knot nematodes can form complex infections with other pathogens, causing more serious economic losses.
The prevention and control of root-knot nematodes are mainly soil fumigants and chemical insecticides, but due to frequent occurrence of problems of non-standard use, harm to human health, environmental pollution and the like, more and more chemical pesticides are prohibited from being used. The search for efficient and environment-friendly root-knot nematode control measures is one of the problems to be solved urgently at present. Therefore, the application of microorganisms to prevent and treat root-knot nematodes has become the key content of domestic and foreign research. The biocontrol bacteria such as Trichoderma spp, Paecilomyces spp, Acremonium spp and the like have obvious control effect on the root-knot nematode.
However, relatively few studies on the mechanisms of biocontrol fungi to control nematodes have limited the application of current biocontrol methods. In view of the situation, researches on the mechanism of controlling and preventing fungi need to be deepened, and the key technology of the researches is to establish a high-efficiency genetic transformation system of the fungi-proofing bacteria. The construction of efficient genetic transformation system of filamentous fungi is generally realized by protoplast transformation mediated by agrobacterium or mediated by polyethylene glycol. The protoplast transformation has the characteristics of high transformation efficiency, simple operation and the like, so that the preparation of the protoplast is the primary problem of genetic transformation. Due to the complex composition and structure of fungal cell walls, there are large differences in the systems and conditions for the preparation of protoplasts. Conditions such as the age of the bacteria, osmotic pressure stabilizer, the type of lyase, the enzymolysis temperature and the like can all influence the enzymolysis reaction in the preparation process of the protoplast, so that the preparation conditions of the protoplast are greatly different. As the cell wall of the Acremonium strictum of the biocontrol fungus of the trichina is thicker, and the quantity of spores is smaller and larger, the mycelia in a conglobation shape are difficult to remove when the mycelia are prepared in the preparation process of the protoplast.
In conclusion, the technical personnel in the field need to solve the problem of how to provide a method for preparing and regenerating the acrosporium alternifolia protoplast of the root-knot nematode biocontrol fungus with simple operation, good repeatability and high protoplast preparation activity.
Disclosure of Invention
In view of the above, the invention provides a method for preparing and regenerating an acremonium cross protoplast of biocontrol fungi of root-knot nematodes.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation and regeneration method of a root-knot nematode biocontrol fungus Acremonium cladosporium protoplast comprises the following steps:
(1) activating a strain;
(2) preparation of spore suspension: inoculating the activated strain into a PDB culture medium, collecting spores after shake cultivation, and preparing spore suspension;
(3) culturing the spore suspension by a shaking table, filtering to obtain mycelium, and repeatedly washing the mycelium to be in a dough shape by using an osmotic pressure stabilizer;
(4) performing enzymolysis on the massive mycelium by using a crasher driselase, filtering, and collecting a protoplast;
(5) and regenerating the protoplast by using a solid regeneration culture medium under the culture condition of dark culture at 25-28 ℃ for 3 d.
Preferably, the strain of step (1) is activated: inoculating the Acremonium alternatum strain stored at the temperature of-80 ℃ to a PDA culture medium, and culturing for 5 days in the dark at the temperature of 25-28 ℃.
The activity of the strain stored at the temperature of minus 80 ℃ is reduced, the strain is inoculated on a PDA culture medium and cultured in the dark for 5 days at the temperature of 28 ℃, the strain can be quickly activated, and the activity of the strain is ensured.
Preferably, the shake culture conditions in step (2) are 25-28 ℃ and 150r/min for 2 d.
Culturing under the condition can quickly obtain a large amount of spores to prepare spore suspension.
Preferably, the concentration of the spore suspension in the step (2) is 1 × 108cfu/ml。
Spore suspension concentration is 1 × 108cfu/ml, sufficient young hyphae can be obtained in a short time for subsequent enzymatic hydrolysis steps.
Preferably, the shake culture conditions in the step (3) are 25-28 ℃ and 150r/min for 36-48 h.
The hypha cell obtained under the culture condition has thin wall and is easy to prepare protoplast by enzymolysis.
Preferably, the osmotic pressure stabilizer in the step (3) is NaCl solution with pH value of 6.5 and concentration of 0.7 mol/l.
The osmotic pressure stabilizer can maintain the balance of the osmotic pressure inside and outside a protoplast cell prepared by the biocontrol fungus Acremonium cladium of the root-knot nematode and ensure the enzymolysis activity of the collapse enzyme driselase.
Preferably, the concentration of the crashing enzyme driselase in the step (4) is 20mg/ml, and the mass-to-volume ratio of the massive mycelium to the crashing enzyme driselase is 1:4 g/ml.
This mass to volume ratio enables a high viability and a high number of protoplasts to be obtained.
Preferably, the enzymolysis condition in the step (4) is 28-30 ℃ and 120r/min enzymolysis for 3-4 h.
The 28 ℃ collapse enzyme driselase can keep higher activity, and the temperature is too high or too low to be beneficial to keeping the activity of the enzyme; the proper increase of the enzymolysis time is beneficial to the generation of the protoplast of the acremonium strictum, but if the enzymolysis time is too long, the yield of the protoplast is reduced, and the enzymolysis time is the optimal enzymolysis time for 3 hours.
Preferably, the solid regeneration medium in step (5) comprises the following components by mass: 0.1% yeast extract, 0.1% enzymatically hydrolyzed casein, 1mol/l sucrose and 0.7% agar powder.
The solid regeneration culture medium can ensure various nutrients required by protoplast regeneration, the concentration of the agar powder is 0.7%, the culture medium with the concentration lower than 0.7% is difficult to solidify, the hardness of the culture medium with the concentration higher than 0.7% is increased, and the regeneration of the protoplast penetrating through the culture medium is not facilitated.
Preferably, the filtration is performed by using sterilized three-layer lens wiping paper.
The three layers of lens wiping paper gaps can filter spores of acrosporium alternifolia of the biocontrol fungus of the root-knot nematode; in the preparation process of the protoplast, the three layers of the mirror wiping paper can filter and separate the protoplast and mycelium which is not fully enzymolyzed to obtain a protoplast solution.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the invention provides the preparation and regeneration method of the root-knot nematode biocontrol fungus Acremonium cladosporium protoplast, which has the advantages of simple operation, good repeatability and high protoplast preparation activity, the regeneration rate of the protoplast reaches 17.78 percent, and a good foundation is laid for the establishment of an A.replicatum genetic transformation system and the research of the biocontrol mechanism of the fungi.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of protoplasts prepared by using the lyase solution A in example 3;
FIG. 2 is a drawing showing protoplasts prepared by using the lyase solution B in example 3;
FIG. 3 is a diagram showing spores and hyphae of protoplasts and genetically transformed strains prepared in example 5, wherein a is the protoplast (indicated by an arrow); b, exciting light to convert conidia of the strain; c, converting hyphae of the strain under excitation light, and measuring the scale to 10 μm.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The required medicament is a conventional experimental medicament purchased from a market channel; the unrecited experimental method is a conventional experimental method, and is not described in detail herein.
Example 1
(1) Strain activation: inoculating Acremonium alternifolia strain stored at-80 ℃ to a PDA culture medium, culturing in the dark at 28 ℃ for 5d, and activating the strain;
(2) preparing spore solution by perforating the edge of the colony cultured in step (1) with 5mm perforator, culturing a bacterial block in PDB culture medium at 25 deg.C and 150r/min for 2d, collecting spore, and preparing into uniform spore suspension with concentration of 1 × 108cfu/mL；
(3) Preparing hypha: taking 100 mu L of the spore suspension liquid obtained in the step (2), culturing for 24, 36, 48, 60 and 72 hours at 25 ℃ and 150r/min in 100mL of PDB culture medium, filtering by using sterilized three-layer lens wiping paper, then repeatedly and fully washing and collecting hyphae by using 0.7mol/L NaCl solution with pH of 6.5, weighing 0.5g of the hyphae in a sterilized 50mL centrifuge tube after the hyphae are washed to be in a conglobate shape, and carrying out the whole operation process on a super clean workbench;
(4) preparation of protoplast: adding 20mg/mL of the breakdown enzyme driselase2mL into the centrifuge tube in the step (3), and carrying out enzymolysis for 3h at 28 ℃ and 120 r/min;
(5) and (4) collecting protoplasts, namely filtering the zymolyte obtained in the step (4) by using sterilized three-layer mirror wiping paper, and collecting the protoplasts.
The statistical protoplast numbers are shown in Table 1.
TABLE 1 Effect of fungus age on Cross shoot proplastosome yield
As shown in the results in Table 1, the yield of protoplasts was the highest when the spore suspension was cultured for 36 hours, and the number of protoplasts reached 2.8 × 107cfu/mL。
Example 2
(1) Strain activation: inoculating Acremonium alternifolia strain stored at-80 ℃ to a PDA culture medium, culturing in the dark at 28 ℃ for 5d, and activating the strain;
(2) preparing spore solution by perforating the edge of the colony cultured in step (1) with 5mm perforator, culturing a bacterial block in PDB culture medium at 25 deg.C and 150r/min for 2d, collecting spore, and preparing into uniform spore suspension with concentration of 1 × 108cfu/mL；
(3) Preparing hypha: taking 100 mu L of the spore suspension in the step (2) to be cultured in 100mL of PDB culture medium at 25 ℃ for 36h at 150r/min, filtering the suspension by using sterilized three-layer mirror paper, then repeatedly and fully washing the suspension by using 0.7mol/L NaCl solution with the pH value of 6.5 to collect hyphae, weighing 0.5g of the hyphae in a sterilized 50mL centrifuge tube after the hyphae are washed to be in a dough shape, and carrying out the whole operation process on a super-clean workbench;
(4) preparation of protoplast: adding 2mL of 20mg/mL of breakdown enzyme driselase into the centrifuge tube in the step (3), carrying out enzymolysis at 28 ℃ and 120r/min, wherein the enzymolysis time is set to be 1, 2, 3, 4, 5 and 6 h;
(5) collecting protoplasts: and (4) filtering the zymolyte obtained in the step (4) by using sterilized three-layer mirror wiping paper to collect the protoplast.
The statistical protoplast numbers are shown in Table 2.
TABLE 2 Effect of enzymolysis time on protoplast yield
As shown in the results in Table 2, the enzyme hydrolysis for 3h gave the highest protoplast yield, with the protoplast number reaching 1.8 × 107cfu/mL。
Example 3
(1) Strain activation: inoculating Acremonium alternifolia strain stored at-80 ℃ to a PDA culture medium, culturing in the dark at 28 ℃ for 5d, and activating the strain;
(2) preparing spore solution by perforating the edge of the colony cultured in step (1) with 5mm perforator, culturing a bacterial block in PDB culture medium at 25 deg.C and 150r/min for 2d, collecting spore, and preparing into uniform spore suspension with concentration of 1 × 108cfu/mL；
(3) Preparing hypha: taking 100 mu L of the spore suspension liquid obtained in the step (2), culturing for 36h at 25 ℃ at 150r/min in 100mL of PDB culture medium, filtering by using sterilized three-layer mirror paper, then repeatedly and fully washing by using a NaCl solution with the pH of 6.5 and the concentration of 0.7mol/L to collect hyphae, weighing 0.5g of the hyphae into a sterilized 50mL centrifuge tube after the hyphae are washed to be in a dough shape, and carrying out the whole operation process on a super clean workbench;
(4) preparation of protoplast: adding lyase A into the centrifuge tube in the step (3): 20mg/mL of the crashase driselase or lyase B: performing enzymolysis on 10mg/mL cellulase and 10mg/mL lywallzyme for 3h at 120r/min, wherein the enzymolysis temperature is set to be 27, 28, 29, 30, 31, 32 and 33 ℃;
(5) collecting protoplasts: and (4) filtering the zymolyte obtained in the step (4) by using sterilized three-layer mirror wiping paper to collect the protoplast.
TABLE 3 influence of enzyme species and enzymolysis temperature on protoplast yield
As can be seen from the results in Table 3, the yield of protoplasts was the highest at an enzymolysis temperature of 28 ℃ and the number of protoplasts reached 3.1 × 10 using the lyase solution A7cfu/mL。
FIG. 1 shows protoplasts prepared from 28 ℃ lyase solution A, and FIG. 2 shows protoplasts prepared from 28 ℃ lyase solution B.
FIG. 1 shows that the number of protoplasts prepared is large, the shape is round, and FIG. 2 shows that the number of protoplasts prepared is small, irregular
Example 4
(1) Strain activation: inoculating Acremonium alternifolia strain stored at-80 ℃ to a PDA culture medium, culturing in the dark at 28 ℃ for 5d, and activating the strain;
(2) preparing spore solution by perforating the edge of the colony cultured in step (1) with 5mm perforator, culturing a bacterial block in PDB culture medium at 25 deg.C and 150r/min for 2d, collecting spore, and preparing into uniform spore suspension with concentration of 1 × 108cfu/mL；
(3) Preparing hypha: placing the spore suspension liquid obtained in the step (2) in 100mL of PDB culture medium, culturing for 36h at 25 ℃ at 150r/min, filtering by using sterilized three-layer lens wiping paper, then repeatedly and fully washing and collecting hyphae by using 0.7mol/L NaCl solution with pH of 6.5, weighing 0.5g of hyphae in a sterilized 50mL centrifuge tube after the hyphae are washed to be in a dough shape, and carrying out the whole operation process on a super-clean workbench;
(4) preparation of protoplast: adding 20mg/mL of the breakdown enzyme driselase2mL into the centrifuge tube in the step (3), and carrying out enzymolysis for 3h at 28 ℃ and 120 r/min;
(5) collecting protoplasts: and (4) filtering the zymolyte obtained in the step (4) by using sterilized three-layer mirror wiping paper to collect the protoplast.
(6) Mixing 0.1mL of protoplast solution with 12mL of 50 deg.C solid regeneration medium (0.1% yeast extract, 0.1% enzyme-hydrolyzed casein, 1M sucrose, 0.7% agar powder) or PDA culture medium, pouring into a culture dish, respectively pouring the same culture medium with agar content of 1% at 50 deg.C, and uniformly covering the culture dish; after solidification, the mixture is placed in an incubator at 28 ℃ for dark culture. Meanwhile, 0.1mL of the above protoplast solution was taken and 1mL of sterile water was added, and the mixture was allowed to stand at room temperature for 30min and cultured in the same manner as a control to offset the error due to the colony formed by the non-protoplast.
The protoplast regeneration rate is calculated as follows:
the regeneration rate (number of regenerated colonies of protoplast-number of regenerated colonies of control group)/total number of protoplasts × 100%.
The results of the protoplast regeneration rate are shown in Table 4.
TABLE 4 Effect of the Medium on regeneration of sporogenous plastids from shoot apicomplexa
From the results in Table 4, the regeneration rates of protoplasts on PDA medium and solid regeneration medium were 10.55% and 17.78%, respectively.
Example 5 genetic transformation of Acremonium alternatum Green fluorescent protein Gene
(1) Strain activation: inoculating Acremonium alternifolia strain stored at-80 ℃ to a PDA culture medium, culturing in the dark at 28 ℃ for 5d, and activating the strain;
(2) preparing spore solution by perforating the edge of the colony cultured in step (1) with 5mm perforator, culturing a bacterial block in PDB culture medium at 25 deg.C and 150r/min for 2d, collecting spore, and preparing into uniform spore suspension with concentration of 1 × 108cfu/mL；
(3) Preparing hypha: placing the spore suspension liquid obtained in the step (2) in 100mL of PDB culture medium, culturing for 36h at 25 ℃ at 150r/min, filtering by using sterilized three-layer lens wiping paper, repeatedly and fully washing by using a NaCl solution with the pH of 6.5 and the concentration of 0.7mol/L to collect hyphae, weighing 0.5g of the hyphae in a sterilized 50mL centrifuge tube after the hyphae are washed to be in a dough shape, and carrying out the whole operation process on an ultra-clean workbench;
(4) preparation of protoplast: adding 2mL of 20mg/mL of breakdown enzyme driselase into the centrifuge tube in the step (3), and carrying out enzymolysis at 28 ℃ for 3h at 120 r/min;
(5) collecting protoplasts, and filtering the enzymatic hydrolysate in the step (4) by using sterilized three-layer mirror wiping paper to obtain filtrate, namely protoplast suspension;
(6) transformation of protoplasts: and (3) genetically transforming the protoplast obtained in the step (5) by a polyethylene glycol-mediated method.
The expression vector plasmid containing the GFP gene was transferred to Acremonium copulatum. The protoplasts were dispensed into 50mL centrifuge tubes at 200. mu.L/tube, HindIII-digested linearized plasmid DNA was added, and the mixture was digested with STC solution (1.2mol/L sorbitol)，50mmol/L CaCl2·2H2O, 10mmol/L Tris-HCl) to 300 μ L, and standing on ice for 20 min; slowly adding 2mL of PTC solution (60% polyethylene glycol 3350, 10mmol/L Tris-ClpH7.5, 50mmol/L calcium chloride) dropwise, and standing on ice for 20 min; adding 30mL of precooled STC into each tube, uniformly mixing, centrifuging at 4000r/min at 4 ℃ for 15 min; discarding the supernatant, adding 3mL of liquid regeneration culture medium (0.1% yeast extract, 0.1% enzymatic hydrolysis casein, 1mol/L sucrose) into each tube, and performing static culture at 28 ℃ for 12-18 h; the culture was poured into a petri dish, 12mL of liquid regeneration medium (temperature was lowered to 50 ℃, preventing scalding of protoplasts) was added and mixed well. The grown mycelia were picked and examined under a fluorescent microscope. Under blue excitation light, the transformants were observed to emit bright green fluorescence, indicating that the GFP gene had been successfully transferred into acremonium cross a.
The prepared protoplasts and spores and hyphae of the genetically transformed strains are shown in FIG. 3.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A preparation and regeneration method of a root-knot nematode biocontrol fungus Acremonium cladosporium protoplast is characterized by comprising the following steps:
(1) activating a strain;
(2) preparation of spore suspension: inoculating the activated strain into a PDB culture medium, collecting spores after shake cultivation, and preparing spore suspension;
(3) culturing the spore suspension by a shaking table, filtering to obtain mycelium, and repeatedly washing the mycelium to be in a dough shape by using an osmotic pressure stabilizer;
(4) performing enzymolysis on the massive mycelium by using a crasher driselase, filtering, and collecting a protoplast;
(5) and regenerating the protoplast by using a solid regeneration culture medium under the culture condition of dark culture at 25-28 ℃ for 3 d.
2. The method for preparing and regenerating the Acremonium chrysosporium protoplast of the biocontrol fungus of the root-knot nematode as claimed in claim 1, wherein the strain in the step (1) is activated: inoculating the Acremonium alternatum strain stored at the temperature of-80 ℃ to a PDA culture medium, and culturing for 5 days in the dark at the temperature of 25-28 ℃.
3. The method for preparing and regenerating the protoplast of Acremonium alternatum as claimed in claim 1, wherein the shake culture conditions in step (2) are 25-28 ℃ and 150r/min for 2 d.
4. The method for preparing and regenerating the protoplast of Acremonium cladosporioides as claimed in claim 1, wherein the concentration of the spore suspension in the step (2) is 1 × 108cfu/ml。
5. The method for preparing and regenerating the Acremonium chrysotium protoplast of the biocontrol fungi of the root-knot nematodes as claimed in claim 1, wherein the shaking culture conditions in the step (3) are 25-28 ℃ and 150r/min for 36-48 h.
6. The method for preparing and regenerating the Acremonium chrysosporium protoplast for biocontrol of root-knot nematode according to claim 1, wherein the osmotic pressure stabilizer in step (3) is NaCl solution with pH value of 6.5 and concentration of 0.7 mol/l.
7. The method for preparing and regenerating the acrosporium alternifolia protoplast of the biocontrol fungus of root-knot nematode as claimed in claim 1, wherein the concentration of the crashing enzyme driselase in the step (4) is 20mg/ml, and the mass-to-volume ratio of the clustered mycelium to the crashing enzyme driselase is 1:4 g/ml.
8. The method for preparing and regenerating the Acremonium chrysotium protoplast of the biocontrol fungi of the root-knot nematodes as claimed in claim 1, wherein the enzymolysis condition in the step (4) is 28-30 ℃ and 120r/min enzymolysis for 3-4 h.
9. The method for preparing and regenerating the Acremonium chrysotium protoplast of the biocontrol fungi of the root-knot nematodes according to claim 1, wherein the solid regeneration culture medium in the step (5) comprises the following components in mass concentration: 0.1% yeast extract, 0.1% enzymatically hydrolyzed casein, 1mol/l sucrose and 0.7% agar powder.
10. The method for preparing and regenerating the Acremonium chrysotium protoplast of the biocontrol fungi of the root-knot nematodes as claimed in any one of claims 1 to 9, wherein the filtration is performed by using sterilized three-layer mirror wiping paper.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010724152.1A CN111705004B (en) | 2020-07-24 | 2020-07-24 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010724152.1A CN111705004B (en) | 2020-07-24 | 2020-07-24 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111705004A true CN111705004A (en) | 2020-09-25 |
| CN111705004B CN111705004B (en) | 2023-04-14 |
Family
ID=72547770
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010724152.1A Active CN111705004B (en) | 2020-07-24 | 2020-07-24 | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111705004B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451110A (en) * | 2013-08-21 | 2013-12-18 | 江西省农业科学院蔬菜花卉研究所 | Method for preparing and regenerating phomopasis asparagi protoplast |
-
2020
- 2020-07-24 CN CN202010724152.1A patent/CN111705004B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103451110A (en) * | 2013-08-21 | 2013-12-18 | 江西省农业科学院蔬菜花卉研究所 | Method for preparing and regenerating phomopasis asparagi protoplast |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111705004B (en) | 2023-04-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111662828B (en) | Metarhizium leylanicum and application thereof | |
| Sun et al. | Transformation of the endochitinase gene Chi67-1 in Clonostachys rosea 67-1 increases its biocontrol activity against Sclerotinia sclerotiorum | |
| Zacchetti et al. | Dynamics of pellet fragmentation and aggregation in liquid-grown cultures of Streptomyces lividans | |
| CN103865948B (en) | A kind of method of fusarium oxysporum sesame protoplastis genetic transformation of PEG mediation | |
| CN113956986B (en) | Method for improving growth and spore-producing ability of insect biocontrol fungi and improving toxicity | |
| CN109609526A (en) | A kind of stripe disease of barley pathogenic gene PgPBS and its application | |
| CN105368866A (en) | Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel | |
| US20140363874A1 (en) | Method for producing laccase by means of liquid fermentation of lentinus edodes | |
| CN102154127B (en) | Genetically modified biocontrol fungus with high intestinal-toxin insecticidal activity and structuring method and application thereof | |
| CN111705004B (en) | Preparation and regeneration method of biocontrol fungus Acremonium cladosporium protoplast of root-knot nematode | |
| CN104293677A (en) | Arthrobotrys oligospora genetic engineering strain with nematode-killing function, and application thereof | |
| Wang et al. | Development of Cordyceps javanica BE01 with enhanced virulence against Hyphantria cunea using polyethylene glycol-mediated protoplast transformation | |
| CN104263660B (en) | A kind of biocontrol trichoderma F18 and its preparation method and application | |
| Andargie et al. | Generation of β-glucuronidase reporter-tagged strain to monitor Ustilaginoidea virens infection in rice | |
| CN112011465A (en) | Preparation and genetic transformation method of deep-sea fungus white siderobia protoplast | |
| CN107236671B (en) | Preparation method of Tilletia foetida protoplast | |
| CN116218730B (en) | Microbial agent for preventing and treating tobacco black shank | |
| CN114958898B (en) | Establishment method and application of PEG-mediated genetic transformation system of aschersonia phaseoli | |
| CN115044512B (en) | Predatory primordium strain and application thereof in biological control of plant diseases | |
| Das et al. | Preparation and regeneration of mycelial protoplasts of Pleurotus florida and P. ostreatus | |
| CN102154294A (en) | Filamentous fungus promoter, terminator and plasmid containing same | |
| Naseema et al. | Isolation and regeneration of protoplasts from the mycelium of Fusarium pallidoroseum–a potential biocontrol agent of water hyacinth [Eichhornia crassipes (Mart.) Solms] | |
| LU500796B1 (en) | Transgenic Strain of Metarhizium anisopliae and Preparation Method and Application thereof | |
| CN110484454B (en) | Culture medium for mass production of Isaria fumosorosea | |
| Petrini et al. | Production and regeneration of protoplasts from Gremmeniella abietina and Ascocalyx abietis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20210127 Address after: No.26 Hangtian Road, Nankai District, Tianjin Applicant after: Tianjin Academy of Agricultural Sciences Address before: 300384 17 kilometers, Jin Jing Road, Xiqing District, Tianjin Applicant before: TIANJIN INSTITUTE OF PLANT PROTECTION |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |