CN113684139A - Ralstonia bainieri and screening method thereof and application of Ralstonia bainieri in degradation of forest waste - Google Patents
Ralstonia bainieri and screening method thereof and application of Ralstonia bainieri in degradation of forest waste Download PDFInfo
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Abstract
The invention provides a Rapex albus strainIrpex lacteusThe strain is numbered as M2-1 and is preserved in China center for type culture Collection, the preservation address is Wuhan, Wuhan university, the preservation number is: CCTCC NO, M2021623, preservation date is: 2021.5.27. the method uses the irpex cacteus to degrade the forest waste, can accelerate the decomposition of the forest waste, and the degraded forest waste can be used for manufacturing microbial fertilizers to enhance the biological fertilizer efficiency.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to irpex cacteus, a screening method thereof and application thereof in degrading forest waste.
Background
In recent years, the industries such as forestry and landscaping in China develop rapidly, the effect is obvious, but the forest waste is increased rapidly. The forest waste is also called as forest residue, and refers to residue after felling, wood processing and timber utilization, and comprises dead branches, fallen leaves, branches, tips, sawdust, shrubs, withered wood, garden trimmings and the like. China produces a large amount of forest waste every year, and the sick and dead trees can affect the health of forests while causing resource waste, so that how to efficiently utilize and treat the forest waste becomes a problem to be faced.
The irpex cacteus is a fungus which mainly parasitizes wood and promotes the wood to decay, and most of the irpex cacteus can degrade broad-leaved trees and coniferous trees. The raking tooth fungus with white capsule invades into the wood material by hypha and spore, and destroys the cell wall of wood fiber by secreting various enzymes, thereby degrading several macromolecular compounds of lignin, cellulose and hemicellulose. A strain capable of efficiently degrading the forest waste is screened out through research, so that the forest waste can be recycled, and resources are saved.
Disclosure of Invention
The invention mainly aims to overcome the defects in the prior art and provide the raking tooth fungus with the leucocytochalgia and the screening method thereof and the application of the raking tooth fungus in degrading forest waste.
The invention provides a Rapex lacteus, the strain number of which is M2-1, and the Rapex lacteus is preserved in China center for type culture Collection, the preservation address is Wuhan university, the preservation number is: CCTCC NO, M2021623, preservation date is: 2021.5.27.
the invention also provides a screening method for screening the irpex leucocephalus strain, which comprises the following steps:
(1) collecting strains: spraying 75% alcohol on pine wood segments with white-bag raking tooth fungus hyphae collected from northern mountain of Nanjing forestry university, sterilizing, volatilizing alcohol, picking hyphae on the wood segments with a sterile scalpel in an ultra-clean workbench, transferring the hyphae to a sterile PDA culture medium, culturing the culture medium in an incubator with 25 ℃ and 40% relative humidity in the dark for 3-5 days, transferring the hyphae to an inclined plane PDA culture medium again when the hyphae grows to two halves of a flat plate, and storing in a refrigerator at 4 ℃ after the hyphae fully grows over the inclined plane; (2) and (3) laccase detection: taking out the strain obtained by culturing in the step (1) from a refrigerator, activating the strain on a basic PDA plate culture medium, beating bacterial colonies in the basic PDA culture medium into fungus cakes by using a 6mm puncher when hyphae grow to two thirds of the plate, inoculating the fungus cakes to the PDA-guaiacol culture medium, culturing for six days, and observing the size of reddish brown color change circles on the culture medium;
(3) lignin peroxidase and manganese peroxidase assays: taking out the strain obtained by culturing in the step (1) from a refrigerator, inoculating the strain on a PDA-aniline blue culture medium, culturing for six days, and observing the size of a fading ring generated on the culture medium;
(4) and (3) cellulase detection: inoculating the strain obtained by culturing in the step (1) into a sodium carboxymethylcellulose culture medium, culturing for six days, dyeing with Congo red, fixing with sodium chloride, and observing the size of a transparent ring generated on the culture medium;
(5) strains which can generate color circles in corresponding culture media are preserved through detection in the step (2), the step (3) and the step (4);
(6) and (3) strain identification: two pairs of universal primers, namely, an Internal transcribed spacer of a fungal ribosomal genome and RNA polymerase 1, are used for carrying out PCR amplification on strain DNA, and then BLAST homologous alignment analysis is carried out on NCBI.
Preferably, the PDA-guaiacol medium in step (2) is prepared by adding guaiacol to the basic PDA medium at a final concentration of 0.5 g/L.
Preferably, the PDA-aniline blue culture medium in the step (2) is prepared by adding aniline blue to the basic PDA culture medium at a final concentration of 0.1 g/L.
Preferably, the sodium carboxymethyl cellulose culture medium in the step (4) comprises 0.25g/L dipotassium hydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate, 5g/L sodium carboxymethyl cellulose, 0.5g/L ammonium sulfate, 15g/L agar powder or 20g/L agar strips.
The invention also provides an application of the irpex leucocephalus strain in degrading forest waste.
The invention also provides a microbial inoculum for degrading forest waste, which comprises the irpex leucocephala strain.
Compared with the prior art, the invention has the beneficial effects that:
1. the method uses the irpex cacteus to degrade the forest waste, can accelerate the decomposition of the forest waste and help people purify the environment.
2. The invention utilizes the irpex cacteus which is a high-efficiency biological compost bacterium, and the degraded forest waste is used for manufacturing a microbial fertilizer to enhance the biological fertilizer efficiency.
Deposit description
The irpex cacteus strain has the strain number of M2-1, and is preserved in China center for type culture Collection (ATCC) at the preservation address of Wuhan university, Wuhan, China, the preservation number is: CCTCC NO: M2021623, preservation date is: 2021.5.27.
the foregoing is only an overview of the technical solutions of the present invention, and in order to more clearly understand the technical solutions of the present invention, the present invention is further described below with reference to the accompanying drawings.
Drawings
FIG. 1 is a graph showing the change of enzyme activity of the strain with time;
wherein 1-A is a graph showing the change of laccase-producing enzyme activity along with time, 1-B is a graph showing the change of lignin peroxidase enzyme activity along with time, and 1-C is a graph showing the change of manganese peroxidase enzyme activity along with time;
FIG. 2 is a graph showing the quality degradation of three wood samples by Rapex alba strains;
FIG. 3 is a graph showing the lignin degradation of three wood samples by Rapex alba strains;
FIG. 4 shows a phylogenetic tree constructed by strain M2-1 based on ITS and RPB1 genes.
Detailed Description
In order to understand the present invention, the following examples are given to further illustrate the present invention. The reagents used in the invention are glucose, absolute ethyl alcohol, agar powder, magnesium sulfate heptahydrate, potassium dihydrogen phosphate, vitamin B1, dipotassium hydrogen phosphate, aniline blue, guaiacol, sodium carboxymethylcellulose, ammonium sulfate, sodium chloride, Congo red, sodium hydroxide, concentrated hydrochloric acid, glacial acetic acid, acetyl bromide and alkali lignin, and the reagents are all domestic analytical purifiers. Laccase kits, manganese peroxidase kits and lignin peroxidase kits are all purchased from Suzhou Keming Biotechnology, Inc.
The basal PDA culture medium comprises: 20g/L glucose, 200g/L potato, 15g/L agar powder or 20g/L agar strips; the PD culture medium is as follows: PDA medium without agar; the ZPDA (comprehensive culture medium) culture medium comprises: 20g/L glucose, 200g/L potato, 6.15g/L magnesium sulfate heptahydrate, 3g/L potassium dihydrogen phosphate, 0.05g/L vitamin B1, 15g/L agar powder, 121 ℃, and adjusting the pH value of the sterilized product in a super clean bench by using 0.5mol/L HCl and 0.5mol/L NaOH after 20 minutes of sterilization; the ZPD culture medium is as follows: ZPDA medium without agar; the aniline blue culture medium: adding 0.1g/L aniline blue into a basic PDA culture medium to prepare an aniline blue culture medium; the PDA-guaiacol culture medium comprises the following components: adding 0.5g/L of guaiacol into a basic PDA culture medium to prepare a guaiacol culture medium; the sodium carboxymethyl cellulose (CMCNa) culture medium: 0.25g/L dipotassium hydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate, 5g/L sodium carboxymethyl cellulose, 0.5g/L ammonium sulfate, 15g/L agar powder or 20g/L agar strips.
The experimental apparatus and equipment used are HH-4 type digital display constant temperature water bath: jie Rui Er electric appliances Co., Ltd, type MIR-553 constant temperature incubator: SANYO Electric Co.ltd, DGG-9070 model Electric heating constant temperature air drying oven: shanghai Sensin laboratory instruments Inc., SCB-1360JS model super clean bench: beijing donghair instruments manufacturing ltd, standard shake culture box: mobilson Biotechnology (China) Ltd., MTI-201B model Multi-Chamber Multi-temperature gradient constant temperature incubator: shanghai Tech chemical science and technology, MDF-U333 type refrigerator: SANYO Electric co. ltd, japan: six instrument factories of full-wavelength microplate reader, Thermo electron corporation, thermoflow voltage-stabilizing electrophoresis, Beijing.
And (3) strain identification: performing PCR amplification on strain DNA by using two pairs of universal primers of a fungal ribosome genome transcribed spacer region (ITS) and RNA polymerase 1(RNA polymerase 1, RPB1) for the collected hyphae, performing BLAST homologous alignment analysis on NCBI, and downloading sequences of similar strains and other kindred species sequences as references;
after multiple Sequence ratios with BioEdit, different gene sequences were concatenated using the configure Sequence program of PhylloSuite 1.2.1 software, and the best TREE building model was established with ModelFinder program, and the maximum likelihood phylogenetic TREE was constructed with IQ-TREE, the results are shown in FIG. 4.
As can be seen from FIG. 4, the hyphae and Irpex lacteus FD-9 are the same branch and have the closest relationship, and the binding morphology is identified as I.lacteus, strain number M2-1.
Example 1
Collecting strains: spraying 75% alcohol on pine wood segments with white-bag raking tooth fungus hyphae collected from northern mountain of Nanjing forestry university, sterilizing, volatilizing alcohol, picking hyphae on the wood segments with a sterile scalpel in an ultra-clean workbench, transferring the hyphae to a sterile PDA culture medium, culturing the culture medium in an incubator with 25 ℃ and 40% relative humidity in the dark for 3-5 days, transferring the hyphae to an inclined plane PDA culture medium again when the hyphae grows to two halves of a flat plate, and storing in a refrigerator at 4 ℃ after the hyphae fully grows over the inclined plane;
example 2
The strain obtained in the step 1 is activated on a basic PDA plate culture medium, when hyphae grow to two thirds of the plate, a colony growing to a culture dish 2/3 in the basic PDA culture medium is beaten into a fungus cake by a 6mm puncher, the fungus cake is inoculated on the PDA-guaiacol culture medium and cultured for six days, and reddish brown color change circles are observed on the culture medium, so that the strain can produce laccase.
Example 3
Lignin peroxidase and manganese peroxidase assays: the strain obtained in the culture of the example 1 is inoculated on a PDA-aniline blue culture medium, the culture is carried out for six days, and the strain can produce lignin peroxidase and manganese peroxidase by observing the fading circle generated on the culture medium.
Example 4
And (3) cellulase detection: the strain obtained by the culture of the example 1 is inoculated in a sodium carboxymethyl cellulose culture medium, cultured for six days, stained by congo red, fixed by sodium chloride, and then the strain is observed to generate a transparent ring on the culture medium, so that the strain can generate the cellulase.
Example 5
The irpex cacteus strain obtained in example 1 is cultured in a PDA culture medium at a constant temperature of 25 ℃, when the diameter of a bacterial colony grows to a culture dish 2/3, the bacterial colony is beaten into a bacterial cake by a 6mm puncher, the bacterial cake at the outer edge of the bacterial colony is selected and connected to a 500mL conical flask to be filled into 200mL PD culture medium, 6 bacterial cakes are connected to each flask, 3 bacterial strains are repeatedly placed in a standard shaking incubator at a temperature of 28 ℃ and a speed of 160r/min for culture for 16d, culture solution is sucked under aseptic conditions from the 4 th day of the beginning of culture, the obtained culture solution is filtered by 4 layers of gauze every sampling time, then the culture solution is centrifuged under the conditions of 6000r/min and 10min, and the centrifuged supernatant is the crude enzyme solution.
The activity of three enzymes was measured using a kit (Suzhou Keming Biotechnology Co., Ltd.) from a crude enzyme solution of the test strain, and the measurement was carried out exactly according to the procedures described in the kit instructions, and the results are shown in FIG. 1.
As can be seen from FIG. 1-A, in the growth process of the strain, the laccase activity is slowly increased firstly, then rapidly increased to reach the peak of the enzyme activity at 12d, the enzyme activity thereafter begins to decrease, the laccase production capacity of the strain M2-1 is better, and the laccase activity is 12U/ml when the peak of the enzyme activity is reached at 12 d; as can be seen from FIG. 1-B, the enzyme-producing activity trend of the lignin peroxidase activity is the same as that of laccase, and the enzyme activity peaks at 12d, and the enzyme-producing activity of the strain M2-1 at 12d is 1.85U/ml; as shown in FIG. 1-C, the enzyme activity of manganese peroxidase is slightly different from that of laccase and lignin peroxidase, and the enzyme production peak is reached at 12d, wherein the enzyme activity is 4.21U/ml.
Example 6
Application of irpex cacteus strain in degradation of forest waste
Putting three kinds of wood sawdust of pinus sylvestris, populus tomentosa and cedar wood into an electric heating constant temperature blast drying oven at 65 ℃ to be dried to constant weight, then respectively weighing 5g of the three kinds of wood sawdust and placing the three kinds of wood sawdust into 240mL of glass tissue culture bottles, weighing each wood sample to be accurate to 0.001g by using an electronic balance, adding 20mL of distilled water into each bottle, uniformly stirring, then placing into a high temperature steam sterilization pot at 121 ℃, and sterilizing for 20 min;
beating the irpex cacteus strains cultured in the example 1 into fungus cakes by using a 6mm puncher, adding 6 fungus cakes into each tissue culture bottle under the aseptic condition, repeating three bottles for each fungus, putting the cells into a sterilized incubator, and standing and culturing at a constant temperature of 28 ℃ under the condition of not adding any exogenous and endogenous nutrient substances to ensure that wood chips are infected by the irpex cacteus strains in the tissue culture bottles;
and (3) culturing for three months in a constant-temperature incubator, taking out the wood sample, slightly scraping surface hyphae, putting the wood sample without the hyphae in an oven at 65 ℃ for drying to constant weight, weighing in an electronic balance, and calculating the mass loss condition of the wood sample after being infected by bacteria, wherein the obtained result is shown in figure 2.
Calculating the formula: the mass loss rate of the wood sample was (W1-W2)/W1X 100% (wherein: W1 represents the oven dry mass of the wood sample before degradation, and W2 represents the oven dry mass of the wood sample after degradation.)
As can be seen from FIG. 2, the decomposing capacities of the irpex cacteus strain M2-1 on three kinds of wood are different, the degradation effect on poplar is the best in the degradation of three kinds of wood samples, the quality loss rate of the strain M2-1 on the degradation of poplar is the best, and reaches 38.53%, the degradation effect of test strains on wood samples of fir and pine is the worst, the quality loss rate of fir is 6.6% and the quality loss rate of pine is 9.27% in the wood samples treated by the strain M2-1.
The lignin content of three wood samples treated by the irpex cacteus strain M2-1 was measured by the acetyl bromide method, and the degradation rate and the mass loss rate of the lignin were calculated as shown in FIG. 3.
As can be seen from FIG. 3, in the wood sample of poplar, after treatment by the strain M2-1, the lignin content was significantly reduced, the lignin degradation rate of M2-1 to poplar was 9.96% at the highest, and the lignin degradation rate of M2-1 to cedar was 4.78% at the lowest.
The harpagophytum leucocytorrhizum strain M2-1 performed well in all three wood samples, with the best degradation effect on poplar. The wood is mainly composed of macromolecular compounds such as lignin, cellulose and hemicellulose, and the three components account for 97-99% of the dry weight of the wood. The contents of the three components are different in coniferous trees and broadleaf trees, the lignin content in the coniferous trees is higher than that in the broadleaf trees, the cellulose content and the hemicellulose content in the broadleaf trees are higher than those in the coniferous trees, the pine trees and the fir trees belong to the coniferous trees, the corrosion resistance of the coniferous trees is higher than that of the poplar trees, and therefore, the lignin degradation effect of the strain M2-1 on the pine trees and the fir trees is not as good as that on the poplar trees.
Appendix: gene sequence of M2-1 strain
ITS:
CGGGGGGAGGTCTACCCTGATTTGAGCTCAGATTGTCAAATGATTGTCTCGGCAAGGAGACGGTTCGAAGCATGAACACCATAAATACTTCAACACCACAGCGCAGATAATTATCACACTGAGGGCGATCCGTAAGATTCACGCTAATGCATTTCAGAGGAGTCGACCGACAAGGGCCGACACAACCTCCAAGTCCAAGCCCGCTAAACCTTCATTACAAAAATTTAGGGGTTGAGAATACCATGAGACTCAAACAGGCATACTCCTCGGAATACCAAGGAGTGCAAGGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACATTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCGAGAGCCAAGAGATCCGTTGCTGAAAGTTGTATATAAATGTGTTATACACAGTTGACATTCTATAACTGAAGCGTTTGTAGTAAAACATAAGAAAGAAAAACGGCTTGTTCAACCGAAGACCTCTCGCGAGATCCTGGAAGCTTCCACCATTTTTTTCTCTTACATAAAGTGCACAGAGGTTAAGAGTGGATGAGCCAGGCGTGCACATGCCTCGTGAGAGGCCAGCTACAACCCGTTCAAAACTCGATAATGATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACGACTTTTACTTCCA
RPB1：
TGGGTGTGAGTCTTTTATACATATAAATCTATTAACCACGTCAAAACATGAACAGCACGTGAGTTGTACCTGTAGAAGCTGCTCGAACTCGGTGATGACGTGAGCCGGAGCACCCTCCTGTTCGCATCGACGGACATTGGCCGAGGCTTTGATGATATCACCTAGCTTGTATGTCAAATCATCTTCGCTACGCATCGTTCCACCATCGACAGCTATACTGGGGCGCACTGGAGGCGGAGGCACGGGTAGAACTGTGAGGATCATCCACTCTGGACGCGCGTACTCGTCTGAGAGGCCGAGAAGATGGAGATCTGAGTCTGACATCTTCTTCAGGGCGGTGTAAACTTCATGTGGAGGGAATAGCCTCTTGTCTGGTTGTAAGCTCTTCACCTCCTATGCTGAGGAATCAGAATGCTGTGAATAAGCTATGACCGCGCGATACAAACCTCGTCGTCATCCTTGGATCGCTTGTATTGGACAAAAAGCTTGAGGCCCTCTTTCCGAATCTGCGGCTGCGCTGCGCCACATCCACCGTGGCCACGTTTGGGCTCGTCACCATCCGCTCCATCTTCCTCCTTGGGCTCGTCCGGTTCGCAGACCATTTTGCCTTTGCAATAGTTCCATACCACCTGCATACGTGCCTTCGGGTCCCGGACGTGTCTGATCTTGTCAGCAAACGCTGAGTCCGACTTACAACGCATTTTCGGTCACACAAGCGTAGTAGTTGAGAACATACAGATCAAGACGTTAGCAATGTTACTAAGAGTATAACTCCTTGCGGTGACAACACCAAAATAAATAGGGGGGCAATGAATGTCGCGTACAAAGTTGCAAGCCCACCCAGATTCACTCGACCTGCGTCACCGGGAGCATCTGGGGCCAGTTATCGCGGCGAGCTTAACCAACGCCGGATATTACTATTTTTCCGCATCCTCGCTCAGCCCCCTTTTCAACACGCAACTACGTGTCCAACC
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> Nanjing university of forestry
<120> Rapex baichii, screening method thereof and application thereof in degrading forest waste
<130> 2021092901-zf-dll
<141> 2021-09-29
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<170> SIPOSequenceListing 1.0
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<213> Irpex lacteus
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cggttcgaag catgaacacc ataaatactt caacaccaca gcgcagataa ttatcacact 120
gagggcgatc cgtaagattc acgctaatgc atttcagagg agtcgaccga caagggccga 180
cacaacctcc aagtccaagc ccgctaaacc ttcattacaa aaatttaggg gttgagaata 240
ccatgagact caaacaggca tactcctcgg aataccaagg agtgcaaggt gcgttcaaag 300
attcgatgat tcactgaatt ctgcaattca cattacttat cgcatttcgc tgcgttcttc 360
atcgatgcga gagccaagag atccgttgct gaaagttgta tataaatgtg ttatacacag 420
ttgacattct ataactgaag cgtttgtagt aaaacataag aaagaaaaac ggcttgttca 480
accgaagacc tctcgcgaga tcctggaagc ttccaccatt tttttctctt acataaagtg 540
cacagaggtt aagagtggat gagccaggcg tgcacatgcc tcgtgagagg ccagctacaa 600
cccgttcaaa actcgataat gatccttccg caggttcacc tacggaaacc ttgttacgac 660
ttttacttcc a 671
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<212> DNA
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tgggtgtgag tcttttatac atataaatct attaaccacg tcaaaacatg aacagcacgt 60
gagttgtacc tgtagaagct gctcgaactc ggtgatgacg tgagccggag caccctcctg 120
ttcgcatcga cggacattgg ccgaggcttt gatgatatca cctagcttgt atgtcaaatc 180
atcttcgcta cgcatcgttc caccatcgac agctatacgt gggcgcactg gaggcggagg 240
cacgggtaga actgtgagga tcatccactc tggacgcgcg tactcgtctg agaggccgag 300
aagatggaga tctgagtctg acatcttctt cagggcggtg taaacttcat gtggagggaa 360
tagcctcttg tctggttgta agctcttcac ctcctatgct gaggaatcag aatgctgtga 420
ataagctatg accgcgcgat acaaacctcg tcgtcatcct tggatcgctt gtattggaca 480
aaaagcttga ggccctcttt ccgaatctgc ggctgcgctg cgccacatcc accgtggcca 540
cgtttgggct cgtcaccatc cgctccatct tcctccttgg gctcgtccgg ttcgcagacc 600
attttgcctt tgcaatagtt ccataccacc tgcatacgtg ccttcgggtc ccggacgtgt 660
ctgatcttgt cagcaaacgc tgagtccgac ttacaacgca ttttcggtca cacaagcgta 720
gtagttgaga acatacagat caagacgtta gcaatgttac taagagtata actccttgcg 780
gtgacaacac caaaataaat aggggggcaa tgaatgtcgc gtacaaagtt gcaagcccac 840
ccagattcac tcgacctgcg tcaccgggag catctggggc cagttatcgc ggcgagctta 900
accaacgccg gatattacta tttttccgca tcctcgctca gccccctttt caacacgcaa 960
ctacgtgtcc aacc 974
Claims (7)
1. The Irpex lacteus strain is M2-1, and is preserved in China center for type culture Collection, with the preservation address of Wuhan university, Wuhan, China, the preservation number: CCTCC NO: M2021623 with preservation date of 2021.5.27.
2. A screening method for screening the rakanka leucotricha strain according to claim 1, comprising the steps of:
(1) collecting strains: spraying 75% alcohol on pine wood segments with white-bag raking tooth fungus hyphae collected from northern mountain of Nanjing forestry university, sterilizing, volatilizing alcohol, picking hyphae on the wood segments with a sterile scalpel in an ultra-clean workbench, transferring the hyphae to a sterile PDA culture medium, culturing the culture medium in an incubator with 25 ℃ and 40% relative humidity in the dark for 3-5 days, transferring the hyphae to an inclined plane PDA culture medium again when the hyphae grows to two halves of a flat plate, and storing in a refrigerator at 4 ℃ after the hyphae fully grows over the inclined plane;
(2) and (3) laccase detection: taking out the strain obtained by culturing in the step (1) from a refrigerator, activating the strain on a basic PDA plate culture medium, beating bacterial colonies in the basic PDA culture medium into fungus cakes by using a 6mm puncher when hyphae grow to two thirds of the plate, inoculating the fungus cakes to the PDA-guaiacol culture medium, culturing for six days, and observing the size of reddish brown color change circles on the culture medium;
(3) lignin peroxidase and manganese peroxidase assays: inoculating the strain obtained by culturing in the step (1) on a PDA-aniline blue culture medium, culturing for six days, and observing the size of a fading ring generated on the culture medium;
(4) and (3) cellulase detection: inoculating the strain obtained by culturing in the step (1) into a sodium carboxymethylcellulose culture medium, culturing for six days, dyeing with Congo red, fixing with sodium chloride, and observing the size of a transparent ring generated on the culture medium;
(5) strains which can generate color circles in corresponding culture media are preserved through detection in the step (2), the step (3) and the step (4);
(6) and (3) strain identification: identifying the strain by using two pairs of genes of a fungal ribosome genome transcribed spacer region Internaltrapbed spacer and RNA polymerase 1, wherein the strain is identified as the Irpex lacteus, and the strain number is M2-1.
3. The method for screening strains of Rapex baileyi according to claim 2, wherein said PDA-guaiacol medium in step (2) is prepared by adding guaiacol to a basal PDA medium at a final concentration of 0.5 g/L.
4. The method for screening Rapex baichaensis strain as claimed in claim 2, wherein the PDA-aniline blue culture medium in step (2) is prepared by adding aniline blue to the basal PDA culture medium to a final concentration of 0.1 g/L.
5. The method for screening Harpagophytum leucocytochaloides strain as claimed in claim 2, wherein the sodium carboxymethylcellulose culture medium in step (4) comprises 0.25g/L dipotassium hydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate, 5g/L sodium carboxymethylcellulose, 0.5g/L ammonium sulfate, 15g/L agar powder or 20g/L agar strips.
6. Use of the rakanka strain of claim 1 for degrading forest waste.
7. A microbial inoculum for degrading forest waste comprising the rakanka leucotricha strain of claim 1.
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| CN115078043A (en) * | 2022-07-08 | 2022-09-20 | 珠海美华医疗科技有限公司 | Fungus staining solution and preparation method thereof |
| CN115078043B (en) * | 2022-07-08 | 2024-03-29 | 珠海美华医疗科技有限公司 | Fungus staining solution and preparation method thereof |
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