CN115340955A - Method for efficiently culturing arbuscular mycorrhizal fungi - Google Patents
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Abstract
The invention relates to a method for efficiently culturing arbuscular mycorrhizal fungi, and belongs to the technical field of microbiology. The invention provides an arbuscular mycorrhizal fungi culture medium which comprises potassium myristate. According to the invention, potassium myristate with a certain concentration is added into the culture medium, so that the germination of parent AMF spores can be directly stimulated, more branched structures can be generated, and the spore density can be improved; the invention combines the application of potassium myristate in the AMF pure culture field with a large-capacity double-chamber separation system, can obtain higher-quality AMF hypha and spores on the basis of the traditional flat plate system, and obviously improves the propagation efficiency; the technical scheme has simple operation steps, can obtain the microbial inoculum with higher spore density and no mixed bacteria pollution in the same culture period, and has wide application prospect in the research field of AMF mechanism.
Description
Technical Field
The invention relates to a method for efficiently culturing arbuscular mycorrhizal fungi, and belongs to the technical field of microbiology.
Background
Arbuscular Mycorrhizal Fungi (AMF) are one of key microbial groups widely distributed in nature and having important ecological functions, and can form mycorrhizal symbionts with nearly 80% of terrestrial plants. Through nutrient trading by symbiosis with AMF, the host plant can utilize the huge hypha network of the host plant to expand the contact range with soil, so that the absorption of the root system to the nutritional elements such as N, P and the like is promoted, and the resistance of the host plant to drought, heavy metal and other environmental stresses can be improved. In return, the host plant provides carbon sources such as sugars and fatty acids to the AMF for its growth. Therefore, AMF is considered to be the microbial group with the most application prospect in the fields of agriculture, forestry, ecological restoration and the like.
The development and optimization of the AMF culture technology are important links for promoting the AMF research process and realizing the large-scale application of the AMF. The existing research proves that AMF is lack of fatty acid synthetase, can not synthesize fatty acid from head, can complete life history only by symbiosis with host plants and obtaining carbon source including fatty acid from host root system under natural environment, belongs to fatty acid auxotroph microorganism of strict nutrient obligate symbiosis, and can not realize pure culture under condition of leaving host. This is a key factor limiting the scientific research and large-scale application of AMF. At present, the AMF propagation technology mainly adopts a mode (namely a pot culture method) of taking sand, soil and the like as carriers and co-culturing with typical AMF host plants (such as clovers, alfalfa and the like); the method is relatively simple to operate and plays an important role in promoting the research and application of AMF. However, the AMF culture obtained by the method also comprises sand, soil, plant roots and other soil microorganisms besides AMF spores and hyphae, and AMF spores and hyphae free of mixed bacteria pollution are difficult to obtain. In addition, this method has (1) a long propagation cycle and requires long-term maintenance (obtaining a large amount of AMF propagules requires that the plant be cultured under specific conditions for about 4 months); (2) the stability of the propagation system is poor (for example, when host plants encounter diseases and insect pests, the yield of AMF propagules is reduced); (3) high requirement on culture conditions (a plant culture room with temperature and humidity control and illumination rhythm control is needed), and the like. These deficiencies severely limit the in-depth research of AMF and also do not facilitate quality control in the large-scale production and application of AMF (e.g., soil microorganisms may contain harmful microorganisms). Hairy root culture is a technology combining genetic engineering and cell engineering developed in the 80 th 20 th century, and is used for inducing plant cells to generate hairy roots by integrating T-DNA contained in Ri plasmid in Agrobacterium rhizogenes (Agrobacterium rhizogenes) into DNA of the plant cells. By constructing a single co-culture system of the hairy roots and the AMF, the method makes it possible to obtain pure AMF hypha and spores without contamination of infectious microbes. The system has the advantages of stability, easiness in propagation, observation and maintenance and the like, and becomes an important technology for preparing a novel AMF microbial inoculum, preserving and cultivating seeds and researching the physiological and molecular mechanisms of AMF. However, the hairy root-AMF co-culture system which isolates external pollution and has single condition faces the following problems in practical application: (1) nutrients of the hairy roots come from the culture medium, while AMF simultaneously obtains nutrients from the hairy roots and the culture medium, and the AMF competes with the nutrients of the hairy roots; (2) the growth rate is slow, and more than 3 months are usually needed according to the culture conditions and the AMF types; (3) the yields of AMF hyphae and spores were low. Therefore, optimizing a single co-culture system to improve the spore propagation efficiency and yield has important value in the research and application fields of AMF.
The double-chamber separation system is a common means for limiting the growth of AMF in general research, and the double-chamber separation system is taken as a culture condition to limit the extension range of the root system of a host plant, so that the symbiotic effect of the AMF and the host plant is enhanced. However, the system is mostly used in potting experiments or bipartite plates at present, and a chamber culture system for constructing the AMF microbial inoculum is not seen at present. The hostless AMF can achieve some degree of non-symbiotic growth under host-free conditions using artificially applied exogenous fatty acids. Wherein, the first and the second end of the pipe are connected with each other, 14 c-labelling experiments confirmed that the carbon in the fatty acid was used for synthesisThe structure of the AMF. However, the technology is not developed, the diameter of the obtained AMF progeny spores can only reach one half of the level of the mother spores, and the symbiosis degree and the growth promoting effect of the AMF progeny spores with the host plants are lower than those of the AMF spores subjected to symbiotic culture.
Disclosure of Invention
The invention aims to provide a method for efficiently culturing arbuscular mycorrhizal fungi, and the method adopts a double-chamber separation system containing potassium myristate with a certain concentration to co-culture hairy roots and AMF, wherein the potassium myristate can improve the growth rate of AMF in the system, and the double-chamber system can greatly improve the yield of AMF.
In order to achieve the purpose, the invention adopts the technical scheme that: an arbuscular mycorrhizal fungi culture medium comprising potassium myristate.
Researches show that AMF in natural environment belongs to soil microorganisms with strict nutrient obligate symbiotic growth, and the dependence on host plant carbon sources determines the particularity that pure culture is difficult to realize; the addition of fatty acid under non-symbiotic culture conditions supplements part of AMF carbon source to a certain extent, but AMF still lacks some unknown nutrients and/or signal substances in the growth and development process, so that AMF grows slowly, the infection efficiency and plant growth promoting effect of the obtained AMF spores are lower than those of the AMF spores obtained under symbiotic culture conditions, and the high-efficiency production of the AMF fungicide is difficult to realize. Through a large number of experiments, the inventor of the application discovers that potassium myristate is added into a culture medium as a stimulating substance and an additional carbon source for inducing growth branches of AMF, on one hand, the defects that the AMF grows slowly under a non-symbiotic AMF culture condition, the diameter of obtained AMF spores is lower than that of AMF spores obtained by symbiotic culture, the infection degree and the plant growth promoting effect are low are overcome, on the other hand, the problems that in a traditional single co-culture plate system, due to the specificity of AMF obligate symbiosis, the propagation period is long are solved, and the propagation rate of the microbial inoculum can be improved to a large extent (the culture time of a double-chamber separation system is reduced by 30% compared with that of a plate).
In a preferred embodiment of the culture medium for arbuscular mycorrhizal fungi according to the present invention, the concentration of potassium myristate in the culture medium is 0.1 to 5mM. According to the research of the invention, when the concentration of the potassium myristate in the culture medium is 0.1-5 mM, the potassium myristate has a good promotion effect on the germination and growth of AMF spores.
The invention also provides application of the culture medium for the arbuscular mycorrhizal fungi in preparation of the arbuscular mycorrhizal fungi agent.
The invention also provides a method for culturing arbuscular mycorrhizal fungi, which adopts a double-chamber separation culture tank for culturing and specifically comprises the following steps:
s1: adding the arbuscular mycorrhizal fungi culture medium into a culture tank;
s2: culturing and propagating parent arbuscular mycorrhizal fungi-hairy root microbial inoculum;
s3: transplanting the parent arbuscular mycorrhizal fungi-hairy root microbial inoculum in the S2 into one chamber of a double-chamber separated culture tank for culture;
s4: dissolving the culture medium by using a sodium citrate solution, carrying out suction filtration, and fixing the volume to obtain a suspension.
According to the research of the invention, the application of potassium myristate in the AMF pure culture field is combined with a large-capacity double-chamber separation system, higher-quality AMF hypha and spores can be obtained on the basis of a traditional flat plate system, and the propagation efficiency is obviously improved.
As a preferred embodiment of the method for culturing arbuscular mycorrhizal fungi of the present invention, the middle of the double-chamber separation culture tank is provided with a separation net with a pore diameter of 35-38 μm.
In a preferred embodiment of the method for culturing arbuscular mycorrhizal fungi of the present invention, the height of the partition net is 1/2 to 2/3 of the depth of the culture tank.
As a preferred embodiment of the method for culturing an arbuscular mycorrhizal fungus according to the present invention, the culture tank in S1 is further autoclaved after the culture medium of the arbuscular mycorrhizal fungus is added.
As a preferred embodiment of the method for culturing an arbuscular mycorrhizal fungus according to the present invention, the arbuscular mycorrhizal fungus in S2 includes Rhizoctonia heterosporum or Rhizoctonia intraradicata.
As a preferred embodiment of the method for culturing arbuscular mycorrhizal fungi of the present invention, the culture conditions in S3 are: culturing at 25 deg.C in dark for 8 weeks.
The invention also provides application of the method for culturing the arbuscular mycorrhizal fungi in preparation of the arbuscular mycorrhizal fungi.
Compared with the prior art, the invention has the following beneficial effects: the invention provides an arbuscular mycorrhizal fungi culture medium, which can directly stimulate parent AMF spores to germinate and generate more branched structures by adding potassium myristate with a certain concentration into the culture medium, and can improve the spore density; the invention combines the application of potassium myristate in the AMF pure culture field with a large-capacity double-chamber separation system, can obtain higher-quality AMF hypha and spores on the basis of the traditional flat plate system, and obviously improves the propagation efficiency; the technical scheme has simple operation steps, can obtain the microbial inoculum with higher spore density and no mixed bacteria pollution in the same culture period, and has wide application prospect in the research field of AMF mechanism.
Drawings
FIG. 1 is a graph of the appearance of AMF root in culture media containing different concentrations of potassium myristate.
Fig. 2 is a graph of spore density changes in various growth nodes for AMFs cultured in media containing different concentrations of potassium myristate, indicating significant differences compared to controls (P < 0.05).
FIG. 3 shows the diameter of AMF microspores cultured in media containing different concentrations of potassium myristate.
FIG. 4 is a schematic view of a dual-chamber separation culture system of example 2.
FIG. 5 is a graph of inoculum yield per weight of medium harvested from a two chamber system culture tank with different letters indicating significant differences (P < 0.05).
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following examples.
Example 1
This example compares the difference in the effect of different concentrations of potassium myristate on the AMF propagation rate by constructing a single AMF and hairy root plate co-culture system, applying four different concentrations of potassium myristate, 0.1, 0.5, 1 and 5mM, respectively, to the culture medium. The specific experimental method is as follows:
(1) Preparing a culture medium: a. m medium was prepared containing the following components in the following concentrations: 731mg/L MgSO 4 ·7H 2 O、80mg/L KNO 3 、65mg/L KCl、4.8mg/L KH 2 PO 4 、288mg/L Ca(NO 3 ) 2 ·4H 2 O、8mg/L Na-Fe-EDTA、0.0024mg/L Na 2 MoO 4 ·2H 2 O, 0.1mg/L thiamine, 0.1mg/L vitamin B 6 0.25mg/L nicotinic acid, 0.75mg/L KI and 6mg/L MnCl 2 ·4H 2 O、2.65mg/L ZnSO 4 ·7H 2 O、1.5mg/L H 3 BO 3 、0.13mg/L CuSO 4 ·5H 2 O, 3mg/L p-hydroxyphenylglycine, 50mg/L inositol, 10g/L sucrose and 3g/L plant gel; b. weighing potassium myristate powder 1.8765g, preparing into 100ml 50mM mother liquor, and heating to dissolve the precipitated powder completely before use; c. adding 0.4ml, 1ml, 4ml and 20ml of potassium myristate mother liquor into 200ml of M culture medium, adjusting pH to 5.5 with hydrochloric acid, and adding 0.6g of plant gel into 200ml of culture medium; d. placing the prepared culture medium and instruments such as scalpel wrapped with tinfoil in a sterilizing pot, and autoclaving at 121 deg.C for 30min;
(2) Propagation and culture of parent AMF-hairy root plate: a. sterilizing the surfaces of the autoclaved culture medium and all instruments by using 75% alcohol, placing the sterilized culture medium and all instruments in an ultra-clean bench after ultraviolet sterilization for 20min, measuring the culture medium in the step (1) by using a sterile injector before the culture medium is cooled, wherein the quantitative volume of the culture medium in each culture dish is 20ml, covering a cover after the culture medium is solidified, placing for later use, and setting 10 times for each concentration; b. selecting heteroclite root cyst mold as a test strain, selecting a well-growing heteroclite root cyst mold-hairy root co-culture flat plate as a microbial inoculum for inoculation, observing the density and distribution condition of spores and hyphae in the flat plate, and partitioning the back surface of the flat plate by using a mark pen in advance; c. soaking the sterilized surgical knife head into 95% alcohol, placing the surgical knife head soaked with the alcohol on an alcohol lamp one by one for burning, and then placing the surgical knife head on a test tube rack for cooling for later use; cutting the parent flat plate with the lines drawn in advance by the cooled scalpel, taking out one of the parent flat plates, placing the cut parent flat plate in a new blank culture medium, sealing the cut parent flat plate by using a sealing film, wiping the used scalpel clean, soaking the scalpel in 95% alcohol, and burning the scalpel again; d. the plate after the propagation needs to be placed in a constant temperature incubator at 25 ℃ to be cultivated for 8 weeks in a dark place.
(3) AMF-observation and harvesting of growth condition of hairy root flat plate: after the propagation is completed, the growth of the AMF in the plate is observed by a body type microscope every 2 weeks and is photographed and recorded, and 6 visual fields (magnification = 78) are photographed by rotating around the parent culture medium during photographing, and the size of each visual field is 18.13X 13.65mm; after photographing was completed, the number of spores in each field was counted by ImageJ, and the diameter of progeny spores was measured. And finally, calculating the number of spores in unit area in each plate and the average diameter, and representing the spore density and the diameter of the progeny spores of the concentration treatment in the growing point.
The results of the experiment are shown in FIGS. 1 to 3. As can be seen from fig. 1, the treatment of the medium containing potassium myristate all had a certain promoting effect on the appearance of the branching-like structure of hyphae outside the roots, and the branching degree of AMF hyphae and the number of secondary spore formation in the medium treated with potassium myristate were significantly higher than those in the control treatment without potassium myristate; as can be seen from fig. 2, the spore density of AMF increased with the increase of the concentration of the potassium myristate treatment in each growth node, and was higher than that of the control group (without potassium myristate), and the AMF secondary spore production amount in the medium treated with potassium myristate was significantly higher than that of the control treatment; as can be seen from fig. 3, the spore diameters of the potassium myristate-treated groups at different concentrations were not significantly different from those of the control group. From the above experimental results, it can be seen that the low-concentration potassium myristate has a significant effect on promoting the generation of branch-shaped structures by the hyphae outside the AMF roots in the initial culture period, and the potassium myristate treatment at each concentration has a significant effect on increasing the density of the AMF spores in the whole growth cycle, and the addition of the potassium myristate does not affect the diameter level of the progeny spores.
Example 2
In the method for culturing arbuscular mycorrhizal fungi of this embodiment, a double-chamber separation culture tank is used for culturing, and the specific method is as follows:
(1) Construction of the two-chamber separation system: a. cleaning a transparent tissue culture tank, shearing nylon nets with apertures of 37 mu m (the height is about 1/2-2/3 of the culture tank body, and the width is enough to reach two sides of the tank body), and firmly adhering the nylon nets to the tank body below and at two sides by using transparent adhesive; b. adding 200ml of M culture medium containing 5mM potassium myristate into the tissue culture tank, adding 0.6g of plant gel, screwing with transparent cover having air hole, placing the tissue culture tank into a sterilization pot, and autoclaving at 121 deg.C for 30min; c. and screwing the sterilized tissue culture pot cover, and cooling at room temperature for later use.
(2) Inoculation, propagation and culture of AMF-hairy roots: a. selecting root cyst mould as a test strain, selecting a root cyst mould-hairy root flat plate with better growth vigor as a microbial inoculum for inoculation, partitioning the flat plate according to the density and distribution condition of spores and hyphae in the flat plate, cutting a culture medium by using a sterile scalpel, picking out the cut culture medium by using the scalpel, and placing the picked culture medium on one side of a prepared double-chamber system tank; b. after inoculation is completed, the cover is screwed and the sealing membrane is pasted, the culture tank is placed in a thermostatic chamber with the temperature of 25 ℃ and is cultured for 6 weeks in a dark place, a schematic diagram of the double-chamber separation culture tank is shown in figure 4, wherein the volume of a single system is 500ml, and is far larger than that of a common culture medium (the volume is 20 ml), so that the transportation and the storage are convenient, and the labor cost is obviously saved.
(3) Harvesting: picking out hairy roots in each tissue culture tank by using tweezers, dissolving a culture medium containing spores and hyphae by using 10mM sodium citrate solution with the volume 10 times that of the culture medium after the hairy roots are completely removed, stirring by using a glass rod to accelerate dissolution, filtering by using a 400-mesh cell sieve after the hairy roots are completely dissolved, then carrying out suction filtration on oversize products to a 400-mesh filter membrane and weighing, and obtaining the net weight of the spores and the hyphae in a single flat plate after subtracting the weight of the filter membrane.
The results of the experiment are shown in FIG. 5. As can be seen from fig. 5, in the control group (0 mM), AMF biomass in the hyphal side medium was significantly higher than that in the hairy root side (P < 0.05); application of medium containing 5mM potassium myristate significantly increased AMF biomass on the hyphal side and hairy root side (P < 0.05). Therefore, the yield of AMF was significantly higher on the hyphal side than on the hairy root side, confirming that the application of the two-chamber system improved the yield of AMF. In addition, addition of potassium fatty acid (5 mM) significantly improved AMF yield both on the hairy root side and on the hyphal side.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. An arbuscular mycorrhizal fungi medium comprising potassium myristate.
2. The arbuscular mycorrhizal fungi medium according to claim 1, wherein the concentration of potassium myristate in the medium is 0.1-5 mM.
3. Use of an arbuscular mycorrhizal fungi culture medium according to any one of claims 1 to 2 for the preparation of arbuscular mycorrhizal fungi.
4. A method for culturing arbuscular mycorrhizal fungi is characterized in that a double-chamber separation culture tank is adopted for culturing, and the method specifically comprises the following steps:
s1: adding an arbuscular mycorrhizal fungi culture medium according to any one of claims 1 to 2 to a culture tank;
s2: culturing and propagating parent arbuscular mycorrhizal fungi-hairy root microbial inoculum;
s3: transplanting the parent arbuscular mycorrhizal fungi-hairy root microbial inoculum in the S2 into one chamber of a double-chamber separated culture tank for culture;
s4: dissolving the culture medium by using a sodium citrate solution, carrying out suction filtration, and fixing the volume to obtain a suspension.
5. The method for cultivating arbuscular mycorrhizal fungi according to claim 4, wherein a separation net with a pore size of 35-38 μm is arranged in the middle of the double-chamber separation culture tank.
6. The method of claim 5, wherein the height of the separation net is 1/2 to 2/3 of the depth of the culture tank.
7. The method of claim 4, wherein the culture tank in S1 is autoclaved after the addition of the culture medium for arbuscular mycorrhizal fungi.
8. The method of claim 4, wherein the arbuscular mycorrhizal fungus in S2 comprises Rhizoctonia heterodera or Rhizoctonia rhizogenes.
9. The method of claim 4, wherein the culturing conditions in S3 are: culturing at 25 deg.C in dark for 8 weeks.
10. Use of the method of culturing arbuscular mycorrhizal fungi according to any one of claims 4 to 9 in the preparation of an arbuscular mycorrhizal fungi inoculant.
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| 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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| 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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