CN114921344A - Method for breeding high-quality morchella esculenta variety by utilizing artificial internal co-biochemical technology - Google Patents

Method for breeding high-quality morchella esculenta variety by utilizing artificial internal co-biochemical technology Download PDF

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CN114921344A
CN114921344A CN202210515335.1A CN202210515335A CN114921344A CN 114921344 A CN114921344 A CN 114921344A CN 202210515335 A CN202210515335 A CN 202210515335A CN 114921344 A CN114921344 A CN 114921344A
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morchella
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CN114921344B (en
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何培新
刘伟
王文升
蔡英丽
曹满堂
陈卓
孙文化
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Sichuan Junyinong Agricultural Technology Co ltd
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Abstract

The invention discloses an artificial internal co-biochemical breeding technology for breeding high-quality morel varieties, which is characterized in that saccharomycetes or bacteria beneficial to artificial cultivation of morel, such as limiting malassezia, aroma rhodosporidium roseum Sporobolomyces, terribacterium, brevundimonas, Methylophilus, Sphingobacterium, flavobacterium and the like, are introduced into morel hypha cells to establish an internal symbiotic relationship between the two parties, and then high-quality morel cultivated varieties with ideal cultivation and production properties such as vigorous growth, high yield, strong stress resistance and the like are screened from the obtained product. The technical operation comprises the process engineering of activated culture of original strains of morchella, introduction of beneficial yeasts or bacteria into morchella hypha cells and identification thereof, determination of culture characteristics and production characteristics of in-vitro co-biochemical morchella, gradual expansion of artificial culture and the like. The technology of the invention opens up a new effective way for breeding the morchella and is beneficial to the sustainable and stable development of the morchella industry.

Description

Method for breeding high-quality morchella esculenta variety by utilizing artificial internal co-biochemical technology
Technical Field
The invention belongs to the technical field of biological breeding, and particularly relates to a method for breeding a high-quality morchella strain by utilizing an artificial internal co-biochemical technology.
Background
Morel (Morel: (A))Morchellaspp.) belongs to Ascomycota (Ascomycota), class Pezizomycetes (Pezizomycetes), order Pezizales (Pezizales), family Morchellacaceae (Morchellaceae), and is an internationally recognized precious fungus for both food and drug use. The morchella esculenta has tender texture and unique flavor, and has multiple physiological activities of oxidation resistance, inflammation resistance, bacteria resistance, immunoregulation, tumor resistance and the like. At present, the outdoor 'soil cultivation mode' of morchella esculenta popularized in large scale in China is successfully applied to Chongqing areas in China in 2012 first and is rapidly popularized to all over the country. According to incomplete statistics, the cultivation area of morchella esculenta in 2021 year in China is 25 ten thousand mu (666.7 m for 1 mu) 2 ) On the left and right sides, they are located in other provinces and cities in China except Hainan province.
The conventional natural breeding is mainly relied on in the present morchella breeding, namely, a large amount of mycelium isolates are obtained by tissue separation, polyspora separation or monospora separation from wild or cultivated ascocarp, and then ideal production strains are screened from the mycelium isolates. However, with the continuous expansion of the artificial cultivation scale and area of morel, and the reasons that the morel is easy to age and degenerate, and the natural morel planting resource is increasingly exhausted, the breeding speed of high-quality cultivation strains (varieties) is far from the pace of the artificial cultivation and development of morel, which is one of the important reasons that the stability of morel cultivation is affected and the loss of mushroom farmers is caused. Therefore, a new efficient breeding technology is sought, the improved breeding of morchella is promoted, and the method has obvious practical significance for stabilizing the production of morchella and obtaining ideal economic benefits of artificial cultivation.
Morchella is a soil-borne bacterium, and soil is the capital nutrition of microorganisms. In the growth and development process of morchella, very complex ecological interaction occurs between morchella and many other microorganisms in soil, including antagonism, competition, symbiosis and the like, wherein the most important is symbiotic relationship. According to the widely accepted concept of symbiosis, the symbiosis is not only a one-to-one mutualistic symbiosis relationship, but also a symbiosis continuum including mutualistic symbiosis, biased symbiosis and antagonism/parasitism. In the symbiotic interaction between microorganisms, the most deeply studied is the bacterial-fungal interaction, i.e., the endophytic bacteria and fungi that colonize in the cytoplasm of fungal hyphae or in the cytoplasm of spores exert important influences on the biology of each other through the types of interactions, such as antagonism, cooperation, synergy, co-habitation and symbiosis.
In recent years, advanced technical means such as omics technology and the like are adopted to represent the change rule of related microbial communities in the growth and development process of the morchella, and some bacteria which possibly have the promotion effect on the growth and development of the morchella are discovered. For example, Zhang et al (2019) studied the diversity of soil bacterial communities in different developmental stages such as the artificial cultivation of six sisters Morchella using replicon sequencing technology and found an unexpectedly high proportion of Pseudomonas (25.30%) at the primordial differentiation stage, suggesting that Pseudomonas may affect Morchella growth (Zhang F, Long L, Hu Z, Yu X, et al. analytes of local molar bacterial community structure and minor element contents in ascocarp and the cubic tissue. Canadian Journal of Microbiology, 2019, 65: 738-. Luet al (2021) detection of goat spiking Using replicon sequencingMorel (A), (B), (C)M. conica) The dynamic change of the bacterial community of the development fruiting body, and the analysis of the co-expression of the transcriptome finds that the bacterial community possibly plays an important role in regulating the expression of the morchella gene; the study found that Flavobacterium: (A)Flavobacterium) Pseudomonas bacteria (A)Pseudomonas) And terribacterium (a)Pedobacter) The growth stage of the morchella conica sporocarp is dominant, and the morchella conica sporocarp plays a role in the development of morchella conica; the research finds several endophytic bacterial communities including grass spirillum, terribacterium, bacillus, and bacillus, and bacillus are included in the group of bacillus species,ArsenicitaleaAnd Duganella bacterium (Duganella bacterium) (II)Duganella) The high association of modules enriched with "cell wall" and "protease regulatory subunit" indicates that a bacterial community may promote the development of fruiting bodies by modulating the expression of specific enzymes involved in cell wall synthesis and proteasome-associated pathways (LuBB, Wu GG, Sun Y, Zhang LS, et alMorchella conica SH. Frontiers in Microbiology, 2021, 12: 682356）。
The research on the influence of the soil microbial community on the growth and development of the morchella is also carried out by adopting a metagenome technology (replicon sequencing and the like). Our research found that the bacterial flora associated with morchella development is terribacillus paradisi: (Parapedobacter) Paenibacillus bacteria (A), (B)Paenibacillus) Anaerobic Bacillus (A), (B), (C)Anoxybacillus) Bacillus (B) and (C)Bacillus) Stenotrophomonas (Stenotrophomonas), etc., which exist in small amount before seeding, are enriched in large amount at the mycelium stage, and are reduced in population amount at the primordial stage and the harvest stage, and may be consumed as nutrients by degradation of morchella at the later stage of morchella mycelium culture. Brevundimonas genus (Brevundimonas) Terribacterium (A), (B)Pedobacter) Methylophilus genus: (Methylophilus genus) ((M))Methylobacillus) Sphingobacterium genus (A), (B), (C)Sphingobacterium) Flavobacterium (F.), (Flavobacterium) Pseudomonas bacteria (A)Pseudomonas) Marseillea (B)Massilia) And Spirosoma (Sw.) RalstoniHerbaspirillum) Duganella bacterium (A. dolastata) ((B.))Duganella) Genus hydrophagia (H.H.)Hydrogenophaga) And the like are enriched in a large amount in the primordial stage,indicating that the compounds are possibly related to promoting the development of primordia and sporocarp of the morchella esculenta.
In foreign countries, artificial endosymbiosis (artificial endosymbiosis) is a category of synthetic biology, which refers to experimental biology or synthetic biology procedures involved in establishing symbiotic relationships in ideal hosts that do not exist in nature. The ultimate goal of artificial co-biochemistry is to construct a novel biosynthesis pathway which can be used in the industries of biofuel production, food and agriculture, fermentation industry, bioremediation and the like; also has potential application value in the production of novel metabolites, biofertilizers, chemicals, enzymes and other bioactive molecules; in addition, it can be used to establish a new life form that can survive in the open space and other celestial bodies or can adapt to the conditions of the earth's great climate change (Puri KM, Butardo Jr V, Sumer H. Evaluation of natural endobiosis for progress Tombysios, 2021, 84: 1-17). However, no research and application report of breeding morchella by using an artificial internal co-biochemical technology is reported at home and abroad.
The invention introduces the microorganisms (beneficial microzyme and bacteria for artificial cultivation of morchella) into the mycelial cells of the morchella cultivation strain, so that the two parties establish a stable endosymbiont relationship, the influence of exogenous beneficial microorganism introduction on growth and development of morchella is researched, an artificial endosymbiont technology is established, and the technology is found to have great popularization and application potential in breeding high-quality strains with high yield, strong resistance and the like of morchella.
Disclosure of Invention
The invention aims to solve the technical problems that the breeding technology of high-quality strains (varieties) for artificially cultivating morchella is single, the speed of fine breed breeding is far beyond the pace of artificial cultivation and development of morchella, the stability of morchella cultivation is influenced, and the production loss of mushroom farmers is caused.
In order to solve the technical problems, the invention adopts the following technical scheme:
the method for breeding the high-quality morchella esculenta variety by utilizing the artificial co-biochemical technology comprises the following steps:
(1) carrying out activated culture on original strains of morchella;
(2) introducing beneficial yeast or bacteria into the toadstool hypha cells;
(3) purifying the morchella strains;
(4) introducing exogenous bacteria or yeasts into the toadstool hypha cells for internal identification;
(5) determining the culture characteristics of the endochemical morchella strains;
(6) measuring the production characteristics of the endochemical morchella strains;
(7) the artificial cultivation of the high-quality strains of the common biochemical morchella is expanded step by step.
Further, the beneficial yeasts and bacteria for artificial cultivation of morchella in the step (1) refer to microorganisms which can promote resistance of morchella to adverse environmental conditions such as heavy metals, soil autotoxins, poor nutrition and the like, improve soil nutrition conditions through nitrification, nitrogen fixation, degradation of organic macromolecules such as carbohydrates and the like, regulate mechanisms such as cell wall synthesis and expression of specific enzymes in proteasome-related pathways through generation of specific secondary metabolites and promote growth of morchella hyphae and development of fruiting bodies, and include microorganisms which limit malassezia (a) ((r))Malassezia restricta) The aroma Rhodosporidium roseum Sporobolomyces (Sporobolomyces) ofRhodosporidiobolus odoratus) Yeast, Geobacillus: (A)Pedobacterspp.), Brevundimonas bacterium (Brevundimonas bacterium: (Brevundimonasspp., Methylophilus bacterium (M.sp.), (Methylobacillusspp., Sphingobacterium (Sphingobacterium) (II)Sphingobacteriumspp.) and Flavobacterium (Flavobacteriumspp.), and the like.
Further, the yeast and bacteria beneficial to the artificial cultivation of the morchella have the following characteristics: culturing the toadstool on a nutrient medium flat plate in a confronting manner, and generating no obvious interspecific competitive characteristics such as antagonistic lines, antagonistic ditches and the like in the colony contact areas of the two parties; the two parts are co-cultured in liquid culture medium, and the biomass of the yeast or bacteria has the characteristics of increasing and then decreasing.
Further, the original strain of morchella esculenta in the step (1) refers to morchella esculenta (F) capable of being artificially cultivatedMorchella importuna) Six sisters of morchella (morchella esculenta)M. sextelata) And seven sisters morchella (morchella esculenta) (C)M. eximia) Nine sisters morchella (Jiu Mei)M. exuberans）、Mel-13、Mel-21 and the like culturable strains or varieties.
Further, the activated culture of the original strain of morel in the step (1) means that the original strain of morel is inoculated on a proper nutrient medium flat plate and is cultured in the dark at a proper temperature (such as 18-25 ℃) until the morel mycelium spreads to the whole flat plate.
Further, the beneficial yeast or bacteria are introduced into the toadstool mycelium cells in the step (2), and a mixed culture method, a microinjection method or a protoplast fusion method can be adopted. The mixed culture method comprises inoculating morchella mycelium blocks on one side of a nutrient medium flat plate, inoculating bacteria or saccharomycete lawn at a proper distance (such as 1-2 cm) from the mycelium blocks by adopting a streaking inoculation method, and culturing the inoculated flat plate at a proper temperature (such as 18-25 ℃) in a dark place until morchella mycelium spreads to the whole flat plate. The microinjection method is to use a micro-operation technology, inject bacteria or yeast cells into the inner part of morchella mycelium cells by using a capillary (with the aperture of about 10 microns), and then culture the plate subjected to injection operation at a proper temperature (such as 18-25 ℃) in a dark place until the morchella mycelium spreads to the whole plate. The protoplast fusion method is that protoplasts of morchella and bacteria or yeast are respectively prepared, then inactivation treatment is carried out, then the protoplast fusion of the two parties is completed by adopting a polyethylene glycol (PEG) mediated fusion method or an electrofusion method, and then the fused protoplasts are cultured on a regeneration culture medium to regenerate.
Further, the purification of the morel strain in the step (3) means that the morel strain introduced with beneficial bacteria or yeasts is inoculated to a nutrient medium flat plate or a test tube inclined plane, is cultured in a dark place at a proper temperature (such as 18-25 ℃), and then morel mycelium fragments growing on the wall of the test tube outside the culture medium or on an inner cover of a culture dish are selected and transferred to a new inclined plane or a new flat plate for culture. Repeating the purification for multiple times (generally more than 3 times), and finally obtaining the morchella mycelium culture with the bacteria or the yeasts entering the inner part of the morchella hypha cells instead of being positioned among the morchella hypha cells.
Further, the exogenous bacteria or yeasts are introduced into the toadstool hypha cells for identification, and the identification can be carried out by adopting a PCR amplification method in combination with one or more of the technologies such as fluorescence in situ hybridization, transmission electron microscopy, scanning electron microscopy and the like; wherein, the PCR amplification method is that total DNA extracted from the morchella culture with the established endosymbiotic relationship is taken as a template, the PCR amplification is carried out by taking the introduced specific primer of bacteria or saccharomycetes as a primer, and the specific band of exogenous bacteria or saccharomycetes exists in the amplification product after the detection of agarose gel electrophoresis; the fluorescence in situ hybridization is to take the specific sequence of the introduced exogenous bacteria or saccharomycetes as a probe, and hybridize with the total DNA extracted from the morchella culture with the established endosymbiotic relationship, and the hybridization result is positive; the transmission electron microscopy refers to observing the toadstool culture with an endosymbiotic relation by adopting a transmission electron microscope, and can see that introduced exogenous bacteria or yeasts exist in toadstool mycelium cells. Scanning electron microscopy refers to observing a morchella culture (hypha cells are torn or crushed) with an endosymbiotic relationship by adopting a scanning electron microscope, and the introduced exogenous bacteria or yeasts exist in the morchella hypha cells.
Further, the determination of the culture characteristics of the endochemical biochemical morchella strain in the step (5) refers to determining the hypha growth speed and the hypha biomass of the morchella strain of endosymbiotic bacteria or yeasts in a proper pH and temperature range of growth by taking the initial morchella strain as a control; determining the influence of heavy metals (chromium, lead, mercury, arsenic, zinc, copper and the like) with different concentrations, soil autotoxic substances (phenolic acid substances, self DNA and the like), extreme temperature, pH and the like on the growth of the toadstool strains of endosymbiotic bacteria or yeasts (determining various stress resistance capabilities of breeding the toadstool strains); the mycelial growth rate and mycelial biomass of the toadstool strains of endosymbiotic bacteria or yeasts are measured in a nitrogen-free medium (for measuring nitrogen fixation capacity) and a low-carbon medium (for measuring stress resistance to poor nutrition).
Further, the determination of the production characteristics of the endochemical morchella strain in the step (6) refers to performing multiple (for example, three different places) small-scale (for example, 5 m of one strain) on the endochemical morchella strain according to the currently general morchella cultivation and management technology 2 ) And (3) artificially cultivating, observing and recording the time and relative strength of fungus cream production, the time and amount of primordium production, the time and amount of young mushroom production, the unit yield of the first mushroom, the unit yield of the second mushroom, the occurrence condition of plant diseases and insect pests, the dry-wet ratio of mushroom bodies and other yield characters, and comprehensively evaluating the production capacity of the bred strains and the potential of further popularization and utilization.
Further, the high-quality morchella cultivation variety (or strain) in the step (7) refers to a high-quality strain (variety) which is obtained by applying an artificial internal co-biochemical technology and has high hypha growth potential, high continuous cropping resistance and saline-alkali resistance, early primordium generation time, high fruiting rate, high yield, good quality and other excellent cultivation and cultivation properties.
Further, the step-by-step artificial cultivation of the high-quality strains of the endochemical morchella in the step (7) is to perform further multi-place (a plurality of different places with representativeness of climate, resources and cultivation modes) and larger area (for example, 100m of each strain) on the high-quality strains with high yield and strong stress resistance of the morchella which are ideal for small-scale cultivation and have potential popularization and application values according to the current universal morchella cultivation management technology 2 The artificial cultivation test of the above), observing, recording and comparatively analyzing the cultivation character, finally screening to obtain the high-quality bacterial strain with vigorous growth, high yield and strong stress resistance, and being used for small-scale demonstration and popularization and large-scale artificial cultivation application.
The invention has the beneficial effects that: 1. the separation, purification and identification of beneficial microzyme or bacteria, the introduction of exogenous beneficial microorganisms into morchella mycelium cells, the identification of the morchella mycelium cells, the establishment of the endosymbiotic relationship, the measurement of the growth and the production characteristics of morchella strains and the like are conventional technical methods in the biological field, so the patent technology has strong operability and is easy to realize; 2. the morchella cultivation strain (variety) which is locally popularized and applied in a large scale is taken as a breeding starting strain, after further breeding is carried out by applying an artificial internal co-biochemical technology, a more ideal cultivation strain (variety) with directly improved growth characteristics and production characteristics can be obtained, the implementation effect of the patent technology is obvious, the implementation time is short, and the cost is low.
Drawings
FIG. 1 is a scanning electron microscope observation image of Morchella mycelium cells, the left image showing Morchella mycelium cells without Malassezia, and the right image showing Morchella mycelium cells transformed with Malassezia.
FIG. 2 is a scanning electron microscope observation image of Morchella mycelium cells, wherein the left image shows Morchella mycelium cells without Agrobacterium and the right image shows Morchella mycelium cells transferred into Agrobacterium.
FIG. 3 is the PCR amplification result for detecting whether the terribacterium is transferred into morchella hypha cells.
FIG. 4 shows the result of PCR amplification for detecting whether Malassezia are transformed into Morchella mycelium cells.
Detailed Description
The present invention will be further described with reference to the following examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention, which is to be given numerous insubstantial modifications and adaptations by those skilled in the art based on the teachings set forth above.
Example 1
The method for breeding the high-quality morchella esculenta by utilizing the artificial internal co-biochemical technology comprises the following steps:
(1) carrying out activated culture on breeding starting morchella strains: selecting morchella (strain preservation number: CCTCC KF 2008790) as a breeding starting strain, inoculating the starting strain to a CYM plate, and performing inversion and light-proof culture at 20 ℃ until the plate is full.
(2) Limiting introduction of malassezia into toadstool hypha cells: streaking restricted malassezia (strain preservation number: ATCC 96810) (streaking width is about 2 cm) on one side of a CYM plate, then inoculating morchella strain blocks at a position 2 cm away from a yeast streaking area, carrying out inverted culture at 20 ℃ for 1-2 weeks, taking morchella cultures growing to different positions of the yeast streaking area, transferring the morchella cultures to a CYM inclined plane, taking a wall-climbing morchella hypha fragment to inoculate a new CYM inclined plane after the inclined plane is full, purifying for 3 times, and storing the inclined plane cultures at 4 ℃.
(3) Confirming that the two parties have established an internal co-biochemical relationship: and confirming that the two parties have established the endosymbiotic relationship by adopting a PCR amplification method and a scanning electron microscope method.
PCR amplification method: the method comprises the steps of collecting mycelium of a purified morchella strain by adopting a method of covering CYM with cellophane, adding liquid nitrogen into the mycelium, fully grinding, and then adopting a method reported in literature (Suntang, Zhao Ming, Liyali, and the like. Puer tea fermentation sample bacteria and fungus DNA simultaneous extraction method research. Chinese agriculture report 2011, 27 (15): 249-: 5'-GCA TAT CAA TAA GCG GAG GAA AAG-3' and Maldown: 5'-GGT CCG TGT TTC AAG ACG G-3' is a primer pair, the 26S rDNA D1/D2 area sequence is amplified by the amplified yeast, the negative control is an amplified system without a template, if the 26S rDNA D1/D2 area sequence of the yeast can be amplified by the purified morchella amplifying system, the result shows that the exogenous restriction malassezia can enter the inside of the morchella hypha cells.
Scanning electron microscopy: taking fresh endosymbiotic morel strains, morel control strains and malassezia cultures, fixing the strains and the cultures overnight at 4 ℃ by glutaraldehyde with the volume fraction of 2.5%, and washing the strains and the cultures with sterile water for 2-3 times, wherein each time lasts for 3 min; performing gradient dehydration by using 25%, 50%, 75%, 85%, 95% and 100% ethanol solution in volume fraction for 15-20 min each time; the dehydrating agent anhydrous ethanol is replaced by 100% isoamyl acetate for 2 times, each time for 15 min. And finally, drying the treated sample in a carbon dioxide critical point dryer. Adhering a conductive adhesive tape on an objective table of a scanning electron microscope, carefully adhering dried hyphal cells on the conductive adhesive tape, adhering the dried hyphal cells to the adhered hyphal cells by using a new conductive adhesive tape, and tearing the new conductive adhesive tape so as to tear the dried cells, wherein the structural characteristics of the interior of the cells can be observed in subsequent observation; and placing the torn sample in gold spraying equipment for gold spraying, and observing and photographing by using a scanning electron microscope.
FIG. 4 shows the result of PCR amplification for detecting whether Malassezia is transformed into Morchella hypha cells. The method reported by literature (Sun Ting, Zhao Ming, Li Yali, etc. Puer tea fermentation sample bacteria and fungus DNA simultaneous extraction method research, Chinese agriculture report, 2011, 27 (15): 249) 253) is adopted to extract the genomic DNA of the Morchella which is transferred into or not transferred into the Malassezia as a template for PCR amplification, and the sequence specific primers (Mal) in the MAT1-1-1F/R and the Malassezia 26S rDNA D1/D2 regions are respectively adopted to carry out mating type gene specific primer pairs up /Mal down ) Performing PCR amplification, wherein target amplification products are MAT1-1-1 gene segments of mating type of morchella esculenta of 412 bp and malassezia 26S rDNA D1/D2 region segments of 620 bp respectively, spotting the amplification products for 1.5% agarose gel electrophoresis, and lanes 13 and 14 are control amplification without a template and have no DNA band; lanes 1, 3, 5, 7, 9 and 11 show the result of amplification of Morchella esculenta without introduction of Malassezia, and only 412 bp of MAT1-1-1 gene fragment of Morchella esculenta mating type was amplified; lane 2, 4, 6, 8, 10 and 12 show the result of amplification of the Morchella esculenta transferred to Malassezia, and only a 620 bp fragment of Malassezia 26S rDNA D1/D2 region was amplified.
And (3) confirming that the two parties have established the endosymbiotic relationship by adopting a PCR amplification method and a scanning electron microscope method. Successfully establishing Morchella strains in symbiotic relationship with Morchella, and observing by scanning electron microscopy to obtain Morchella cells in Morchella mycelium cells (see right figure of figure 1); adopts Malassezia 26S rDNA D1/D2 region sequence specific primer (Mal) up /Mal down ) PCR amplification is carried out, the amplification product is spotted on 1.5% agarose gel for electrophoresis analysis, and the target DNA fragment of malassezia 26S rDNA D1/D2 region with 620 bp can appear (see the results of lanes 2, 4, 6, 8, 10 and 12 in the attached figure 4). If the PCR amplification method and the scanning electron microscopy observation show positive results, the maraca is indicatedThe chromobacterium successfully establishes an endosymbiotic relation with the morchella.
(4) And (3) determining culture characteristics of the endo-biochemical strain: measuring hypha growth speed and hypha biomass of the Morchella strains of the endosymbiont yeasts in a proper pH and temperature range of growth by taking the initial Morchella strains as a control; determining the influence of heavy metals (chromium, lead, zinc, copper, etc.) and mandelic acid (soil phenolic acid autotoxic substance) on the growth of morchella strains of endosymbiont yeasts; the mycelial growth rate and mycelial biomass of the strains of the endocytotic yeasts were determined in nitrogen-free medium (determination of nitrogen fixation capacity) and in low-carbon medium (determination of stress resistance to nutritional deficiencies). The determination shows that the mycelium biomass of the toadstool strain of the endosymbiotic restricted malassezia is obviously higher than that of the control toadstool at the temperature of 30 ℃, and the resistance of the endosymbiotic toadstool strain to high temperature is obviously enhanced; hyphal biomass was significantly higher on 1/4 and 1/8 CYM media than controls, indicating significantly enhanced resistance to poor nutrient environments; establishment of symbiosis relationship to heavy metal ZnCl 2 、Pb（NO 3 ） 2 And the adaptability of phenolic acid autotoxic substance mandelic acid is obviously improved.
(5) And (3) measuring the production character of the endo-biochemical strain: subjecting Morchella esculenta control strain and strains for establishing endosymbiotic relationship to three-site experimental cultivation (each strain is 3 m) 2 ) Observing and recording the yield properties such as the time and the relative strength of the morchella cream, the time and the amount of primordia, the time and the amount of young mushroom, the unit yield of the first batch of mushroom and the like, and finding that the morchella cream growth condition of the morchella strain establishing the endosymbiotic relationship with the malassezia is superior to that of a control strain; the original mushroom is multiple, the differentiation is rapid, the fruiting time is advanced, and the quantity of young mushrooms is far higher than that of control strains; compared with a control strain, the yield of the co-biochemical strain in the morchella is improved by 3.5 times, and the good yield increase potential is shown.
Example 2
The method for breeding the high-quality morchella esculenta variety by utilizing the artificial internal co-biochemical technology comprises the following steps:
(1) and (3) carrying out activated culture on the breeding starting morchella strain: selecting morchella (strain preservation number: CCTCC KF 2008793) as a breeding starting strain, inoculating the starting strain to a CYM plate, and performing inversion and light-proof culture at 20 ℃ until the plate is full.
(2) Introducing the agrobacterium into the toadstool hypha cells: inoculating agrobacterium (such as agrobacterium terricola with CGMCC (China general microbiological culture collection number) 1.15993) on one side of a CYM flat plate (the streaking width is about 2 cm), then inoculating morchella seed blocks at a position 2 cm away from a yeast streaking area, carrying out inverted culture at 20 ℃ for 1-2 weeks, taking morchella cultures growing to different positions of the bacteria streaking area, transferring the morchella cultures to a CYM inclined plane, taking a wall-climbing morchella hypha fragment to inoculate a new CYM inclined plane after the inclined plane is full, purifying for 3 times, and storing the inclined plane cultures at 4 ℃.
(3) Confirming that the two parties have established an internal co-biochemical relationship: and (3) confirming that the two parties have established the endosymbiotic relationship by adopting a PCR amplification method and a scanning electron microscope method.
PCR amplification method: collecting mycelium of purified morchella strain by covering CYM plate with cellophane, adding liquid nitrogen to the mycelium, fully grinding, extracting genomic DNA of morchella into which terribacterium has been transferred or which has not been transferred as template for PCR amplification by a method reported in literature (Sun Ting, Zhao Ming, Li Yao Li, etc.. Puer tea fermentation sample bacteria and fungus DNA simultaneous extraction method research, Chinese agriculture report 2011, 27 (15): 249-, the universal primers 27F 5'-AGA GTT TGA TCC TGG CTC AG-3' and 1492R 5'-TAC GAC TTA ACC CCA ATC GC-3' are used as a primer pair to amplify the bacterial 16S rDNA sequence, an amplification system without a template is used as a negative control, if the purified morchella amplification system can amplify the 16S rDNA sequence of the bacteria, the exogenous terreus probably enters the inner part of the morchella hypha cells.
Scanning electron microscopy: taking a fresh endosymbiotic morel strain, a morel control strain and a bacterial culture, fixing the strains at 4 ℃ overnight by using glutaraldehyde with the volume fraction of 2.5%, and washing the strains for 2-3 times by using sterile water for 3 min each time; carrying out gradient dehydration for 15-20 min each time by using ethanol solutions with volume fractions of 25%, 50%, 75%, 85%, 95% and 100%; the dehydrating agent anhydrous ethanol is replaced by 100% isoamyl acetate for 2 times, each time for 15 min. And finally, drying the treated sample in a carbon dioxide critical point dryer. Adhering a conductive adhesive tape on an objective table of a scanning electron microscope, carefully adhering dried hyphal cells on the conductive adhesive tape, then using a new conductive adhesive tape to attach the dried hyphal cells opposite to the adhered hyphal cells, and tearing the hyphal cells so as to tear the dried cells, wherein the structural characteristics of the interior of the cells can be observed in the subsequent observation; and placing the torn sample in gold spraying equipment for gold spraying, and observing and taking a picture by using a scanning electron microscope.
FIG. 3 is the PCR amplification result for detecting whether the terribacterium is transferred into morchella hypha cells. The method reported by the literature (sunting, Zhaoming, Liyali, etc., the method for simultaneously extracting bacteria and fungal DNA of a Pu' er tea fermentation sample is adopted for research, Chinese agronomy report, 2011, 27 (15): 249) 253) to extract genomic DNA of morchella which is transferred into terribacterium or is not transferred into terribacterium as a template for PCR amplification, 27F/1492R and MAT 1-1F/R are respectively subjected to PCR amplification by adopting a bacterial 16S rDNA universal primer pair and a morchella mating type gene specific primer pair, target amplification products are respectively about 1500 bp bacterial 16S rDNA fragments and 412 bp morchella mating type MAT1-1-1 gene fragments, 1.5 percent agarose gel electrophoresis is carried out on amplification products in a spotting way, 1 st, lanes 9 and 10 are control amplifications without template, without any DNA band; lanes 2, 4, 6 and 8 show the amplification results of Morchella esculenta without Agrobacterium, and only 412 bp of Morchella esculenta mating type MAT1-1-1 gene fragment was amplified; lanes 3, 5 and 7 show the amplification results of the E.coli transformed with the terribacterium, and only 16S rDNA band of about 1500 bp is amplified.
And (3) confirming that the two parties have established the endosymbiotic relationship by adopting a PCR amplification method and a scanning electron microscope method. Successfully establishing the toadstool strain in the symbiotic relationship between the agrobacterium and the toadstool, and observing through scanning electron microscopy, wherein agrobacterium cells can be observed in toadstool mycelium cells (shown in a right picture of a figure 2); PCR amplification is carried out by using an agrobacterium 16S rDNA sequence specific primer pair (27F/1492R), and an amplification product is spotted on 1.5% agarose gel for electrophoresis analysis, so that about 1500 bp of an agrobacterium 16S rDNA target DNA fragment can be generated (see lane 3, 5 and 7 results of figure 3). If positive results appear in the PCR amplification method and the scanning electron microscopy, the result shows that the agrobacterium successfully establishes the endosymbiont relationship with the morchella.
(4) And (3) determining culture characteristics of the endo-biochemical strain: measuring hypha growth speed and hypha biomass of the Morchella esculenta strain of the endosymbiotic bacteria in a proper pH and temperature range by taking the initial Morchella esculenta strain as a control; determining the influence of heavy metals (chromium, lead, zinc, copper, etc.) and mandelic acid (soil phenolic acid autotoxic substance) on the growth of the Morchella esculenta of endosymbiotic yeast; the mycelial growth rate and mycelial biomass of the strains of the endocytotic yeasts were determined in nitrogen-free medium (determination of nitrogen fixation capacity) and in low-carbon medium (determination of stress resistance to nutritional deficiencies). The determination shows that the mycelium biomass of the toadstool strain of the endosymbiotic agrobacterium is obviously higher than that of the control toadstool at the temperature of 30 ℃, which shows that the resistance of the endosymbiotic toadstool strain to high temperature is obviously enhanced; hyphal biomass was significantly higher on 1/4 and 1/8 CYM media than controls, indicating significantly enhanced resistance to poor nutrient environments; the mycelium grows in the nitrogen-free culture medium, and the establishment of the endosymbiotic relation is shown to endow the morchella with the nitrogen fixation capacity; establishment of intergrowth relationship to heavy metal CuCl 2 、Pb（NO 3 ） 2 And the adaptability of phenolic acid autotoxic substance mandelic acid is obviously improved.
(5) And (3) measuring the production traits of the endosymbiont strain: subjecting Morchella esculenta control strain and strains for establishing endosymbiotic relation to simultaneous experimental cultivation in three places (each strain is 3 m) 2 ) The yield traits of the frost producing time and relative strength, primordium producing time and amount, young mushroom producing time and amount, first-crop mushroom unit yield and the like are observed and recorded, and the morchella strain establishing the endosymbiont relationship with malassezia is found to be superior to the starting strain in the aspects of frost producing, primordium growing, fruiting time and fruiting number, the endosymbiont morchella yield is 6.2 times higher than that of the starting strain, and good yield increasing potential is shown.
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, and such changes and modifications are within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

1. The method for breeding the high-quality morchella esculenta variety by utilizing the artificial co-biochemical technology is characterized by comprising the following steps of: introducing yeast or bacteria beneficial to artificial cultivation of morchella into the inside of morchella hypha cells, purifying for multiple times, detecting and confirming that an endosymbiotic relation is established between the yeast and the morchella, and screening a high-quality morchella cultivation variety with ideal cultivation and production properties from the toadstool.
2. The method for breeding the morchella esculenta high-quality variety according to claim 1, comprising the following steps:
(1) carrying out activated culture on original strains of morchella;
(2) introducing beneficial yeast or bacteria into the toadstool hypha cells;
(3) purifying the endosymbiotic morchella strain;
(4) introducing exogenous bacteria or yeasts into the toadstool hypha cells for internal identification;
(5) determining the culture characteristics of the endochemical morchella strains;
(6) and (3) determining the production characteristics of the endochemical morchella strains.
3. The method for breeding the morchella esculenta high-quality variety by using the artificial co-biochemical technology according to claim 2, wherein the method comprises the following steps: the original strains of morchella in the step (1) comprise morchella ladder, morchella hexameica, morchella seventeensis, morchella nonameica, Mel-13 and Mel-21 which can be artificially cultured; the activated culture of the original strain of the morchella in the step (1) is to inoculate the strain preserved at low temperature to a nutrient medium plate and culture the strain at 18-25 ℃ in the dark until the morchella mycelium spreads to the whole plate.
4. The method for breeding the high-quality morchella esculenta variety by utilizing the artificial internal co-biochemical technology according to claim 1 or 2, which is characterized in that: beneficial saccharomycetes and bacteria for artificial cultivation of morchella and morchella are oppositely cultured on a nutrient medium flat plate, obvious interspecific competition characteristics are not generated in a colony contact area of the two parties, the two parties are co-cultured in a liquid medium, and the biomass of the saccharomycetes or bacteria has the characteristics of increasing firstly and then decreasing.
5. The method for breeding the high-quality morchella esculenta variety by utilizing the artificial internal co-biochemical technology according to claim 1 or 2, which is characterized in that: the yeasts beneficial to artificial cultivation of the morchella comprise malassezia californica and rhodosporidium roseum, and the bacteria beneficial to artificial cultivation of the morchella comprise terribacterium, brevundimonas, methylophilus, sphingobacterium and flavobacterium.
6. The method for breeding the high-quality morchella esculenta variety by using the artificial internal co-biochemical technology according to claim 2, which is characterized in that: and (2) introducing beneficial yeasts or bacteria into the toadstool hypha cells by adopting a mixed culture method, a microinjection method or a protoplast fusion method.
7. The method for breeding the high-quality morchella esculenta variety by using the artificial internal co-biochemical technology according to claim 2, which is characterized in that: and (3) purifying the endosymbiotic morel strain in the step (3) is to inoculate the beneficial bacteria or yeast-introduced morel strain into a nutrient medium flat plate or a test tube inclined plane, carry out light-proof culture at 18-25 ℃, then pick up morel mycelium fragments growing out of the culture medium on the test tube wall or on the inner cover of a culture dish, transfer the morel mycelium fragments to a new inclined plane or flat plate for culture, repeat purification for many times in such a way, and finally obtain a morel mycelium culture in which the bacteria or the yeast enter the morel mycelium cells but are not positioned among the morel mycelium cells.
8. The method for breeding the morchella esculenta high-quality variety by using the artificial co-biochemical technology according to claim 2, wherein the method comprises the following steps: and (4) introducing exogenous bacteria or yeasts into the toadstool hypha cells for identification by combining a PCR amplification method with one or more of fluorescence in-situ hybridization, transmission electron microscopy or scanning electron microscopy.
9. The method for breeding the high-quality morchella esculenta variety by using the artificial internal co-biochemical technology according to claim 2, which is characterized in that: the culture character determination of the co-biochemical morel strain in the step (5) is to determine the hypha growth speed and hypha biomass of the morel strain of endosymbiotic bacteria or saccharomycetes in a proper pH and temperature range by taking the initial morel strain as a reference; determining the influence of heavy metals with different concentrations, autotoxic substances in soil, extreme temperature and pH on the growth of the toadstool strains of endosymbiotic bacteria or yeasts; and (3) determining the hypha growth speed and hypha biomass of the toadstool strains of endosymbiotic bacteria or yeasts in a nitrogen-free culture medium and a low-carbon culture medium.
10. The method for breeding the morchella esculenta high-quality variety by using the artificial co-biochemical technology according to claim 2, wherein the method comprises the following steps: the determination of the production characteristics of the endochemical morchella strain in the step (6) refers to the fact that the endochemical morchella strain is artificially cultivated on a large scale in multiple places according to the currently general morchella cultivation management technology, the time and the relative strength of fungus frost production, the time and the amount of primordia production, the time and the amount of young mushroom production, the unit yield of first-batch mushrooms, the unit yield of second-batch mushrooms, the occurrence condition of plant diseases and insect pests and the dry-wet ratio of mushroom bodies are observed and recorded, and the production capacity of breeding strains and the potential of further popularization and utilization are comprehensively evaluated.
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