A mycorrhizal fungus agent capable of improving cold resistance of durian and a preparation method thereof

Abstract

The application provides a kind of cluster root fungus agent for improving the cold resistance of durian and a preparation method thereof, and belongs to the technical field of microbial inoculant.The preparation method of the cluster root fungus agent comprises the following steps: collecting subfrigid zone plant rhizosphere soil, and screening cluster root fungus spores in the soil;inoculating the cluster root fungus spores into sand mixed culture medium, then planting white clover or ryegrass and other plants to carry out enrichment culture and obtain culture;mixing the culture with cutting substrate, and cutting one-year-old durian branches to carry out rooting culture and first low-temperature culture;storing the root system of cutting seedlings in cold storage, crushing, mixing with cutting substrate, cutting one-year-old durian branches to carry out second low-temperature culture, and collecting the root system of cutting seedlings and rhizosphere soil to obtain the cluster root fungus agent.The cluster root fungus agent can induce durian to produce low-temperature resistant protective enzymes, thereby improving the cold resistance of durian and providing a new idea for cultivating durian cold-resistant varieties.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial inoculants, and particularly relates to a cluster root fungus inoculant capable of improving the cold resistance of durians and a preparation method thereof. BACKGROUND

[0002] Durians are known as the "King of Fruits" and are highly favored by consumers due to their high nutritional value and unique flavor. However, durians are a tropical fruit and are very sensitive to low temperatures, and low-temperature environments can severely affect the growth and fruiting of durians. Studies have shown that durians cannot grow normally in an environment with an air temperature below 15℃, and the minimum temperature they can tolerate is about 5℃. At present, although there are sporadic plantings in Hainan, Guangxi and other places, and a small amount of domestic durians are sold on the market, they still cannot meet the market demand for durian fresh fruits and their derivatives. Therefore, it is of great significance to cultivate cold-resistant durian varieties to expand the planting area and increase the yield of durians.

[0003] The research and cultivation of cold-resistant durian varieties have lagged far behind market demand at home and abroad. Currently, the only available literature is on the physiological mechanism of cold resistance and comparative analysis of cold-resistant varieties, and no invention patents on the cultivation of cold-resistant durian varieties have been found. Therefore, it is of great strategic significance to cultivate cold-resistant durian varieties as soon as possible for the durian industry. SUMMARY

[0004] Therefore, the present application aims to provide a cluster root fungus inoculant capable of improving the cold resistance of durians and a preparation method thereof, so as to solve the problem of low-temperature sensitivity of existing durian germplasm.

[0005] The present application provides a preparation method of a cluster root fungus inoculant, comprising the following steps:

[0006] 1) Collecting the rhizosphere soil of sub-cold zone plants, and screening the cluster root fungus spores in the soil;

[0007] 2) Inoculating the cluster root fungus spores into a sand soil culture medium, then planting plants for enrichment culture and obtaining a culture;

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

[0009] 4) Cold-storing and crushing the root system of the cutting seedlings, mixing with the cutting substrate, cutting one-year-old durian branches for second low-temperature culture to obtain cutting seedlings, and collecting the root system and rhizosphere soil of the cutting seedlings to obtain the cluster root fungus inoculant.

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

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

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

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

[0014] Preferably, the temperature of the refrigeration of step 4) is 5-8 DEG C, and the refrigeration time is 30-40 days.

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

[0016] The arbuscular mycorrhizal fungus inoculum provided by the application has a spore concentration of greater than 100 spores / 10 g of dry soil.

[0017] The application also provides application of the arbuscular mycorrhizal fungus inoculum in improving cold resistance of durians, wherein the arbuscular mycorrhizal fungus inoculum is inoculated into the rhizosphere of durian plants.

[0018] The application also provides application of the arbuscular mycorrhizal fungus inoculum in cultivating durian cold-resistant varieties.

[0019] Compared with the prior art, the application has the following beneficial effects: the preparation method of the arbuscular mycorrhizal fungus inoculum provided by the application screens out arbuscular mycorrhizal fungi that can infect durian root systems, can still have germination ability after cold storage in durian root systems, and can successfully infect durian root systems again by mutual action of arbuscular mycorrhizal fungi and durian cutting seedling root systems and low-temperature culture; the arbuscular mycorrhizal fungus inoculum can induce durians to produce low-temperature-resistant protective enzymes and osmotic adjustment substances, thereby improving the cold resistance of durians; and the application provides a new idea for cultivating durian cold-resistant varieties. DETAILED DESCRIPTION

[0020] The application provides a preparation method of arbuscular mycorrhizal fungus agent, comprising the following steps: 1) collecting the rhizosphere soil of sub-frigid zone plants, and screening arbuscular mycorrhizal fungus spores in the soil; 2) inoculating the arbuscular mycorrhizal fungus spores into a sand-soil culture medium, then planting plants for enrichment culture and obtaining a culture; 3) mixing the culture with a cutting substrate, cutting year-old durian branches for rooting culture, then performing first low-temperature culture to obtain cutting seedling roots; 4) mixing the cutting seedling roots after cold storage and crushing with the cutting substrate, cutting year-old durian branches for second low-temperature culture to obtain cutting seedlings, and collecting the roots and rhizosphere soil of the cutting seedlings to obtain the arbuscular mycorrhizal fungus agent.

[0021] In the application, the rhizosphere soil of sub-frigid zone plants is collected, and arbuscular mycorrhizal fungus spores in the soil are screened. In the application, the sub-frigid zone is preferably the Heihe region of China, and the plants preferably include grape trees or pear trees. In the specific implementation process of the application, the rhizosphere soil of grape trees or pear trees in the Heihe region of China is collected. The screening is performed by using a wet screening decantation-sucrose density gradient centrifugation method.

[0022] After obtaining the screened arbuscular mycorrhizal fungus spores, the arbuscular mycorrhizal fungus spores are inoculated into a sand-soil mixed culture medium, then plants are subjected to enrichment culture and a culture is obtained. In the application, the inoculation amount of the arbuscular mycorrhizal fungus spores is preferably 40-60 spores / 10 g of dry sand, the mass ratio of sand to soil in the sand-soil mixed culture medium is (10-8):(1-3), preferably 9:2, and the water content of the sand-soil mixed culture medium is preferably 25%-35%, more preferably 28%-32%. In the application, the plants include white clover and / or ryegrass. The application does not have 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 80-100 days, more preferably 85-95 days, and most preferably 90 days.

[0023] The application mixes the culture with a cutting substrate after the enrichment culture is finished, then roots the cut year-old durian branches, and then carries out the first low-temperature culture to obtain the cutting seedling root system. In the application, the culture is a total culture including arbuscular mycorrhizal fungal spores, sand and arbuscular mycorrhizal fungal spore-infected plant roots. In the application, the cutting substrate includes grass carbon soil, river sand, perlite and vermiculite; the mass ratio of the grass carbon soil, river sand, perlite and vermiculite is preferably (1-3):(2-4):(1-3):(2-4), and further preferably 2:3:2:3; the cutting substrate is preferably subjected to sterilization treatment before use, and the sterilization treatment is preferably 121℃ steam intermittent sterilization, and the sterilization times are preferably 1-3 times, and more preferably 3 times. In the application, the mass ratio of the culture and the cutting substrate is preferably (1-1.5):50, and preferably (1.1-1.3):50. The application carries out rooting culture after cutting, and the daytime temperature of the rooting culture is preferably 26-30℃, and further preferably 27-29℃, and the nighttime temperature of the rooting culture is preferably 18-22℃, and further preferably 19-21℃; the time of the rooting culture is preferably 85-95d, and further preferably 88-92d.

[0024] The application irrigates the chitosan oligosaccharide solution every 2-4d, and preferably every 3d, during the rooting culture stage after cutting; the concentration of the chitosan oligosaccharide solution is preferably 13wt%-18wt%, and further preferably 14wt%-17wt%, and the irrigation amount of the chitosan oligosaccharide solution is preferably 15-25ml / plant, and further preferably 18-22ml / plant. In the application, the chitosan oligosaccharide used has a degree of polymerization of 25-40, and the irrigation of the chitosan oligosaccharide solution has the effects of water retention, auxiliary spore adhesion to the root epidermis and inhibition of the growth of other microorganisms except AM fungal spores.

[0025] The application carries out the first low-temperature culture to obtain the cutting seedling root system after the rooting culture. In the application, the temperature of the first low-temperature culture is preferably 15-18℃, and the time of the first low-temperature culture is preferably 35-40d. After the cutting seedling root system is obtained, the root system is preferably washed with running water for 3-5min, and then the root system surface moisture is dried at room temperature.

[0026] The invention obtains the cutting seedling root system, and then mixes the cutting seedling root system after refrigeration and crushing with a cutting substrate to obtain a cutting seedling by second low-temperature culture of a year-old durian branch, and obtains the arbuscular mycorrhizal fungus inoculum by collecting the root system and rhizosphere soil of the cutting seedling. In the invention, the refrigeration temperature is preferably 5-8℃, and the refrigeration time is preferably 30-40d. In the invention, the refrigerated cutting seedling root system is successfully infected with arbuscular mycorrhizal fungus hyphae and intraradical spores, and the cutting seedling root system is used to eliminate spores that cannot infect durian root system, and the purpose of refrigeration is to induce 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 cutting seedling root system is crushed, and the crushing is preferably performed by a crusher. Then the crushed cutting seedling root system is mixed with the cutting substrate, and the mass ratio of the crushed cutting seedling root system to the cutting substrate is preferably (1-1.5):1000, and more preferably (1.1-1.4):1000. The year-old durian branch in the invention is not particularly limited, and a conventional year-old durian branch in the art can be used.

[0027] In the invention, the daytime temperature of the second low-temperature culture is preferably 14-16℃, and more preferably 15℃, the nighttime temperature of the second low-temperature culture is preferably 9-11℃, and more preferably 10℃, and the time of the second low-temperature culture is preferably 120-150d. After the second low-temperature culture is completed, the root system and rhizosphere soil of the cutting seedling are collected to obtain the arbuscular mycorrhizal fungus inoculum.

[0028] The invention provides the arbuscular mycorrhizal fungus inoculum, and the arbuscular mycorrhizal fungus spore concentration in the arbuscular mycorrhizal fungus inoculum is greater than 100 / 10g of dry soil.

[0029] The invention also provides the application of the arbuscular mycorrhizal fungus inoculum in improving the cold resistance of durian, and the arbuscular mycorrhizal fungus inoculum is inoculated into the rhizosphere of the durian plant.

[0030] The invention also provides the application of the arbuscular mycorrhizal fungus inoculum in cultivating durian cold-resistant varieties.

[0031] The arbuscular mycorrhizal fungus inoculum provided by the invention can effectively improve the cold resistance of durian, so that the durian can still grow normally under low temperature of 5-10℃ for 30d.

[0032] The technical solutions provided by the invention are described in detail below in combination with examples, but they should not be understood as limitations on the protection scope of the invention.

[0033] Example 1

[0034] Gather the grape rhizosphere soil in Heihe region of China, screen out the arbuscular mycorrhizal fungal spores in the soil, screen by wet screening decanting-sucrose density gradient centrifugation method, without classification and identification, then inoculate the arbuscular mycorrhizal fungal spores in the sand soil mixed culture medium (the mass ratio of sand and soil is 10:3, the water content is 30%), the inoculation amount of arbuscular mycorrhizal fungal spores is 45 spores / 10g dry sand, plant white clover and then carry out enrichment and propagation culture for 90d.

[0035] Mix the culture of the propagated arbuscular mycorrhizal fungus agent (including spores, sand soil and infected plant roots) with the cutting medium (the mass ratio of grass carbon soil, river sand, perlite and vermiculite is 2:3:2:3, intermittent sterilization by steam at 121℃ for two times) according to the weight ratio of 1.5:50, then cut the year-old tender branches of durian (variety: cat mountain king) in the medium. After cutting, irrigate each cutting seedling with 20 mL of chitosan oligosaccharide solution (chitosan oligosaccharide degree of polymerization is 25-40) with a concentration of 15% every 3d, and maintain the environmental temperature at 28℃ during the day and 20℃ at night.

[0036] After 90d of rooting of the cutting seedlings, control the environmental temperature at 15℃ for 35d, then collect the root system of the cutting seedlings, pay attention to wash the root system with running water for 5min, then dry the surface water of the root system at room temperature, place it in the refrigerator and maintain the temperature at 5℃ for 40d;

[0037] Take out the above refrigerated root system, crush it with a crusher, then mix it into the cutting medium (the mass ratio of grass carbon soil, river sand, perlite and vermiculite is 2:3:2:3, intermittent sterilization by steam at 121℃ for two times) after intermittent sterilization by steam at 121℃ for two times according to the weight ratio of 1:1000, then cut the year-old tender branches of durian in the medium, irrigate each cutting seedling with 20 mL of chitosan oligosaccharide solution (chitosan oligosaccharide degree of polymerization is 25-40) with a concentration of 15% every 3d after cutting, and maintain the environmental temperature at 15℃ during the day and 10℃ at night.

[0038] After 150d of rooting of the above cutting seedlings, take out the root system and its rhizosphere soil, dry them, and measure the concentration of arbuscular mycorrhizal fungal spores, which is greater than 100 / 10g dry soil, to obtain the arbuscular mycorrhizal fungal agent.

[0039] Example 2

[0040] Gather the pear rhizosphere soil in Heihe region of China, screen out the arbuscular mycorrhizal fungal spores in the soil, screen by wet screening decanting-sucrose density gradient centrifugation method, without classification and identification, then inoculate the arbuscular mycorrhizal fungal spores in the sand soil mixed culture medium (the mass ratio of sand and soil is 9:2, the water content is 35%), the inoculation amount of arbuscular mycorrhizal fungal spores is 55 spores / 10g dry sand, plant rye grass and then carry out enrichment and propagation culture for 90d.

[0041] The culture of the mycorrhizal fungus agent after propagation (including spores, sand and infected plant roots, etc.) is mixed with the cutting substrate (grass charcoal, river sand, perlite and vermiculite in a mass ratio of 2:4:3:4, 121℃ intermittent steam sterilization twice) at a weight ratio of 1:50, and then the year-old tender branches of durian (variety: cat mountain king) are cut in the substrate. After cutting, each cutting seedling is irrigated with 15ml of chitosan oligosaccharide solution (chitosan oligosaccharide degree of polymerization is 25-40) with a concentration of 18% every 3d, and the environmental temperature is maintained at 28℃ during the day and 20℃ at night.

[0042] After 95d of rooting of the cutting seedlings, the environmental temperature is controlled at 15℃ for 35d, then the root system of the cutting seedlings is collected, and the root system is washed with running water for 3min, then the root system is dried at room temperature after the surface water is dried, and then placed in a refrigerator at a temperature of 8℃ for 35d;

[0043] The above refrigerated roots are taken out, crushed with a crusher, and mixed into the cutting substrate (grass charcoal, river sand, perlite and vermiculite in a mass ratio of 2:4:3:4, 121℃ intermittent steam sterilization twice) at a weight ratio of 1.5:1000 after 121℃ intermittent sterilization twice, then the year-old tender branches of durian are cut in the substrate, and after cutting, each cutting seedling is irrigated with 15ml of chitosan oligosaccharide solution (chitosan oligosaccharide degree of polymerization is 25-40) with a concentration of 18% every 3d, and the environmental temperature is maintained at 15℃ during the day and 10℃ at night.

[0044] After 100d of rooting of the above cutting seedlings, the root system and its rhizosphere soil are taken out and dried, and the concentration of arbuscular mycorrhizal fungal spores is greater than 100 / 10g of dry soil to obtain the arbuscular mycorrhizal fungal agent.

[0045] Example 3

[0046] The rhizosphere soil of grape in Heihe area of China is collected, and the arbuscular mycorrhizal fungal spores in the soil are screened out by wet screening and sucrose density gradient centrifugation method, without classification and identification, then the arbuscular mycorrhizal fungal spores are inoculated in the sand mixed culture medium (the mass ratio of sand and soil is 8:1, and the water content is 28%), and the inoculation amount of arbuscular mycorrhizal fungal spores is 60 spores / 10g of dry sand, and the ryegrass is planted for enrichment and propagation culture for 90d.

[0047] The culture of the mycorrhizal fungus agent after propagation (including spores, sand and infected plant roots, etc.) is mixed with the cutting substrate (grass carbon soil, river sand, perlite and vermiculite in a mass ratio of 2:3:2:3, 121℃ intermittent steam sterilization twice) at a weight ratio of 1.3:50, and then the year-old tender branches of durian (variety: Cat Mountain King) are cut into the substrate. After cutting, each cutting seedling is irrigated with a 13% chitosan solution (chitosan degree of polymerization 25-40) at a concentration of 25 mL every 3 days, and the environmental temperature is maintained at 28℃ during the day and 20℃ at night.

[0048] After 90 days of rooting of the cuttings, the environmental temperature is controlled at 15℃ for 40 days, then the root system of the cuttings is collected, and the root system is washed with running water for 5 minutes, then the root system is dried at room temperature to remove surface moisture and placed in a refrigerator at 5℃ for 40 days;

[0049] The above refrigerated roots are taken out, crushed with a crusher, and mixed into the cutting substrate (grass carbon soil, river sand, perlite and vermiculite in a mass ratio of 2:3:2:3, 121℃ intermittent steam sterilization twice) at a weight ratio of 1.5:1000, then the year-old tender branches of durian are cut into the substrate, and after cutting, each cutting seedling is irrigated with a 13% chitosan solution (chitosan degree of polymerization 25-40) at a concentration of 25 mL every 3 days, and the environmental temperature is maintained at 15℃ during the day and 10℃ at night.

[0050] After 150 days of rooting of the above cuttings, the root system and its rhizosphere soil are taken out and dried, and the concentration of arbuscular mycorrhizal fungal spores is greater than 100 per 10g of dry soil to obtain an arbuscular mycorrhizal fungal agent.

[0051] Experimental Example 1

[0052] The arbuscular mycorrhizal fungal agents obtained by Examples 1-3 are mixed with the cutting substrate (grass carbon soil, river sand, perlite and vermiculite in a mass ratio of 2:3:2:3, 121℃ intermittent steam sterilization twice) at a mass ratio of 1:50 to prepare culture media A, B and C; the control culture medium D is the cutting substrate. Each culture medium is set up for 30 cutting treatments.

[0053] Year-old durian tender branches (variety: Cat Mountain King), 15cm long, 1-2cm in diameter, with the downward end of the branch inserted into the culture medium, are placed in a greenhouse at 25-28℃, normal watering, and cultured for 90 days. Then placed at 15℃ for 30 days, and then at 10℃ for 30 days. The growth rate of each culture medium cutting seedling is observed and recorded, and the results are shown in Table 1.

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

[0055] In the 25~28℃ stage, the growth states of the cutting seedlings in culture media A~D are basically consistent, and there is no significant difference, in the 15℃ culture stage, the growth states of the cutting seedlings in culture media A~C are obviously better than that in culture medium D, in the 10℃ culture stage, the cutting seedlings in culture medium D do not grow and all wither and die, while the cutting seedlings in culture media A~C can grow normally and the growth states are basically consistent with that in the 15℃ stage.

[0056] Table 1: average growth rate of durian cutting seedlings in different culture stages

[0057]

[0058] From the above examples, it can be seen that the arbuscular mycorrhizal fungus agent provided by the present application can improve the cold resistance of durian, so that the durian can still grow normally after 30d at low temperature of 5~10℃.

[0059] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing an arbuscular mycorrhizal fungal inoculant to improve the cold resistance of durian, comprising the following steps: 1) Collect rhizosphere soil from subarctic plants and sieve out arbuscular mycorrhizal fungal spores from the soil; 2) Inoculate arbuscular mycorrhizal fungal spores into a sand-soil mixed culture medium, then plant plants to enrich the culture and obtain the culture. 3) After mixing the culture with the cutting substrate, rooting culture was carried out on one-year-old durian branches, and then the root system of the cuttings was obtained by first low-temperature culture. 4) After the roots of the cuttings are refrigerated and crushed, they are mixed with the cutting substrate. One-year-old durian branches are then subjected to a second low-temperature culture to obtain cuttings. The roots and rhizosphere soil of the cuttings are collected to obtain arbuscular mycorrhizal fungi inoculant. Step 3) The daytime temperature for the rooting culture is 26~30℃, and the nighttime temperature for the rooting culture is 18~22℃; the rooting culture time is 85~95 days. Step 3) The temperature of the first low-temperature culture is 15-18℃, and the culture time is 35-40 days; Step 3) After the cuttings are planted, water them with chitosan oligosaccharide solution every 2 to 4 days. The concentration of the chitosan oligosaccharide solution is 13wt% to 18wt%, and the amount of chitosan oligosaccharide solution watered is 15 to 25 ml / plant. Step 4) The refrigeration temperature is 5-8℃, and the refrigeration time is 30-40 days; In step 4), the daytime temperature of the second low-temperature culture is 14-16℃, the nighttime temperature of the second low-temperature culture is 9-11℃, and the duration of the second low-temperature culture is 120-150 days.

2. The preparation method according to claim 1, characterized in that, Step 2) The inoculation amount of arbuscular mycorrhizal fungi spores is 40-60 spores / 10 g dry sand, and the mass ratio of sand to soil in the sand-soil mixed culture medium is (10-8):(1-3); the enrichment culture time is 80-100 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 medium to the cutting substrate in step 3) is 1~1.5:

50.

4. The arbuscular mycorrhizal fungal inoculant prepared by the preparation method according to any one of claims 1 to 3, characterized in that, The concentration of arbuscular mycorrhizal fungal spores in the inoculant is greater than 100 spores per 10g dry soil.

5. The application of the arbuscular mycorrhizal fungal inoculant prepared by the preparation method according to any one of claims 1 to 3, or the arbuscular mycorrhizal fungal inoculant according to claim 4, in improving the cold resistance of durian, characterized in that, The arbuscular mycorrhizal fungal agent was inoculated into the rhizosphere of durian plants.

6. The application of the arbuscular mycorrhizal fungal agent prepared by the preparation method according to any one of claims 1 to 3 or the arbuscular mycorrhizal fungal agent according to claim 4 in the cultivation of cold-resistant durian varieties.

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