Application of retinol in promoting the synthesis and / or growth of pine plant-milkissima symbiotic system

Abstract

The present invention belongs to the technical field of fungi / mycorrhiza cultivation, and specifically relates to the application of retinol in promoting the synthesis and / or growth of a pine plant-milk mushroom symbiotic system. The present invention adds retinol to the pine plant-milk mushroom symbiotic system, that is, after the milk mushroom hyphae are inoculated into the root of the pine plant, retinol is added around the root system, which can increase the mycorrhizal tip diameter of the ectomycorrhizal seedlings, increase the contact area between the mycorrhiza and the soil or the cultivation medium, and then increase the mycorrhiza absorption of phosphorus, promote the growth of the pine plant, and improve the quality of the mycorrhizal seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of fungi / mycorrhiza cultivation, and in particular relates to the application of retinol in promoting the synthesis and / or growth of a pine plant-milkissima symbiotic system. Background Art

[0002] Lactarius is a fungus belonging to the Basidiomycetes, Hymeromycetes, Agaricales, Russula family, and Lactarius genus. Lactarius resources are abundant and widely distributed throughout the world, most of which are found in temperate regions. There are about 400 species worldwide, and more than 150 species have been reported in the literature. Its fruiting body is a delicious delicacy on the table, deeply loved by the public, and has considerable economic benefits. There are 140 species of Lactarius fungi reported in my country, and about 40 of them are edible.

[0003] Artificial ecological cultivation of mycorrhizal edible fungi utilizes the symbiotic relationship between trees and fungi to achieve artificial cultivation and multi-year mushroom production through aseptic seedling cultivation, mycorrhizal synthesis, mycorrhizal seedling transplanting, plantation establishment and post-management. This model combines afforestation and starts to produce mushrooms when the seedlings are just grown. After that, continuous harvests can be made for 15-50 years. It is a rare sustainable development model in vegetation restoration and transformation of medium and low-yield fields.

[0004] In 2000, Alexis Guerin-Laguette et al. first cultivated the fruiting bodies of Lactarius pineus under laboratory conditions. Subsequently, researchers from Spain and New Zealand continued to improve the method of mycelial mycorrhization, developed a lower-cost and more practical technology, and were able to synthesize a large number of mycorrhizal seedlings in the greenhouse. The mycorrhizal rate and mycorrhizal structure of mycorrhizal seedlings are indicators for evaluating the quality of mycorrhizal seedlings. The mycorrhizal function has an important impact on the cultivation of host plants in plantations and even mushroom production. However, there are few studies on ectomycorrhiza in this field, and the factors that affect the mycorrhizal rate and mycorrhizal structure are unknown, which cannot effectively promote the synthesis and growth of ectomycorrhiza of Lactarius pineus. Summary of the invention

[0005] The purpose of the present invention is to provide an application of retinol in promoting the synthesis and / or growth of a pine plant-milkissima symbiotic system, wherein the retinol can promote the growth of pine plants and ectomycorrhizal seedlings and improve the quality of the ectomycorrhizal seedlings.

[0006] The present invention provides the use of retinol in promoting the synthesis and / or growth of a pine plant-milkissima symbiotic system.

[0007] Preferably, the pine plant-Lactarius symbiotic system includes the Yunnan pine-Lactarius symbiotic system.

[0008] Preferably, the cultivating comprises promoting the growth of pine plants and / or the growth of ectomycorrhizal seedlings.

[0009] Preferably, the promoting the growth of ectomycorrhizal seedlings comprises increasing the diameter of the mycorrhizal tips of the ectomycorrhizal seedlings.

[0010] Preferably, the promotion of pine plant growth includes one or more of promoting the dry weight of the aboveground part, promoting the dry weight of the underground part, promoting the absorption of phosphorus by the aboveground part and promoting the absorption of phosphorus by the underground part.

[0011] The present invention also provides a method for synthesizing pine plant-lactarius ectomycorrhizal seedlings, comprising: adding a retinol organic solvent aqueous solution to the root system of a pine plant seedling inoculated with lactarius mycelium, and then cultivating the seedlings to obtain the pine plant-lactarius ectomycorrhizal seedlings.

[0012] Preferably, the concentration of the retinol organic solvent aqueous solution is 0.01-0.1 mM / L; the solvent of the retinol organic solvent aqueous solution is an organic solvent aqueous solution; and the volume percentage of the organic solvent aqueous solution is 0.1%.

[0013] Preferably, the dosage of the retinol organic solvent aqueous solution is 10 mL / plant.

[0014] Preferably, the pine seedlings inoculated with Lactarius mycelium are pine seedlings having 5 or more lateral roots.

[0015] The present invention also provides application of the synthesis method described in the above technical solution in the cultivation of lactarius.

[0016] Beneficial effects:

[0017] The present invention provides an application of retinol in promoting the synthesis and / or growth of a pine plant-milk mushroom symbiotic system. The present invention adds retinol to the pine plant-milk mushroom symbiotic system, that is, after the milk mushroom hyphae are inoculated into the root of the pine plant, retinol is added around the root system, which can increase the mycorrhizal tip diameter of the ectomycorrhizal seedlings, increase the contact area between the mycorrhiza and the soil or the cultivation medium, and then increase the mycorrhiza absorption of phosphorus, promote the growth of the pine plant, and improve the quality of the mycorrhizal seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figure 1 These are photos and slices of the mycorrhizal tips of the ectomycorrhizal seedlings in Example 1 under a microscope. DETAILED DESCRIPTION

[0020] The present invention provides the use of retinol in promoting the synthesis and / or growth of a pine plant-lactariofugal symbiotic system, more preferably the use of retinol in promoting the synthesis and growth of a pine plant-lactariofugal symbiotic system.

[0021] In the present invention, the pine plants in the pine plant-lactarius symbiotic system preferably include yunnan pine, and the lactarius preferably include pine lactarius; that is, the pine plant-lactarius symbiotic system of the present invention more preferably includes yunnan pine-lactarius symbiotic system.

[0022] The method of promoting the growth of the pine plant-milkissina symbiotic system of the present invention preferably includes promoting the growth of pine plants and / or the growth of ectomycorrhizal seedlings, and more preferably includes promoting the growth of pine plants and the growth of ectomycorrhizal seedlings; the method of promoting the growth of ectomycorrhizal seedlings preferably includes increasing the mycorrhizal tip diameter of the ectomycorrhizal seedlings; the method of promoting the growth of pine plants preferably includes promoting the dry weight of the aboveground part, promoting the dry weight of the underground part, promoting the absorption of phosphorus by the aboveground part and promoting the absorption of phosphorus by the underground part, and further preferably includes promoting the dry weight of the aboveground part of pine plants and / or promoting the absorption of phosphorus by the underground part, and more preferably includes promoting the dry weight of the aboveground part of the plant and promoting the absorption of phosphorus by the underground part.

[0023] The present invention also provides a method for synthesizing pine plant-lactarius ectomycorrhizal seedlings, comprising: adding a retinol organic solvent aqueous solution to the root system of a pine plant seedling inoculated with lactarius mycelium, and then cultivating the seedlings to obtain the pine plant-lactarius ectomycorrhizal seedlings.

[0024] The present invention preferably prepares a lactarius solid mycelium agent, and the preparation method of the lactarius solid mycelium agent preferably comprises the following steps:

[0025] The milk mushroom fruiting body block tissue is cultured on an m+p solid culture medium to obtain milk mushroom mycelia, wherein the milk mushroom mycelia grows on the culture medium;

[0026] The block culture medium with the lactarius hyphae is continuously cultured in a bacterial agent culture matrix to obtain a lactarius solid mycelium agent.

[0027] The present invention preferably cultures the milk mushroom fruiting body block tissue on the m+p solid culture medium to obtain milk mushroom mycelia, and the milk mushroom mycelia grows on the culture medium. The milk mushroom fruiting body block of the present invention is preferably the tissue inside the milk mushroom fruiting body away from the surface after disinfection. The present invention does not specifically limit the size of the milk mushroom fruiting body block, and the milk mushroom fruiting body block of conventional size in the art can be used. The m+p solid culture medium of the present invention is preferably based on 12g / LPDA as the basic culture medium, and also includes 10g / L glucose, 0.5g / L yeast extract, 0.5g / L maltose extract, 0.15g / L MgSO4, 50mg / L vitamin B1, and 1.5mL / L 1% (w / v) ferric citrate. The mycelia culture of the present invention is preferably dark culture, and the temperature of the mycelia culture is preferably 20-25°C, more preferably 23°C; the time is preferably 6-8 weeks. The mycelia culture of the present invention is preferably carried out in an incubator. The present invention does not specifically limit the model and source of the incubator, and a conventional incubator in the art can be used.

[0028] After obtaining the lactarius hyphae, the present invention preferably continues to culture the block culture medium with the lactarius hyphae in a bacterial agent culture matrix to obtain a lactarius solid mycelium agent. The area of ​​the block culture medium with the lactarius hyphae in the present invention is preferably 50 to 150 mm 2 / piece, more preferably 100mm 2 / piece.

[0029] The bacterial agent culture matrix of the present invention preferably includes a formula matrix and an m+p liquid culture medium; the volume ratio of the formula matrix to the m+p liquid culture medium is preferably 250:75. The formula matrix of the present invention preferably includes vermiculite, perlite, peat and pine bark, and the volume ratio of the vermiculite, perlite, peat and pine bark is preferably 4:2:2:1. The m+p liquid culture medium of the present invention preferably uses 12g / LPDB as the basic culture medium, and also includes 10g / L glucose, 0.5g / L yeast extract, 0.5g / L maltose extract, 0.15g / LMgSO4, 50mg / L vitamin B1, and 1.5mL / L 1% (w / v) ferric citrate.

[0030] The continued culture of the present invention is preferably carried out in a breathable culture device, and the breathable culture device preferably includes a plastic bottle with a PTFE membrane in the bottle cap. The volume ratio of the block culture medium of the milk mushroom mycelium of the present invention to the breathable culture device is 8 to 10 blocks: 300 mL. The volume of the bacterial agent culture matrix of the present invention is preferably 70 to 90% of the breathable culture device, more preferably 80%. The temperature of the continued culture of the present invention is preferably 20 to 25°C, more preferably 23°C; the time is preferably 8 to 10 weeks, more preferably 8 weeks.

[0031] Before continuing the culture, the present invention preferably sterilizes the formula matrix and the breathable culture device; the sterilization temperature of the formula matrix is ​​preferably 121° C., and the time is preferably 90 minutes; the sterilization temperature of the breathable culture device is preferably 121° C., and the time is preferably 30 minutes.

[0032] The present invention preferably cultivates pine seedlings, and the method for cultivating pine seedlings preferably comprises:

[0033] The sterilized pine plant seeds are cultured in a formulated substrate to obtain pine plant seedlings.

[0034] The present invention preferably disinfects pine seeds, and the disinfection method preferably includes: soaking the pine seeds in hydrogen peroxide for 5 to 15 minutes, washing the soaked seeds with sterile water, and obtaining sterilized pine seeds. The soaking time of the present invention is preferably 10 minutes; the number of sterile water washing is preferably 3 times. The present invention does not specifically limit the specific method of sterile water washing, and the conventional washing method in the art can be used.

[0035] After obtaining the sterilized pine seeds, the present invention cultures the sterilized pine seeds in a formula matrix to obtain pine seedlings. The formula matrix of the present invention is preferably the same as the formula matrix used in the preparation process of the above-mentioned lactarius solid mycelium agent, and will not be described in detail. The culture time of the present invention is preferably 10 to 12 weeks; the lighting is preferably natural lighting; the temperature is preferably 20 to 30°C; and watering is preferably done once a week; the lateral roots of the obtained pine seedlings are preferably ≥5, and more preferably >10.

[0036] After obtaining the lactarius solid mycelium agent and the pine seedlings, the present invention preferably transplants the pine seedlings into a seedling raising device containing a formula matrix. The volume of the formula matrix of the present invention is preferably 1 / 3 of the volume of the seedling raising device. The present invention has no special restrictions on the transplanting process, and the conventional transplanting process in the art can be used. The seedling raising device of the present invention is preferably a seedling raising box.

[0037] After the transplanting, the present invention adds the solid mycelium agent of lactarius to the root system of the pine plant seedlings. The volume of the solid mycelium agent of lactarius of the present invention is preferably 1 / 10 of the volume of the seedling raising device.

[0038] After the adding, the present invention preferably fills the seedling raising device with the formula matrix, and then performs post-inoculation culture to obtain pine seedlings inoculated with lactarius mycelium. The total volume ratio of the lactarius solid mycelium agent to the formula matrix of the present invention is preferably 1:9. The temperature of the post-inoculation culture of the present invention is preferably 20 to 30°C, more preferably 25°C; the time is preferably 60 to 180 days, more preferably 90 days. The dosage of the lactarius solid mycelium agent of the present invention is preferably 60 mL / plant.

[0039] After obtaining the pine seedlings inoculated with lactarius hyphae, the present invention adds the retinol organic solvent aqueous solution to the root system of the pine seedlings inoculated with lactarius hyphae, and then cultivates the seedlings to obtain the pine-lactarius ectomycorrhizal seedlings. The concentration of the retinol organic solvent aqueous solution of the present invention is preferably 0.01-0.1mM / L, more preferably 0.1mM / L; the solvent of the retinol organic solvent aqueous solution is preferably ethanol aqueous solution or methanol aqueous solution, more preferably ethanol aqueous solution; the volume percentage of the organic solvent aqueous solution is preferably 0.1%. The dosage of the retinol organic solvent aqueous solution of the present invention is preferably 10mL / plant. The temperature for cultivating the seedlings of the present invention is preferably 20-30°C, more preferably 25°C; the time is preferably 2-6 months, more preferably 3 months.

[0040] The present invention adds retinol to the pine plant-milk mushroom symbiotic system, that is, after the milk mushroom hyphae are inoculated into the root of the pine plant, retinol is added around the root system, which can increase the mycorrhizal tip diameter of the ectomycorrhizal seedlings, increase the contact area between the mycorrhiza and the soil or the cultivation medium, and then increase the mycorrhiza to absorb phosphorus, promote the growth of the pine plant, and improve the quality of the mycorrhizal seedlings.

[0041] Based on the above technical advantages, the present invention also provides the application of the synthesis method described in the above technical scheme in the cultivation of milk mushrooms, providing technical support for the cultivation and planting of milk mushrooms.

[0042] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] A method for synthesizing ectomycorrhizal seedlings of Yunnan pine and pine lactarius, comprising the following steps:

[0045] 1. Preparation of solid mycelium agent of Pulsatilla truncatula: disinfect the surface of fresh Pulsatilla truncatula, cut the tissue inside the fruiting body of Pulsatilla truncatula away from the surface with a scalpel in a clean bench, cut it into small pieces and inoculate it on a sterilized m+p solid culture medium, wherein the components of the m+p solid culture medium are: 12g / LPDA culture medium, 10g / L glucose, 0.5g / L yeast extract, 0.5g / L maltose extract, 0.15g / L MgSO4, 50mg / L vitamin B1, 1.5mL / L 1% (w / v) ferric citrate; after culturing in a 23°C incubator for 6-8 weeks, well-grown Pulsatilla truncatula mycelium is obtained.

[0046] Cut the well-grown Lactarius mycelium and culture medium into pieces of about 100 mm 2 small pieces; prepare a formula matrix by mixing vermiculite: perlite: peat: pine bark in a volume ratio of 4:2:2:1; sterilize the formula matrix at 121°C for 90 minutes, put 250mL of the sterilized formula medium into a 300mL sterilizable plastic bottle, the bottle cap contains a PTFE membrane for air permeability, and then add 75mL of m+p liquid medium, wherein the components of the m+p liquid medium are: 12g / L PDB medium, 10g / L glucose, 0.5g / L yeast extract, 0.5g / L maltose extract, 0.15g / L MgSO4, 50mg / L vitamin B1, 1.5mL / L 1% (w / v) ferric citrate; sterilize at 121°C for 30min; 2 The mycelium blocks were evenly placed in a plastic culture bottle and cultured in the dark at 23°C for 8 to 10 weeks to obtain a solid mycelium agent of Lactarius pine.

[0047] 2. Cultivation of Yunnan pine seedlings: Soak the Yunnan pine seeds in hydrogen peroxide for 10 minutes, wash them three times with sterile water, and evenly sow them in the sterilized above-mentioned formula matrix (vermiculite: perlite: peat: pine bark volume ratio is 4:2:2:1). After cultivating for about 10 to 12 weeks under natural light and 20-30℃ conditions, Yunnan pine seedlings are obtained. When the Yunnan pine seedlings have ≥5 lateral roots, they can be used to inoculate synthetic mycorrhizal seedlings.

[0048] 3. Yunnan pine-Lactarius inoculation: take the Yunnan pine seedlings in step 2, transplant them individually into a square seedling box with a volume of about 600 mL, fill the bottom of the seedling box with 200 mL of sterilized formula matrix, then put the Yunnan pine seedlings into the seedling box, add 60 mL of the solid lactarius mycelium agent in step 1 directly into the seedling box to fully contact the root system of the Yunnan pine seedlings, then fill the seedling box with the formula matrix, and cultivate the Yunnan pine seedlings in a glass greenhouse under natural light at 20-30° C. for 90-180 days.

[0049] 4. Adding retinol: After adding the solid mycelium agent of Lactarius pine in step 3, add 0.1 mM / L retinol ethanol aqueous solution (the solvent is 0.1% ethanol aqueous solution by volume) around the root system of the Yunnan pine seedlings at a dosage of 10 mL / plant, and continue to cultivate in the glass greenhouse under the conditions in step 3.

[0050] Comparative Example 1

[0051] The conditions in Example 1 were used to synthesize the ectomycorrhizal seedlings of Yunnan pine and Lactarius pine, except that in step 4, the same dosage of 0.1% by volume ethanol aqueous solution was added around the root system of the Yunnan pine seedlings.

[0052] Test Example 1

[0053] Samples were taken from the Yunnan pine seedlings in Example 1 and Comparative Example 1 cultured in a glass greenhouse for 3 months, and the mycorrhizae were photographed under a microscope. Figure 1 As shown, the left picture is a photo taken under a microscope, and the right picture is a slice picture. The mature mycorrhizal root tip and frozen section were taken to measure the diameter of the mature zone of the mycorrhizal root tip;

[0054] The root tissues of the above-ground and underground parts of Yunnan pine seedlings were taken, the fresh weight was weighed, and they were dried at 60°C for 72 hours to constant weight. The dry weight was weighed, and the moisture content of the needles was calculated to be about 50% based on the fresh / dry weight data before and after drying. The total phosphorus content was measured by grinding into powder; the results are shown in Table 1.

[0055] It should be noted that the data in Table 1 are derived from: three independent repeated experiments were carried out according to the methods in Example 1 (treatment group) and Comparative Example 1 (control group), the time interval between the two independent experiments was 2 months, the treatment group and the control group were carried out simultaneously in each independent repeated experiment, and the treatment group and the control group in each independent experiment contained 8 biological replicates; the mycorrhizal tip diameter is the total average value of the diameters of 5 randomly selected mycorrhizal tips of each seedling in each independent repeated experiment, the phosphorus absorption is the average value of 5 randomly selected seedlings (representing 5 replicates, and seedlings were selected in 3 independent repeated experiments) from the samples of all repeated experiments, and the dry weight is the average value of 8 randomly selected seedlings (representing 8 replicates, and seedlings were selected in 3 independent repeated experiments) from the samples of all repeated experiments. The data in Table 1 are the average values ​​of three independent repeated experiments.

[0056]

[0057] It can be concluded from Table 1 that compared with Comparative Example 1, the mycorrhizal tip diameter, the phosphorus absorption of the aboveground and underground parts, and the dry weight of the aboveground and underground parts in Example 1 are all improved, especially the mycorrhizal tip diameter, the phosphorus absorption of the underground part, and the dry weight of the aboveground part are significantly improved compared with Comparative Example 1.

[0058] It can be concluded from the above examples that retinol can promote the growth of pine plants and ectomycorrhizae in the pine plant-Lactarius symbiotic system, and promote the formation and growth of pine plant-Lactarius ectomycorrhizal seedlings.

[0059] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for synthesizing ectomycorrhizal seedlings of Yunnan pine and pine lactarius, characterized in that: include: After adding the retinol organic solvent aqueous solution to the root system of the Yunnan pine seedlings inoculated with the mycelium of Lactarius pine, the mycorrhizal seedlings were cultivated to obtain the Yunnan pine-Lactarius pine ectomycorrhizal seedlings with increased mycorrhizal tip diameter; The concentration of the retinol organic solvent aqueous solution is 0.01-0.1 mM / L; the dosage of the retinol organic solvent aqueous solution is 10 mL / plant.

2. The synthesis method according to claim 1, characterized in that The solvent of the retinol organic solvent aqueous solution is an organic solvent aqueous solution; the volume percentage of the organic solvent aqueous solution is 0.1%.

3. The synthesis method according to claim 1, characterized in that The Yunnan pine seedlings inoculated with Lactarius pine hyphae are Yunnan pine seedlings with lateral roots of ≥5.

4. Application of the synthesis method according to any one of claims 1 to 3 in the cultivation of Pleurotus ostreatus.

5. The use of the synthesis method according to any one of claims 1 to 3 in promoting the growth of the Yunnan pine-Lactarius pine symbiotic system, characterized in that: The promoting the growth of the Yunnan pine-Lactarius pine symbiotic system includes promoting the growth of Yunnan pine and / or the growth of ectomycorrhizal seedlings; The method of promoting the growth of Yunnan pine includes promoting the absorption of phosphorus by the above-ground part and / or promoting the absorption of phosphorus by the underground part.

6. The use according to claim 5, characterized in that: The method of promoting the growth of ectomycorrhizal seedlings includes increasing the diameter of the mycorrhizal root tips of the ectomycorrhizal seedlings.

7. The use according to claim 5, characterized in that: The promoting the growth of Yunnan pine also includes promoting the dry weight of the above-ground part and / or promoting the dry weight of the underground part.

Images