CN111165266A - Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture - Google Patents
Method for continuously expanding propagation of arbuscular mycorrhizal fungal spores through compartment culture Download PDFInfo
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- CN111165266A CN111165266A CN202010017883.2A CN202010017883A CN111165266A CN 111165266 A CN111165266 A CN 111165266A CN 202010017883 A CN202010017883 A CN 202010017883A CN 111165266 A CN111165266 A CN 111165266A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a method for continuously expanding propagation of arbuscular mycorrhizal fungal spores by chamber cultivation, which is characterized in that a cultivation chamber is divided into a plurality of isolation chambers, a lower-layer substrate, a middle-layer substrate and an upper-layer substrate are filled in the isolation chambers, and interval continuous cultivation, inoculation and separation are carried out; the life history of the arbuscular mycorrhizal fungi is 90 days, after the host plants in each isolation room are cultured for 120 days, the host plants are killed by shading treatment to generate a large number of arbuscular mycorrhizal fungi spores, so that the fresh arbuscular mycorrhizal fungi spores can be obtained by taking the ratio of 120 days per interval to the number of isolation rooms.
Description
Technical Field
The invention relates to a method for expanding propagation of arbuscular mycorrhizal fungal spores, in particular to a method for continuously expanding propagation of arbuscular mycorrhizal fungal spores by cell culture, and belongs to the field of microbiology.
Background
The arbuscular mycorrhizal fungi are obligate living nutrition symbiotic saccule mycorrhizal fungi, can not be purely cultured in vitro, only can infect living host plant root systems through arbuscular mycorrhizal fungi hypha to form a symbiotic system, then can produce arbuscular mycorrhizal fungi spores, the arbuscular mycorrhizal fungi spores are dormant bodies of the arbuscular mycorrhizal fungi, the arbuscular mycorrhizal fungi hypha can germinate when the arbuscular mycorrhizal fungi encounter host plants, and then infect the host plant root systems to form the symbiotic system again. The arbuscular mycorrhizal fungi can form symbiont with more than 80% of land vascular bundle plants, and hyphae of the arbuscular mycorrhizal fungi can extend out of the area range reached by the growth of the root system of the host plant in drought-poor soil to provide mineral elements such as nitrogen, phosphorus and the like and water for the host plant. In addition, the ectorhizopus produced by arbuscular mycorrhizal fungi spores can form a hypha net between host plants, and the hypha net can carry out material transportation and information transmission between the host plants. Therefore, the research on the symbiotic mechanism of the arbuscular mycorrhizal fungi and the host plant and the function of the arbuscular mycorrhizal fungi on the host plant and the ecosystem are wide, and the stress resistance of the host plant can be improved through mycorrhization of the root system of the host plant in production. However, the number of active wild arbuscular mycorrhizal fungi spores under the field condition is small, the requirements of production and application cannot be met, and the separation and identification are complicated. After the arbuscular mycorrhizal fungi and the root system of the host plant form a symbiotic system, if the arbuscular mycorrhizal fungi host is dead, a large amount of arbuscular mycorrhizal fungi spores are generated by the arbuscular mycorrhizal fungi hyphae, and the possibility is provided for artificial propagation of the arbuscular mycorrhizal fungi spores.
The main methods for obtaining arbuscular mycorrhizal fungi spores at present are a single-pot culture method and a double-culture system method. The single-pot culture method is to plant host plant, inoculate arbuscular mycorrhizal fungal spore, infect host plant root system after spore germination, and achieve spore propagation by host plant growth. For example, the method for enriching arbuscular mycorrhizal fungi spores by layered culture disclosed by the national intellectual property office (CN106190944B) can enrich a large amount of arbuscular mycorrhizal fungi spores, but lacks continuity, and needs a long time for multiple propagation because the growth period of a host plant and the production period of the arbuscular mycorrhizal fungi spores are long. The dual culture method is characterized in that host plants on a culture medium are inoculated with spores of germinating arbuscular mycorrhizal fungi under an aseptic condition, then the hyphae of the arbuscular mycorrhizal fungi invade into the root system to form symbiont, and the arbuscular mycorrhizal fungi are cultured through the growth of the root system to finally obtain the arbuscular mycorrhizal fungi spores. At present, no simple and practical method for continuously obtaining arbuscular mycorrhizal fungi spores exists.
Disclosure of Invention
The invention aims to overcome the defect of obtaining arbuscular mycorrhizal fungi spores by the conventional method, and discloses a method for continuously propagating the arbuscular mycorrhizal fungi spores by chamber culture.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a. preparing a culture room: the culture pot body is equally divided into a plurality of isolation chambers by using an isolation plate, the height of the isolation plate is consistent with the depth of the culture pot body, the upper part of each isolation plate is respectively provided with a rectangular groove, the bottom end of the culture pot body is provided with a plurality of drain holes, the bottom of the isolation chamber is filled with a lower-layer matrix with good water absorbability, water retentivity and air permeability, the lower-layer matrix is filled with a middle-layer matrix with larger particle size and poor water absorbability and water retentivity, filter cloth is respectively paved on the middle-layer matrix, mixed matrix with the volume ratio of 1:1 of vermiculite to white sand is respectively filled on the filter cloth until the culture pot body is filled, the filling thickness of the lower-layer matrix and the middle-layer matrix is respectively two fifths and one tenth of the depth of the culture pot body to form the culture chambers;
b. firstly, sowing host plant seeds in any one of isolation chambers, inoculating arbuscular mycorrhizal fungal spores which are separated and identified from the field or artificially propagated in the vicinity of root systems after the host plant seeds grow into seedlings, continuously propagating the arbuscular mycorrhizal fungal spores through the host plant in the isolation chambers, wherein the number of days at intervals is the ratio of 120 to the number of the isolation chambers, and sowing the host plant seeds in adjacent isolation chambers successively according to the arrangement sequence of the isolation chambers so as to grow into seedlings without inoculating the arbuscular mycorrhizal fungal spores;
c. pouring Hoagland nutrient solution with higher phosphorus concentration into the tray every 15 days, pouring Hoagland nutrient solution with lower phosphorus concentration into the upper-layer matrix every 30 days, and simultaneously watering the upper-layer matrix and the tray to keep the water content of the matrix at 50-60 percent so as to ensure the growth of host plants and arbuscular mycorrhizal fungi hyphae;
d.after 120 days, shading the host plants in the isolation room sowed for the first time to ensure that the host plants die, wherein the number of days per interval is the ratio of 120 to the number of the isolation rooms, shading the host plants in other isolation rooms in sequence according to the original sowing sequence to ensure that the host plants die, removing the upper-layer substrate in any isolation room after the host plants in any isolation room die, collecting the middle-layer substrate and the lower-layer substrate, and decanting by using a wet sieve to obtain arbuscular mycorrhizal fungal spores;
e. and (3) after taking out the substrate in any one isolation chamber, keeping the upper substrate for recycling, preparing a new culture chamber according to the steps, sowing and inoculating host plants according to the original sowing sequence again, and obtaining the arbuscular mycorrhizal fungal spores required by repeated and repeated operation.
The culture room is divided into a plurality of isolation rooms, and the continuous culture, inoculation and separation are carried out at intervals, and because the arbuscular mycorrhizal fungi can infect the root system of the host plant to generate a fungi silk net and the fungi silk net is diffused and infected, only one-time inoculation is needed, the root system of the newly-germinated seedling of the arbuscular mycorrhizal fungi spore in the adjacent isolation room can be infected by the arbuscular mycorrhizal fungi spore, so that the method has continuity compared with a single-pot culture method; the life history of the arbuscular mycorrhizal fungi is about 90 days, after the host plants in each isolation room are cultured for 120 days, the host plants are killed by shading treatment to generate a large number of arbuscular mycorrhizal fungi spores, so that the fresh arbuscular mycorrhizal fungi spores can be obtained by taking the ratio of 120 days per interval to the number of isolation rooms.
Drawings
FIG. 1 is a schematic sectional front view of a culture chamber of the present invention.
FIG. 2 is a schematic top view of a culture chamber according to the present invention.
The reference numerals are explained below:
the culture pot comprises a culture pot body 1, a partition plate 2, a drain hole 3, a lower-layer substrate 4, a middle-layer substrate 5, filter cloth 6, an upper-layer substrate 7 and a tray 8.
Detailed Description
The present invention will be described in detail with reference to the following examples and accompanying drawings:
a. preparing a culture room: selecting a circular culture pot body (1), equally dividing the culture pot body (1) into four isolation chambers by using two crossed isolation plates (2), wherein the height of each isolation plate (2) is consistent with the depth of the culture pot body (1), a rectangular groove is formed in the upper part of each isolation plate (2), a gap between each isolation plate (2) and the culture pot body (1) is sealed by using glass cement, four drain holes (3) are formed in the bottom end of the culture pot body (1), white sand (4) with the diameter of about 2mm is filled in the bottom of each isolation chamber, glass beads (5) with the diameter of 4mm are filled in the white sand (4), filter cloth (6) with the diameter of 45 mu m is laid on each glass bead (5), the filter cloth (6) is tightly attached to the rectangular groove in the isolation plates (2), mixed matrix (7) with the volume ratio of 1:1 of vermiculite to the white sand is filled in the culture pot body (1) is respectively filled in the filter cloth (6), and the white sand (4) and the glass beads (5) are respectively filled in the thickness of two fifths of the depth of And one tenth, forming a culture chamber, below which a tray (8) is placed;
b. firstly, sowing host plant seeds in any one of the isolation chambers, inoculating arbuscular mycorrhizal fungal spores separated and identified from the field near the root system after the host plant seeds grow into seedlings, continuously propagating the arbuscular mycorrhizal fungal spores through the host plant in the isolation chambers, and sowing the host plant seeds in adjacent isolation chambers in a clockwise direction at intervals of 30 days so as to enable the host plant seeds to grow into seedlings without inoculating the arbuscular mycorrhizal fungal spores;
c. pouring phosphorus concentration of 200 mu M.L in the tray every 15 days-1The Hoagland nutrient solution is poured into a mixed matrix at intervals of 30 days, wherein the phosphorus concentration is 1 mu M.L-1The Hoagland nutrient solution is watered into the mixed matrix and the tray at the same time, and the water content of the matrix is kept between 50 and 60 percent;
d.120 days later, shading the host plants in the first sowed isolation room to ensure that the host plants die, shading the host plants in other three isolation rooms according to the sowing sequence in the isolation rooms at intervals of 30 days to ensure that the host plants die, removing the mixed matrix in any isolation room after the host plants die, collecting glass beads and white sand, and obtaining arbuscular mycorrhizal fungal spores by using a wet sieve decantation method;
e. and (3) after taking out the substrate in any one isolation chamber, keeping the mixed substrate for recycling, preparing a new culture chamber according to the steps, sowing and inoculating host plants according to the original sowing sequence again, and obtaining the arbuscular mycorrhizal fungal spores required by repeated cyclic and reciprocating operations.
Claims (2)
1. The method for continuously expanding propagation of arbuscular mycorrhizal fungal spores by cell culture is characterized by comprising the following steps of:
a. preparing a culture room: the culture pot body (1) is equally divided into a plurality of isolation chambers by a partition plate (2), the height of the partition plate (2) is consistent with the depth of the culture pot body (1), the upper part of each partition plate (2) is respectively provided with a rectangular groove, the bottom end of the culture pot body (1) is provided with a plurality of drain holes (3), the bottom of each isolation chamber is filled with a lower-layer substrate (4) with good water absorbability, water retentivity and air permeability, the lower-layer substrate (4) is filled with a middle-layer substrate (5) with larger particle size and poor water absorbability and water retentivity, filter cloth (6) is respectively paved on the middle-layer substrate (5), the filter cloth (6) is respectively filled with a mixed substrate (7) of vermiculite and white sand with the volume ratio of 1:1 until the culture pot body (1) is filled, the filling thicknesses of the lower-layer substrate (4) and the middle-layer substrate (5) are respectively two fif, a culture chamber is formed, the culture chamber can be cylindrical or rectangular or other shapes, and a tray (8) is arranged below the culture chamber;
b. firstly, sowing host plant seeds in any one of isolation chambers, inoculating arbuscular mycorrhizal fungal spores which are separated and identified from the field or artificially propagated in the vicinity of root systems after the host plant seeds grow into seedlings, continuously propagating the arbuscular mycorrhizal fungal spores through the host plant in the isolation chambers, wherein the number of days at intervals is the ratio of 120 to the number of the isolation chambers, and sowing the host plant seeds in adjacent isolation chambers successively according to the arrangement sequence of the isolation chambers so as to grow into seedlings without inoculating the arbuscular mycorrhizal fungal spores;
c. pouring Hoagland nutrient solution with higher phosphorus concentration into the tray every 15 days, pouring Hoagland nutrient solution with lower phosphorus concentration into the upper-layer matrix every 30 days, and simultaneously watering the upper-layer matrix and the tray to keep the water content of the matrix at 50-60 percent so as to ensure the growth of host plants and arbuscular mycorrhizal fungi hyphae;
d.after 120 days, shading the host plants in the isolation room sowed for the first time to ensure that the host plants die, wherein the number of days per interval is the ratio of 120 to the number of the isolation rooms, shading the host plants in other isolation rooms in sequence according to the original sowing sequence to ensure that the host plants die, removing the upper-layer substrate in any isolation room after the host plants in any isolation room die, collecting the middle-layer substrate and the lower-layer substrate, and decanting by using a wet sieve to obtain arbuscular mycorrhizal fungal spores;
e. and (3) after taking out the substrate in any one isolation chamber, keeping the upper substrate for recycling, preparing a new culture chamber according to the steps, sowing and inoculating host plants according to the original sowing sequence again, and obtaining the arbuscular mycorrhizal fungal spores required by repeated and repeated operation.
2. The method for continuous multiplication of arbuscular mycorrhizal fungal spores in a chamber culture according to claim 1, wherein:
a. preparing a culture room: selecting a circular culture pot body (1), equally dividing the culture pot body (1) into four isolation chambers by using two crossed isolation plates (2), wherein the height of each isolation plate (2) is consistent with the depth of the culture pot body (1), a rectangular groove is formed in the upper part of each isolation plate (2), a gap between each isolation plate (2) and the culture pot body (1) is sealed by using glass cement, four drain holes (3) are formed in the bottom end of the culture pot body (1), white sand (4) with the diameter of about 2mm is filled in the bottom of each isolation chamber, glass beads (5) with the diameter of 4mm are filled in the white sand (4), filter cloth (6) with the diameter of 45 mu m is laid on each glass bead (5), the filter cloth (6) is tightly attached to the rectangular groove in the isolation plates (2), mixed matrix (7) with the volume ratio of 1:1 of vermiculite to the white sand is filled in the culture pot body (1) is respectively filled in the filter cloth (6), and the white sand (4) and the glass beads (5) are respectively filled in the thickness of two fifths of the depth of And one tenth, forming a culture chamber, below which a tray (8) is placed;
b. firstly, sowing host plant seeds in any one of the isolation chambers, inoculating arbuscular mycorrhizal fungal spores separated and identified from the field near the root system after the host plant seeds grow into seedlings, continuously propagating the arbuscular mycorrhizal fungal spores through the host plant in the isolation chambers, and sowing the host plant seeds in adjacent isolation chambers in a clockwise direction at intervals of 30 days so as to enable the host plant seeds to grow into seedlings without inoculating the arbuscular mycorrhizal fungal spores;
c. pouring phosphorus concentration of 200 mu M.L in the tray every 15 days-1The Hoagland nutrient solution is poured into a mixed matrix at intervals of 30 days, wherein the phosphorus concentration is 1 mu M.L-1The Hoagland nutrient solution is watered into the mixed matrix and the tray at the same time, and the water content of the matrix is kept between 50 and 60 percent;
d.120 days later, shading the host plants in the first sowed isolation room to ensure that the host plants die, shading the host plants in other three isolation rooms according to the sowing sequence in the isolation rooms at intervals of 30 days to ensure that the host plants die, removing the mixed matrix in any isolation room after the host plants die, collecting glass beads and white sand, and obtaining arbuscular mycorrhizal fungal spores by using a wet sieve decantation method;
e. and (3) after taking out the substrate in any one isolation chamber, keeping the mixed substrate for recycling, preparing a new culture chamber according to the steps, sowing and inoculating host plants according to the original sowing sequence again, and obtaining the arbuscular mycorrhizal fungal spores required by repeated cyclic and reciprocating operations.
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Cited By (6)
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| CN111972265A (en) * | 2020-07-20 | 2020-11-24 | 中国农业科学院深圳农业基因组研究所 | Mikania micrantha puccinia expanding propagation method |
| CN112514710A (en) * | 2020-11-30 | 2021-03-19 | 山西农业大学 | Arbuscular mycorrhizal fungi continuous type propagation incubator |
| CN112586222A (en) * | 2020-11-30 | 2021-04-02 | 赣州久创科技有限公司 | System for expanding propagation of arbuscular mycorrhizal fungi by taking alfalfa as host |
| CN113462517A (en) * | 2021-06-09 | 2021-10-01 | 东北师范大学 | Multilayer chambered device for continuously expanding propagation of arbuscular mycorrhizal fungal spores |
| CN114657074A (en) * | 2022-04-15 | 2022-06-24 | 北京市农林科学院 | Culture method for efficiently propagating arbuscular mycorrhizal fungal spores in layered manner by using matrix nutrients |
| CN116640672A (en) * | 2023-05-30 | 2023-08-25 | 华南农业大学 | Culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111972265A (en) * | 2020-07-20 | 2020-11-24 | 中国农业科学院深圳农业基因组研究所 | Mikania micrantha puccinia expanding propagation method |
| CN112514710A (en) * | 2020-11-30 | 2021-03-19 | 山西农业大学 | Arbuscular mycorrhizal fungi continuous type propagation incubator |
| CN112586222A (en) * | 2020-11-30 | 2021-04-02 | 赣州久创科技有限公司 | System for expanding propagation of arbuscular mycorrhizal fungi by taking alfalfa as host |
| CN112586222B (en) * | 2020-11-30 | 2022-01-25 | 赣州久创科技有限公司 | System for expanding propagation of arbuscular mycorrhizal fungi by taking alfalfa as host |
| CN113462517A (en) * | 2021-06-09 | 2021-10-01 | 东北师范大学 | Multilayer chambered device for continuously expanding propagation of arbuscular mycorrhizal fungal spores |
| CN114657074A (en) * | 2022-04-15 | 2022-06-24 | 北京市农林科学院 | Culture method for efficiently propagating arbuscular mycorrhizal fungal spores in layered manner by using matrix nutrients |
| CN116640672A (en) * | 2023-05-30 | 2023-08-25 | 华南农业大学 | Culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants |
| CN116640672B (en) * | 2023-05-30 | 2024-04-12 | 华南农业大学 | Culture method for inducing AM fungi to generate mycelium and secondary spores in vitro by utilizing root secretions of leguminous plants |
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