CN113403204B - Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method - Google Patents

Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method Download PDF

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CN113403204B
CN113403204B CN202110518816.3A CN202110518816A CN113403204B CN 113403204 B CN113403204 B CN 113403204B CN 202110518816 A CN202110518816 A CN 202110518816A CN 113403204 B CN113403204 B CN 113403204B
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arbuscular mycorrhizal
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朱红惠
刘晓迪
冯曾威
姚青
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses a method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application thereof. Potassium myristate, strigolactone, methyl jasmonate and peptone are added into a culture medium of arbuscular mycorrhizal fungi. Through creative exploration experiments, the invention obviously improves the production of the arbuscular mycorrhizal fungi spores by adding potassium myristate, strigolactone, methyl jasmonate and peptone into the spores of the arbuscular mycorrhizal fungi, and the spore quantity produced by single mother spores of the arbuscular mycorrhizal fungi in the addition treatment is 9 times that produced by single mother spores of the arbuscular mycorrhizal fungi without the addition treatment. The addition of potassium myristate, strigolactone, methyl jasmonate and peptone can significantly increase the production of arbuscular mycorrhizal fungal spores under non-symbiotic conditions.

Description

Method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi and application of method
The technical field is as follows:
the invention belongs to the technical field of microbial culture, and particularly relates to a method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi.
Background art:
the arbuscular mycorrhizal fungi is a living nutritional symbiotic fungus widely existing in soil, belongs to glomus sacculus subgenus, and can establish a symbiotic relationship with 80% of roots of land 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.
The existing method for propagating arbuscular mycorrhizal fungi spores needs to establish a symbiotic relationship with plants as a premise. At present, the expanding propagation of arbuscular mycorrhizal fungi spores is mainly carried out in two ways: firstly, sorghum and clover combination (or other plant combination) is used as a host plant, and is propagated in a certain proportion of soil, river sand, diatomite and other matrixes, so that the obtained microbial inoculum takes the soil as a carrier, and a larger plant cultivation space is needed in the cultivation process; 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. In addition, the culture process only needs to be carried out in a constant-temperature incubator, not only is the occupied area small and the space utilization rate high, but also the limitation of external environments (such as illumination, temperature and the like) is avoided.
Spores are important 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 number of the arbuscular mycorrhizal fungi is an important factor in the propagation technology of the arbuscular mycorrhizal fungi, and the method is also 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 infection rate, the spore yield and the expanding propagation efficiency of the arbuscular mycorrhizal fungi are improved. However, the method is carried out under non-sterilization conditions, and the obtained arbuscular mycorrhizal fungal spores carry mixed bacteria, so that the requirements of many scientific research and multi-science researches on aseptic spores cannot be met.
The prior art (application number is 201911320670.0) discloses an application of abscisic acid in promoting the spore production of arbuscular mycorrhizal fungi, and the abscisic acid is added into spores of the arbuscular mycorrhizal fungi, so that the abscisic acid can directly act on the spores of the arbuscular mycorrhizal fungi, and the spore number of the arbuscular mycorrhizal fungi is obviously increased. The application of gibberellin-deficient mutants in promoting the sporulation of arbuscular mycorrhizal fungi (application No. CN 202010718350.7) significantly improves the spore yield of the arbuscular mycorrhizal fungi by inoculating the arbuscular mycorrhizal fungi with hairy roots of the gibberellin-deficient mutants of tomato, and significantly improves the spore number of the arbuscular mycorrhizal fungi under normal pH conditions (pH 6.5) and acidic conditions (pH 4.5) with the hairy roots of the gibberellin-deficient mutants. The spores propagated by the method are sterile, and are favorable for further research on arbuscular mycorrhizal fungi in scientific research. However, the above method needs to take root systems as host plants, and spores can be produced only after the arbuscular mycorrhizal fungi establish symbiotic relationship with the host plants, so the steps are complicated, and the production period of the spores is long.
Therefore, the research on the expanding propagation method of the arbuscular mycorrhizal fungi, which has the advantages of simple expanding operation, short period, high expanding propagation efficiency, small dependence on external environment and capability of obviously increasing the spore yield under the non-symbiotic condition, is of great significance, and is a great step forward in the aspect of realizing pure culture of the arbuscular mycorrhizal fungi.
The invention content is as follows:
the technical problem to be solved by the invention is to overcome the defects and shortcomings of the existing arbuscular mycorrhizal fungi propagation technology and provide a method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi.
The method for promoting non-symbiotic spore production of the arbuscular mycorrhizal fungi comprises the step of adding potassium myristate, strigolactone, methyl jasmonate and peptone into a culture medium of the arbuscular mycorrhizal fungi.
The second purpose of the invention is to provide a preparation for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi, which is prepared by adding potassium myristate, strigolactone, methyl jasmonate and peptone to a culture medium of the arbuscular mycorrhizal fungi.
Preferably, in the culture medium, the potassium myristate is added at a concentration of 0.5mM, the strigolactone is added at a concentration of 100nM, the methyl jasmonate is added at a concentration of 1. Mu.M, and the peptone is added at a concentration of 1mg/L.
Preferably, the culture medium is: contains per liter: mgSO (MgSO) 4 ·7H 2 O：739mg；KNO 3 ：76mg；KCl：65mg；Ca(NO 3 ) 2 ·4H 2 O：359mg；KH 2 PO 4 ：4.1mg；MnSO 4 ·4H 2 O：2.45mg；ZnSO 4 ·7H 2 O：0.28mg；H 3 BO 3 ：1.85mg；CuSO 4 ·5H 2 O：0.22mg；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O：0.034mg；NaMoO 4 ·2H 2 O:0.0024mg; thiamine hydrochloride (VB) 1 ): 1mg; pyridoxine hydrochloride (VB) 6 ): 0.9mg; nicotinic acid: 1mg; calcium pantothenate: 0.9mg; vitamin B 12 :0.4mg; biotin: 0.0009mg; naFeEDTA:8mg; sucrose: 1g of a compound; plant gel: 2.5g; glucose: 1g of a compound; peptone: 1mg; potassium myristate: 0.5mmol; strigolactone: 100nmol; methyl jasmonate: 1 mu mol, and the solvent is water.
Preferably, the arbuscular mycorrhizal fungus is heteroclite radiculospora DAOM197198.
The third purpose of the invention is to provide the application of the potassium myristate, the strigolactone, the methyl jasmonate and the peptone in promoting the non-symbiotic spore production of the arbuscular mycorrhizal fungi.
Preferably, potassium myristate, strigolactone, methyl jasmonate and peptone are added to the culture medium of the arbuscular mycorrhizal fungi.
The invention has the following beneficial effects:
through creative exploration experiments, the invention obviously improves the production of the arbuscular mycorrhizal fungi spores by adding potassium myristate, strigolactone, methyl jasmonate and peptone into the spores of the arbuscular mycorrhizal fungi, and the spore quantity produced by single mother spores of the arbuscular mycorrhizal fungi in the addition treatment is 9 times that produced by single mother spores of the arbuscular mycorrhizal fungi without the addition treatment. The addition of potassium myristate, strigolactone, methyl jasmonate and peptone can significantly increase the production of arbuscular mycorrhizal fungal spores under non-symbiotic conditions.
Based on the above, the invention provides a preparation and a method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi, the method can expand propagation of the arbuscular mycorrhizal fungi in a non-symbiotic system, the expansion efficiency is high, the spore production of the arbuscular mycorrhizal fungi can be obviously increased, and the preparation and the method also have the advantages of simple and convenient operation, low cost, short period and the like; therefore, the method has wide application prospect in promoting the spore production of the arbuscular mycorrhizal fungi or preparing a preparation for promoting the spore production of the arbuscular mycorrhizal fungi.
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FIG. 1 shows the sporulation of the Pholiospora heteroclita DAOM197198 in a non-symbiotic culture system in example 1; wherein, A is sporulation on modified MSR medium; b is sporulation on basal MSR medium. White arrows indicate the mother spores, and black (a) or gray (B) arrows indicate the sporozoites produced by the mother spores.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Effect of different media on sporulation of arbuscular mycorrhizal fungi under non-symbiotic conditions
1. Experimental methods
In this embodiment, a model strain of arbuscular mycorrhizal fungi (heteroclite radiculospora DAOM 197198) is selected to study the influence of an improved MSR culture medium on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions, and specific experimental methods and experimental results are respectively as follows:
(1) Strigolactone (0.3 mg) was weighed out and dissolved in 1mL of acetone to prepare a 1mM strigolactone solution.
(2) 11.4mg of methyl jasmonate was weighed and dissolved in 5ml of absolute ethanol to prepare a 10mM methyl jasmonate solution.
(3) 1.33g of potassium myristate was weighed and dissolved in 10ml of double distilled water to prepare a 0.5M potassium myristate solution.
(4) The strigolactone solution and the methyl jasmonate solution were filter sterilized with 0.22 μm organic phase of organic nylon (nylon) 66. The potassium myristate solution was filter-sterilized with a 0.22 μm inorganic Polyethersulfone (PES) filter.
(5) The formula of the MSR basal medium is (the addition amount in 1L water): mgSO (MgSO) 4 ·7H 2 O：739mg；KNO 3 ：76mg；KCl：65mg；Ca(NO 3 ) 2 ·4H 2 O：359mg；KH 2 PO 4 ：4.1mg；MnSO 4 ·4H 2 O：2.45mg；ZnSO 4 ·7H 2 O：0.28mg；H 3 BO 3 ：1.85mg；CuSO 4 ·5H 2 O：0.22mg；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O：0.034mg；NaMoO 4 ·2H 2 O:0.0024mg; thiamine hydrochloride (VB) 1 ): 1mg; pyridoxine hydrochloride (VB) 6 ): 0.9mg; nicotinic acid: 1mg; calcium pantothenate: 0.9mg; vitamin B 12 :0.4mg; biotin: 0.0009mg; naFeEDTA:8mg; sucrose: 10g of a mixture; plant gel: 2.5g. According to the components and contents, uniformly mixing the components, adjusting the pH value: 5.5, sealing the mouth and then sterilizing for 15min at the high temperature of 121 ℃. Thereby obtaining an MSR medium.
(6) Configuration of modified MSR medium: in 1L MSR culture medium, the sucrose addition amount is changed to 1g, and then 1g of glucose and 1mg of peptone are added, and other components are consistent with those of the basic MSR culture medium, the pH is adjusted to 5.5, and the MSR culture medium is sterilized at 121 ℃ for 15min under high pressure after sealing. Then, 100. Mu.L of strigolactone solution of 1mM, 100. Mu.L of methyl jasmonate solution of 10mM and 1mL of potassium myristate solution of 0.5M after filtration sterilization were added to the culture medium sterilized at high temperature and high pressure on a clean bench so that the final use concentrations of potassium myristate, strigolactone and methyl jasmonate were 0.5mM, 100nM and 1. Mu.M, respectively, and the mixture was poured into a petri dish for use after being mixed uniformly.
(7) Adding solvent (acetone, anhydrous ethanol and double distilled water) with the same amount as the improved MSR culture medium into the MSR culture medium sterilized at high temperature and high pressure on a clean bench, mixing well, and pouring into a culture dish for later use.
(8) Half of the culture on the spore culture medium of the heterosporum DAOM197198 propagated in the hairy root-arbuscular mycorrhizal fungi dual-culture system is completely transferred into a 50mL centrifuge tube, a 3-fold volume of 10mM sodium citrate solution with pH6.0 is added, the solution is filtered by a 25-micron nylon membrane after shaking on a shaking table (the shaking rotation speed is 120rpm, the temperature is 25 ℃) for 1h, and the spare spores are obtained.
(9) The spores were scattered by a dissecting needle under a stereomicroscope, inoculated into a medium, about 50 spores were inoculated into each dish, 5 times for each treatment, sealed with a sealing film, and placed in an incubator for dark culture (culture temperature 25 ℃).
(10) After dark culture for 6 weeks in a constant temperature incubator at 25 ℃, the dishes are 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-heterosporulation DAOM197198 nongenic culture system in this example 1 is shown in fig. 1, and it can be seen that the heterosporulation DAOM197198 produces a large amount of new spores in the non-symbiotic culture system of the modified MSR medium added with potassium myristate, strigolactone, methyl jasmonate and peptone.
The results of the effect of the modified MSR medium on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions are shown in table 1, and it can be seen that the number of sporozoites produced by a single mother spore of arbuscular mycorrhizal fungi under non-symbiotic conditions treated with the modified MSR medium supplemented with potassium myristate, strigolactone, methyl jasmonate, and peptone is 9 times greater than that treated with the base MSR medium, indicating that the modified MSR medium significantly increases the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions.
TABLE 1 Effect of different treatments under non-symbiotic conditions on the production of arbuscular mycorrhizal fungal spores
Treatment group Number of sporozoites produced by a single mother spore
Basic MSR medium 0.65±0.08
Improved MSR culture medium 5.47±0.62***
Note: ". Indicates that there was a significant difference in the independent sample T-test at the level of P.ltoreq.0.001.
Comparative example 1 Effect of Potassium myristate on spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions
1. Experimental methods
In this embodiment, a model strain of arbuscular mycorrhizal fungi (heteroclite-root cyst-mildew DAOM 197198) is selected to study the influence of the improved potassium myristate on the spore production of arbuscular mycorrhizal fungi under non-symbiotic conditions, and the specific experimental methods and experimental results are as follows:
(1) 1.33g of potassium myristate was weighed, dissolved in 10ml of double distilled water to prepare a 0.5M potassium myristate solution, and the potassium myristate solution was subjected to filtration sterilization with a 0.22 μ M inorganic phase Polyethersulfone (PES) filter.
(2) The formula of the MSR basic culture medium is (the addition amount in 1L): mgSO (MgSO) 4 ·7H 2 O：739mg；KNO 3 ：76mg；KCl：65mg；Ca(NO 3 ) 2 ·4H 2 O：359mg；KH 2 PO 4 ：4.1mg；MnSO 4 ·4H 2 O：2.45mg；ZnSO 4 ·7H 2 O：0.28mg；H 3 BO 3 ：1.85mg；CuSO 4 ·5H 2 O：0.22mg；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O：0.034mg；NaMoO 4 ·2H 2 O:0.0024mg; thiamine hydrochloride (VB) 1 ): 1mg; pyridoxine hydrochloride (VB) 6 ): 0.9mg; nicotinic acid: 1mg; calcium pantothenate: 0.9mg; vitamin B 12 :0.4mg; biotin: 0.0009mg; naFeEDTA:8mg; sucrose: 10g, 2.5g of plant gel and water as a solvent. According to the components and the content, the components are uniformly mixed, and the pH value is adjusted to 5.5. Sealing, and sterilizing at 121 deg.C under high pressure for 15min.
(3) And pouring the MSR culture medium on a clean bench into a culture dish, cooling and solidifying for later use as a control.
(4) When the MSR culture medium is cooled to about 40 ℃, 0.5M potassium myristate (myr) solution which is sterilized by filtration is added to ensure that the final use concentration of the potassium myristate is 0.5mM respectively, and the mixture is poured into a culture dish for later use after being mixed uniformly.
(5) Half of the culture on the spore culture medium of the Heterospora heteroclita DAOM197198 propagated in the hairy root-arbuscular mycorrhizal fungi dual-culture system is completely transferred to a 50mL centrifuge tube, a 3-fold volume of a 10mM sodium citrate solution with pH6.0 is added, the solution is shaken on a shaking table (the shaking rotation speed is 120rpm, the temperature is 25 ℃) for 1h, and then the solution is filtered by a nylon membrane with the thickness of 25 mu m to obtain the reserved spores.
(6) The spores were dispersed by a dissecting needle under a stereomicroscope, inoculated into MSR medium, inoculated into about 50 spores per dish, treated 5 times each, sealed with a sealing film, and placed in a constant temperature incubator for dark culture (culture temperature 25 ℃).
(7) After dark culture for 6 weeks in a constant temperature incubator at 25 ℃, the dishes are 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
From table 2 it can be seen that the addition of potassium myristate to MSR medium significantly promoted the formation of non-symbiotic spores, each mother spore producing 1.6 sporozoites whereas on MSR medium without potassium myristate only 0.7 sporozoites were produced per mother spore, with spore yield 2.4 times higher per mother spore after potassium myristate treatment (myr) than without (MSR). But was inferior to the modified MSR medium of example 1, which was 9 times the treatment of the basal MSR medium.
TABLE 2 Effect of Potassium myristate on arbuscular mycorrhizal fungal spore production under non-symbiotic conditions
Treatment group Sporozoites derived from a single mother sporeNumber of children
MSR 0.67±0.14
MSR with added myr 1.58±0.32*
Note: "+" indicates that there was a significant difference in the independent sample T-test at the level P.ltoreq.0.05.
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 (5)

1. A method for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi is characterized in that potassium myristate, strigolactone, methyl jasmonate and peptone are added into a culture medium of the arbuscular mycorrhizal fungi; in the culture medium, the adding concentration of potassium myristate is 0.5mM, the adding concentration of strigolactone is 100nM, the adding concentration of methyl jasmonate is 1 MuM, the adding concentration of peptone is 1mg/L, and the arbuscular mycorrhizal fungi is heterorhizospora heteroclita.
2. The method of claim 1, wherein the medium comprises, per liter: mgSO (MgSO) in vitro 4 ·7H 2 O：739 mg；KNO 3 ：76 mg；KCl：65 mg；Ca(NO 3 ) 2 ·4H 2 O：359 mg；KH 2 PO 4 ：4.1 mg；MnSO 4 ·4H 2 O： 2.45 mg；ZnSO 4 ·7H 2 O：0.28 mg；H 3 BO 3 ：1.85 mg；CuSO 4 ·5H 2 O：0.22 mg；(NH 4 ) 6 Mo 7 O 24 ·4H 2 O：0.034 mg；NaMoO 4 ·2H 2 O:0.0024mg; thiamine hydrochloride (VB) 1 ): 1mg; pyridoxine hydrochloride (VB) 6 ): 0.9mg; nicotinic acid: 1mg; calcium pantothenate: 0.9mg; vitamin B 12 :0.4mg; biotin: 0.0009mg; naFeEDTA:8mg; sucrose: 1g of a compound; plant gel: 2.5g; glucose: 1g of a compound; peptone: 1mg; potassium myristate: 0.5mmol; strigolactone: 100nmol; methyl jasmonate: 1. mu mol, and the solvent is water.
3. The method according to claim 1 or 2, wherein the arbuscular mycorrhizal fungus is a heteroradiculospora DAOM197198.
4. A preparation for promoting non-symbiotic spore production of arbuscular mycorrhizal fungi is characterized in that potassium myristate, strigolactone, methyl jasmonate and peptone are added into a culture medium of the arbuscular mycorrhizal fungi, wherein the addition concentration of the potassium myristate is 0.5mM, the addition concentration of the strigolactone is 100nM, the addition concentration of the methyl jasmonate is 1 mu M, and the addition concentration of the peptone is 1mg/L, and the arbuscular mycorrhizal fungi is Heterospora heteroclitoria.
5. The application of potassium myristate, strigolactone, methyl jasmonate and peptone in promoting non-symbiotic spore production of arbuscular mycorrhizal fungi is characterized in that the potassium myristate, the strigolactone, the methyl jasmonate and the peptone are added into a culture medium of the arbuscular mycorrhizal fungi; in a culture medium, the adding concentration of potassium myristate is 0.5mM, the adding concentration of strigolactone is 100nM, the adding concentration of methyl jasmonate is 1 muM, the adding concentration of peptone is 1mg/L, and the arbuscular mycorrhizal fungus is heteromycorrhizal neurospora.
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