Arbuscular mycorrhizal fungus inoculant capable of improving cold resistance of durian and preparation method of arbuscular mycorrhizal fungus inoculant

Abstract

The invention provides an arbuscular mycorrhizal fungus inoculant for improving cold resistance of durian and a preparation method of the arbuscular mycorrhizal fungus inoculant, and belongs to the technical field of microbial inoculants.The preparation method of the arbuscular mycorrhizal fungus inoculant comprises the following steps that subfrigid zone plant rhizosphere soil is collected, and arbuscular mycorrhizal fungus spores in the soil are screened out; arbuscular mycorrhizal fungal spores are inoculated to a sandy soil mixed culture medium, then plants such as white clover or ryegrass are planted for enrichment culture, and a culture is obtained; mixing the culture with a cutting medium, and performing rooting culture and first low-temperature culture on annual durian branches; and refrigerating and crushing root systems of the cutting seedlings, mixing the crushed root systems with a cutting medium, cutting annual durian branches for second low-temperature culture, and collecting the root systems and rhizosphere soil of the cutting seedlings to obtain the arbuscular mycorrhizal fungus fungicide. The arbuscular mycorrhizal fungus inoculant can induce durian to generate low-temperature-resistant protective enzyme, so that the cold resistance of the durian is improved, and a new thought is provided for cultivating durian cold-resistant varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, and in particular relates to an arbuscular mycorrhizal fungal agent capable of improving the cold resistance of durian and a preparation method thereof. Background Art

[0002] Durian is known as the "king of fruits". It has high nutritional value and unique flavor and is deeply loved by consumers. As the world's largest durian consumer market, China consumes nearly two million tons of fresh durian every year. However, durian is a tropical fruit and is very sensitive to low temperatures. Low temperature environments will seriously affect the growth and fruiting of durian. Studies have shown that durian cannot grow normally in an environment with a temperature below 15°C, and the lowest temperature it can tolerate is around 5°C. Although there are sporadic plantings in Hainan, Guangxi and other places, and a small amount of domestic durian is on the market, it is far from meeting the market demand for fresh durian and its derivatives. Therefore, cultivating cold-resistant durian varieties is of great significance for expanding the planting area of ​​durian and increasing its yield.

[0003] The research and cultivation of cold-resistant durian varieties at home and abroad lag far behind market demand. The only literature available at present is the comparative analysis of its cold-resistant physiological mechanism and cold-resistant varieties, and no invention patents for the cultivation of cold-resistant durian varieties have been found. In 2023, a 30-year-old durian tree that survived frost was found in Mengla County, Xishuangbanna, Yunnan, but it is still unknown whether it is a cold-resistant variety. Therefore, it is of great strategic significance for the durian industry to cultivate cold-resistant durian varieties as soon as possible. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an arbuscular mycorrhizal fungal agent that can improve the cold resistance of durian and a preparation method thereof, so as to solve the problem that the existing durian germplasm is sensitive to low temperature.

[0005] The present invention provides a method for preparing an arbuscular mycorrhizal fungal agent, comprising the following steps:

[0006] 1) Collect rhizosphere soil of subarctic plants and screen out arbuscular mycorrhizal fungal spores in the soil;

[0007] 2) inoculating the arbuscular mycorrhizal fungi spores into a sandy soil culture matrix, then planting plants for enrichment culture and obtaining a culture;

[0008] 3) after mixing the culture with the cutting medium, cutting one-year-old durian branches for rooting culture, and then performing a first low-temperature culture to obtain the root system of the cutting seedlings;

[0009] 4) Refrigerate and crush the root system of the cutting seedlings and mix them with the cutting medium, cut the one-year-old durian branches for a second low-temperature culture to obtain the cutting seedlings, and collect the root system and rhizosphere soil of the cutting seedlings to obtain the arbuscular mycorrhizal fungal agent.

[0010] Preferably, in step 2), the inoculation amount of arbuscular mycorrhizal fungi spores is 40 to 60 spores / 10 sand-soil mixed culture matrix, and the mass ratio of sand to soil in the sand-soil mixed culture matrix is ​​(10 to 8): (1 to 3); the time of the enrichment culture is 80 to 100 days; and the planted plants include white clover and / or ryegrass.

[0011] Preferably, the mass ratio of the culture medium to the cutting medium in step 3) is (1-1.5):50; the daytime temperature of the rooting culture in step 3) is 26-30°C, and the nighttime temperature of the rooting culture is 18-22°C; the time of the rooting culture is 85-95d.

[0012] Preferably, in step 3), the temperature of the first low-temperature culture is 15-18° C., and the time of the first low-temperature culture is 35-40 days.

[0013] Preferably, after the cutting in step 3), chitosan oligosaccharide solution is irrigated every 2 to 4 days, the concentration of the chitosan oligosaccharide solution is 13 wt% to 18 wt%, and the irrigation amount of the chitosan oligosaccharide solution is 15 to 25 ml / plant.

[0014] Preferably, the refrigeration temperature in step 4) is 5 to 8° C., and the refrigeration time is 30 to 40 days.

[0015] Preferably, in step 4), the daytime temperature of the second low-temperature culture is 14-16° C., the nighttime temperature of the second low-temperature culture is 9-11° C., and the duration of the second low-temperature culture is 120-150 days.

[0016] The invention provides the arbuscular mycorrhizal fungal agent, wherein the concentration of arbuscular mycorrhizal fungal spores in the arbuscular mycorrhizal fungal agent is greater than 100 per 10g of dry soil.

[0017] The present invention also provides the use of the arbuscular mycorrhizal fungal agent in improving the cold resistance of durian, and the arbuscular mycorrhizal fungal agent is inoculated into the rhizosphere of the durian plant.

[0018] The present invention also provides the use of the arbuscular mycorrhizal fungal agent in cultivating cold-resistant durian varieties.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the preparation method of the arbuscular mycorrhizal fungal agent provided by the present invention, by interacting the arbuscular mycorrhizal fungi with the root system of durian cutting seedlings, and culturing at low temperature, screens out the arbuscular mycorrhizal fungi that can infect the durian root system, still has the ability to germinate after being refrigerated in the durian root system, and can successfully infect the durian root system again; the arbuscular mycorrhizal fungal agent described in the present invention can induce durian to produce protective enzymes and osmotic regulating substances that resist low temperatures, thereby improving the cold resistance of durian; and provides a new idea for breeding cold-resistant durian varieties. DETAILED DESCRIPTION

[0020] The invention provides a method for preparing an arbuscular mycorrhizal fungal agent, comprising the following steps: 1) collecting rhizosphere soil of subarctic plants, and screening out arbuscular mycorrhizal fungal spores in the soil; 2) inoculating the arbuscular mycorrhizal fungal spores into a sandy soil culture matrix, then planting plants for enrichment culture and obtaining a culture; 3) mixing the culture with a cutting medium, cutting one-year-old durian branches for rooting culture, and then performing a first low-temperature culture to obtain a cutting seedling root system; 4) refrigerating and crushing the cutting seedling root system, mixing it with the cutting medium, cutting one-year-old durian branches for a second low-temperature culture to obtain a cutting seedling, and collecting the root system and rhizosphere soil of the cutting seedling to obtain the arbuscular mycorrhizal fungal agent.

[0021] In the present invention, rhizosphere soil of subarctic plants is collected, and arbuscular mycorrhizal fungal spores in the soil are screened out. In the present invention, the subarctic zone is preferably the Heihe region of China, and the plants preferably include grapevines or pear trees. In the specific implementation of the present invention, rhizosphere soil of grapevines or pear trees in the Heihe region of China is preferably collected; the screening is performed by wet sieving decantation-sucrose density gradient centrifugation.

[0022] After obtaining the screened arbuscular mycorrhizal fungal spores, the present invention inoculates the arbuscular mycorrhizal fungal spores into a sand-soil mixed culture matrix, and then the plant is enriched and cultured to obtain a culture. In the present invention, the inoculation amount of the arbuscular mycorrhizal fungal spores is preferably 40 to 60 spores / 10g dry sand, and the mass ratio of sand to soil in the sand-soil mixed culture matrix is ​​(10 to 8): (1 to 3), preferably 9:2; the water content of the sand-soil mixed culture matrix is ​​preferably 25% to 35%, more preferably 28% to 32%; in the present invention, the plant includes white clover and / or ryegrass. The present invention has no special limitation on the temperature of the enrichment culture, and the natural environment temperature can be used; the time of the enrichment culture is preferably 80 to 100 days, more preferably 85 to 95 days, and most preferably 90 days.

[0023] After the enrichment culture is completed, the present invention mixes the culture with the cutting medium, cuts the annual durian branches for rooting culture, and then performs the first low-temperature culture to obtain the cutting seedling root system. In the present invention, the culture is a total culture of plant roots infected with arbuscular mycorrhizal fungal spores, sandy soil, and arbuscular mycorrhizal fungal spores. In the present invention, the cutting medium includes peat soil, river sand, perlite and vermiculite; the mass ratio of the peat soil, river sand, perlite and vermiculite is preferably (1-3:): (2-4): (1-3): (2-4), and more preferably 2:3:2:3; the cutting medium is preferably sterilized before use, and the sterilization treatment is preferably 121°C steam intermittent sterilization, and the number of sterilizations is preferably 1-3 times, and more preferably 3 times. In the present invention, the mass ratio of the culture medium to the cutting medium is preferably (1-1.5):50, preferably (1.1-1.3):50. In the present invention, rooting culture is performed after cutting, and the daytime temperature of the rooting culture is preferably 26-30°C, more preferably 27-29°C, and the nighttime temperature of the rooting culture is preferably 18-22°C, more preferably 19-21°C; the time of the rooting culture is preferably 85-95d, more preferably 88-92d.

[0024] According to the present invention, after the cutting, in the rooting culture stage, the chitosan oligosaccharide solution is irrigated once every 2 to 4 days, preferably once every 3 days; the concentration of the chitosan oligosaccharide solution is preferably 13wt% to 18wt%, more preferably 14wt% to 17wt%, and the irrigation amount of the chitosan oligosaccharide solution is preferably 15 to 25ml / plant, more preferably 18 to 22ml / plant. In the present invention, the degree of polymerization of the chitosan oligosaccharide used is 25 to 40, and the role of irrigating the chitosan oligosaccharide solution is to help the spores adhere to the root epidermis while retaining water, and inhibit the growth of other microorganisms except AM fungal spores.

[0025] The present invention performs a first low-temperature culture after the rooting culture to obtain the root system of the cutting seedlings. In the present invention, the temperature of the first low-temperature culture is preferably 15 to 18°C, and the time of the first low-temperature culture is preferably 35 to 40 days. After obtaining the root system of the cutting seedlings, the present invention preferably rinses the root system with running water for 3 to 5 minutes, and then dries the surface moisture of the root system at room temperature.

[0026] After obtaining the root system of the cutting seedling, the root system of the cutting seedling is refrigerated, crushed and mixed with the cutting medium, and the cutting seedling is cut into one-year-old durian branches for a second low-temperature culture to obtain the cutting seedling, and the root system and rhizosphere soil of the cutting seedling are collected to obtain the arbuscular mycorrhizal fungal agent. In the present invention, the temperature of the refrigeration is preferably 5-8°C, and the time of the refrigeration is preferably 30-40d. In the present invention, the refrigerated cutting seedling root system contains arbuscular mycorrhizal fungal hyphae and root endospores that successfully infect the durian root system, and the cutting seedling root system is used to eliminate the strain spores that cannot infect the durian root system. The purpose of the refrigeration is to induce the arbuscular mycorrhizal fungi to produce secondary metabolites and protective enzymes that can resist low temperature in the durian root environment. After the refrigeration is completed, the present invention is crushed, and the crushing is preferably carried out using a crusher. Then the crushed cutting seedling root system is mixed with the cutting medium, and the mass ratio of the crushed cutting seedling root system to the cutting medium is preferably (1-1.5): 1000, and more preferably (1.1-1.4): 1000. The present invention has no special limitation on the one-year-old durian branch, and the conventional one-year-old durian tender branch in the art can be used.

[0027] In the present invention, the daytime temperature of the second low-temperature culture is preferably 14-16°C, more preferably 15°C, the nighttime temperature of the second low-temperature culture is preferably 9-11°C, more preferably 10°C; the time of the second low-temperature culture is preferably 120-150 days. After the second low-temperature culture is completed, the root system and rhizosphere soil of the cutting seedlings are collected to obtain the arbuscular mycorrhizal fungal agent.

[0028] The invention provides the arbuscular mycorrhizal fungal agent, wherein the concentration of arbuscular mycorrhizal fungal spores in the arbuscular mycorrhizal fungal agent is greater than 100 per 10g of dry soil.

[0029] The present invention also provides the use of the arbuscular mycorrhizal fungal agent in improving the cold resistance of durian, and the arbuscular mycorrhizal fungal agent is inoculated into the rhizosphere of the durian plant.

[0030] The present invention also provides the use of the arbuscular mycorrhizal fungal agent in cultivating cold-resistant durian varieties.

[0031] The arbuscular mycorrhizal fungal agent provided by the invention can effectively improve the cold resistance of durian, so that the durian can still maintain normal growth after 30 days at a low temperature of 5 to 10°C.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] Grape rhizosphere soil was collected from Heihe area of ​​China, and the arbuscular mycorrhizal fungal spores in the soil were screened out. The spores were screened by wet sieving decantation-sucrose density gradient centrifugation without classification and identification. Then, the arbuscular mycorrhizal fungal spores were inoculated into a sandy soil mixed culture matrix (the mass ratio of sand to soil was 10:3, and the water content was 30%). The inoculation amount of the arbuscular mycorrhizal fungal spores was 45 spores / 10g dry sandy soil. After planting white clover, enrichment and propagation culture was carried out for 90 days.

[0035] The culture of the arbuscular mycorrhizal agent after propagation and cultivation (including spores, sandy soil and infected plant roots, etc.) was mixed with a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:3:2:3, 121°C steam intermittent sterilization twice) at a weight ratio of 1.5:50, and then the annual tender branches of durian (variety: Mao Shan Wang) were cut into the medium. After cutting, each cutting seedling was irrigated with 20 mL of a 15% chitosan oligosaccharide solution (chitosan oligosaccharide polymerization degree of 25-40) every 3 days, and the ambient temperature was maintained at 28°C during the day and 20°C at night.

[0036] 90 days after the above-mentioned cuttings took root, the ambient temperature was controlled at 15°C for 35 days, and then the roots of the cuttings were collected. The roots were rinsed with running water for 5 minutes, and then the roots were dried at room temperature and placed in a refrigerator, maintained at 5°C for 40 days;

[0037] The root system after refrigeration is taken out, crushed with a crusher, and then mixed into a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:3:2:3, 121°C steam intermittent sterilization twice) at a weight ratio of 1:1000, and then the annual durian shoots are cut into the medium, and after cutting, each cutting seedling is irrigated with 20 ml of a 15% chitosan oligosaccharide solution (chitosan oligosaccharide polymerization degree is 25-40) every 3 days, and the ambient temperature is maintained at 15°C during the day and 10°C at night.

[0038] After the above-mentioned cuttings have taken root for 150 days, the roots and the rhizosphere soil are taken out and dried, and the concentration of arbuscular mycorrhizal fungi spores is measured to be greater than 100 / 10g dry soil to obtain an arbuscular mycorrhizal fungi agent.

[0039] Example 2

[0040] Rhizosphere soil of pear trees in Heihe area of ​​China was collected, and the arbuscular mycorrhizal fungal spores in the soil were screened out. The wet sieving decantation-sucrose density gradient centrifugation method was used for screening, and no classification and identification were required. Then, the arbuscular mycorrhizal fungal spores were inoculated into a sandy soil mixed culture matrix (the mass ratio of sand to soil was 9:2, and the water content was 35%). The inoculation amount of arbuscular mycorrhizal fungal spores was 55 spores / 10g dry sandy soil. After planting ryegrass, enrichment and expansion culture was carried out for 90 days.

[0041] The culture of the arbuscular mycorrhizal agent after propagation and cultivation (including spores, sandy soil and infected plant roots, etc.) was mixed with a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:4:3:4, 121°C steam intermittent sterilization twice) at a weight ratio of 1:50, and then the annual tender branches of durian (variety: Mao Shan Wang) were cut into the medium. After cutting, each cutting seedling was irrigated with 15 ml of a chitosan oligosaccharide solution with a concentration of 18% (the degree of polymerization of chitosan oligosaccharide was 25-40) every 3 days, and the ambient temperature was maintained at 28°C during the day and 20°C at night.

[0042] 95 days after the above-mentioned cuttings took root, the ambient temperature was controlled at 15°C for 35 days, and then the roots of the cuttings were collected. The roots were rinsed with running water for 3 minutes, and then the roots were dried at room temperature and placed in a refrigerator, maintained at 8°C for 35 days;

[0043] The root system after refrigeration is taken out, crushed with a crusher, and then mixed into a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:4:3:4, 121°C steam intermittent sterilization twice) at a weight ratio of 1.5:1000, and then the annual durian shoots are cut into the medium, and after cutting, each cutting seedling is irrigated with 15 ml of 18% chitosan oligosaccharide solution (chitosan oligosaccharide polymerization degree is 25-40) every 3 days, and the ambient temperature is maintained at 15°C during the day and 10°C at night.

[0044] 100 days after the above-mentioned cuttings have taken root, the roots and rhizosphere soil thereof are taken out and dried, and the concentration of arbuscular mycorrhizal fungi spores is measured to be greater than 100 / 10g dry soil to obtain an arbuscular mycorrhizal fungi agent.

[0045] Example 3

[0046] Grape rhizosphere soil was collected from Heihe area of ​​China, and the arbuscular mycorrhizal fungal spores in the soil were screened out. The wet sieving decantation-sucrose density gradient centrifugation method was used for screening, and no classification and identification were required. Then, the arbuscular mycorrhizal fungal spores were inoculated into a sandy soil mixed culture matrix (the mass ratio of sand to soil was 8:1, and the water content was 28%). The inoculation amount of the arbuscular mycorrhizal fungal spores was 60 spores / 10g dry sandy soil. After planting ryegrass, the enrichment and propagation culture was carried out for 90 days.

[0047] The culture of the arbuscular mycorrhizal agent after propagation and cultivation (including spores, sandy soil and infected plant roots, etc.) was mixed with a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:3:2:3, 121°C steam intermittent sterilization twice) at a weight ratio of 1.3:50, and then the annual tender branches of durian (variety: Mao Shan Wang) were cut into the medium. After cutting, each cutting seedling was irrigated with 25 mL of a 13% chitosan oligosaccharide solution (chitosan oligosaccharide polymerization degree of 25-40) every 3 days, and the ambient temperature was maintained at 28°C during the day and 20°C at night.

[0048] 90 days after the above-mentioned cuttings took root, the ambient temperature was controlled at 15°C for 40 days, and then the roots of the cuttings were collected. The roots were rinsed with running water for 5 minutes, and then the roots were dried at room temperature and placed in a refrigerator, maintained at 5°C for 40 days;

[0049] The root system after refrigeration is taken out, crushed with a crusher, and then mixed into a cutting medium (peaty soil, river sand, perlite and vermiculite mass ratio of 2:3:2:3, 121°C steam intermittent sterilization twice) at a weight ratio of 1.5:1000, and then the annual durian shoots are cut into the medium, and after cutting, each cutting seedling is irrigated with 25 ml of a 13% chitosan oligosaccharide solution (chitosan oligosaccharide polymerization degree is 25-40) every 3 days, and the ambient temperature is maintained at 15°C during the day and 10°C at night.

[0050] After the above-mentioned cuttings have taken root for 150 days, the roots and the rhizosphere soil are taken out and dried, and the concentration of arbuscular mycorrhizal fungi spores is measured to be greater than 100 / 10g dry soil to obtain an arbuscular mycorrhizal fungi agent.

[0051] Experimental Example 1

[0052] The arbuscular mycorrhizal fungi prepared in Examples 1 to 3 were mixed with cutting substrates (peaty soil, river sand, perlite and vermiculite in a mass ratio of 2:3:2:3, and sterilized twice with steam at 121°C) in a mass ratio of 1:50 to prepare culture media A, B and C; the control culture media D was the cutting substrate. 30 cutting treatments were set for each culture media.

[0053] One-year-old durian shoots (variety: Musang King) with a shoot length of 15 cm and a diameter of 1-2 cm were cut obliquely at the downward end and inserted into the culture medium. They were placed in a greenhouse at 25-28°C and watered normally for 90 days. They were then placed at 15°C for 30 days and then at 10°C for 30 days. The growth rate of the cuttings in each culture medium was observed and recorded. The results are shown in Table 1.

[0054] Growth rate of each stage (%) = [(plant height at the end of the stage - plant height at the beginning of the stage) / plant height at the beginning of the stage] × 100%

[0055] At 25-28°C, the growth status of the cuttings in the culture media A-D was basically the same, with no significant difference. At 15°C, the growth of the cuttings in the culture media A-C was significantly better than that in the culture media D. At 10°C, the cuttings on the culture media D did not grow, but all dried up and died. The cuttings in the culture media A-C were able to grow normally, and the growth was basically the same as that at 15°C. It can be seen that the arbuscular mycorrhizal fungal agent provided by the present invention can improve the cold resistance of durian.

[0056] Table 1 Average growth rate of durian cutting seedlings at different culture stages

[0057]

[0058] It can be seen from the above examples that the arbuscular mycorrhizal fungal agent obtained by the preparation method of the arbuscular mycorrhizal fungal agent provided by the present invention can improve the cold resistance of durian, so that durian can maintain normal growth after 30 days at a low temperature of 5-10°C.

[0059] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an arbuscular mycorrhizal fungal agent for improving the cold resistance of durian, comprising the following steps: 1) Collect rhizosphere soil of subarctic plants and screen out arbuscular mycorrhizal fungal spores in the soil; 2) inoculating arbuscular mycorrhizal fungi spores into a sand-soil mixed culture matrix, then planting plants for enrichment culture and obtaining cultures; 3) after mixing the culture with the cutting medium, cutting one-year-old durian branches for rooting culture, and then performing a first low-temperature culture to obtain the root system of the cutting seedlings; 4) Refrigerate and crush the root system of the cutting seedlings and mix them with the cutting medium, cut the one-year-old durian branches for a second low-temperature culture to obtain the cutting seedlings, and collect the root system and rhizosphere soil of the cutting seedlings to obtain the arbuscular mycorrhizal fungal agent.

2. The preparation method according to claim 1, characterized in that: Step 2) The inoculation amount of the arbuscular mycorrhizal fungi spores is 40 to 60 spores / 10g dry sandy soil, the mass ratio of sand to soil in the sandy soil mixed culture matrix is ​​(10 to 8): (1 to 3); the enrichment culture time is 80 to 100 days; the planted plants include white clover and / or ryegrass.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the culture material to the cutting medium in step 3) is 1-1.5:50; the daytime temperature of the rooting culture in step 3) is 26-30° C., and the nighttime temperature of the rooting culture is 18-22° C.; the time of the rooting culture is 85-95 days.

4. The preparation method according to claim 3, characterized in that: Step 3) The temperature of the first low-temperature culture is 15-18°C, and the time of the first low-temperature culture is 35-40 days.

5. The preparation method according to claim 4, characterized in that: Step 3) After the cutting, watering with chitosan oligosaccharide solution every 2 to 4 days, the concentration of the chitosan oligosaccharide solution is 13wt% to 18wt%, and the watering amount of the chitosan oligosaccharide solution is 15 to 25 ml / plant.

6. The preparation method according to claim 1, characterized in that: Step 4) The refrigeration temperature is 5 to 8° C. and the refrigeration time is 30 to 40 days.

7. The preparation method according to claim 6, characterized in that: In step 4), the daytime temperature of the second low-temperature culture is 14-16° C., the nighttime temperature of the second low-temperature culture is 9-11° C., and the duration of the second low-temperature culture is 120-150 days.

8. The arbuscular mycorrhizal fungal agent prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The concentration of arbuscular mycorrhizal fungi spores in the arbuscular mycorrhizal fungi agent is greater than 100 spores / 10g dry soil.

9. Use of the arbuscular mycorrhizal fungal agent prepared by the preparation method according to any one of claims 1 to 7 or the arbuscular mycorrhizal fungal agent according to claim 8 in improving the cold resistance of durian, characterized in that: The arbuscular mycorrhizal fungal agent is inoculated into the rhizosphere of the durian plant.

10. Use of the arbuscular mycorrhizal fungal agent prepared by the preparation method according to any one of claims 1 to 7 or the arbuscular mycorrhizal fungal agent according to claim 8 in cultivating cold-resistant durian varieties.

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