CN111073842A - Application of abscisic acid in promoting arbuscular mycorrhizal fungi spore production - Google Patents
Application of abscisic acid in promoting arbuscular mycorrhizal fungi spore production Download PDFInfo
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Abstract
The invention discloses application of abscisic acid in promoting arbuscular mycorrhizal fungi to produce spores. According to the invention, the abscisic acid is added into the spores of the arbuscular mycorrhizal fungi, the abscisic acid can directly act on the spores of the arbuscular mycorrhizal fungi, the production of the spores of the arbuscular mycorrhizal fungi is obviously improved, the addition of the abscisic acid also can obviously improve the production of the spores of the arbuscular mycorrhizal fungi under symbiotic conditions, and the abscisic acid can be widely used for promoting the spores of the arbuscular mycorrhizal fungi; the invention also provides a preparation and a method for promoting the spore production of the arbuscular mycorrhizal fungi, the method can propagate the arbuscular mycorrhizal fungi in an aseptic condition, has high propagation efficiency, can obviously increase the spore production of the arbuscular mycorrhizal fungi, is simple and convenient to operate, has low cost and short period, and has wide popularization and application prospects.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, it relates to the use of abscisic acid in promoting sporulation of arbuscular mycorrhizal fungi.
Background
Arbuscular Mycorrhizal Fungi (AMF) are symbiotic fungi that are living nutritional and inhabit in soil, belong to the subphylum gloeostereum, and can establish symbiotic relationship with the root systems of most terrestrial plants. After the arbuscular mycorrhizal fungi and the plants establish a symbiotic relationship, the root systems of the crops can be greatly promoted to absorb mineral nutrients such as phosphorus and nitrogen, the stress resistance of the crops is improved, for example, the tolerance of the crops to nutrient impoverishment, drought, heavy metal toxicity, pH value stress, plant diseases and insect pests and the like is improved, the growth vigor of the crops is improved, and further, the yield of the crops is improved, and the quality of the crops is improved. Therefore, the arbuscular mycorrhizal fungi have great application potential in agricultural production.
Since the arbuscular mycorrhizal fungi have not been purely cultured so far, and cannot sporulation under a non-symbiotic state (i.e. a state of not symbiotic with plants), the propagation of arbuscular mycorrhizal fungi spores needs to be premised on establishment of symbiotic relationship. At present, the expanding propagation of arbuscular mycorrhizal fungi spores is mainly carried out in two ways: firstly, a sorghum and clover combination (or other plant combinations) is used as a host plant, and the host plant is propagated in a matrix such as soil, river sand, diatomite and the like in a certain proportion, so that the obtained microbial inoculum takes the soil as a carrier; secondly, establishing a dual culture system of arbuscular mycorrhizal fungi and hairy roots (such as carrots and tomatoes) under the aseptic conditions of a culture dish and the like, and propagating spores and the like under the aseptic conditions. Because spores obtained by the hairy root double culture system are sterile, the method is widely applied in the fields of scientific research, production and the like.
Spores are propagules of arbuscular mycorrhizal fungi, and the number of the spores is an important index for quantifying the quality of the microbial inoculum. Therefore, how to increase the spore production of the arbuscular mycorrhizal fungi is an important factor in the propagation technology of the arbuscular mycorrhizal fungi, and is an important way for effectively improving the quality of the microbial inoculum.
The prior art (application number is 201710058144.6) discloses an expanding propagation method of arbuscular mycorrhizal fungi, which uses corn, marigold and clover as host plants, water and fertilizer are applied in the growth process of the host plants to expand the arbuscular mycorrhizal fungi, and measures of no fertilization, reduced watering, slight wilting or severe wilting of the plants and watering to cause the plants to suffer from drought stress are adopted in the later stage of expanding propagation, so that the arbuscular mycorrhizal fungi have high infection rate, high fungus yield and high expanding propagation efficiency. However, the method is carried out under non-sterilization conditions, and the obtained arbuscular mycorrhizal fungi spores carry mixed fungi, so that the requirements of many scientific researches cannot be met, and in addition, the spore production of the arbuscular mycorrhizal fungi is still to be improved.
Therefore, the research on the expanding propagation method of the arbuscular mycorrhizal fungi, which has high expanding propagation efficiency and can obviously increase the sporulation yield under the aseptic condition, is of great significance.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the existing arbuscular mycorrhizal fungi propagation technology and provide application of abscisic acid in promoting arbuscular mycorrhizal fungi to produce spores.
The invention aims to provide application of abscisic acid in promoting arbuscular mycorrhizal fungi to produce spores.
The invention also aims to provide the application of the abscisic acid in preparing a preparation for promoting the sporulation of the arbuscular mycorrhizal fungi.
It is still another object of the present invention to provide an agent for promoting sporulation of arbuscular mycorrhizal fungi.
It is still another object of the present invention to provide a method for promoting sporulation of arbuscular mycorrhizal fungi.
The above purpose of the invention is realized by the following technical scheme:
according to the research of the invention, the abscisic acid is added into the spores of the arbuscular mycorrhizal fungi, and the abscisic acid can directly act on the spores of the arbuscular mycorrhizal fungi, so that the production of the spores of the arbuscular mycorrhizal fungi is obviously improved; the addition of abscisic acid in the plant root system has no obvious difference between the production amount of arbuscular mycorrhizal fungal spores and the production amount of arbuscular mycorrhizal fungal spores without addition of abscisic acid; compared with the method without adding abscisic acid, the addition of abscisic acid can obviously improve the spore production of arbuscular mycorrhizal fungi under symbiotic conditions; therefore, the following applications should be within the scope of the present invention:
application of abscisic acid in promoting arbuscular mycorrhizal fungi to produce spores.
Application of abscisic acid in preparation of a preparation for promoting sporulation of arbuscular mycorrhizal fungi.
Preferably, the arbuscular mycorrhizal fungus is Rhizophagus oryzae (Rhizophagus irregularis).
More preferably, the rhizospora heteroclidinium is rhizospora heteroclidinium DAOM 197198.
Based on the application, the invention provides a preparation for promoting the sporulation of arbuscular mycorrhizal fungi, and the preparation comprises abscisic acid.
The invention also provides a method for promoting the sporulation of the arbuscular mycorrhizal fungi, and the abscisic acid is mixed with the arbuscular mycorrhizal fungi.
Preferably, the method is: adding abscisic acid into a hairy root-arbuscular mycorrhizal fungi dual culture system.
Preferably, the arbuscular mycorrhizal fungus is rhizosporangium heteroclidum.
More preferably, the rhizospora heteroclidinium is rhizospora heteroclidinium DAOM 197198.
The invention has the following beneficial effects:
through creative exploration experiments, the abscisic acid is added into spores of the arbuscular mycorrhizal fungi, and can directly act on the spores of the arbuscular mycorrhizal fungi, so that the production of the spores of the arbuscular mycorrhizal fungi is obviously improved; compared with the method without adding abscisic acid, the spore production of the arbuscular mycorrhizal fungi under the symbiotic condition with the addition of abscisic acid is 4.5 times that of the arbuscular mycorrhizal fungi without the addition of abscisic acid, and the spore production of the arbuscular mycorrhizal fungi under the symbiotic condition can be obviously improved by adding the abscisic acid;
based on the above, the invention provides a preparation and a method for promoting the spore production of arbuscular mycorrhizal fungi, the method can propagate the arbuscular mycorrhizal fungi under the aseptic condition, has high propagation efficiency, can obviously increase the spore production of the arbuscular mycorrhizal fungi, and has the advantages of simple and convenient operation, low cost and short period; therefore, the abscisic acid has wide application prospect in promoting the spore production of the arbuscular mycorrhizal fungi or preparing preparations for promoting the spore production of the arbuscular mycorrhizal fungi.
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FIG. 1 is a schematic representation of the two-compartment culture dish used in example 1 and its experimental treatment; wherein, the figure (A) is a shape diagram of a two-compartment culture dish; (B) the figure is a schematic of the experimental treatment of example 1.
FIG. 2 shows sporulation in the tomato hairy root-Heterorhizopus heteroclidinus DAOM197198 double culture system of example 2; wherein, the picture (A) is the sporulation observed by a stereomicroscope; (B) the figure shows the sporulation observed by a biological microscope.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Effect of abscisic acid on spore production by arbuscular mycorrhizal fungi under non-symbiotic conditions
In this embodiment, a model strain of arbuscular mycorrhizal fungi (heteroclite radiculospora DAOM197198) is selected to study the influence of abscisic acid on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions, and the specific experimental methods and experimental results are respectively as follows:
1. experimental methods
The manufacturing method of the two-compartment culture dish in the embodiment comprises the following steps: a1.2 cm x 8.3cm aluminum plate is adhered to the middle of a 9cm culture dish by glass to prepare a uniform culture dish with two compartments, namely a Root Compartment (RC) for culturing tomato hairy roots and a Spore Compartment (SC) for inoculating and culturing arbuscular mycorrhizal fungi to obtain spores. And after the glass cement is completely solidified, cleaning the culture dish, sterilizing for 30min at the high temperature of 121 ℃ under high pressure for 2 times, and drying for later use.
The formula of the MSR culture medium is (the addition amount in 1L): MgSO (MgSO)4·7H2O：739mg；KNO3：76mg；KCl：65mg；Ca(NO3)2·4H2O：359mg；KH2PO4：4.1mg；MnSO4·4H2O：2.45mg；ZnSO4·7H2O：0.28mg；H3BO3：1.85mg；CuSO4·5H2O：0.22mg；(NH4)6Mo7O24·4H2O：0.034mg；NaMoO4·2H2O: 0.0024 mg; thiamine hydrochloride (VB)1): 1 mg; pyridoxine hydrochloride (VB)6): 0.9 mg; nicotinic acid: 1 mg; calcium pantothenate: 0.9 mg; vitamin B12: 0.4 mg; biotin: 0.0009 mg; NaFeEDTA: 8 mg.
(1) Preparing MSR culture medium according to the formula, adding 10g/L sucrose, adjusting pH of the solution to 5.5, and subpackaging in conical flasks; adding 2.5g/L plant gel into each conical flask, sealing, and sterilizing at 121 deg.C for 15 min;
(2) weighing 0.0132g of abscisic acid powder, placing in a 10mL centrifuge tube, dissolving with 100 μ L of 1M NaOH, diluting to 5mL, and making into 10-2M abscisic acid solution, filtering and sterilizing with 0.22 μ M inorganic phase Polyethersulfone (PES) filter membrane, and diluting abscisic acid solution with sterile water to 10%-4M；
(3) The 10-stage configuration of 1ml/L of the above-described medium was added to the sterilized MSR medium according to 3 different treatments (root chamber (RC) and Spore Chamber (SC) medium addition abscisic acid conditions are denoted as RC/SC: no abscisic acid addition/no abscisic acid addition (-ABA/-ABA), abscisic acid addition/no abscisic acid addition (+ ABA/-ABA), no abscisic acid addition/abscisic acid addition (-ABA/+ ABA)), respectively-4M abscisic acid solution to make the final concentration of abscisic acid in the culture medium be 10-7M, after being uniformly mixed, pouring the mixture into two 9 cm-compartment culture dishes, wherein 20mL of each culture dish is placed on an ultra-clean workbench after ultraviolet sterilization, and cooling the culture dishes to 50 ℃ by blowing air for later use after solidification;
(4) transferring half of culture on spore culture medium of Heterosporangium heteroclita DAOM197198 propagated in a hairy root-arbuscular mycorrhizal fungi dual-culture system into a 50mL centrifuge tube, adding 3 times of 10mM sodium citrate solution with pH6.0, shaking on a shaking table (shaking rotation speed of 120rpm and temperature of 25 ℃) for 1h, and filtering the solution by using a nylon membrane with the thickness of 25 mu m to obtain spare spores;
(5) dispersing spores with dissecting needle under stereomicroscope, inoculating into MSR culture medium, inoculating about 100 spores per culture medium, sealing with sealing film, and culturing in constant temperature incubator at 25 deg.C in dark.
After dark culture in an incubator at 25 ℃ for 12 weeks, the dishes were taken out, placed upside down under a stereomicroscope for observation and counted for the number of newly formed spores in the Root Chamber (RC) and Spore Chamber (SC) on each dish.
The two-compartment culture dish used in this example 1 and a schematic thereof are shown in FIG. 1.
2. Results of the experiment
The results of the influence of abscisic acid on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions are shown in table 1, and it can be seen that the addition of abscisic acid can improve the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions, and especially that the addition of abscisic acid (-ABA/+ ABA) in the Spore Chamber (SC) can significantly improve the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions; whereas when abscisic acid (+ ABA/-ABA) was added in the Root Compartment (RC), there was no significant difference between the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions and the spore production of the group treated without addition of abscisic acid (-ABA/-ABA).
From the above results, it can be seen that abscisic acid can significantly increase the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions, and is a result of direct action of abscisic acid on arbuscular mycorrhizal fungi spores, rather than a result of action of abscisic acid on arbuscular mycorrhizal fungi through the root system.
TABLE 1 Effect of abscisic acid on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions
| Treatment (RC/SC) | Spore production |
| -ABA/-ABA | 27.4±6.3a |
| +ABA/-ABA | 28.5±3.0a |
| -ABA/+ABA | 63.6±5.7b |
Note: "RC" and "SC" refer to root compartment and spore compartment, respectively, and the same lower case letters indicate that there was no significant difference at the 5% level for Tukey HSD assay.
Example 2 Effect of abscisic acid on the production of spores of arbuscular mycorrhizal fungi under symbiotic conditions
1. Experimental methods
The culture dish used in this example was a normal glass culture dish, and the formulation of the MSR medium used was the same as that of the MSR medium used in example 1.
(1) Preparing MSR culture medium according to the formula, adding 10g/L sucrose, adjusting pH of the solution to 5.5, and subpackaging in conical flasks; adding 2.5g/L plant gel into each conical flask, sealing, and sterilizing at 121 deg.C for 15 min;
2) weighing 0.0132g of abscisic acid powder, placing in a 10mL centrifuge tube, dissolving with 100 μ L of 1M NaOH, diluting to 5mL, and making into 10-2M abscisic acid solution, filtering and sterilizing with 0.22 μ M inorganic phase Polyethersulfone (PES) filter membrane, and diluting abscisic acid solution with sterile water to 10%-4M；
(3) The method comprises providing an ABA group (MSR group) containing abscisic acid in MSR medium and a non-abscisic acid group (MSR group), wherein the ABA group is prepared by adding 1ml/L of 10 of the above-mentioned components to the MSR medium after sterilization-4M abscisic acid solution to make the final concentration of abscisic acid in the culture medium be 10-7M, uniformly mixing, and pouring into a culture dish;
(4) transferring half of culture on spore culture medium of Heterosporangium heteroclita DAOM197198 propagated in a hairy root-arbuscular mycorrhizal fungi dual-culture system into a 50mL centrifuge tube, adding 3 times of 10mM sodium citrate solution with pH6.0, shaking on a shaking table (shaking rotation speed of 120rpm and temperature of 25 ℃) for 1h, and filtering the solution by using a nylon membrane with the thickness of 25 mu m to obtain spare spores;
(5) dispersing spores by using a dissecting needle under a stereomicroscope, inoculating the spores into MSR culture medium, inoculating about 100 spores into each culture medium, sealing by using a sealing film, and placing in a constant-temperature incubator for dark culture (the culture temperature is 25 ℃);
(6) after 5 days of culture, inoculating about 5 strips of tomato hairy roots which are 3cm away from spores and are transformed into 'Xinjinfeng No. 1' by using agrobacterium rhizogenes ACCC10060, sealing the tomato hairy roots by using a sealing film, and continuously culturing the tomato hairy roots in an incubator in the dark (the culture temperature is 25 ℃).
After culturing for 8 weeks in a 25 ℃ incubator at a constant temperature in the dark, the dishes were taken out, placed upside down under a stereomicroscope for observation and counted for the number of newly formed spores in each dish.
2. Results of the experiment
The sporulation of the tomato hairy root-heteroclite sporangium DAOM197198 double culture system in this example 2 is shown in FIG. 2, and it can be seen that after abscisic acid is added to the sterile hairy root-arbuscular mycorrhizal fungi double culture system, a large amount of new spores are generated by the heteroclite sporangium DAOM 197198.
The results of the influence of abscisic acid on the spore production of arbuscular mycorrhizal fungi under symbiotic conditions are shown in table 2, and it can be seen that the spore production of arbuscular mycorrhizal fungi under symbiotic conditions with addition of abscisic acid is 4.5 times that without addition of abscisic acid compared with no addition of abscisic acid, indicating that the addition of abscisic acid significantly improves the spore production of arbuscular mycorrhizal fungi under symbiotic conditions.
TABLE 2 Effect of abscisic acid on the spore production of arbuscular mycorrhizal fungi under symbiotic conditions
| Treatment group | Number of spores |
| ABA | 822.25±441.12* |
| MSR | 183.75±183.75 |
Note: "+" indicates significant differences in T-test at the 5% level.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. Application of abscisic acid in promoting arbuscular mycorrhizal fungi to produce spores.
2. Application of abscisic acid in preparation of a preparation for promoting sporulation of arbuscular mycorrhizal fungi.
3. Use according to claim 1 or 2, wherein the arbuscular mycorrhizal fungus is heterothecium radicicola.
4. Use according to claim 3, wherein the Rhizoctonia heteroclita is Rhizoctonia heteroclita DAOM 197198.
5. An agent for promoting sporulation of arbuscular mycorrhizal fungi, wherein the agent comprises abscisic acid.
6. A method for promoting sporulation of arbuscular mycorrhizal fungi, characterized in that abscisic acid is mixed with arbuscular mycorrhizal fungi.
7. The method according to claim 6, characterized in that the method is: adding abscisic acid into a hairy root-arbuscular mycorrhizal fungi dual culture system.
8. The method according to claim 6 or 7, wherein the arbuscular mycorrhizal fungus is Rhizoctonia heterodera.
9. The method of claim 8, wherein the Rhizoctonia heteroclita is Rhizoctonia heteroclita DAOM 197198.
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| 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 |
| CN112385507A (en) * | 2020-11-16 | 2021-02-23 | 南京农业大学 | Seedling culture medium containing arbuscular mycorrhiza and preparation method and application thereof |
| 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 |
| CN115029293A (en) * | 2022-06-08 | 2022-09-09 | 南京大学 | Method for efficiently propagating ascosphaera radiculosa spores through hairy roots of carrots |
| CN117844648A (en) * | 2023-12-26 | 2024-04-09 | 安徽农业大学 | Method for improving growth and infection activity of arbuscular mycorrhizal fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115029293A (en) * | 2022-06-08 | 2022-09-09 | 南京大学 | Method for efficiently propagating ascosphaera radiculosa spores through hairy roots of carrots |
| CN117844648A (en) * | 2023-12-26 | 2024-04-09 | 安徽农业大学 | Method for improving growth and infection activity of arbuscular mycorrhizal fungi |
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Application publication date: 20200428 |