CN116640672B - Culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants - Google Patents
Culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants Download PDFInfo
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- CN116640672B CN116640672B CN202310623580.9A CN202310623580A CN116640672B CN 116640672 B CN116640672 B CN 116640672B CN 202310623580 A CN202310623580 A CN 202310623580A CN 116640672 B CN116640672 B CN 116640672B
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Classifications
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- A—HUMAN NECESSITIES
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- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention discloses a culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants. The multi-layer sandwich type sandwich membrane culture system can effectively isolate the infection of the special-shaped rhizopus sporulation spores to the root system of the astragalus sinicus, and under the condition of isolated bodies, the special-shaped rhizopus sporulation can absorb root secretion and promote the hypha growth and secondary spore formation of the special-shaped rhizopus sporulation. Meanwhile, the nutrient solution simulating root system secretion components can induce the oocyst dysmorphism to generate secondary spores under the condition of isolated body. Therefore, the method can propagate AM fungi under the in-vitro condition without infecting host plant root systems, has high propagation efficiency, can separate pure spores, is convenient for scientific research, and has the advantages of simple operation, short period and the like; therefore, the method can be used for accurately identifying morphological characteristics and strain classification of wild AM fungi, expanding a arbuscular mycorrhizal fungi germplasm resource library, and providing a simpler, more convenient and feasible propagation method for the foundation and application research of arbuscular mycorrhizal fungi.
Description
Technical Field
The invention relates to the technical field of isolated culture of non/difficultly-cultured mycorrhizal fungi, in particular to a culture method for inducing oomycorrhizal fungi of arbuscular mycorrhizal fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of astragalus sinicus of leguminous plants.
Background
Mycorrhizal fungi are widely distributed in natural ecosystems and can form mycorrhizal symbiota with more than 90% of plants (Shi et al, 2023; genie et al, 2020); mycorrhiza has irreplaceable ecological significance in promoting energy flow and substance exchange between organisms in a natural ecosystem, evolution and distribution of the organisms, maintaining stability of the ecosystem, maintaining ecological balance and sustainable development (Liu Runjin and Chen Yinglong, 2007). The mycorrhizal fungi can be used as a microbial inoculum product for serving agriculture and forestry production, promotes the green development of agriculture and forestry, and has very important social significance (Feng Gu, etc. 2010). According to the great strategic demands of the microorganisms such as energy and protein, the functions of the culturable microorganisms are required to be developed, and the biological resources of the microorganisms which are not/difficultly cultivated are further excavated to serve the production and life of human beings.
There are thousands of microbial groups of bacteria, fungi, etc. in the rhizosphere soil of agroforestry plants, and reciprocal co-evolution between plants and rhizosphere microbial communities (Trivedi et al 2020). Arbuscular mycorrhizal (Arbuscular Mycorrhizal, AM) fungi are an important group of plant rhizosphere microorganisms, capable of forming symbiotes with more than 72% of terrestrial plants (Rich et al, 2021; genre et al, 2020). Plants provide AM fungi with fatty acids and sugars to complete their life history (Jiang et al, 2017; an et al, 2019), whereas AM fungi promote the absorption and utilization of nutrients and moisture such as phosphorus, nitrogen by plants (Zhang et al, 2023; xie et al, 2022; wang et al, 2020; li et al, 2013). At the same time, AM fungi also recruit beneficial microbiota and in cooperation with hyphal microorganisms effectively improve rhizosphere micro-ecology (Wang et al, 2023; li et al, 2023; wang et al, 2020), thereby promoting healthy growth of plants. Although AM fungi have a remarkable pro-active effect, they belong to a class of soil fungi that are difficult to culture, thus limiting the development and utilization of AM fungal resources.
In the absence of host plants, the AM fungus has a short growth period of only weeks, and a subsequent life history cannot be completed. Dormant AM fungal spores store large amounts of fat droplets, proteins and glycogen. Similar to plant seeds, the hydrolysis of these lipids in AM fungal spores produces energetic compounds to sustain metabolic activity and cell division and DNA synthesis. Although AM fungi are obligate biotrophic microorganisms, there is evidence that they have a degree of saprophytic nutrition, can grow to a certain extent under ex vivo conditions, and even have the ability to break down complex organisms. These evidence suggest that the early growth of AM fungi is not entirely dependent on the host plant providing nutrition. When the host plant root system exists, the host plant root system can release specific secretion substances for promoting the growth of AM fungal hypha and induce the expression of genes related to energy metabolism, growth and development of AM fungal in vivo. The root exudates are various chemical substances released into soil by plant roots, and the main sources are decomposition of root surface cells and cell contents, release of organic matters, microbial modification, plant root action and products thereof. Wherein the compounds with smaller molecular weight have small specific weight in plant root secretions, but are very rich in variety, and have stronger biological activity, such as amino acid, organic acid, saccharide and phenolic acid secondary metabolites; the root secretion of the plant can stimulate the germination of AM fungi and the growth of the germ tube hyphae, and can induce the AM fungi to produce isolated mycelium and secondary spores.
However, the related foundation and application foundation research of arbuscular mycorrhizal fungi biology at present need a huge amount of spores as materials and microbial inoculum support, but a large gap exists between AM fungi as materials and the industry demand, and the situation of supply and demand occurs. Therefore, it is critical to solve the problem to explore culture conditions under which AM fungi produce isolated mycelium and secondary sporulation under ex vivo conditions.
Reference is made to:
feng Gu, zhang Fusuo, li Xiaolin, zhang Junling, gaijing apple. (2010) arbuscular mycorrhizal fungi function and control in agricultural production [ J ].
Soil journal 47:995-1004.
Liu Runjin, chen Yinglong (2007) mycorrhiza school [ M ]. Beijing: science Press.
An J,Zeng T,Ji C,de Graaf S,Zheng Z,Xiao TT,Deng X,Xiao S,Bisseling T,Limpens E,Pan Z.
(2019)A Medicago truncatula SWEET transporter implicated in arbuscule maintenance during arbuscular mycorrhizal symbiosis[J].New Phytologist.224:396-408.
Genre A,Lanfranco L,Perotto S,Bonfante P.(2020)Unique and common traits in mycorrhizal symbioses[J].Nature Reviews Microbiology.18:649-660.
Jiang Y,Wang W,Xie Q,Liu N,Liu L,Wang D,Zhang X,Yang C,Chen X,Tang D,Wang E.(2017)
Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi[J].
Science.356:1172-1175.
Li T,Hu YJ,Hao ZP,Li H,Wang YS,Chen BD.(2013)First cloning and characterization of two functional aquaporin genes from an arbuscular mycorrhizal fungus Glomus intraradices[J].
New Phytologist.197:617-630.
Li X,Zhao R,Li D,Wang G,Bei S,Ju X,An R,Li L,Kuyper TW,Christie P,Bender FS,Veen C,van der Heijden MGA,van der Putten WH,Zhang F,Butterbach-Bahl K,Zhang J.(2023)
Mycorrhiza-mediated recruitment of complete denitrifying Pseudomonas reduces N2O emissions from soil[J].Microbiome.11:45.doi:10.1186/s40168-023-01466-5.
Rich MK,Vigneron N,Libourel C,Keller J,Xue L,Hajheidari M,Radhakrishnan GV,Le Ru A,
Diop SI,Potente G,Conti E,Duijsings D,Batut A,Le Faouder P,Kodama K,Kyozuka J,Sallet E,Bcard G,Rodriguez-Franco M,Ott T,Bertrand-Michel J,Oldroyd GED,Szvnyi P,Bucher M,Delaux PM.(2021)Lipid exchanges drove the evolution of mutualism during plant terrestrialization[J].Science.372:864-868.
Shi J,Wang X,Wang E.(2023)Mycorrhizal symbiosis in plant growth and stress adaptation:
from genes to ecosystems[J].Annual Review of Plant Biology.doi:
10.1146/annurev-arplant-061722-090342.
Trivedi P,Leach JE,Tringe SG,Sa T,Singh BK.(2020)Plant-microbiome interactions:from community assembly to plant health[J].Nature Reviews Microbiology.18:607-621.Wang G,Jin Z,George TS,Feng G,Zhang L.(2023)Arbuscular mycorrhizal fungi enhance plant phosphorus uptake through stimulating hyphosphere soil microbiome functional profiles for phosphorus turnover[J].New Phytologist.doi:10.1111/nph.18772.
Wang S,Chen A,Xie K,Yang X,Luo Z,Chen J,Zeng D,Ren Y,Yang C,Wang L,Feng H,Lpez-Arredondo DL,Herrera-Estrella LR,Xu G.(2020)Functional analysis of the Os NPF4.5
nitrate transporter reveals a conserved mycorrhizal pathway of nitrogen acquisition in plants[J].
Proceedings of the National Academy of Sciences,USA.117:16649-16659.
Xie X,Lai W,Che X,Wang S,Ren Y,Hu W,Chen H,Tang M.(2022)A SPX domain-containing phosphate transporter from Rhizophagus irregularis handles phosphate homeostasis at symbiotic interface of arbuscular mycorrhizas.New Phytologist.234:650-671.
Zhang Shuyuan,Nie Yuying,Fan Xiaoning,Wei Wei,Chen Hui,Xie Xianan,Tang Ming.(2023)A transcriptional activator from Rhizophagus irregularis regulates phosphate uptake and homeostasis in AM symbiosis during phosphorous starvation.Frontiers in Microbiology.13:1114089.doi:10.3389/fmicb.2022.1114089.
Disclosure of Invention
In order to overcome the limitations and defects of AM fungi propagation technology in the prior art, the invention aims to provide a culture method for inducing the oocyst of the arbuscular mycorrhizal fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants.
The invention provides an in-vitro culture method for inducing AM fungi to produce secondary spores by using the root secretions of the milk vetch living bodies of leguminous plants, provides reference and reference for further using a pure culture method to expand mycorrhizal fungi strains (strains) which are not/difficultly cultured, and simultaneously provides powerful technical support for accurately identifying the types of AM fungi in a natural ecological system, expanding an AM fungi germplasm resource library, the foundation of mycorrhizal biology, application research and the like.
The aim of the invention is achieved by the following technical scheme:
a culture method for inducing oocyst mold of arbuscular mycorrhizal fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants comprises the following steps:
s1, selecting seeds of leguminous plants for surface sterilization, and placing the seeds in a dark condition for treatment until the seeds sprout radicles with the length of 0.8-1.2 cm (preferably 1 cm), transplanting the radicles, and obtaining seedlings after more lateral roots grow;
s2, pretreating arbuscular mycorrhizal fungi spores, including surface sterilization, average distribution quantity and dark environment culture;
s3, setting a multi-layer sandwich membrane-sandwiched culture system to carry out potting test: injecting one part of spores pretreated in the step S2 onto a microporous filtering film, covering the spores with the microporous filtering film, sealing the edges of the filtering film with a strong adhesive to form a sandwich structure, respectively placing seedlings obtained in the step S1 on two sides of the sandwich structure, and clamping plants with two identical microporous filtering films again to form a multi-layer sandwich membrane-clamping culture system; vertically inserting a multi-layer sandwich membrane-sandwiched culture system into the center of a flowerpot filled with quartz sand, exposing the diameter of a microporous filter membrane to the outside by 8-10 mm for adding water and nutrient solution;
s4, preparing a nutrient solution simulating root system secretion components according to the components of root system secretion of the leguminous plants, wherein the nutrient solution is used for the growth of isolated hyphae and the formation of secondary spores of arbuscular mycorrhizal fungi;
s5, placing the multi-layer sandwich membrane-sandwiched culture system in the step S3 in a fungus culture room for culture, and performing potting test, wherein each pot is watered once every 3 days; the nutrient solution for simulating root system secretion in the step S4 is not added or added, and the nutrient solution for simulating root system secretion is injected into the middle spore-containing interlayer every other day every basin; culturing for more than 5 weeks, and collecting mycelium and secondary spores in vitro.
In the culture method, the leguminous plant is astragalus sinicus;
the step S1 specifically comprises the following steps:
selecting uniformly sized milk vetch seeds, sterilizing the surfaces of the milk vetch seeds in a 1% sodium hypochlorite solution containing Tween 20 for 15-20 min (preferably 15 min), flushing the milk vetch seeds with sterile water for three times, and culturing the milk vetch seeds in dark at 25 ℃ for 24h until the seeds absorb water to swell; discarding the waste liquid, flushing 3 times by using sterile water, then laying the seeds on an MS solid culture medium to germinate, and carrying out dark treatment for 10-12 hours until the seeds germinate to obtain radicles with the length of 0.8-1.2 cm (preferably 1 cm); and transferring and cultivating, and obtaining the astragalus sinicus seedlings for transplanting when more lateral roots grow out.
In the step S2, the arbuscular mycorrhizal fungi are oocyst dysmorphism; further, rhizopus morphous (Rhizophagus irregularis DAOM 197198).
The step S2 specifically includes the following steps:
1) Oocyst dysmorphism (r.irregulation DAOM 197198) was placed into cell culture plates for surface sterilization: washing spores with 0.05g/mL Tween 20, soaking the spores with 0.02g/mL chloramine T solution for 2 times, washing the spores with sterile water for 3 times each time for 10-15 min (preferably 10 min), and taking 200mg/L streptomycin and 100mg/L gentamicin according to the volume ratio of the streptomycin to the gentamicin of 1:2, mixing to obtain a streptomycin and gentamicin mixed solution, soaking for 7-10 min (preferably 7 min), rinsing with sterile water for 7-8 times, and preserving for later use;
2) Dispersing the spores subjected to surface sterilization under a stereoscopic microscope by using a dissecting needle, taking 50 parts of the spores as one part, respectively injecting the spores into a bacteria culture plate containing sterile water, and culturing the spores in a dark way to induce the spores to generate germination tubes;
the temperature of the dark culture in the step 2) is 25 ℃;
in the step S3, the number of spores in one part is 40-50; preferably 50.
In the step S3, the diameter of the microporous filtering membrane is 50mm, and the pore diameter is 0.8 mu m.
In the step S3, two seedlings obtained in the step S1 are respectively put on two sides of the sandwich structure;
in the step S3, the diameter of the quartz sand is 0.7-1 mm; before use, the product needs to be pickled and sterilized; specifically, 2mol/L HCl is used for soaking for 24 hours, then secondary pure water is used for washing and pickling quartz sand to be neutral, deionized water is used for washing for 3-5 times, and the quartz sand is dried in the shade; then sterilizing by moist heat, and drying for standby.
The conditions of the wet heat sterilization are 121 ℃ wet heat sterilization for 20min.
The temperature of the drying is 50 ℃.
In the step S3, the flowerpot is a square flowerpot with the length of 6 multiplied by 6cm, and the bottom of the flowerpot is provided with an exhaust hole.
Preferably, in step S4, the nutrient solution for simulating root system secretion comprises the following components by taking water as a solvent, adjusting the pH to 5.5-5.7, and each liter:
TABLE 1
In the step S5, the amount of each watering of each basin is 100mL;
in the step S5, the dosage of the nutrient solution simulating the root system secretion component is 1 mL/time;
in step S5, the culture is preferably performed for 5 to 9 weeks.
In step S5, the conditions for the cultivation are that the ambient conditions are a temperature of 26℃and a relative humidity of 60%.
Compared with the prior art, the invention has the following advantages and effects:
(1) According to the characteristic that AM fungi growth depends on stimulation of plant root secretions, the plant root secretions comprise various fatty acids, organic nitrogen, plant hormones and the like by taking astragalus sinicus as an example as a starting point; in a near-natural state, simulating root secretion components, taking fatty acid (such as myristic acid) as a carbon source and an energy source, taking peptone as a nitrogen source, and additionally adding plant hormone substances to prepare a nutrient solution simulating root secretion components; the method utilizes the milk vetch living root system secretion or the milk vetch living root system secretion and the nutrient solution simulating the root system secretion, and then utilizes the multi-layer sandwich membrane-sandwiched culture system to induce the generation of secondary spores and mycelia under the in-vitro non-symbiotic condition.
(2) The multi-layer sandwich-type sandwich membrane culture system can effectively isolate the infection of the oocyst spores of the arbuscular mycorrhizal fungi and the oocyst spores of the dysmorphism rhizopus of the arbuscular mycorrhizal fungi to the root system of the milk vetch, and can absorb root secretions and promote the hypha growth and secondary spore formation of the oocyst spores under the condition of isolated bodies. Meanwhile, the nutrient substances such as fatty acid (myristic acid), secretion hormone (strigolactone and jasmonic acid), organic nitrogen (peptone) and the like are added into the nutrient solution simulating root secretion components, so that the oocyst mould can be induced to produce the secondary spores under the condition of isolated bodies. Provides a new method for promoting the AM fungi to generate secondary spores under non-symbiotic conditions and also provides a new technology for enrichment and separation culture research of mycorrhizal fungi which are not/difficultly cultured.
(3) The method can propagate AM fungi under the in-vitro condition without infecting host plant root systems, has high propagation efficiency, can separate pure spores, is convenient for scientific research, and has the advantages of simple operation, short period and the like; therefore, the morphological characteristics and strain classification of the wild AM fungi can be accurately identified, the arbuscular mycorrhizal fungi germplasm resource library is expanded, and a simpler, more convenient and feasible propagation method is provided for the foundation and application research of the arbuscular mycorrhizal fungi.
Drawings
FIG. 1 is a schematic diagram of a root exudate collection apparatus for collecting and metabolomic analysis of Astragalus sinicus root exudates;
FIG. 2 is a diagram of an apparatus for inducing AM fungal growth from living plant root exudates and simulated root exudates nutrient solution in plant "accompanying" mode; a is a schematic diagram; b is a physical diagram, the sandwich device is extracted for one period within 14 days to observe the hypha density of spore germination, generate secondary spore condition, and observe 4 periods.
Fig. 3 is a graph of the induction of the production of secondary spores by the apocynum venetum by "accompanying" and nutrient solution treatment of milk vetch (i.e. milk vetch root exudate-nutrient solution treatment): the method comprises the steps of utilizing a multilayer sandwich method, and generating secondary spores after in vitro culture of the rhizopus stolonifer for 3 weeks (A-C), 5 weeks (D-F), 7 weeks (G-I) and 9 weeks (J-O); black arrows indicate microspores (Mother spores); white arrows indicate Secondary spores (Secondary spores). The scale bars in the figures are all 100. Mu.m.
Fig. 4 is a graph of the induction of apocynum venetum to produce isolated mycelium by "accompanying" and nutrient solution treatment of milk vetch (i.e., milk vetch root exudate-nutrient solution treatment): dyeing by Trypan Blue (Trypan Blue) after inducing the hyphae of the Saponaria heteromorphic Saponaria by the Astragalus root secretion; wherein A-D represents the obvious germination tubes and branching hyphae of AM fungi after 3 weeks of culture; E-H shows that after 5 weeks of culture, hypha density is increased under the uninterrupted induction of root secretion, and secondary spores with smaller diameters appear; I-L represents an increase in diameter of the secondary spores after 7 weeks of culture; M-P represents that after 9 weeks of culture, a compact mycelium net starts to form, and the number and diameter of the secondary spores show a trend of increasing; black arrows indicate the Mother spores (Mother spores), white arrows indicate the Secondary spores (Secondary spores), and gray arrows indicate the branching hyphae (branched hyphae), all scale bars being 100 μm.
Fig. 5 is a graph of the "accompanying" and nutrient solution treatments of milk vetch (i.e. milk vetch root exudate-nutrient solution treatment) for 3 weeks (a), 5 weeks (B), 7 weeks (C) and 9 weeks (D) after which the root segments of milk vetch were observed not to be infested with AM fungi, rhizopus dyscrasii.
FIG. 6 is a schematic diagram of the induction of a secondary spore by Rhizopus dysmorphis by a simulated root system secretion component nutrient solution (i.e., nutrient solution treatment group): the nutrition liquid is only used for generating secondary spore after 3 weeks (A-C), 5 weeks (D-F), 7 weeks (G-I) and 9 weeks (J-L) of in vitro culture of the rhizopus heteromorphic mould under the condition of no plant accompanying; black arrows indicate microspores (Mother spores); white arrows indicate Secondary spores (Secondary spores). The scale bars in the figures are all 100. Mu.m. Myr-k+GR24+MeJA represents a nutrient solution simulating the components of root system secretions; mock represents water as a blank.
FIG. 7 is a diagram showing the "accompanying" and nutrient solution treatment of Astragalus sinicus (i.e. Astragalus sinicus root secretion-nutrient solution treatment) induced branching of the AM fungus Rhizopus vulgare filaments; data points represent 8 replicates of the same batch.
FIG. 8 is a graph showing the number of secondary spores produced by AM fungal Rhizopuidambaris induced by nutrient solution (Nutrient solution)/nutrient solution+Astragalus root secretion (AS) (AS+ Nutrient solution); data points represent 3 duplicate samples corresponding to the same lot.
FIG. 9 is a plot of nutrient solution (Nutrient solution)/nutrient solution+Astragalus root secretion (AS) (AS+ Nutrient solution) inducing the production of secondary spores by the AM fungus Rhizopus delemar; data points represent 3 duplicate samples corresponding to the same lot.
FIG. 10 is a graph showing the number of secondary spores produced by the AM fungus Rhizopuidambaris induced by Astragalus sinicus root secretion (AS)/nutrient solution (Nutrient solution) +Astragalus sinicus root secretion (AS); data points represent 3 duplicate samples corresponding to the same lot.
FIG. 11 is a diagram showing the magnitude of Astragalus sinicus root secretion (AS)/nutrient solution (Nutrient solution) +Astragalus sinicus root secretion (AS) inducing the production of secondary spores by AM fungus Rhizopuschia dyscrasii; data points represent 3 duplicate samples corresponding to the same lot.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 construction of root system secretion collecting apparatus schematically shown in FIG. 1
1) Preparing an MS solid culture medium, taking a 200mL system as an example, adding 1g of MS culture medium dry powder, 0.6g of agar and 3g of sucrose, adjusting the pH to 5.8, sealing, placing the mixture at a high temperature of 121 ℃ for sterilization for 15min, slightly cooling, and pouring the mixture into a flat plate for later use;
2) Selecting uniformly sized Astragalus sinicus seeds, sterilizing the surfaces of the Astragalus sinicus seeds in a 1% sodium hypochlorite solution containing Tween 20 for 15min, washing the Astragalus sinicus seeds with sterile water for three times, and placing the Astragalus sinicus seeds in a dark incubator at 25 ℃ until the seeds absorb water; discarding waste liquid, flushing for 3 times again by using sterile water, then laying seeds on an MS solid culture medium for germination, carrying out dark treatment for 10-12 hours until the seeds sprout to give radicle with the length of 1cm, transferring to a culture room for cultivation, obtaining astragalus sinicus seedlings after more lateral roots grow (about 7 days), and transplanting;
3) Taking 1000-1200 g of quartz sand with the diameter of about 0.7-1 mm, soaking the quartz sand for 24 hours by using 2mol/L HCl, washing and pickling the quartz sand to be neutral by using secondary pure water, and washing the quartz sand by using deionized water for 3-5 times; glass beads (diameter 1 mm) were treated in the same manner; drying the quartz sand and glass beads after pickling in the shade respectively, bagging (or an aluminum box or a vessel), placing the quartz sand and the glass beads in an autoclave (121 ℃ for 1 h) for moist heat sterilization, cooling, and placing the quartz sand and the glass beads in a 50 ℃ oven for drying for standby; after a sterilized square flowerpot (6 multiplied by 6 cm) (the lower part of which is provided with an exhaust hole) is sleeved with a sterile plastic bag for treatment, quartz sand and glass beads are sequentially paved, and deionized water is added to wet gravel;
4) And (3) using part of the astragalus sinicus plants for analyzing the components of root secretions, culturing the astragalus sinicus seedlings for 30 days, pulling out the whole plants, discarding the whole plants, collecting quartz sand matrixes of the whole plants, and extracting the root secretions in the quartz sand by adopting a water extraction method. The extraction steps are as follows: soaking quartz sand in distilled water for 6-8 hr, filtering the soaking liquid with nylon net cloth, and collecting and combining the filtering liquid; washing the quartz sand matrix with 95% ethanol, filtering the quartz sand matrix with nylon mesh cloth, and collecting and mixing filtrates; collecting all the filtrate into 50mL centrifuge tubes, centrifuging at 4000rpm for 5min, and collecting supernatant; concentrating the supernatant in a freeze dryer (Christ, germany), mixing the freeze-dried powder of the multi-tube root system secretion to 20-50 mg, packaging, and detecting the whole components of the root system secretion of the milk vetch by adopting a non-target metabolome strategy.
EXAMPLE 2 construction of potting System A schematic illustration of the construction of the potting System is shown in FIG. 2A
1) Oocyst dysmorphism (r.irregulation DAOM 197198) was placed into 24-well cell culture plates for surface sterilization: washing spores with 0.05g/mL Tween 20, soaking the spores in 0.02g/mL chloramine T solution for 2 times, 10min each time, washing the spores with sterile water for 3 times, and taking 200mg/L streptomycin and 100mg/L gentamycin according to the volume ratio of the streptomycin to the gentamycin of 1:2, mixing to obtain a streptomycin and gentamicin mixed solution, soaking for 7min, rinsing with sterile water for 7-8 times, and standing at 4 ℃ for later use;
2) Dispersing the spores subjected to surface sterilization under a stereoscopic microscope by using a dissecting needle, taking 50 parts of spores as one part, respectively injecting the spores into 12-hole bacterial culture plates containing sterile water, and placing the plates in a dark incubator at 25 ℃ to induce the spores to generate germination tubes;
3) Sterilizing quartz sand at 121deg.C under high pressure for 20min, oven drying, and placing into square flowerpot (6×6cm);
4) Injecting one part of spores (50) onto a microporous filter membrane (with the diameter of 50mm, the aperture of 0.8 mu m and an organic system), covering the microporous filter membrane with the microporous filter membrane, sealing the edges of the filter membrane with a strong adhesive to form a sandwich structure, respectively placing two sprouted astragalus sinicus seedlings on two sides of the sandwich structure, and clamping plants with the two identical microporous filter membranes to form a multilayer sandwich membrane-clamping culture system; the "multi-layered sandwich" membrane-sandwiched culture system was inserted vertically into the center of a square flowerpot, exposing about one fifth of the diameter of the filter membrane to the outside (physical diagram is shown in fig. 2B) for the addition of water and nutrient solution.
EXAMPLE 3 Induction of isolated mycelium
1) Preparing myristic acid stock solution:
a. preparing 40% fatty acid-free bovine serum albumin solution: the phosphate buffer is preheated to 55 ℃, 12g of fatty acid-free bovine serum albumin (fatty acid-free BSA) is measured, the mixture is placed in a 50mL centrifuge tube, 30mL of phosphate buffer solution is added, the mixture is directly placed in a high-speed centrifuge for centrifugation at 10000rpm for 60min without shaking or even mixing, and the complete dissolution can be achieved (shaking or shaking can lead BSA to form blocky crystals and not dissolve), thus 30mL of 40% (m/v) fatty acid-free bovine serum albumin solution is obtained, and the appearance is a brownish yellow clear sample.
b. 40mM potassium myristate saponification solution was prepared: 0.4g (0.01 mol) of NaOH was dissolved in 100mL of deionized water to prepare 100mL of a 0.1mol/L NaOH solution, and 30mL was taken. 0.319g (1.2 mmol) of potassium myristate (Myr-k) was added to 30mL of NaOH solution, and the mixture was placed in a water bath at 75℃to carry out sufficient saponification for about 30 minutes, and finally, the mixture was colorless, transparent and clear, to obtain 30mL of 40mM potassium myristate saponified solution. The temperature is maintained and the next operation is continued.
c. The preserved potassium myristate saponification solution is rapidly added into the fatty acid-free bovine serum albumin solution to obtain 60mL 20mM potassium myristate+20% fatty acid-free bovine serum albumin solution, namely myristic acid storage solution. Shaking up properly, standing at a certain temperature (below 55deg.C) to assist dissolution for 30min (after cooling at room temperature, observing the property as brown yellow clear sample, no difference with the original solution property, no solid or flocculent impurity, stable property, filtering and sterilizing the stored liquid with organic nylon membrane of 0.22 μm organic phase on an ultra clean bench, and storing in refrigerator at 4deg.C for a long time.
2) Sucking 1 μL of strigolactone concentrate (with concentration of 10 mM) to 100mL of water, preparing 100nM strigolactone mother liquor, filtering with 0.22 μm organic phase organic nylon membrane for sterilization, and preserving for later use;
3) Sucking 23.6 mu L of methyl jasmonate (0.998 g/mL 98%) to 100mL of water, preparing 100 mu M methyl jasmonate mother liquor, filtering and sterilizing with an organic nylon membrane of 0.22 mu M organic phase, and preserving for later use;
4) On an improved yeast basic (SC) culture medium, adding nutrition required by the growth and secondary spore formation of three mycorrhizal fungi hyphae, including fatty acid (myristic acid), plant hormone (strigolactone (GR 24) and methyl jasmonate (MeJA)) and organic nitrogen (peptone), and providing carbon source, nitrogen source, energy, growth factor and other external nutrient substances for AM fungi; the formulation of the nutrient solution simulating the root system secretion component (1L of solvent is water) is (see table 1):
TABLE 1
Regulating pH to 5.5, sealing, sterilizing at 121deg.C under high temperature and high pressure for 15min, cooling, subpackaging into 1mL tube with 1.5mL centrifuge tube on an ultra-clean workbench, and placing several tubes into a refrigerator at 4deg.C for preservation;
5) The "multi-layered sandwich" sandwich culture system obtained in example 2 was placed in a fungus culture chamber in sequence for cultivation under the environmental conditions of 26℃and 60% relative humidity; about 100mL of water was poured per basin set up, once every 3 days; 1 mL/time of the nutrient solution for simulating root system secretion components is added, and the nutrient solution for simulating root system secretion components is injected into the middle spore-containing tunica media every other day by the basin device; culturing for 9 weeks. Wherein, set up the grouping: milk vetch root system secretion-nutrient solution treatment group: the milk vetch is used for accompanying and injecting the nutrient solution simulating the root system secretion component; astragalus root exudates treatment group: a nutrient solution containing milk vetch for accompanying and not injecting simulated root system secretion components; nutrient solution treatment group: no milk vetch 'accompanies' but has injection of the nutrient solution simulating root system secretion components; each set was set with 3 replicates.
6) After the time delay experiment is carried out and cultured for 1 week, the membrane clamping device is extracted in a period of 14 days, the membrane clamping device is taken out and placed in a culture dish to be slightly wetted by sterile water, the density of sporogenic hyphae and the occurrence of secondary spores are observed and counted under a stereoscopic microscope, and 4 periods are observed.
Wherein the diameter size of the secondary spores is observed and measured under a stereoscopic microscope (Nikon SMZ18, japan); when the diameter of the sporozoites reaches 70-80%, the secondary spores can be separated and used for subsequent tests such as secondary spore germination activity detection, cell nucleus staining and the like.
The mycelium production was observed ex vivo by Trypan Blue staining. The preparation method of the Trypan Blue dye liquor comprises the following steps of: lactic acid: water = 1:1:1, and then adding 0.05% trypan blue to prepare a dye solution. Taking out the microporous filtering film, placing the microporous filtering film in a glass culture dish, directly sucking 500-800 mu L trypan blue dye liquor by using a liquid-transferring gun, uniformly injecting the whole filtering film, and placing the whole filtering film under a stereoscopic microscope to observe hyphae.
Analysis of results:
in example 1, astragalus sinicus (A.sinicus) of Leguminosae was used as a host plant for Saprolegnia heteromorphic (R.irregularis DAOM 197198), and root secretions thereof were extracted; the components of the astragalus root secretions are resolved by adopting a non-targeted metabolome method, and a subsequent multilayer sandwich membrane-sandwiched culture system is utilized to construct an in-vitro culture system of the heteromorphic rhizopus. Wherein the root secretion of Astragalus sinicus mainly comprises fatty acid, amino acid and short peptide, polyketone, alkaloid, carbohydrate, terpenoid, etc. (see table 2 in detail), and the root secretion component simulating nutrient solution is prepared according to the main component (see example 3) for generating mycelium and secondary spore under the condition of in vitro.
Table 2 shows the metabolic components of the root secretion of Astragalus sinicus
Note that: MS2name is a substance name qualitatively obtained through secondary mass spectrum matching; MS2score is a qualitative scoring value of the secondary mass spectrum matching, and the closer the scoring value is to 1, the higher the reliability of the representing matching result; level is the ranking of identified substances, B (i): reliable matching based on a standard database MS/MS; b (ii): reliable matching based on the simulation database MS/MS; rt is the median retention time, representing the retention time of the peak in all samples; mz is the median mass-to-charge ratio, representing the mass-to-charge ratio of this peak in all samples.
Culturing for 9 weeks under the "accompanying" and nutrient solution treatment of herba astragali Melilotoidis (i.e. the treatment of radix astragali Melilotoidis secretion-nutrient solution); after 1 week of culture, the filters were withdrawn every 2 weeks and observed for the production of isolated hyphae and secondary spores by Rhizopus delemar. Experimental results show that after 9 weeks of in vitro culture, the astragalus root secretions and the nutrient solution of the simulated root secretion components comprising fatty acid (myristic acid), organic nitrogen (peptone) and plant hormone (GR 24 strigolactone and methyl jasmonate) can induce the oocyst dysmorphiae to generate enough mycelium and secondary spores with larger diameter (55-100 mu M) (shown in figures 3 and 4). It can be seen that the oocyst dysenteriae can germinate and grow hyphae and generate secondary spores under non-symbiotic conditions under the action of the nutrient solution of the simulated root system secretion components added with potassium myristate, strigolactone, methyl jasmonate and peptone.
Example 2 plant "accompanying" ex vivo culture system simulating the root secretions of milk vetch, to verify that only the root secretions of milk vetch act on the spores themselves through the filter membrane, the root segments of milk vetch plants in the "sandwich method" were stained with 0.05% trypan blue, and the results showed that the root segments after 3 weeks (a), 5 weeks (B), 7 weeks (C) and 9 weeks (D) of culture were free of AM fungal infection, and that the stimulation of the elongation of the wire of the calico sporangium dysmorphicum and the occurrence of the production of secondary spores could be due to the induction of root secretions, not the spore production results caused after the AM fungal calico sporangium dysmorphicum infection of the plants (as shown in fig. 5). Under the double induction of the root secretion of the milk vetch and the nutrient solution simulating the root secretion, the mycelium of the microspore is obviously branched. Experimental results show that the average length of the sporozoites in the third week in the initial stage is less than 10mm, and the length density of the sporozoites is obviously increased after 9 weeks of culture. It can be seen that the continued action of the Astragalus sinicus root secretions and nutrient solution promotes the continued branching of AM fungal hyphae to form a hyphal network (see FIGS. 4 and 7).
The treatment conditions in example 3 were a milk vetch (AS) root system secretion-nutrient solution treatment group, a milk vetch root system secretion treatment group and a nutrient solution treatment, respectively. The astragalus root secretion treatment group is insufficient for growth of the special-shaped rhizopus as the root growth of the astragalus root secretion treatment group is limited by the interlayer film in the middle and later stages; the nutrient solution treatment group lacks living plants to uninterruptedly output root secretions, the nutrient solution components are only used for supplying the special-shaped rhizopus, and other conditions except the conditions are consistent with the milk vetch root secretion treatment group. Experimental results show that the physiological conditions of the culture medium are consistent with those of a Astragalus sinicus root secretion-nutrient solution treatment group, and the Astragalus sinicus root secretion and the nutrient solution simulating root secretion components can induce the dysmorphism rhizocyst mould to produce isolated mycelium and secondary spores after 9 weeks. The number of secondary spores in the nutrient solution group did not reach the effect of in vivo plant induction, and the secondary spore diameter was maintained at 20 μm until the ninth week (see fig. 6). The number of secondary spores and the average diameter of the secondary spores are obviously better than those of the nutrient solution treatment group under the auxiliary induction of the astragalus root secretion-nutrient solution treatment group, which shows that the secretion components of the plant root system are far more than those of the nutrient solution, the secondary spores can stimulate the enlargement of the heteromorphic rhizocyst mould (as shown in fig. 8 and 9), and the number of the secondary spores and the hypha length of the secondary spores of the astragalus root secretion group are obviously equal to those of the astragalus root secretion-nutrient solution treatment group in the first 5 weeks, and the secondary spores are gradually separated from the astragalus root secretion treatment group due to the subsequent support of the nutrient solution (as shown in fig. 10 and 11).
In conclusion, the astragalus root secretion can effectively stimulate the AM fungi dysmorphism root cyst mould to generate secondary spores under the in-vitro condition, proves that the root secretion is important for the in-vitro growth of the AM fungi and the generation of the secondary spores, and provides an effective culture method for enrichment and separation culture research of the mycorrhizal fungi which are difficult to culture and are symbiotic with the obligate living bodies.
In the description of the present specification, the description with reference to the terms "a fact example", "a specific example", etc., means that a specific feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one example of the invention. The embodiments of the invention disclosed above are intended only to help illustrate the invention. The examples are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (8)
1. Inducing arbuscular mycorrhizal fungi dysmorphism root cyst mould by utilizing astragalus root secretionRhizophagus irregularis) Ex vivo productionThe culture method of the secondary spores is characterized by comprising the following steps:
s1, selecting astragalus sinicus seeds for surface disinfection, and placing the seeds under a dark condition for treatment until the seeds sprout radicle with the length of 0.8-1.2 cm, transplanting the radicle, and obtaining seedlings after more lateral roots grow;
s2, pretreating the oomycete spores of the arbuscular mycorrhizal fungi dysmorphism rhizopus, wherein the pretreatment comprises surface sterilization, average distribution quantity and dark environment culture;
s3, setting a multi-layer sandwich membrane-sandwiched culture system to carry out potting test: injecting one part of spores pretreated in the step S2 onto a microporous filtering film, covering the spores with the microporous filtering film, sealing the edges of the filtering film with a strong adhesive to form a sandwich structure, respectively placing seedlings obtained in the step S1 on two sides of the sandwich structure, and clamping plants with two identical microporous filtering films again to form a multi-layer sandwich membrane-clamping culture system; vertically inserting a multi-layer sandwich membrane-sandwiched culture system into the center of a flowerpot filled with quartz sand, exposing the diameter of a microporous filter membrane to the outside by 8-10 mm for adding water and nutrient solution;
s4, preparing a nutrient solution simulating root secretion components according to the components of the astragalus root secretion, wherein the nutrient solution is used for forming in-vitro secondary spores of the oocyst dysmorphism of the arbuscular mycorrhizal fungi;
s5, placing the multi-layer sandwich membrane-sandwiched culture system in the step S3 in a fungus culture room for culture, and performing potting test, wherein each pot is watered once every 3 days; the nutrient solution for simulating root system secretion in the step S4 is not added or added, and the nutrient solution for simulating root system secretion is injected into the middle spore-containing interlayer every other day every basin; culturing for more than 5 weeks, and collecting secondary spores in vitro;
in the step S4, the formula of the nutrient solution for simulating root secretion components adopts water as a solvent, and the pH is adjusted to 5.5-5.7, wherein each liter of nutrient solution comprises the following components: mgSO (MgSO) 4 ･7H 2 O：731 mg，KNO 3 ：80 mg，KCl：65 mg，KH 2 PO 4 ：4.8 mg，Ca(NO 3 ) 2 ･4H 2 O：288 mg，Fe(III)-EDTA：8 mg，MnCl 2 ･4H 2 O：3 mg，ZnSO 4 ･7H 2 O：1.3 mg，H 3 BO 3 ：1.5 mg，CuSO 4 ･5H 2 O：0.065 mg，Na 2 MoO 4 ･2H 2 O:0.0012 mg, KI:0.75 mg, MES fatty acid methyl ester sulfonate 10mM, glucose 1g, glycine 3 mg, pyridoxine-HCl pyridoxine hydrochloride 0.1 mg,Nicotinic acid nicotinic acid 0.5 mg, myo-inositol 50mg, thiamine-HCl thiamine hydrochloride 10 mg, peptone 1g, potassium myristate 200. Mu.M, strigolactone 0.1 nM, methyl jasmonate 0.1 nM.
2. The culture method according to claim 1, wherein:
in the step S2, the arbuscular mycorrhizal fungi are cyst mould dysmorphisRhizophagus irregularis）DAOM 197198。
3. The culture method according to any one of claims 1 to 2, wherein:
in the step S3, the number of spores in one part is 40-50;
in the step S3, the diameter of the microporous filter membrane is 50mm, and the pore diameter is 0.8 mu m;
in the step S3, two seedlings obtained in the step S1 are respectively put on two sides of the sandwich structure;
in the step S3, the diameter of the quartz sand is 0.7-1 mm;
in step S3, the flowerpot is a square flowerpot with a size of 6×6cm, and the bottom of the flowerpot is provided with an exhaust hole.
4. The culture method according to any one of claims 1 to 2, wherein:
in the step S5, the amount of each watering of each basin is 100mL;
in the step S5, the dosage of the nutrient solution simulating the root system secretion component is 1 mL/time;
in step S5, the conditions for the cultivation are that the ambient conditions are a temperature of 26℃and a relative humidity of 60%.
5. The culture method according to any one of claims 1 to 2, wherein:
in step S5, the culture is performed for 5 to 9 weeks.
6. The culture method according to claim 1 or 2, wherein:
the step S1 specifically comprises the following steps:
selecting uniformly sized milk vetch seeds, carrying out surface disinfection in a 1% sodium hypochlorite solution containing Tween 20 for 15-20 min, washing with sterile water for three times, and culturing in dark at 25 ℃ until the seeds swell due to water absorption; discarding the waste liquid, flushing for 3 times by using sterile water, then laying the seeds on an MS solid culture medium to germinate, and carrying out dark treatment for 10-12 h until the seeds germinate to obtain radicle with the length of 0.8-1.2 cm; and transferring and cultivating, and obtaining the astragalus sinicus seedlings for transplanting when more lateral roots grow out.
7. The culture method according to claim 2, wherein:
the step S2 specifically includes the following steps:
1) The special-shaped rhizopus is preparedRhizophagus irregularis) DAOM 197198, loading into cell culture plates for surface sterilization: cleaning spores by using 0.05g/mL Tween 20, soaking the spores in 0.02g/mL chloramine T solution for 2 times, washing the spores with sterile water for 3 times each time for 10-15 min, and taking 200mg/L streptomycin and 100mg/L gentamicin according to the volume ratio of the streptomycin to the gentamicin of 1:2, mixing to obtain a streptomycin and gentamicin mixed solution, soaking for 7-10 min, rinsing with sterile water for 7-8 times, and preserving for later use;
2) The spores after surface sterilization were scattered under a stereoscopic microscope with a dissecting needle, and each 50 parts were individually injected into a bacterial culture plate containing sterile water and dark-cultured to induce spores to produce germination tubes.
8. The culture method according to any one of claims 1 to 2, wherein:
in the step S3, the quartz sand is required to be pickled and sterilized before being used; specifically, after soaking 24h by using 2mol/L HCl, washing and pickling quartz sand to be neutral by using secondary pure water, washing 3-5 times by using deionized water, and drying in the shade; then sterilizing by moist heat, and drying for standby.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586755A (en) * | 2017-10-09 | 2018-01-16 | 南京农业大学 | One kind promotes AMF spore rapid propagation method |
| CN111165266A (en) * | 2020-01-08 | 2020-05-19 | 华南农业大学 | Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture |
| CN111909855A (en) * | 2020-07-23 | 2020-11-10 | 华南农业大学 | Application of gibberellin-deficient mutant in promoting arbuscular mycorrhizal fungi spore production |
| CN113403204A (en) * | 2021-05-12 | 2021-09-17 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method |
| CN113528697A (en) * | 2021-07-12 | 2021-10-22 | 华南农业大学 | Heterorhizosporangium root symbiosis early marker gene and application thereof |
| CN114774289A (en) * | 2022-04-13 | 2022-07-22 | 安徽农业大学 | Method for improving spore production quality and efficiency of arbuscular mycorrhizal fungi |
| CN115340955A (en) * | 2022-08-31 | 2022-11-15 | 华南师范大学 | Method for efficiently culturing arbuscular mycorrhizal fungi |
| CN116082076A (en) * | 2022-10-25 | 2023-05-09 | 中国农业大学 | Phosphate fertilizer synergist for promoting crop growth and phosphorus absorption |
-
2023
- 2023-05-30 CN CN202310623580.9A patent/CN116640672B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107586755A (en) * | 2017-10-09 | 2018-01-16 | 南京农业大学 | One kind promotes AMF spore rapid propagation method |
| CN111165266A (en) * | 2020-01-08 | 2020-05-19 | 华南农业大学 | Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture |
| CN111909855A (en) * | 2020-07-23 | 2020-11-10 | 华南农业大学 | Application of gibberellin-deficient mutant in promoting arbuscular mycorrhizal fungi spore production |
| CN113403204A (en) * | 2021-05-12 | 2021-09-17 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method |
| CN113528697A (en) * | 2021-07-12 | 2021-10-22 | 华南农业大学 | Heterorhizosporangium root symbiosis early marker gene and application thereof |
| CN114774289A (en) * | 2022-04-13 | 2022-07-22 | 安徽农业大学 | Method for improving spore production quality and efficiency of arbuscular mycorrhizal fungi |
| CN115340955A (en) * | 2022-08-31 | 2022-11-15 | 华南师范大学 | Method for efficiently culturing arbuscular mycorrhizal fungi |
| CN116082076A (en) * | 2022-10-25 | 2023-05-09 | 中国农业大学 | Phosphate fertilizer synergist for promoting crop growth and phosphorus absorption |
Non-Patent Citations (15)
| Title |
|---|
| AM真菌孢子萌发及其与白三叶草双重培养研究初报;邵菊芳等;中国农学通报;20080805(第08期);第287-291页 * |
| AM真菌孢子萌发及其与紫云英转化根双重培养体系建立的研究;曹玲;中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑;20120215(第02期);第1-61页 * |
| AM真菌离体双重培养研究进展;冉海燕等;山地农业生物学报;20160428(第02期);第62-67页 * |
| Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus;Sachiko Tanaka等;COMMUNICATIONS BIOLOGY;20220112;第05卷;第1-9页 * |
| Plant sesquiterpenesinduce hyphal branching in arbuscular mycorrhizal fungi;Kohki Akiyama等;Nature;第435卷;第824-827页 * |
| Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids;Hiromu Kameoka等;Nature Microbiology;20191031;第04卷;第1654-1660页 * |
| 丛枝菌根真菌与宿主植物识别共生的分子机制;郝晓红等;草地学报(第05期);第800-804页 * |
| 丛枝菌根真菌响应桉树茄科雷尔氏菌侵染进程基因的筛选与鉴定;黄迪等;微生物学通报;第50卷;第815-831页 * |
| 基于多组学方法研究AMF共生诱导的玉米根系分泌物变化及变化机理;马俊卿;中国优秀博士学位论文全文库（电子期刊）农业科技辑(第02期);第1-207页 * |
| 影响丛枝菌根真菌孢子萌发的几种因素研究;董昌金等;植物营养与肥料学报;09(第04期);第1.2.1、1.2.2、2.2及3节 * |
| 根系分泌物对紫云英油菜间作的响应;王亚君等;应用生态学报;第32卷;摘要及第3节 * |
| 植物激素调控丛枝菌根发育的作用机制研究进展;王浩等;微生物学通报;第49卷;第4448−4466页 * |
| 植物组织培养物和根系对VA菌根孢子萌发的影响研究;王鸿等;四川农业大学学报;12(第03期);第3.2节 * |
| 球囊霉属几种AM真菌孢子表面消毒与萌发的研究;董昌金等;菌物系统(第03期);第410-416页 * |
| 离体培养环境下AM真菌次生孢子产生条件的优化;冉海燕;中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑;20170315(第03期);第1-76页 * |
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