CN117363686A - Detection culture medium combination for needle mushroom strain degradation, detection method and application - Google Patents
Detection culture medium combination for needle mushroom strain degradation, detection method and application Download PDFInfo
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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Abstract
The invention belongs to the technical field of strain detection, and particularly relates to a flammulina velutipes strain degradation detection culture medium combination, a detection method and application. The detection medium combination comprises a culture medium containing H 2 O 2 Any two or more of PDA medium containing NaCl, PDA medium containing SDS, and PDA medium containing CR; containing H 2 O 2 H in PDA Medium of (C) 2 O 2 The addition amount of the PDA culture medium is 0.9-1.3 ml/1L; the addition amount of NaCl in the PDA culture medium containing NaCl is 15.5-19.5 g/1L relative to the PDA culture medium, and the addition amount of SDS in the PDA culture medium containing SDS is 0.03-0.07 g/1L relative to the PDA culture medium; the amount of CR added to the PDA medium is 0.1-0.5 g/1L. The culture medium combination provided by the invention is used for detecting whether the flammulina velutipes strain is degenerated or not, and can reduce the degeneration of the strain caused in flammulina velutipes production to a certain extentAnd the resulting yield decreases and quality decreases.
Description
Technical Field
The invention belongs to the technical field of strain detection, and particularly relates to a flammulina velutipes strain degradation detection culture medium combination, a detection method and application.
Background
Flammulina velutipes (Flammulina velutipes) aliases such as winter mushrooms, agrocybe aegerita, broussonetia, etc., belonging to the genus Eumycota, basidiomycotina, hymenomycetes, agaricales, tricholomataceae, and Lysimachia. All cultivated needle mushrooms in china, japan and korea are named f.filiformi. Needle mushrooms are widely distributed in nature in china, japan, russia, europe, north america, australia, etc. The North Heilongjiang, the south to the Yunnan, the east to the Xinjiang are all suitable for the growth of flammulina velutipes. In China, the cultivation history of flammulina velutipes is very long, the yield is over ten thousand tons, and the flammulina velutipes is the country with the largest yield in the world. The flammulina velutipes fruiting body is delicious in taste and rich in nutrition, and is rich in proteins, amino acids, vitamins, unsaturated fatty acids, dietary fibers and the like which are necessary for human beings. It contains not only rich nutrients, but also various bioactive substances. The traditional medicine considers that the flammulina velutipes has the effects of cold property, salty taste, smoothness, liver entering, stomach entering, intestine channel entering, benefiting the liver, intestines and stomach, improving wisdom, resisting tumor and the like, is suitable for hepatitis, gastrointestinal ulcer, hypertension, cholesterol reduction, infantile mental retardation and the like, and is a dual-purpose fungus for medicine and food.
The strain is a basic material for microbiology and life science research, and especially related industrial production such as antibiotics, amino acids, brewing and the like by utilizing microorganisms is not separated from strain resources. Therefore, the method maintains the excellent properties of the strain, prevents the degradation of the strain, is an important component of microbiological research and microorganism breeding, and is a solid foundation for sustainable development and virtuous circle of the edible fungus industry. Degradation refers to the phenomenon that the strain is gradually lost in the subculture or preservation process due to variation of the strain under the normal culture condition due to the influence of various factors, and is a gradual change process from quantitative to qualitative change, and finally, the yield and quality are reduced. The strain degradation is caused by a plurality of comprehensive factors such as strain impurity, mixed strain pollution, environmental discomfort, improper preservation and the like, and is also the combination of the genetic characteristics of the strain and the molecular regulation effect thereof. Strain degeneration generally refers to the phenomenon in which one or more physiological traits and morphological features of a strain gradually decline or disappear after a prolonged passaging. Common bacterial decay is morphologically manifested as a reduction in conidia or a change in colony color. Physiologically, it is often referred to as a decrease in the ability of a strain to ferment, and some strains have decreased ability to resist phage. High-yield mutant strains obtained by mutation breeding often exhibit wild-type traits and the like. However, the actual degradation of the strain must be distinguished from morphological and physiological variations in the strain caused by environmental changes. If the trace elements are absent from the medium, the number of spores is reduced and the color of spores is changed. In addition, temperature, pH, and different carbon and nitrogen sources all cause strain changes. But these phenomena disappear as soon as normal conditions are restored. In addition, contamination with bacteria can also cause strain degradation artifacts. Therefore, it is necessary to correctly judge whether or not the degradation occurs to find a correct solution. Bacterial degeneration generally occurs gradually from quantitative to qualitative, as well as decreased yields and associated characteristic changes throughout the population, and does not refer to a change in individual cells.
In the industrial cultivation process of flammulina velutipes, the problems of mycelium aging, strain degradation, uneven fruiting, delayed fruiting, serious yield reduction and the like often occur. Wherein, strain degradation can lead to the phenomena of mycelium activity reduction, fruiting body quality and yield reduction, and the like, and is an important factor for restricting the development of flammulina velutipes. The causes of strain degeneration are many, including karyotype change, gene mutation, virus infection of asexual propagules of strains, decrease of intracellular enzyme synthesis ability, etc., and in addition, due to factors such as genetic diversity of strain itself, malnutrition, unlimited passage, long-term cryopreservation, etc. (Li Yajiao and Guo Jiufeng, 2018). In the process of industrial cultivation of flammulina velutipes, virus infection is one of the main reasons for bacterial degradation. Many times of research at home and abroad prove that the flammulina velutipes infected with viruses can have the phenomena of weakened hypha growth and brown fruiting bodies, and the virus diseases are not easy to detect due to long latent period, insignificant disease symptoms and the like of the viruses, and huge losses are caused once the viruses burst. Therefore, it is important to monitor the degradation of flammulina velutipes strains.
The existing degradation method of the edible fungus strain comprises pollution prevention, passage control, environment suitability, periodic fungus separation and the like. Although it is reported that strain degradation of flammulina velutipes can be distinguished by decolorization capability of a special culture medium containing bromothymol blue based on acid-base change after strain growth and metabolism, strain degradation of flammulina velutipes is still currently evaluated by a fruiting test before large-scale production as a biological detection method of a universal and reliable index. These detection methods have the problem that the strain activity and excellent properties cannot be evaluated rapidly, simply and effectively. Therefore, a novel method for detecting the degradation of the flammulina velutipes strain is needed to be used for effectively evaluating the activity and the excellent properties of the flammulina velutipes strain, and further evaluating the yield and the quality of flammulina velutipes influenced by the strain.
Disclosure of Invention
In order to solve the problems that the strain vitality and the excellent properties cannot be evaluated rapidly, simply and effectively in the prior art, the invention adopts the following technical scheme:
the invention provides a detection culture medium combination for needle mushroom strain degradation, which comprises a culture medium containing H 2 O 2 Any two or more of PDA medium containing NaCl, PDA medium containing SDS, and PDA medium containing CR;
containing H 2 O 2 H in PDA Medium of (C) 2 O 2 The addition amount of the PDA culture medium is 0.9-1.3 ml/1L;
the addition amount of NaCl in the PDA culture medium containing NaCl is 15.5-19.5 g/1L relative to the PDA culture medium;
the addition amount of SDS in the PDA culture medium containing SDS is 0.03-0.07 g/1L relative to the PDA culture medium;
the amount of CR added to the PDA medium is 0.1-0.5 g/1L.
Preferably, the PDA culture medium consists of the following materials in percentage by mass:
20% of potatoes;
glucose 2%;
agar 1.6%;
distilled water makes up 100%.
Preferably, the said H-containing 2 O 2 The PDA culture medium comprises the following components in percentage by weight: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 0.9-1.3 ml/L H 2 O 2 。
Preferably, the PDA culture medium containing NaCl comprises the following components in percentage by weight: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 15.5-19.5 g/1LNaCl.
Preferably, the PDA culture medium containing SDS comprises the following components in percentage by weight: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar and 0.03-0.07 g/L SDS.
Preferably, the composition of the CR-containing PDA culture medium and its final concentration are: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 0.1-0.5 g/L CR.
The invention also provides application of the flammulina velutipes strain degradation detection culture medium combination in detecting edible fungus strain degradation.
Preferably, the strain degeneration refers to the physiological property or morphological feature of the strain of the edible fungus is reduced after the strain is preserved for more than or equal to 3 times; wherein, the physiological property decline comprises the reduction of the growth and fermentation capacity of the strain; the morphological feature decline includes slow hyphal growth, reduction of conidia, and change of colony color.
Preferably, the edible fungi comprise flammulina velutipes.
The invention provides a method for detecting needle mushroom strain degradation by utilizing a combination of detection culture mediums for needle mushroom strain degradation, which comprises the following steps:
s1, inoculating an original strain of flammulina velutipes and a flammulina velutipes strain to be detected to any one or any two or more than two culture mediums of a detection culture medium combination and a PDA culture medium respectively, and placing the culture mediums on a 20-25 ℃ incubator for illumination culture, wherein the PDA culture medium is used as a control;
and S2, after culturing for 5-7 days, observing the hypha growth condition of the needle mushroom original strain and the needle mushroom strain to be detected on different detection culture mediums, and if the growth of the needle mushroom strain to be detected on the detection culture mediums is obviously slower than that of the original needle mushroom strain, indicating that the needle mushroom strain to be detected has a degradation phenomenon.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a detection culture medium for detecting the degradation of flammulina velutipes strains, a detection method and application thereof. Meanwhile, the detection method can be used for rapidly, simply and effectively evaluating the activity and the excellent properties of the flammulina velutipes strain, and further evaluating the yield and the quality of flammulina velutipes influenced by the strain.
2. The invention develops a detection culture medium for detecting strain degradation of flammulina velutipes and a detection method based on the differential expression analysis of transcriptome and based on the biochemical response difference of adversity stress. The detection method can be used for evaluating the yield and quality of the flammulina velutipes strain, and provides a set of simple, convenient and effective detection method for the degradation of the flammulina velutipes strain.
3. The invention analyzes degradation mechanism of flammulina velutipes strain at transcriptome level, determines stress resistance of the strain from physiological and biochemical level, and finally performs fruiting culture of the strain to identify yield, length and cap size. Provides an identification index for identifying the degeneration of the strain in the future.
Drawings
FIG. 1 is a phylogenetic tree constructed based on ITS sequences.
FIG. 2 shows the vegetative growth of mycelia according to the present invention in which flammulina velutipes strain F11 and flammulina velutipes strain F17 are grown on the mycelia of PDA plates; b is a growth curve on a PDA plate;
FIG. 3 is an analysis of different expressed genes in the present invention, wherein the Differentially Expressed Genes (DEGs) are determined based on |log2Ration| >1 and P value < 0.05; black dots represent significantly regulated (up-regulated or down-regulated) genes, grey dots represent non-significantly regulated genes;
FIG. 4 shows GO classification of DEGs after GO enrichment analysis in the present invention;
FIG. 5 is a Go enrichment analysis of DEGs in the present invention;
FIG. 6 KEGG classification of DEGs after enrichment analysis in the present invention;
FIG. 7 is a KEGG enrichment analysis of DEGs in the present invention;
FIG. 8 shows the growth difference of different stress indexes of mycelia in the invention, wherein A is the colony growth diameter of flammulina velutipes strain F11 and flammulina velutipes strain F17 in PDA culture medium; b is needle mushroom strain F11 and needle mushroom strain F17 in a content of 1mMH 2 O 2 Colony growth diameter in PDA medium; c is the colony growth diameter of flammulina velutipes strain F11 and flammulina velutipes strain F17 in PDA culture medium containing 0.3M NaCl; d is the colony growth diameter of flammulina velutipes strain F11 and flammulina velutipes strain F17 in PDA culture medium containing 0.005% SDS; e is the colony growth diameter of flammulina velutipes strain F11 and flammulina velutipes strain F17 in PDA culture medium containing 300 mug/ml CR;
FIG. 9 shows the difference in the quality of the growth yield in the present invention, wherein A is the cultivation of Flammulina velutipes; b is the dry weight and fresh weight of flammulina velutipes; c is needle mushroom fruiting body; d is the length of flammulina velutipes fruiting body; e is needle mushroom cap; f is the diameter of the needle mushroom cap.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1
1. Material
Test strain: the flammulina velutipes strain used in the invention is obtained by separating flammulina velutipes from industrial cultivated flammulina velutipes commodity (the mother strain adopted by flammulina velutipes commodity is referred to flammulina velutipes HS-JG01 disclosed in patent with the authority number of CN 103283608B). Wherein the flammulina velutipes strain F11 is a primary strain, and is obtained by separating from commercial products of industrial cultivation of flammulina velutipes mother seeds. The flammulina velutipes strain F17 is a offspring strain obtained after 6 times of continuous passage of the flammulina velutipes strain F11, and the time of each passage is 7-9 days.
The invention carries out strain identification on flammulina velutipes strain F11 and flammulina velutipes strain F17, and comprises the following specific steps:
placing flammulina velutipes strain F11 into PDB for liquid culture, collecting hypha, extracting DNA by adopting a CTAB method, and carrying out PCR amplification by utilizing universal primers ITS1 and ITS4, wherein the nucleotide sequence of ITS1 is shown as SEQ ID No. 1: 5'-TCCGTAGGTGAACCTGCGG-3'; the nucleotide sequence of ITS4 is shown in SEQ ID No. 2: 5'-TCCTCCGCTTATTGATATGC-3'. The PCR amplification procedure was: the pre-denaturation was carried out at 94℃for 4 min, 35 cycles consisting of 94℃for 30 seconds, 53℃for 30 seconds, 72℃for 45 seconds, extension at 72℃for 10 min and storage at 4 ℃. Amplified PCR products were purified and sent to Shanghai Biotechnology Co.Ltd for sequencing. After sequencing, performing Blast comparison on the sequencing result in an NCBI database to obtain a database sequence, and performing cluster analysis on the sequencing result sequence and the database sequence through MEGA software. Wherein, the mass percentages of the components in the formula of the PDB are as follows: 20% of potato, 2% of glucose and 100% of distilled water.
The results showed that the sequence of flammulina velutipes strain F11 was highly similar to the flammulina velutipes sequence in the database (FIG. 1). Accordingly, both flammulina velutipes strain F11 and flammulina velutipes strain F17 which are serially passaged are flammulina velutipes (Flammulina filiformis) and are used for subsequent studies.
2. Needle mushroom strain culture
After the flammulina velutipes strain F11 and flammulina velutipes strain activation F17 are activated, a 5mm puncher is used for preparing fungus cakes, the fungus cakes are respectively inoculated on a PDA culture medium and a detection culture medium, the culture is carried out in a 23 ℃ incubator under illumination, and the colony morphology and the hypha growth condition are observed.
Colony morphology: on days 3, 5 and 7, the colony morphology of flammulina velutipes strain F11 and flammulina velutipes strain F17 was observed and photographed and recorded (see FIG. 2).
Hypha growth: after 3 days of inoculation, the colony diameters of flammulina velutipes strain F11 and flammulina velutipes strain F17 were measured by a crisscross method, and the hypha growth rate was calculated.
Wherein, the formula of the PDA culture medium (1L) is as follows: 200g of potato, 20g of glucose, 16g of agar and 1000mL of distilled water.
The mycelium growth rates of the flammulina velutipes strain F11 and the flammulina velutipes strain F17 subjected to subculture on the PDA culture medium are statistically consistent, and the flammulina velutipes strain F11 and the flammulina velutipes strain F17 have similar growth trends (shown in figure 2). The results show that the mycelium growth rates of the flammulina velutipes strain F11 and the flammulina velutipes strain F17 subjected to subculture are basically consistent under the conventional culture conditions, and the flammulina velutipes strain F11 and the flammulina velutipes strain F17 have similar growth trends, so that the two strains have no obvious difference in mycelium nutrition growth and colony phenotype.
3. Differential functional analysis of transcript levels
Flammulina velutipes strain F11 and Flammulina velutipes strain F17 total RNA were extracted by Invitrogen PureLink RNA extraction kit (Thermo Fisher, shanggai, china). The RNA concentration of the samples was determined by Nanodrop NC2000 (Thermo Scientific, shanghai, china) while checking the integrity and quality of the RNA samples by agarose electrophoresis and Agilent 2100Bioanalyzer (AgilentTechnologies, USA), a cDNA library was constructed and tested by the Illumina Hiseq4000 platform of person company (Shanghai, china).
The original reading of each sample was checked after sequencing to be between 42,497,308 and 46,010,500, a Q20 value above 98%, a Q30 value above 94% and a percentage of ambiguous bases less than 0.01%. The average number of pure readings obtained after mass filtration is 42,306,531; the percentage of pure readings is 94.17% -94.36%, and the average is 94.24%; this indicates good sequencing quality. Clean reads were assembled from the head to give transcripts and monogenes, 34149 transcripts and 12385 monogenes, respectively, with maximum length of 12863bp, average length of 2064 and 1844, respectively. The above results indicate that sequencing data is reasonable between different biological repeats and that sequencing data can be used for further analysis.
The raw reads were filtered after sequencing to remove the aptamer and low quality reads, and the obtained clean reads were assembled de novo into longer isocratic groups in the overlap region using the Trinity platform (Mortazavi et al, 2008). Subsequent analysis was performed using clean data. Single genes were annotated by alignment with the NR, GO, KEGG, eggNOG, swiss-Prot and Pfam databases. Wherein, in the NR database, a total of 9994 single genes were successfully annotated, accounting for 80.69% of all assembled single genes; in the KEGG and GO databases, 3584 and 3703 genes were annotated, accounting for 28.94% and 29.9% of all genes, respectively; in the Pfam, eggNOG and Swissprot databases 5921-7304 genes, respectively, were annotated accounting for 47.81-58.97% of all genes. Furthermore, the results of eggNOG showed that 7304 monomer genes were classified into 26 classes.
The expression level of each single gene was normalized to a map reading per kilobase fragment per million (FPKM) using RSEM (Li and Dewey, 2011). Screening of Differentially Expressed Genes (DEGs) using DESeq2 (Anders and Huber, 2010) resulted in a double change and a p-value corrected for False Discovery Rate (FDR) of 0.05 or less. A scatter plot is generated using an Rbaseplot function for representing up-and down-regulated DEGs. Based on the conditions of |log2ration| >1 and P value <0.05, differential Expression Genes (DEGs) were determined, and 280 up-regulated genes and 352 down-regulated genes between the two strains of flammulina velutipes strain F11 and flammulina velutipes strain F17 were obtained in total (as shown in fig. 3).
The DEGs were functionally annotated according to GO and KEGG pathway analysis, and GO and KEGG pathway enrichment analysis was performed using clusterif iotaler in R (Yu et al 2012). The results indicate that GO analysis classifies DEGs into three classes, molecular Function (MF), biological Process (BP) and Cellular Component (CC) (FIG. 4), where oxidoreductase activity (GO: 0016491) is the most enriched type of DEGs (25 DEGs, 15 of which are up-regulated and 10 of which are down-regulated) (as shown in FIG. 5). Furthermore, KEGG analysis showed that DEGs had a matching relationship in 61 pathways (as shown in fig. 6), where peroxisome (ko 04146) was one of the KEGG pathways containing the most DEGs and higher enrichment factors, which played a key role in redox signaling and lipid homeostasis (as shown in fig. 7).
Therefore, the annotation and enrichment analysis of the DEGs in the GO and KEGG channels show that the DEGs participate in the regulation of adversity stress such as active oxygen and the like through the comparison analysis of the transcription level between the flammulina velutipes strain F11 and the flammulina velutipes strain F17, and can be related to strain degeneration.
4. Physiological and biochemical level differential analysis
According to the analysis results of the transcriptome, the oxidation-reduction resistance and the molecular regulation level of different strains are different. On this basis, the following physiological and biochemical tests were set up:
test strain: the flammulina velutipes strain F11 and the flammulina velutipes strain F17.
The invention activates flammulina velutipes strain F11 and flammulina velutipes strain F17 simultaneously, and respectively uses the flammulina velutipes strain F11 and flammulina velutipes strain F17 in PDA culture medium and 1mM hydrogen peroxide (H) 2 O 2 ，H 2 O 2 3%) by volume, PDA medium containing 0.3M sodium chloride (NaCl), PDA medium containing 0.005% Sodium Dodecyl Sulfate (SDS), and PDA medium containing 300. Mu.g/mL Congo Red (CR) were incubated at 23℃with light, and colony morphology and hypha growth rate were observed at 5d and 7 d. Wherein, PDA culture medium can be replaced by PDB culture medium, the PDB culture medium has the same composition as PDA culture medium, but no agar is added.
The results showed (FIG. 8) that as the number of passages of the strain increased (e.g., between flammulina velutipes strain F11 and flammulina velutipes strain F17, 6 passages), neither flammulina velutipes strain F11 nor flammulina velutipes strain F17 showed a significant phenotypic difference in colony morphology and hypha growth rate on PDA medium. However, the hyphae of flammulina velutipes strain F11 and flammulina velutipes strain F17 cultivated on the above different detection media showed significant differences in growth rate at both 5 days and 7 days, and the specific results were as follows:
at 5 days, at 1mM H 2 O 2 The growth speed of flammulina velutipes strain F17 on the PDA culture medium is 54.9% slower than that of flammulina velutipes strain F11; on PDA culture medium containing 0.3M NaCl, flammulina velutipes strain F17 has 19.5% slower growth rate than flammulina velutipes strain F11 hypha; flammulina velutipes strain F17 grows 25.9% slower than flammulina velutipes strain F11 hypha on PDA medium containing 0.005% SDS; flammulina velutipes strain F17 showed a 55.7% slower hyphal growth rate than Flammulina velutipes strain F11 on PDA medium containing 300. Mu.g/ml CR. At 7 days, 1mM H was contained 2 O 2 Flammulina velutipes on PDA culture mediumThe growth speed of the mycelium of the strain F17 is 38.0% slower than that of the mycelium of the flammulina velutipes strain F11; on PDA culture medium containing 0.3M NaCl, flammulina velutipes strain F17 has 37.2% slower growth rate than flammulina velutipes strain F11 hypha; flammulina velutipes strain F17 grows 16.7% slower than flammulina velutipes strain F11 hypha on PDA medium containing 0.005% SDS; flammulina velutipes strain F17 showed a 50.9% slower hyphal growth rate than Flammulina velutipes strain F11 on PDA medium containing 300. Mu.g/ml CR.
In summary, the assay medium combinations set up according to transcriptome analysis (four media including containing 1mM H 2 O 2 Any one or more of PDA medium containing 0.3M NaCl, PDA medium containing 0.005% SDS, and PDA medium containing 300. Mu.g/mL CR) can be used to distinguish the difference in growth phenotype of flammulina velutipes strain F11 and flammulina velutipes strain F17, and thus presumably can be used to detect the degradation phenomenon of flammulina velutipes.
5. Yield and quality character difference analysis of fruiting
In order to verify whether the above detection medium combination effectively detects the occurrence of strain degeneration of flammulina velutipes, a fruiting (fruiting body cultivation) test is performed on flammulina velutipes strain F11 and flammulina velutipes strain F17, and the specific steps are as follows:
the formula of the culture material is 68% of corncob, 16% of cotton seed hull, 15% of bran and 1% of lime. The water content of the material is 64-65%. After mixing materials according to the formula, 10 bottles of polypropylene plastic bottles with the diameter of 8cm are adopted, the mixture is autoclaved for 2 hours at the temperature of 121 ℃, and the flammulina velutipes strain F11 and the flammulina velutipes strain F17 are inoculated on a culture medium according to the inoculation amount of 5% under the aseptic condition. Culturing for about 20 days after inoculation, controlling the relative humidity of air to be 85% -90% after fungus scratching, reducing the culture temperature to 7-10 ℃ to promote bud growth, and harvesting after about 27 days when the fungus cover begins to be flat and the bud can be developed after proper scattered illumination. Measuring the biomass of fruiting body, and observing and recording the morphological characteristics (such as length of fruiting body and size of fruiting cap) to evaluate the yield and quality of Flammulina velutipes (Fr.) Sing on a large scale of degenerated strain detected by examining the culture medium.
The results showed (FIG. 9) that the fresh weight and dry weight of fruiting body of flammulina velutipes strain F11 were 11.1% higher than those of flammulina velutipes strain F17, the length of the mushroom stem was 15.6% different, and the diameter of the cap was 32.1%. These results showed that although there was no difference in hyphal growth of flammulina velutipes strain F11 and flammulina velutipes strain F17 on PDA common medium (fig. 2), the difference in stress resistance exhibited based on the hyphal growth on the test medium was remarkable (fig. 8), and the biomass and quality of the fruiting body formed were different (fig. 9). Therefore, by utilizing a transcriptome analysis method, key adjustment factors related to the degradation of the flammulina velutipes strain are discovered, and the combination of the detection culture mediums is set according to the key adjustment factors, so that the method can be used for detecting the degradation of the flammulina velutipes strain, and can provide scientific reference and technical guarantee for the yield and quality of flammulina velutipes and sustainable large-scale production of flammulina velutipes.
The results show that the detection culture medium combination provided by the invention can be used for detecting whether the flammulina velutipes strain is degenerated or not, and the risk of yield and quality degradation of flammulina velutipes caused by the degeneration of the strain can be monitored and controlled to a certain extent.
Comparative example 1
A method for detecting degradation of flammulina velutipes strains (a conventional biological detection method) comprises the following steps:
an empirical test was used in which the strains to be tested (the same strains as in example 1: flammulina velutipes strain F11 and flammulina velutipes strain F17) were inoculated into PDA medium and their colony morphology and hypha growth biological phenotype were used as main references.
The bacterial colony morphology and growth condition are detected by taking the experience of staff as a judgment basis, and the specific method is as follows: before fruiting test, judging whether the strain is degenerated or not, mainly based on subjective experience of people. And then, carrying out a conventional fruiting test according to the result, and observing fruiting conditions of the strain after culturing for about 50 days so as to evaluate whether the strain is found to be degenerated.
The scientific index reference of the method for detecting the degradation of the flammulina velutipes strain in comparative example 1 is extremely low. The judgment of whether the strain is degenerated or not is carried out before the fruiting test, and is mainly based on subjective experience of people, but not based on scientific data indexes. And then, carrying out a conventional fruiting test, culturing for about 50 days, and then observing fruiting conditions of the strain, so as to evaluate whether the strain is degraded or not, and carrying out subsequent industrial production. Compared with the detection method provided in the embodiment 1 of the application, the detection method takes a long time (at least about 50 days), and can not realize rapid, simple and efficient activity evaluation and excellent properties of the flammulina velutipes strain, and the degradation level of the strain can not be detected.
The detection medium combination and the detection method using the detection medium provided by the application adopt the data information based on molecular adjustment, and the set detection medium combination (four kinds of culture mediums comprise 1mM H 2 O 2 PDA medium containing 0.3M NaCl, PDA medium containing 0.005% sds, PDA medium containing 300 μg/ml CR, and PDA medium), were placed in a 23 ℃ incubator for light culture, and after 5-7 days, differences in hypha growth phenotype of the different strains on the test medium combination were observed. If the strain to be detected is found to grow obviously slower than the original strain, the strain to be detected is shown to degenerate. The method of the application takes a short time (5-7 days are expected), can realize rapid, simple and efficient evaluation of the activity of the flammulina velutipes strain, and detects the degradation level of the strain.
The invention provides a detection culture medium for detecting the degradation of flammulina velutipes strains, and a detection method and application thereof. Meanwhile, the detection method can be used for rapidly, simply and effectively evaluating the activity and excellent properties of the flammulina velutipes strain, and further evaluating the yield and quality of flammulina velutipes influenced by the strain.
The invention develops a detection culture medium and a detection method for detecting strain degradation of flammulina velutipes based on a transcriptome differential expression analysis as a theoretical basis and based on a biochemical response difference of adversity stress. The detection method can be used for evaluating the yield and quality of the flammulina velutipes strain, and provides a set of simple, convenient and effective detection method for the degradation of the flammulina velutipes strain.
The invention analyzes the degradation mechanism of flammulina velutipes strain at transcriptome level, determines the stress resistance of the strain from physiological and biochemical level, and finally performs fruiting culture on the strain to identify the fruiting body yield, the mushroom stem length and the mushroom cap size formed by the strain, thereby providing practical identification indexes and references for the subsequent identification of the degradation of the strain.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A detection culture medium combination for needle mushroom strain degradation is characterized by comprising a culture medium containing H 2 O 2 Any two or more of PDA medium containing NaCl, PDA medium containing SDS, and PDA medium containing CR;
containing H 2 O 2 H in PDA Medium of (C) 2 O 2 The addition amount of the PDA culture medium is 0.9-1.3 ml/1L;
the addition amount of NaCl in the PDA culture medium containing NaCl is 15.5-19.5 g/1L relative to the PDA culture medium;
the addition amount of SDS in the PDA culture medium containing SDS is 0.03-0.07 g/1L relative to the PDA culture medium;
the amount of CR added to the PDA medium is 0.1-0.5 g/1L.
2. The flammulina velutipes strain degradation detection medium combination according to claim 1, characterized in that the PDA medium consists of the following materials in percentage by mass:
20% of potatoes;
glucose 2%;
agar 1.6%;
distilled water makes up 100%.
3. The combination of detection media for needle mushroom strain degradation according to claim 1, wherein the combination contains H 2 O 2 The PDA culture medium comprises the following components in percentage by weight: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 0.9-1.3 ml/L H 2 O 2 。
4. The combination of culture media for detecting degradation of needle mushroom strains according to claim 1, wherein the PDA culture media containing NaCl comprises the following components in percentage by weight: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 15.5-19.5 g/1LNaCl.
5. The combination of detection media for degradation of needle mushroom strains according to claim 1, wherein the PDA medium containing SDS comprises the following components in the final concentration: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar and 0.03-0.07 g/L SDS.
6. The combination of culture media for detecting degradation of needle mushroom strains according to claim 1, wherein the composition of the CR-containing PDA culture medium and the final concentration thereof are as follows: 190-210 g/L potato, 18-22 g/L glucose, 14-18 g/L agar, 0.1-0.5 g/L CR.
7. The use of the flammulina velutipes strain degradation detection medium combination of claim 1 in detecting edible fungus strain degradation.
8. The use according to claim 7, wherein the bacterial degradation means that the physiological properties or morphological characteristics of the strain of the edible fungus are reduced after the preservation of the bacterial strain at a passage number of not less than 3 times; wherein, the physiological property decline comprises the reduction of the growth and fermentation capacity of the strain; the morphological feature decline includes slow hyphal growth, reduction of conidia, and change of colony color.
9. The use according to claim 7, wherein the edible mushrooms comprise flammulina velutipes.
10. A method for detecting needle mushroom strain degradation by using the combination of the needle mushroom strain degradation detection medium according to claim 1, comprising the steps of:
s1, inoculating an original strain of flammulina velutipes and a flammulina velutipes strain to be detected to any one or any two or more than two culture mediums of a detection culture medium combination and a PDA culture medium respectively, and placing the culture mediums on a 20-25 ℃ incubator for illumination culture, wherein the PDA culture medium is used as a control;
and S2, after culturing for 5-7 days, observing the hypha growth condition of the needle mushroom original strain and the needle mushroom strain to be detected on different detection culture mediums, and if the growth of the needle mushroom strain to be detected on the detection culture mediums is obviously slower than that of the original needle mushroom strain, indicating that the needle mushroom strain to be detected has a degradation phenomenon.
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