A method to improve the growth and infection activity of arbuscular mycorrhizal fungi

Abstract

The invention discloses a method for improving the growth and infection activity of arbuscular mycorrhizal fungi, belongs to the field of microbial culture, and according to the living habits of arbuscular mycorrhizal fungi spores, abiotic stress is respectively carried out in the spore vegetative growth period and the reproductive growth period, wherein the abiotic stress is temperature stress or ultraviolet stress, wherein the temperature stress is 4°C or 37°C stress treatment for 3-6 days, and the ultraviolet stress is ultraviolet irradiation for 5-20 minutes. The present invention uses the method of temperature and ultraviolet to carry out abiotic stress in the vegetative growth period and the reproductive growth period to improve the activity in terms of spore yield and infection intensity, which is efficient and low-cost, simple to operate and easy to promote. It is helpful to increase the production amount of microbial agent and reduce the usage amount of arbuscular mycorrhizal fungi microbial agent, reduce production costs, and provide help for the large-scale production of microbial agent.

Description

Technical Field

[0001] The invention relates to the technical field of microbial cultivation, and in particular to a method for improving the growth and infection activity of arbuscular mycorrhizal fungi. Background Art

[0002] Arbuscular mycorrhizal fungi (AMF) represent a very promising agroecological tool to reduce the amount of fertilizers and pesticides needed for food production. Therefore, obtaining a large number of pure and highly active arbuscular mycorrhizal fungal spores is particularly important for the development of AMF inoculants. The most commonly used in vitro dual culture system for cultivating AMF spores is to produce contamination-free AMF spores. However, the culture dishes are sealed to retain moisture and reproduced in darkness and stable temperature, lacking environmental stimulation, resulting in low spore production and activity. In the natural environment, there are not only continuous fluctuations in temperature and humidity, but also changes in pH, and it is also affected by a variety of other abiotic factors. Whether these abiotic factors affect the growth of AMF and mycorrhizal responses has not yet been studied. The sharp reduction in host diversity and the continuous replanting of a single host (most commonly carrots) may affect the function and genetic diversity of cultured AMF. Then the low activity of domesticated spores may not be able to interact with other isolates in nature.

[0003] The existing methods for increasing the spore production of arbuscular mycorrhizal fungi in the dual isolation system in the prior art mainly improve the spore activity from the aspects of host plants and signal molecules, for example, by changing the culture conditions and adding exogenous substances: such as Strigolactones (SLs), lipochito-oligosaccharides, polyethylene glycol, plant-derived tobacco water, etc. Changing the culture conditions can increase the early spore germination rate, but the activity in terms of spore production rate and infection intensity is not significantly improved, and the addition of exogenous substances increases the cost of researching microbial agent products and is inconvenient to operate. Summary of the invention

[0004] The purpose of the present invention is to provide a rapid, efficient and low-cost method for improving spore activity in view of the problem of low spore activity in a double in vitro culture system, which has important application value for the development of microbial agents.

[0005] The present invention is achieved through the following technical solutions:

[0006] The invention provides a method for improving the growth and infection activity of arbuscular mycorrhizal fungi. According to the life habits of arbuscular mycorrhizal fungi spores, abiotic stress is respectively applied in the spore vegetative growth period and the reproductive growth period. The abiotic stress is temperature stress or ultraviolet stress. The temperature stress is 4°C or 37°C stress treatment for 3-6 days, and the ultraviolet stress is ultraviolet irradiation for 5-20 minutes.

[0007] As a further optimization scheme of the present invention, the vegetative growth period refers to the time when hyphae grow one month after the spores colonize the host, and the abiotic stress during the vegetative growth period is used to increase the spore yield of arbuscular mycorrhizal fungi.

[0008] As a further optimization scheme of the present invention, the abiotic stress condition for increasing the spore yield during the vegetative growth period is 4° C. stress for 6 days or ultraviolet stress for 5 minutes or 20 minutes.

[0009] As a further optimization scheme of the present invention, the planting host in the vegetative growth period is carrot root

[0010] As a further optimization scheme of the present invention, the reproductive growth period refers to the time when spores grow two months after the spores infect the host, and the abiotic stress during the reproductive growth period is used to improve the infection ability of the spores of arbuscular mycorrhizal fungi.

[0011] As a further optimization scheme of the present invention, the abiotic stress conditions for improving the spore infection ability during the reproductive growth period are 4° C. stress for 3 days, or 37° C. stress for 6 days, or ultraviolet stress for 10 or 20 minutes.

[0012] As a further optimization scheme of the present invention, the infected host during the reproductive growth period is corn.

[0013] Compared with the prior art, the present invention has the following advantages: the present invention uses temperature and ultraviolet light to apply abiotic stress during the vegetative growth period and reproductive growth period to increase the activity of spore production and infection intensity, is highly efficient and inexpensive, and is easy to operate and popularize. It helps to increase the production of microbial agents and reduce the use of arbuscular mycorrhizal fungal agents, thereby reducing production costs and providing assistance for the large-scale production of microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the amount of mycelium before stress; Figure A is the control group, Figure BI is the treatment group, among which Figure B is the 373 group, Figure C is the ZW10 group, Figure D is the 43 group, Figure E is the 376 group, Figure F is the 46 group, Figure G is the ZW15 group, Figure H is the ZW5 group, and Figure I is the ZW20 group;

[0015] Figure 2is the spore production after stress; Figure A is the control group, Figure BI is the treatment group, Figure B is the 373 group, Figure C is the ZW10 group, Figure D is the 43 group, Figure E is the 376 group, Figure F is the 46 group, Figure G is the ZW15 group, Figure H is the ZW5 group, and Figure I is the ZW20 group;

[0016] Figure 3 The effect of stress on spore production; in the figure: no * indicates P>0.01; P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001, all significant analyses are comparisons between the treatment group and the control group;

[0017] Figure 4 The following are comparisons of spore size after stress; Figure A shows the spore size of the control group, and Figure B shows the spore size of the ZW10 treatment group;

[0018] Figure 5 Comparison of spore wall thickness after stress; Figure A is the spore wall thickness of the control group, and Figure B is the spore wall thickness of the 43 treatment group; the area between the two arrows indicates the spore wall thickness, and the area within the circle indicates the spore contents;

[0019] Figure 6 It is a bar graph of spore size and spore wall thickness after stress;

[0020] Figure 7 Figure 2 is a diagram of corn root infection; Figure A is the spore infection result of the control group, Figure B is the spore infection result after stress, Figure C is the infection effect of the stressed spore root, the arrow in Figure D indicates the hyphae in the root, and the circle indicates the arbuscular structure.

[0021] Figure 8 These are the results of the effects of stress on spore infection rate and spore infection intensity. DETAILED DESCRIPTION

[0022] The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used are commercially available products unless otherwise specified.

[0023] The purpose of this embodiment is to provide a fast, efficient and low-cost method for improving spore activity in the dual in vitro culture system, which has important application value for the development of microbial agents. The specific method includes: according to the life habits of the spores, abiotic stress is applied during the spore vegetative growth period and reproductive growth period, respectively. The experiment sets the control group to be cultured at 25°C (recorded as group 25), the experimental group to be stressed at 4°C for 3d (recorded as group 43), 4°C for 6d (recorded as group 46), 37°C for 3d (recorded as group 373), 37°C for 6d (recorded as group 376), and irradiated under ultraviolet light for 5min, 10min, 15min, and 20min respectively (recorded as groups ZW5, ZW10, ZW15, and ZW20, respectively). The spore growth is observed regularly, and the number of spores, spore size, spore wall thickness, and corn root infection intensity are measured after 3 months of co-cultivation.

[0024] The materials and formulations used in this embodiment include:

[0025] MSR medium

[0026]

[0027] Note: When adding MSR trace mother solution (1000×) and iron salt mother solution (100×), operate in a clean bench. Use NaOH to adjust the pH to

[0028] 5.8, add 3.5g / L plant gel, sterilize in a 121℃ high temperature autoclave. After sterilization, place it on the sterilized clean bench, wait until it drops to a suitable temperature, add 1mL / L MSR vitamin mother solution (1000×), mix well and pour into MSR solid culture medium plate.

[0029]

[0030]

[0031] Iron salt mother liquor (100×)

[0032]

[0033] MSR vitamin mother solution (1000×)

[0034]

[0035] Hoagland trace stock solution (1000×)

[0036]

[0037] Iron salt mother liquor (500×)

[0038]

[0039] Hoagland Nutrient Solution

[0040]

[0041]

[0042] Low phosphorus trace stock solution (1000×)

[0043]

[0044] Low Phosphorus Hoagland Nutrient Solution

[0045]

[0046] FAA fixative

[0047]

[0048] 0.05% Trypan blue dye

[0049]

[0050] Alkaline hydrogen peroxide

[0051]

[0052] Lactic acid glycerol

[0053]

[0054] Example 1 Subculture of spores

[0055] 1.1 Preparation before subculture

[0056] All instruments (inoculation knife, tweezers, cooling bucket, etc.) are sterilized at 121℃ for 20 minutes. The prepared culture medium is placed in a 121℃ high-temperature sterilizer and sterilized for 20 minutes. When the culture medium cools to about 60℃, divide the culture medium into circular culture dishes with a radius of 8cm, about 40L / dish. When the culture medium solidifies, subculture. In order to reduce the contamination rate, all sterilized instruments need to be sterilized in an inoculation sterilizer at a temperature of 300℃, and placed in a cooling bucket for cooling before use.

[0057] 1.2 Subculture operation

[0058] Carry out subculture in a clean bench, select white and tender carrot roots with many branches, and inoculate them on new MSR solid culture medium with tweezers, trying to keep the growth state of the original roots unchanged. Select arbuscular mycorrhizal fungi spore plates of the same batch with roughly the same amount of spores. Take out the spores and culture medium at the same time and put them into an empty culture dish to mix evenly. Each time, pick roughly the same amount and inoculate it 1-2 cm away from the carrot root tip, trying to let the hairy roots surround the spores, so as to shorten the time for the hyphae to invade the hairy roots and speed up the establishment of the symbiotic system. After inoculation, make a mark, including the spore name and inoculation date.

[0059] Example 2 Abiotic stress treatment and growth performance detection during the spore vegetative growth period

[0060] 2.1 Abiotic stress treatment during spore vegetative growth

[0061] In order to reduce the error, spore plates of the same batch, with similar growth of carrot hairy roots, cultured for about 40 days and similar mycelium amount, were selected. Four spore plates were used for each treatment group. Marking was done by randomly selecting 4 locations at different positions on each spore plate and drawing an area of ​​1 cm 2 The control group was kept in a constant temperature incubator at 25°C for dark culture. The treated groups (43, 46, 373, 376 groups) were kept in a 4°C refrigerator for dark culture for 3 days and 6 days, and in a 37°C incubator for dark culture for 3 days and 6 days when the spores grew hyphae one month later. The treated groups (ZW5, ZW10, ZW15, ZW20 groups) were kept in the same constant temperature incubator at 25°C for dark culture for 50 days after the spores grew hyphae one month later.

[0062] 2.2 Measurement of spore count

[0063] The number of spores in the grid was counted under a stereomicroscope. The data were recorded and analyzed using Prism software.

[0064] 2.4 Analysis of the effects of stress treatment on increasing spore production

[0065] Temperature and UV stress were applied to spores in the vegetative growth stage. Mycelial growth status before spore stress ( Figure 1 ), the amount of mycelium in each treatment group was similar. After 50 days of culture, the spores matured, turned yellow, and the number tended to be stable ( Figure 2 ). Data analysis revealed that compared with the control group (spore count 345 / cm 3 ), at 4°C for 6 days (spore count: 662 / cm 3 ), irradiated under ultraviolet conditions for 5 min (spore count: 877 / cm3 ) and irradiated under UV conditions for 20 min (the number of spores was 962 / cm 3 )The number of spores increased significantly ( Figure 3 However, the number of spores in the 4℃ stress for 3 days and 37℃ stress for 6 days decreased significantly compared with the control group ( Figure 3 There was no significant difference in the number of spores when the stress was 37℃ for 3 days and when the stress was 10min and 15min under UV irradiation ( Figure 3 ). In summary, the number of spores of fungal spores that are growing in nutrition was significantly increased by abiotic stress treatment at 4°C for 6 days, UV irradiation for 5 minutes, and UV irradiation for 20 minutes. Abiotic stress significantly increases the spore production, which provides great help for the application of microbial agent products.

[0066] Example 3 Abiotic stress treatment during the spore reproductive growth period and infection performance detection

[0067] 3.1 Abiotic stress treatment during the spore reproductive growth period

[0068] Select the control group in step 2.1 that has grown spores but no mature spores and has been dark cultured in a constant temperature incubator at 25℃ for 2 months. Place the spores in a 4℃ refrigerator for dark culture for 3d and 6d, and incubate them in a 37℃ incubator for 3d and 7d, and place them under a UV lamp at a distance of 50cm for 5min, 10min, 15min, and 20min respectively. Draw 4 1cm×1cm squares randomly on the spore plate for later observation. The spores are cultured for 3 months.

[0069] 3.2 Acquisition of infected hosts

[0070] 3.2.1 Disinfection

[0071] The host of infection is the commercial seeds of Annong 591 corn. Clean the surface coating, soak the seeds in pure water, and wash them several times after soaking until the water no longer changes color. Operate in a clean bench, place the seeds in a plastic conical flask, add freshly prepared 12% (v / v) NaClO and disinfect for 5 minutes, disinfect twice. Then wash with sterile water 4-5 times, 5 minutes each time. Every time you change the disinfectant and sterile water, you need to change the conical flask to reduce the infection rate.

[0072] 3.2.2 Germination paper

[0073] Soak the germination paper in 0.1% (v / v) carbendazim for 30 minutes, place the sterilized corn kernels on the line in the middle and upper part of the germination paper, and put about 8-10 corn kernels on each germination paper. Slowly roll up the germination paper and fix it with a rubber band. Put the rolled corn germination paper in a beaker, pour about 20mL of pure water, and then put it in a light incubator for 3-7 days. Observe the growth of the seeds once a day, add pure water to the beaker in time, and obtain the host seedlings.

[0074] 3.3 Infection

[0075] 3.3.1 Matrix

[0076] One week before sowing, mix vermiculite, sand and perlite in a ratio of 6:1:1. Sterilize the mixed matrix at 121℃ for 40 minutes and set aside.

[0077] 3.3.2 Preparation of spore suspension

[0078] In a clean bench, use a scalpel to take the stressed spores into a 1.5 mL centrifuge tube and dissolve them with sodium citrate. After dissolution, use a pipette to transfer the spores into a centrifuge tube filled with sterile water, with about 100 spores per tube.

[0079] 3.3.3 Vaccination

[0080] (1) Disinfection before inoculation: Before transplanting, each planting pot needs to be wiped with alcohol for sterilization and a filter membrane should be placed at the bottom to reduce contamination by foreign bacteria.

[0081] (2) Inoculation: First, pour 1 / 2 of the substrate into the pot, mix about 100-200 spores with one-sixth of the substrate and place them in the middle of the pot. Next, select seedlings of uniform robustness, plant three seedlings evenly in each pot, sprinkle about 100-200 spore suspensions on the roots of each seedling, and finally attach a layer of quartz sand. Pour 1mL of pure water into each pot, insert a bamboo stick, and attach a film. There are a total of 9 treatment groups, each with three replicates, for a total of 27 pots. Disposable sterile gloves must be worn when transplanting, and gloves must be changed between each treatment.

[0082] (5) Management: After 3-7 days, when the corn seedlings are about to touch the film, lift the film and remove the bamboo sticks. The planting time is November. Keep the indoor temperature at 18-25℃ every day and open the windows for ventilation at around 10 o'clock in the morning. In the first month of planting, water each pot with 1L of Hogland nutrient solution per week. In the second month, water each pot with 1L of low-phosphorus Hogland nutrient solution per week. Plant for a total of 60 days.

[0083] 3.4 Measurement of spore structure

[0084] Measurement of spore diameter: Under a stereo microscope at 120×, all spores in the grid were collected, and then the diameter of each mature spore was measured using CAD software. Approximately 300 spores were measured for each treatment group.

[0085] Measurement of spore wall thickness: sterilize the sodium citrate solution, scalpel, and sterile water centrifuge tube at 121°C for 20 minutes. Operate in a clean bench, place the sterilized scalpel in an inoculation sterilizer at 300°C for sterilization, and then cool it for use. Use a scalpel to cut the spores in the grid and place them in centrifuge tubes containing sodium citrate. After sodium citrate dissolves the culture medium, replace the sodium citrate solution with sterile water. The dissolved spores are entangled with the hyphae, so they can be seen with the naked eye. Mark them and place them in a 4°C refrigerator for later use. Use a pipette to transfer the spores from the centrifuge tube to a slide, and use a dissecting needle to separate the spores from the hyphae under a stereo microscope. Cover with a coverslip, press the coverslip steadily and moderately, the spore contents flow out, and sterile water is injected from the side to disperse the contents near the spore wall. This operation covers the spore wall to prevent the contents from flowing out, affecting observation. Under a microscope, the spore wall can be clearly observed. Under a microscope at 400 times magnification, collect images of the spore wall of each spore, and measure approximately 300 spores in each treatment group. Save the collected images, and use CAD software to measure the thickness of the spore wall of each spore.

[0086] 3.5 Measurement of spore infectivity

[0087] Carefully remove the seedlings from the substrate to reduce root damage, and wash them in a 60cm×120cm blue basin until the residual substrate on the seedlings is removed. Wipe the moisture on the roots, place them on a black curtain and take photos to record the root status. After taking photos, immediately put them in water to prevent root cell damage. Randomly take the upper, middle and lower parts of the roots, stain them using trypan blue staining, and prepare slides. About 300 roots were observed in each group, and the infection rate was counted under a microscope.

[0088] The method of trypan blue staining and slide preparation comprises the following steps:

[0089] (1) Fixation: Select thin and tough roots, wash them, dry them with filter paper, cut them into 1 cm long root segments, place them in a glass bottle, add FAA fixative to immerse the root segments, and fix them for more than 4 hours.

[0090] (2) Transparency: Take out 2g of root segments from the FAA fixative, rinse with water and drain, place in a 50mL beaker, add 10% (w / v) KOH to immerse the root segments. The immersion time and heating degree vary with the thickness and hardness of the roots. Usually heat in a water bath at 90°C for 1h. After heating, pour out the KOH and gently rinse the root sample with clean water several times (do not stir vigorously until the water is no longer yellow, drain the water, and place in a 50mL beaker. (Roots with darker colors should be decolorized with alkaline hydrogen peroxide after washing).

[0091] (3) Staining: Lactated acid glycerol method: Add 5% lactic acid solution to the cup, soak for 5 minutes, and pour out the lactic acid. Add 0.05% trypan blue dye and soak in a 90℃ water bath for 30 minutes (determine the time according to the condition of the roots). Pour out the excess dye, add lactated acid glycerol and soak several times until most of the excess dye in the roots is removed. The root segments need to be kept in lactated acid glycerol during the staining process.

[0092] (4) Preparation: Use tweezers to take out the stained root segments and arrange them neatly on a clean glass slide. Place 10 root segments on one slide. Then add 2-3 drops of lactic acid to the slide, put a cover slip in, and gently squeeze the root segments for easy observation. Observe under a microscope and calculate the statistics.

[0093] Statistical method for root infection rate:

[0094] The root infection rate was graded using the Trouvelot method, which divided the AM fungal infection rate in plant root segments into five levels (N0=0, N1 <1%, N2 <10%, N3 <50%, N4 <70%, N5 >90%). F% was the infection rate (Frequency of mycorrhiza in the root system), and M% was the intensity of mycorrhiza infection in the entire host root system (Intensity of the mycorrhiza in the root system).

[0095] F% = (number of root segments infected by AMF in the sample / total number of root segments in the sample) × 100

[0096] M% = (95×N5+70×N4+30×N3+5×N2+N1) / total number of root segments of the sample

[0097] 3.6 Conclusion

[0098] 3.6.1 Analysis of the changes in spore structure after stress treatment

[0099] When the spores in the reproductive growth stage were treated with temperature and UV stress, the spore morphology changed, which was manifested in the thickness of the spore wall, the size of the spores and the content of the spore contents ( Figure 4, 5 ). Through statistical analysis of spore diameter data, it was found that the average spore diameter of the control group 25 was 133.016μm, and the average spore diameters of the treatment groups 43 and ZW5 were 133.979μm and 133.182μm, respectively, which were not significantly different from the control group. The average diameters of the treatment groups 46, 373, 376, and ZW15 were 126.502μm, 123.415μm, 124.667μm, and 133.182μm, respectively, and the spores became smaller than those in the control group; the average spore diameters of the treatment groups ZW10 and ZW20 were 144.196μm and 139.105μm, respectively, which were significantly different from those in the control group, and the spore diameters increased ( Figure 6 A). Figure 4 It can be visually seen that the spores increase in size after stress.

[0100] The statistical analysis of spore wall thickness showed that the average spore wall thickness of the control group 25 was 2.972 μm, while the average spore wall thickness of the treatment groups ZW20, 373, and ZW5 were 2.830 μm, 3.080 μm, and 3.087 μm, respectively, which were not significantly different from the control group. The average spore wall thickness of the treatment groups 46, ZW10, and ZW15 were 2.346 μm, 2.70 μm, and 2.511 μm, respectively, which were reduced compared with the treatment groups. The average spore wall thickness of the treatment groups 43 and 376 were 3.505 μm and 3.167 μm, respectively, which were significantly different from the control group, and the spore wall was thicker ( Figure 6 B). The relationship between spore size and spore wall thickness was observed and analyzed, and it was found that there was no positive correlation between spore diameter and spore wall thickness. Therefore, the increase in spore size may be caused by the thickening of the spore wall and the increase in spore contents.

[0101] 3.6.2 Analysis of the results of improving the symbiotic rate of spores with corn after stress treatment

[0102] Through the corn pot experiment, the infection rate and infection intensity of spores were calculated. The corn roots stained with trypan blue staining method clearly showed the hyphae and arbuscules in the roots after AMF and corn roots coexisted. Figure 7 ). Infection rate analysis showed that ( Figure 8 A): The infection rate of the control group 25 was 29.1%, and the infection rates of the treatment groups 373, ZW5, ZW15, 46, ZW20, ZW10, 43, and 376 were 49.8%, 51.1%, 55.1%, 56.2%, 62.3%, 62.8%, 65.7%, and 76.3%, respectively; the infection rates of the treatment groups were all higher than those of the control group, and the infection rate of the treatment group 376 was the highest, about 2.6 times that of the control group. Infection intensity data analysis ( Figure 8B): The infection intensity of the control group 25 was 8.9%, and the infection rates of the treatment groups 373, 46, ZW5, ZW15, ZW20, ZW10, and 376 were 27.1%, 30.9%, 31.6%, 33.8%, 34.4%, 36.7%, and 40.7%, respectively. The infection intensity of the treatment groups was higher than that of the control group, and the infection intensity of the treatment group 376 was the highest, which was 3.6 times higher than that of the control group.

[0103] The infection rate and infection intensity of spores are the most intuitive indicators for checking spore activity. From the data analysis of spore size, spore wall thickness, infection rate and infection intensity, it can be found that the spore morphology of treatment groups 43, ZW10, and 376 changed, and the infection rate and infection intensity increased, indicating that changes in spore morphology can improve spore activity; among them, the infection rate and infection intensity of treatment group 376 were higher than those of treatment group ZW10 with enlarged spores and treatment group 43 with the highest spore wall thickening, indicating that the moderate increase in spore wall thickness has the best effect on improving spore activity.

[0104] The infection degree, number and size of hyphae and arbuscules in the control group were relatively small ( Figure 7 A). After the stress, the spores are infected and the number of hyphae in the root is large and intertwined, and a single arbuscule fills the entire cell ( Figure 7 B). Figure 7 Comparison between A and B shows that the spores are more abundant after stress than those without stress. Figure 7 C is the infection status of the whole root after stress. Figure 7 D. The circle represents a branch, which is like the "lungs" of humans, continuously supplying nutrients to the plant.

[0105] The above is a detailed implementation method and a specific operation process of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above embodiment.

Claims

1. A method for improving the growth and infection activity of arbuscular mycorrhizal fungi, characterized in that: According to the life habits of arbuscular mycorrhizal fungi spores, abiotic stress is applied during the spore vegetative growth period and reproductive growth period, respectively. The vegetative growth period refers to the time when hyphae grow one month after the spores colonize the host, and the reproductive growth period refers to the time when spores grow two months after the spores infect the host; Specifically, the abiotic stress condition for increasing spore yield during the vegetative growth period is 4° C. stress for 6 days, and the abiotic stress condition for increasing spore infection ability during the reproductive growth period is 4° C. stress for 3 days, or 37° C. stress for 6 days.

2. A method for improving the growth and infection activity of arbuscular mycorrhizal fungi according to claim 1, characterized in that: The colonization host during the vegetative growth period is carrot root.

3. A method for improving the growth and infection activity of arbuscular mycorrhizal fungi according to claim 1, characterized in that: The host of infection during the reproductive growth period is corn.

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