A method for quickly separating white rot fungi infected by miscellaneous bacteria

Abstract

The present invention discloses a method for rapidly separating white rot fungi infected by miscellaneous fungi, belonging to the technical field of microbial culture. This method mainly includes four steps: spore liquid preparation, isolation and purification culture, identification culture, and scale-up culture. Different from traditional methods such as streak separation and addition of inhibitors, this method mainly conducts restrictive culture on common miscellaneous fungi such as Penicillium and Trichoderma in production applications through a white rot fungi selective medium, and rejuvenates the nutrition of the target strain. This method is simple to operate, has a high success rate, effectively reduces the time cost, and further improves the production efficiency. In addition, the white rot fungi separated and purified by this method have high activity and stable quality.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial culture, and specifically relates to a method for rapidly isolating white rot fungi infected by miscellaneous bacteria. Background Art

[0002] Due to the need for the sustainable and coordinated development of human economy and society and the construction of an environment-friendly society, lignocellulose, as a renewable biomass resource, has extremely high production capacity and economic value due to its rich source and ecological friendliness. At present, there are problems such as low raw material utilization rate and high production energy consumption ratio in the biorefinery of lignocellulose biomass. Mainly because of the physical and chemical properties and structural characteristics of lignocellulose, it is difficult to achieve an ideal yield without lignin removal. As a white filamentous fungus of the Basidiomycetes class, white rot fungi are one of the microorganisms with great application potential in the field of biorefinery due to their unique biodegradation enzyme system and strong degradation ability. White rot fungi can invade wood through mycelia, use lignin as a carbon source and release protein enzymes such as manganese peroxidase, lignin peroxidase, and laccase. While breaking the natural barrier in lignocellulose, they release polysaccharides such as cellulose, hemicellulose, and pectin, thereby completely degrading the main components in lignocellulose into CO2 and H2O to achieve the efficient and high-value application of biomass energy. In addition, white rot fungi, as a common and widely distributed edible mushroom, including more than 200 varieties such as Pleurotus ostreatus, Lentinula edodes, and Coriolus versicolor, also have extremely high edible and medicinal values.

[0003] During the subculture preservation, inoculation, and cultivation of white rot fungi, it is easy to cause contamination by miscellaneous bacteria due to reasons such as contamination of the mother strain, unqualified sterilization effect of the culture material and container, improper inoculation operation, and environmental pollution of the inoculation. The miscellaneous bacteria compete with white rot fungi for nutrients and secrete harmful substances, competitively killing the mycelia of white rot fungi and affecting the growth of the target strain and the yield of the target product. In the production and application of white rot fungi, the most common invading miscellaneous bacteria are mainly molds such as Trichoderma, Penicillium, Aspergillus, and Mucor.

[0004] At present, for the strains infected by miscellaneous bacteria, especially the mother strains and original strains used in various productions, the most effective method is to purify them by adding exogenous additives such as antibiotics and inhibitors. However, the addition of antibiotics and pesticides poses a risk of causing drug resistance in miscellaneous bacteria. For the strains used in food industrial production, the use of chemical agents such as antibiotics and pesticides brings environmental threats and bioaccumulation effects, and it is easy for the toxicity to eventually accumulate in the human body, causing health and safety hazards. In addition, although the use of plant-derived fungicides such as terpenoids, hydroxyanthraquinones, and thioesters, and plant extracts such as garlic, onion, and ginger can effectively inhibit the growth activity of miscellaneous bacteria and reduce ecological and health risks, they are prone to inhibiting the activity and growth of white rot fungi. Moreover, the optimal antibacterial concentrations and ratios of different plant extracts for different molds are different, and screening is required for different contamination situations, with a relatively high application cost and long time consumption. Summary of the Invention

[0005] The object of the present invention is to provide a method for quickly separating white rot fungi infected by miscellaneous bacteria. By inoculating the infected white rot fungi on a selective medium and restricting the nutrients such as nitrogen source, carbon source, inorganic salts and vitamins in the medium, the growth of miscellaneous bacteria such as Trichoderma is inhibited and the growth of the target strain is facilitated. After the purified white rot fungi cells are identified and cultured and propagated, the purpose of pure culturing white rot fungi in a relatively short time is achieved. This method is simple and easy to operate, can quickly separate white rot fungi from the infected strains, has a high success rate, and greatly improves the separation and purification efficiency.

[0006] To achieve the above object, the technical solution of the present invention sequentially includes the following steps:

[0007] A method for quickly separating white rot fungi infected by miscellaneous bacteria sequentially includes the following steps:

[0008] (1) Spore liquid preparation: Take fresh spores on the surface of the infected white rot fungi under sterile conditions, place them in sterile water, and shake well.

[0009] (2) Isolation and purification culture: Inoculate the spore liquid obtained in step (1) into a selective medium and culture it in the dark at a constant temperature until hyphae appear on the surface of the medium.

[0010] The method described above,

[0011] The components of the selective medium used in step (2) include: MgSO4·7H2O 0.5 - 0.55 g, CaCl2 0.1 - 0.2 mg, FeSO4·7H2O 0.4 - 0.5 mg, ZnSO4·7H2O 1.4 - 1.5 mg, Na2HPO4·2H2O 0.12 - 0.13 g, KH2PO4 0.51 - 0.52 g, CuSO4·5H2O 0.17 - 0.18 mg, C4H4O6K2 0.1 - 0.15 g, glucose 1 - 1.2 g, VB1 0.1 - 0.2 mg, agar 20 - 21 g, pure water 1000 mL.

[0012] The method described above,

[0013] In step (2), it is cultured in the dark in a constant temperature incubator at 29°C - 31°C until hyphae appear on the surface of the medium.

[0014] The method described above,

[0015] Carry out identification culture on the hyphae obtained in step (2): Pick the hyphae after purification culture in step (2) and streak inoculate them on the guaiacol-PDA medium.

[0016] The method described above,

[0017] The described guaiacol-PDA medium comprises: 46-46.1 g of glucose potato agar medium, 0.3-1 mL of guaiacol, and 1000 mL of pure water.

[0018] In the described method, pick the mycelia after culturing in step (2) and inoculate them into the identification medium containing guaiacol. After culturing at 29-31 °C for 3-10 days, observe the guaiacol-PDA plate to determine the growth of the colonies. If the colonies are uniform, then conduct microscopic examination on the mycelia and spores of the strain to determine whether it is a white-rot fungus. If it does not conform to the colony characteristics of white-rot fungi, repeat the operations in steps (1) and (2) and perform isolation and purification culture again.

[0019] In the described method, the colony characteristics of white-rot fungi conforming to those on the identification medium containing guaiacol are as follows: the surface mycelia of the colony are filamentous or flocculent aerial mycelia, the color is thick white, and a brownish-red color development circle is formed around the colony.

[0020] In the described method, the microscopic examination method is as follows: pick a small amount of mycelia with a sterile inoculation loop and place them on a glass slide with a drop of pure water, and stain them with 1% methylene blue staining solution. After covering with a cover glass, observe under a microscope. The microscopic characteristics of white-rot fungi under microscope examination are: the strain has a developed mycelium, and most are multinucleate, few septate, and without clamp connections.

[0021] In the described method, expand and culture the strain identified by the guaiacol-PDA medium: pick a single colony with an obvious color development circle after identification culture, inoculate it in blocks into the PDA medium and statically culture it; observe whether the germinated mycelia are contaminated with miscellaneous bacteria. If not, the purification is successful.

[0022] In the described method, inoculate the identified white-rot fungus into the PDA medium and culture it at 29 °C - 31 °C for 5-7 days for expansion; the components of the PDA medium include 46-46.1 g of glucose potato agar medium and 1000 mL of pure water.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] (1) Compared with the traditional dilution method and plate streaking, the present invention adopts three-step culture of isolation and purification culture, identification culture, and expansion culture. In the implementation cases, the white-rot fungi can be successfully isolated and purified in one round of culture, without the need for multiple repeated gradient dilutions of the bacterial suspension and streaking separation, avoiding the repeated consumption of human and material resources due to measures such as determining the dilution concentration of the bacterial suspension and plate streaking. It takes a short time, is simple and easy to operate, and can greatly improve the efficiency of strain purification.

[0025] (2) Compared with adding antibiotics or other microbial inhibitors, this method restricts the cultivation of white-rot fungi infected by contaminating bacteria. While avoiding the growth of contaminating bacteria, it screens out highly active white-rot fungal strains and greatly reduces potential risks such as antibiotic resistance and inhibition of the target strain's activity, facilitating the maintenance of the stability of the target strain's traits.

[0026] (3) After selective cultivation by this method, the single colonies of the target strain are identified and cultivated, avoiding the possibility of impure strains and facilitating subsequent pure cultivation. In addition, through two steps of the guaiacol method and microscopic examination, comprehensive identification is carried out from aspects such as colony morphological characteristics, color reaction of the guaiacol-PDA medium, and microscopic morphology. On the premise of ensuring the accuracy of strain extraction, the reagents and equipment required for identification are easily obtainable, the method is simple and effective, and the technical and facility requirements for identification are reduced. Description of the Drawings

[0027] Figure 1 Purification diagram of the culture medium for the inoculation of Coriolus versicolor infected by Trichoderma

[0028] Figure 2 Color reaction within 30 minutes after inoculating the purified Coriolus versicolor infected by Trichoderma into the guaiacol-PDA medium

[0029] Figure 3 Identification culture diagram of the purified Coriolus versicolor infected by Trichoderma

[0030] Figure 4 Microscopic examination diagram of the purified Coriolus versicolor infected by Trichoderma

[0031] Figure 5 Propagation diagram of the purified Coriolus versicolor infected by Trichoderma

[0032] Figure 6 Secondary purification diagram of the purified Coriolus versicolor infected by Trichoderma after 7 days of separation and purification by the traditional method

[0033] Figure 7 Purification diagram of the culture medium for the inoculation of Coriolus versicolor infected by Aspergillus

[0034] Figure 8 Identification culture diagram of the purified Coriolus versicolor infected by Aspergillus

[0035] Figure 9 Microscopic examination diagram of the purified Coriolus versicolor infected by Aspergillus

[0036] Figure 10 Propagation diagram of the purified Coriolus versicolor infected by Aspergillus

[0037] Figure 11 Purification diagram of the culture medium for the inoculation of Grifola frondosa infected by Trichoderma

[0038] Figure 12 Identification and culture diagram of Grifola frondosa infected by Trichoderma after separation and purification;

[0039] Figure 13 Microscopic examination diagram of Grifola frondosa infected by Trichoderma after separation and purification;

[0040] Figure 14 Propagation diagram of Grifola frondosa infected by Trichoderma after inoculation, separation and purification;

[0041] Figure 15 Streak separation and purification diagram of Coriolus versicolor infected by Trichoderma on PDA medium;

[0042] Figure 16 Secondary purification diagram of Coriolus versicolor infected by Trichoderma after 2 days of separation and purification culture;

[0043] Figure 17 Secondary purification diagram of Coriolus versicolor infected by Trichoderma on corn flour medium;

[0044] Figure 18 Separation and purification diagram of Coriolus versicolor infected by Trichoderma on corn flour medium added with 1% garlic extract;

[0045] Figure 19 Separation and purification diagram of Coriolus versicolor infected by Trichoderma on corn flour medium added with 0.5% garlic extract;

[0046] Figure 20 Flow schematic diagrams of Comparative Examples 1 to 5. Specific implementation solutions

[0047] In order to better clarify the specific technical solutions and implementation modes and fully reflect the advantages and effects of the present invention, the following is described with reference to the accompanying drawings in the embodiments of the present invention. The content described in the embodiments is only used to illustrate the present invention and should not and will not limit the present invention described in detail in the claims. In addition, unless otherwise specified, the reagents and experimental methods used in the following embodiments are all conventional products and operation methods in the art.

[0048] Example 1

[0049] A purification method for rapidly separating white-rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0050] (1) Preparation of spore liquid: The white rot fungus Coriolus versicoLor infected by Trichoderma (the contaminated Coriolus versicolor strains stored in the laboratory, direct morphological observation shows that the development morphology of the contaminated strain is white hyphae, which gradually turns green in the later stage and dyes the culture medium yellow, which is consistent with the culture change trend of Trichoderma reesei strains stored in the laboratory. Cross contamination during operation is suspected, and further microscopic examination is performed, which is consistent with the spore morphology of Trichoderma reesei) is activated and inoculated into sterile PDA solid culture medium (PDA is potato dextrose agar culture medium) and cultured at 30°C for 5 days. Under sterile conditions, fresh spores are scraped from the surface of the white rot fungus with a sterile inoculation loop and placed in sterile water to prepare a spore suspension. The spore suspension is shaken for 10 minutes to make the spores evenly distributed.

[0051] (2) Isolation and purification culture: Take 0.5g MgSO4·7H2O, 0.1mg CaCl2, 0.4mg FeSO4·7H2O, 1.4mg ZnSO4·7H2O, 0.125g Na2HPO4·2H2O, 0.519g KH2PO4, 0.175mg CuSO4·5H2O, 0.1g C4H4O6K2, 1g glucose, 0.1mg VB1, 20g agar, add 1000mL pure water, shake and mix, and sterilize at 121℃ for 30min. Under sterile conditions, cool to 50-60℃ and pour into a plate. After cooling and solidification, it becomes a white rot fungus selection medium. Use a sterile inoculation loop to dip a small amount of the spore solution prepared in step (1) and inoculate it into the selection medium ( Figure 1 ) and cultured at 30°C. Observe regularly and culture until white hyphae appear on the surface of the culture medium.

[0052] (3) Identification culture: Take 46g of glucose potato agar medium, 1mL of guaiacol, add 1000mL of pure water, shake and mix, and sterilize at 121℃ for 30min. Under sterile conditions, cool to 50-60℃ and pour into a plate. After cooling and solidification, it becomes guaiacol-PDA medium. Use a sterile inoculation loop to pick up the mycelium purified and cultured in step (2), and inoculate it into the guaiacol-PDA medium. The mycelium should be white mycelium with good growth. The inoculated guaiacol-PDA medium will show obvious pink color change within 30min ( Figure 2 ), that is, the isolated and purified Coriolus versicolor has extremely strong vitality and laccase production ability. After culturing at 30°C for 5 days, the colony morphology was white short hairs, with uniform morphology, and a brown-red color circle around the colony ( Figure 3)。Using a sterile inoculation loop, pick a small amount of mycelium and place it on a glass slide with a drop of pure water. Then stain it with 1% methylene blue staining solution, cover the cover glass, and observe it under a microscope. Under microscopic examination, the strain has well-developed mycelium, and fungal hyphae with the characteristics of the clamp connection of Trametes versicolor can be seen. Figure 4 )。

[0053] (4) Multiplication culture: Pick a single colony with an obvious color development circle after the identification culture in step (3), inoculate it in a PDA medium by cutting into pieces, culture it at 30 °C for 5 days, and continuously observe whether the germinated mycelium is contaminated with miscellaneous bacteria to obtain purified white rot fungi with strong viability after multiplication culture. Figure 5 )。By the traditional streaking isolation method, after secondary purification, the whole Trametes versicolor is green and still mixed with Trichoderma infectans, and the purification is not successful. Figure 6 )。

[0054] Example 2

[0055] A purification method for quickly separating white rot fungi infected by Aspergillus niger, and the specific implementation steps are as follows:

[0056] (1) Spore suspension preparation: Activate and inoculate Trametes versicolor (Coriolus versicoLor), a white rot fungus infected by Aspergillus niger (obtained by mixed culture of laboratory-reserved Aspergillus niger strains and Trametes versicolor) into a sterilized PDA medium, and culture it at 30 °C for 5 days. Under sterile conditions, use a sterile inoculation loop to scrape fresh spores from the surface of the white rot fungus and place them in sterile water to prepare a spore suspension. Shake the spore suspension evenly for 10 min to make the spores evenly distributed.

[0057] (2) Isolation and purification culture: Take 0.5 g of MgSO4·7H2O, 0.1 mg of CaCl2, 0.4 mg of FeSO4·7H2O, 1.4 mg of ZnSO4·7H2O, 0.125 g of Na2HPO4·2H2O, 0.519 g of KH2PO4, 0.175 mg of CuSO4·5H2O, 0.1 g of C4H4O6K2, 1 g of glucose, 0.1 mg of VB1, and 20 g of agar, add 1000 mL of pure water, shake and mix well, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour the plate. After cooling and solidifying, it becomes a selective medium for white rot fungi. Use a sterile inoculation loop to dip a small amount of the spore suspension prepared in step (1) and inoculate it into the selective medium Figure 7 ) and culture it at 30 °C. Observe regularly until white mycelium appears on the surface of the medium.

[0058] (3) Identification and culture: Take 46 g of glucose potato agar medium and 0.3 mL of guaiacol, add 1000 mL of pure water, mix well by shaking, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour into plates. After cooling and solidifying, it becomes guaiacol-PDA medium. Use a sterile inoculation loop to pick the mycelium after purification culture in step (2) and inoculate it into the guaiacol-PDA medium. The mycelium should be well-growing white mycelium. After culturing the inoculated guaiacol-PDA medium at 30 °C for 3 days, colonies with a white short villous shape, uniform morphology, and a brownish-red color development ring around the colonies are obtained ( Figure 8 ). Use a sterile inoculation loop to pick a small amount of mycelium, place it on a glass slide with a drop of pure water, stain it with 1% methylene blue staining solution, cover with a cover glass, and observe under a microscope. Under microscopic examination, the strain has a well-developed mycelium, and fungal hyphae with the characteristics of Trametes versicolor clamp connections can be seen ( Figure 9 ).

[0059] (4) Propagation culture: Pick a single colony with an obvious color development ring after identification and culture in step (3), cut and inoculate it into PDA medium, culture at 30 °C for 5 days, and continuously observe whether the germinated mycelium is contaminated with miscellaneous bacteria to obtain purified white-rot fungi with strong viability after propagation culture ( Figure 10 ).

[0060] Example 3

[0061] A purification method for rapidly separating white-rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0062] (1) Spore suspension preparation: Activate and inoculate the white-rot fungus Grifola frondosa infected by Trichoderma into sterilized PDA medium and culture at 30 °C for 5 days. Under sterile conditions, use a sterile inoculation loop to scrape fresh spores from the surface of the white-rot fungus and place them in sterile water to prepare a spore suspension. Shake the spore suspension evenly for 10 min to make the spores evenly distributed.

[0063] (2) Separation, purification and cultivation: Take 0.5 g of MgSO4·7H2O, 0.1 mg of CaCl2, 0.4 mg of FeSO4·7H2O, 1.4 mg of ZnSO4·7H2O, 0.125 g of Na2HPO4·2H2O, 0.519 g of KH2PO4, 0.175 mg of CuSO4·5H2O, 0.1 g of C4H4O6K2, 1 g of glucose, 0.1 mg of VB1, and 20 g of agar, add 1000 mL of pure water, mix well by shaking, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour plates. After cooling and solidifying, it becomes a selective medium for white rot fungi. Dip a sterile inoculation loop into a small amount of the spore solution prepared in step (1) and inoculate it into the selective medium ( Figure 11 ). Cultivate at 30 °C. Observe regularly until white mycelia appear on the surface of the medium.

[0064] (3) Identification cultivation: Take 46 g of glucose potato agar medium and 0.3 mL of guaiacol, add 1000 mL of pure water, mix well by shaking, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour plates. After cooling and solidifying, it becomes a guaiacol - PDA medium. Use a sterile inoculation loop to pick the mycelia after purification cultivation in step (2) and inoculate them into the guaiacol - PDA medium. The mycelia should be well - growing white mycelia. After culturing the inoculated guaiacol - PDA medium at 30 °C for 10 days, a colony morphology with white short villous shape, uniform morphology, and a brown - red color - developing circle around the colony is obtained ( Figure 12 ). Use a sterile inoculation loop to pick a small amount of mycelium, place it on a glass slide with a drop of pure water, stain it with 1% methylene blue stain solution, cover it with a cover glass, and observe it under a microscope. Under microscope examination, the strain conforms to the characteristics of white rot fungi with well - developed mycelia, relatively straight, mostly multinucleate, few septa, and no clamp connections ( Figure 13 ).

[0065] (4) Propagation cultivation: Pick a single colony with an obvious color - developing circle after identification cultivation in step (3), inoculate it in blocks into the PDA medium, cultivate at 30 °C for 5 days, and continuously observe whether the germinated mycelia are contaminated with miscellaneous bacteria to obtain purified white rot fungi after propagation cultivation and with strong viability ( Figure 14 ).

[0066] Comparative Example 1

[0067] A purification method for separating white rot fungi infected by Trichoderma, the specific implementation steps are as follows:

[0068] (1) Spore liquid preparation: Activate and inoculate the white-rot fungus Coriolus versicolor infected by Trichoderma into a sterilized PDA medium, and culture it at 30 °C for 5 days. Under aseptic conditions, scrape fresh spores from the surface of the white-rot fungus with a sterile inoculation loop and place them in sterile water to prepare a spore suspension. Shake the spore suspension well for 10 min to evenly distribute the spores.

[0069] (2) Isolation and purification culture: Dip a small amount of the spore liquid prepared in step (1) with a sterile inoculation loop and inoculate it into a sterilized PDA medium, and streak inoculate it in three zones to obtain as many single colonies as possible. Culture it at 30 °C and observe regularly. On the second day of culture, white hyphae begin to appear. On the third day of culture, green spores appear in the middle of the white hyphae. Cultured until the seventh day, the colony distribution is uneven, and there is white hyphal distribution on the left side of the plate ( Figure 15 ), and the strain is still contaminated by Trichoderma.

[0070] (3) Secondary purification culture: Two days after the isolation and purification culture in step (2), pick the white hyphae with a sterile inoculation loop and streak inoculate them into a sterilized PDA medium. Culture it at 30 °C for 7 days. The colony morphology is round, the whole is green, and there is a white hyphal growth zone around the colony. The strain is still contaminated by Trichoderma ( Figure 16 ), and the purification is not successful.

[0071] Comparative Example 2

[0072] A purification method for isolating white-rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0073] (1) Spore liquid preparation: Activate and inoculate the white-rot fungus Coriolus versicolor infected by Trichoderma into a sterilized PDA medium, and culture it at 30 °C for 5 days. Under aseptic conditions, scrape fresh spores from the surface of the white-rot fungus with a sterile inoculation loop and place them in sterile water to prepare a spore suspension. Shake the spore suspension well for 10 min to evenly distribute the spores.

[0074] (2) Isolation and purification culture: Dip a small amount of the spore liquid prepared in step (1) with a sterile inoculation loop and inoculate it into a sterilized PDA medium, and streak inoculate it in three zones to obtain as many single colonies as possible. Culture it at 30 °C and observe regularly. On the second day of culture, white hyphae begin to appear. On the third day of culture, green spores appear in the middle of the white hyphae. Cultured until the seventh day, the colony distribution is uneven, and there is white hyphal distribution on the left side of the plate, and the strain is still contaminated by Trichoderma.

[0075] (3) Secondary purification culture: After 7 days of separation and purification culture in step (2), use a sterile inoculation loop to pick the suspected white hyphae of white rot fungi in the colony, and try to avoid the green spore part as much as possible. Use a sterile inoculation loop to pick the white hyphae and streak inoculate them into the sterilized PDA medium. Culture at 30 °C for 7 days. The colony morphology is round, the center is green, and there is a white hyphal growth zone around the colony. The strain is still contaminated by Trichoderma ( Figure 6 ), and the purification is not successful.

[0076] Comparative Example 3

[0077] A purification method for separating white rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0078] (1) Spore suspension preparation: Activate and inoculate the white rot fungus Coriolus versicoLor infected by Trichoderma into the sterilized PDA medium, and culture at 30 °C for 5 days. Under sterile conditions, use a sterile inoculation loop to scrape fresh spores from the surface of the white rot fungus and place them in sterile water to prepare a spore suspension. Shake the spore suspension evenly for 10 min to make the spores evenly distributed.

[0079] (2) Preparation of corn meal medium: Take 5 g of corn meal, 5 g of peptone, 2.5 g of yeast extract, 35 g of glucose, 0.5 g of MgSO4, 1 g of KH2PO4, 0.05 g of VB1, and 20 g of agar, add 1000 mL of pure water, shake and mix well, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour plates.

[0080] (3) Separation and purification culture: Use a sterile inoculation loop to dip a small amount of the spore suspension prepared in step (1) and inoculate it into the sterilized corn meal medium, and streak inoculate in three zones to obtain as many single colonies as possible. Culture at 30 °C and observe regularly. On the 2nd day of culture, white hyphae start to appear. On the 3rd day of culture, green spores appear in the middle of the white hyphae. On the 7th day of culture, the colony morphology is round, the center is green, and there is a white hyphal growth zone around the colony. The strain is still contaminated by Trichoderma and the purification is not successful.

[0081] (4) Secondary purification culture: After 7 days of separation and purification culture in step (2), use a sterile inoculation loop to pick the white hyphae, and use a sterile inoculation loop to pick the white hyphae and streak inoculate them into the sterilized corn meal medium. Culture at 30 °C for 7 days. The colony morphology is round, the center is green, and there is a white hyphal growth zone around the colony. The strain is still contaminated by Trichoderma ( Figure 17 ), and the purification is not successful.

[0082] Comparative Example 4

[0083] A purification method for separating white rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0084] (1) Spore liquid preparation: Activate and inoculate the white-rot fungus Coriolus versicolor infected by Trichoderma into a sterilized PDA medium, and culture it at 30 °C for 5 days. Under sterile conditions, scrape fresh spores from the surface of the white-rot fungus with a sterile inoculation loop and place them in sterile water to prepare a spore suspension. Shake the spore suspension evenly for 10 min to make the spores evenly distributed.

[0085] (2) Preparation of garlic extract: Peel fresh garlic, wash and dry it, and cut it into small pieces. Put the garlic pieces into a blender and crush them, then squeeze the mashed garlic with 5 layers of gauze to obtain a crude garlic extract. Put the crude garlic extract into a centrifuge and centrifuge for 10 min at a speed of 3500 r / min, and take the supernatant.

[0086] (3) Isolation, purification and culture: Take 5 g of corn flour, 5 g of peptone, 2.5 g of yeast extract, 35 g of glucose, 0.5 g of MgSO4, 1 g of KH2PO4, 0.05 g of VB1, and 20 g of agar, add 1000 mL of pure water, shake and mix well, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour the plate, add 1% of the garlic extract, and shake and mix well. After cooling and solidifying, dip a small amount of the spore liquid prepared in step (1) with a sterile inoculation loop and streak inoculate it into the medium in three zones to obtain as many single colonies as possible. Culture at 30 °C and observe regularly. After culturing for 10 days, there is no change on the surface of the medium. After culturing for more than 40 days, a thin white mycelium spreads on the plate, the edge of the mycelium is yellowish-black, and the strain is in a declining state ( Figure 18 ).

[0087] Control Example 5

[0088] A purification method for separating white-rot fungi infected by Trichoderma, and the specific implementation steps are as follows:

[0089] (1) Spore liquid preparation: Activate and inoculate the white-rot fungus Coriolus versicolor infected by Trichoderma into a sterilized PDA medium, and culture it at 30 °C for 5 days. Under sterile conditions, scrape fresh spores from the surface of the white-rot fungus with a sterile inoculation loop and place them in sterile water to prepare a spore suspension. Shake the spore suspension evenly for 10 min to make the spores evenly distributed.

[0090] (2) Preparation of garlic extract: Peel fresh garlic, wash and dry it, and cut it into small pieces. Put the garlic pieces into a blender and crush them, then squeeze the mashed garlic with 5 layers of gauze to obtain a crude garlic extract. Put the crude garlic extract into a centrifuge and centrifuge for 10 min at a speed of 3500 r / min, and take the supernatant.

[0091] (3)Isolation, purification and cultivation: Take 5 g of corn flour, 5 g of peptone, 2.5 g of yeast extract, 35 g of glucose, 0.5 g of MgSO4, 1 g of KH2PO4, 0.05 g of VB1, and 20 g of agar. Add 1000 mL of pure water, mix well by shaking, and sterilize at 121 °C for 30 min. Under sterile conditions, cool to 50 - 60 °C and pour the plate. Add 0.5% garlic extract and shake well. After cooling and solidifying, dip a small amount of the spore solution prepared in step (1) with a sterile inoculation loop and streak inoculate it into the culture medium in three zones to obtain as many single colonies as possible. Incubate at 30 °C and observe regularly. On the second day of cultivation, light green hyphae appear in the center of the plate ( Figure 19 ). After 10 days of cultivation, the whole plate is covered with Trichoderma. The colony morphology is round, and there are sporadic white hyphae covering the center of the colony. The strain is still contaminated with Trichoderma and the purification is not successful. Comparative Examples 1 to 5 are only for simply comparing the operations ( Figure 20 ) and effects of different isolation and purification methods to better reflect the implementation advantages of this method.

[0092] The above embodiments only represent several implementation modes of the present invention, and the description is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A method for quickly separating white rot fungi infected by miscellaneous bacteria, characterized in that, The following steps are included in sequence: (1)Sporulation fluid preparation: Fresh spores on the surface of the infected white-rot fungus are taken under sterile conditions and placed in sterile water, and shaken and mixed evenly. The contaminating fungus is Trichoderma or Aspergillus niger, and the white-rot fungus is Coriolus versicolor ( Coriolus versicolor ); (2) Isolation and purification culture: inoculate the spore solution obtained in step (1) into a selective culture medium and culture it in a light-proof and constant temperature environment until hyphae appear on the surface of the culture medium; The components of the selection medium used in step (2) are: MgSO4·7H2O 0.5-0.55g, CaCl2 0.1-0.2mg, FeSO4·7H2O 0.4-0.5mg, ZnSO4·7H2O 1.4-1.5mg, Na2HPO4·2H2O 0.12-0.13g, KH2PO4 0.51-0.52g, CuSO4·5H2O 0.17-0.18mg, C4H4O6K2 0.1-0.15g, glucose 1-1.2g, VB1 0.1-0.2mg, agar 20-21g, and pure water 1000mL; (3) Identification culture: The mycelium obtained after purification and culture in step (2) was streaked and inoculated into a guaiacol-PDA medium, wherein the guaiacol-PDA medium comprises: 46-46.1 g of glucose potato agar medium, 0.3-1 mL of guaiacol, and 1000 mL of pure water; (4) Propagation and culture: Pick out single colonies with obvious color circles after identification and culture, inoculate them into PDA culture medium and culture them statically; observe whether the germinated mycelium contains foreign bacteria. If not, purification is successful.

2. A method for quickly separating white rot fungi infected by miscellaneous bacteria, characterized in that, The following steps are included in sequence: (1) Spore solution preparation: Fresh spores on the surface of the infected white-rot fungus are taken under sterile conditions and placed in sterile water, and shaken and mixed evenly. The contaminating fungus is Trichoderma, and the white-rot fungus is Grifola frondosa ( Griflola frondosa ) (2) Isolation and purification culture: inoculate the spore solution obtained in step (1) into a selective culture medium and culture it in a light-proof and constant temperature environment until hyphae appear on the surface of the culture medium; The components of the selection medium used in step (2) are: MgSO4·7H2O 0.5-0.55g, CaCl2 0.1-0.2mg, FeSO4·7H2O 0.4-0.5mg, ZnSO4·7H2O 1.4-1.5mg, Na2HPO4·2H2O 0.12-0.13g, KH2PO4 0.51-0.52g, CuSO4·5H2O 0.17-0.18mg, C4H4O6K2 0.1-0.15g, glucose 1-1.2g, VB1 0.1-0.2mg, agar 20-21g, and pure water 1000mL; (3) Identification culture: The mycelium obtained after purification and culture in step (2) was streaked and inoculated into a guaiacol-PDA medium, wherein the guaiacol-PDA medium comprises: 46-46.1 g of glucose potato agar medium, 0.3-1 mL of guaiacol, and 1000 mL of pure water; (4) Propagation and culture: Pick out single colonies with obvious color circles after identification and culture, inoculate them into PDA culture medium and culture them statically; observe whether the germinated mycelium contains foreign bacteria. If not, purification is successful.

3. The method according to claim 1, characterized in that Step (2) Place the culture medium in a constant temperature incubator at 29°C-31°C in the dark and culture until hyphae appear on the surface of the culture medium.

4. The method according to claim 1, wherein Pick the mycelium after purification and culture in step (2) and streak-inoculate it on the guaiacol-PDA medium. After culturing at 29-31 °C for 3-10 days, observe the guaiacol-PDA plate to determine the colony growth situation. The colonies are uniform, and the mycelium and spore morphology of the strain are examined under a microscope to determine that it is a white-rot fungus.

5. The method according to claim 4, characterized in that, The microscopic examination method is as follows: Use a sterile inoculation loop to pick a small amount of mycelium and place it on a glass slide with a drop of pure water, and stain it with 1% methylene blue staining solution. After covering the cover glass, observe it under a microscope.

6. The method according to claim 1, wherein Expand and culture the strain identified by the guaiacol-PDA medium: Expand and culture it at 29 °C - 31 °C for 5-7 days; the components of the PDA medium include 46-46.1 g of glucose potato agar medium and 1000 mL of pure water.

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