Mycorrhizal cultivation method for improving stress resistance of pinus khasys seedlings

Abstract

The invention relates to a mycorrhizal cultivation method for improving the stress resistance of pinus khasys seedlings, and belongs to the technical field of agricultural planting, the cultivation method comprises the following steps: (1) compounding fibrous belly fungi, bolete and russule virens into a microbial inoculum, and culturing to prepare microcapsules; (2) taking turf, vermiculite, humus soil and perlite as substrates, and adding a microcystis agent and a water-retaining agent; (3) the seeds are disinfected and sown, and seedlings are domesticated through light intensity gradient, water stress and temperature fluctuation; 4, during transplanting, abscisic acid is used for treating root systems, organic fertilizer is pre-filled, and mycorrhizal activation promoting liquid is perfused, through the synergistic effect of composite mycorrhizal fungi and stress domestication, the stress resistance, the survival rate and the afforestation adaptability of pinus khasys seedlings are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of agricultural planting, in particular to a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiyavar.langbianensis seedlings. BACKGROUND

[0002] Pinus kesiyavar.langbianensis is a key tree species for ecological afforestation in the dry-hot valleys and karst regions of Southwest China, mainly distributed in dry-hot valleys and karst mountainous areas in Yunnan, Guangxi and other places. There are three characteristics in this kind of area: concentrated rainy season, long dry season, and large diurnal temperature difference. The surface temperature in summer can reach above 50℃, and it may suddenly freeze in winter. The wood of Pinus kesiyavar.langbianensis is a high-quality pulp raw material, and the turpentine can be used as a chemical material. More importantly, it can firmly hold the soil in the stone cracks and prevent soil erosion. However, in the prior art, the traditional fungicide will die in large numbers when it encounters drought and high temperature, and the root fungus grows rapidly, easily occupying the living space of the green and red Russula, and finally the synergistic effect is weakened. Directly planting bare roots during traditional transplanting will lead to weakened resistance of the seedlings and easy infection of the seedlings, and the industry often uses rooting powder to promote activation, and uses hormones to stimulate root growth during transplanting, but the new roots are fragile and are more easily attacked by diseases, and the use of single fungicide embedding, wrapping the fungus with sodium alginate, will still fail due to internal nutrient depletion during drought.

[0003] In summary, there is an urgent need in the market to develop a new microbial fertilizer preparation method for citrus disease control and its application. SUMMARY

[0004] The present application provides a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiyavar.langbianensis seedlings to solve the problems raised in the background art.

[0005] To solve the above technical problems, the present application discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiyavar.langbianensis seedlings, which comprises the following steps: (1) mixing the Rhizopogon fulvum, Boletus complex fungicide and green and red Russula fungicide, culturing them in PDMY solid culture medium, mixing them with sodium alginate solution, and preparing micro-mycelia fragments by high-pressure homogenization; mixing them with chitooligosaccharide solution, humic acid slow-release particles, potassium dihydrogen phosphate and magnesium sulfate, adding calcium chloride solution for solidification, and preparing microcapsules by soaking in salicylic acid solution; (2) mixing and sterilizing grass charcoal, vermiculite, humus soil and perlite, adding microcapsule fungicide and polyacrylamide to prepare the substrate; (3) disinfecting the Pinus kesiyavar.langbianensis seeds and sowing them in the substrate, controlling the temperature, humidity and light; before transplanting, performing light intensity gradient acclimation, water stress acclimation and temperature fluctuation acclimation; (4) transplanting treatment: when the seedlings reach 20 cm, immerse the nutrient cup in abscisic acid solution, pre-fill the planting hole with organic fertilizer and sulfur powder, irrigate the rhizosphere with a promoting liquid containing methyl jasmonate and chitooligosaccharide, and cover the seedlings with a mixture of pine needles and rice husk to keep the soil moist and suppress weeds.

[0006] Further, the nutrient agent comprises biochemical potassium fulvate, molasses fermentation powder, seaweed powder and rare earth nitrate.

[0007] Further, the Rhizopogon complex microbial agent, the Russula variabilis microbial agent and the Rhizopogon complex microbial agent are mixed at a weight ratio of (55-65):(20-30):(10-20).

[0008] Further, the grass carbon, the vermiculite, the humus soil and the perlite are mixed at a volume ratio of (30-45):(25-35):(15-25):(5-15) to sterilize, and the microcapsule microbial agent and the polyacrylamide are added to prepare the substrate.

[0009] Further, 45-55 g / L of the microcapsule microbial agent and 2.5-3.5 g / L of the polyacrylamide are added.

[0010] Further, before transplanting, light intensity gradient acclimation is performed, the light is increased by 5000 lux to 35000 lux per day, water stress acclimation is performed, the water content of the substrate is reduced to 30% in stages, and temperature fluctuation acclimation is performed as 35℃ during the day and 12℃ at night.

[0011] Further, the PDMY solid culture medium contains 500 ml / L of potato juice, 1 g / L of yeast extract, 2.1 g / L of malt extract powder, 5 g / L of glucose and 16 g / L of agar.

[0012] Further, the high-pressure homogenization condition is 80 MPa pressure for 3 cycles.

[0013] Further, the microcapsule solidification condition comprises using a 1.5% concentration calcium chloride solution, a peristaltic pump flow rate of 5 ml / min, a needle diameter of 0.7 mm, and a solidification time of 20 min; the sterilization condition is 115-130℃ high-pressure steam sterilization for 60 min; the seed treatment in step (3) is 0.5% potassium permanganate soaking for 30 min and 25℃ warm water soaking for 12 hours; and the light intensity acclimation period in step (3) is 7 days, and the light is increased by 5000 lux per day.

[0014] Further, the rhizosphere activation liquid in step (4) contains 0.1 mmol / L methyl jasmonate and 0.05% chitinous oligosaccharide; the planting hole in step (4) is pre-filled with 200 g of decomposed organic fertilizer and 100 g of sulfur powder; the seedling container in step (3) is a 300 ml black nutrient cup, 2 seeds are sown in each cup, and the soil covering depth is 1.5 cm; and the seedling environment control in step (3) is 25℃ during the day and 18℃ at night, the humidity is 70%, the light intensity is 5000 lux, and the water content of the substrate is maintained at 60% of the field water holding capacity.

[0015] Compared with the prior art, the application provides a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings, and has the following beneficial effects:

[0016] 1. This application is approved: By encapsulating sodium alginate in microcapsules and inducing treatment with salicylic acid, the colonization and survival rate of compound mycorrhizal fungi is improved. Furthermore, the salicylic acid pretreatment activates the expression of mycelial stress resistance genes, enhancing their activity under stress conditions. The dual effects of physical protection and chemical induction of microcapsules enhance the synergistic effect of mycorrhizal fungi survival rate and stress resistance induction.

[0017] 2. This application improves the synthesis of stress-resistant proteins in seedlings and shortens the seedling recovery period after afforestation through the triple synergistic domestication of light intensity gradient, water stepwise reduction and diurnal temperature difference. It has excellent effect of accurately simulating afforestation adversity through compound stress domestication.

[0018] 3. This application forms a three-level protection system of "physical barrier (ABA membrane) - chemical signal (jasmonic acid) - nutrient induction (chitin)" by treating abscisic acid film during transplanting and irrigating with methyl jasmonate / chitin oligosaccharide activating solution, thereby reducing the electrolyte leakage rate of the root system under drought stress.

[0019] 4. The combination of peat moss, vermiculite, humus, and perlite with polyacrylamide water-retaining agent improves the water-holding capacity of the substrate. At the same time, the microencapsulated bacterial agent can continuously release active substances under the support of humic acid slow-release granules. The combination of the substrate pore structure and water-retaining agent enhances water and fertilizer retention. The humic acid slow-release granules extend the functional period of the bacterial agent.

[0020] 5. Pre-filling planting holes with sulfur powder creates an antibacterial microenvironment, which, together with the chitin oligosaccharides in the mycorrhizal activating solution, reduces the incidence of soil-borne diseases by 80% and promotes mycelial infection efficiency. Sulfur powder inhibits pathogens, while chitin oligosaccharides induce systemic disease resistance in plants. This application has the synergistic effect of inhibiting soil-borne diseases and mycorrhizal symbiosis. Attached Figure Description

[0021] Figure 1 The seedlings in Example 1 of this application were cultivated using a mycorrhizal culture method to enhance the stress resistance of Pinus kesina seedlings. Detailed Implementation

[0022] The preferred embodiments of this application are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit this application.

[0023] In addition, the descriptions such as "first", "second", etc. in the present application are only for the purpose of description, and do not mean to indicate the order or sequence, nor to limit the present application, which are merely to distinguish the components or operations described by the same technical terms, and cannot be understood as indicating or implying the relative importance of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first", "second" can explicitly or implicitly include at least one of the features. In addition, the technical solutions and technical features of various embodiments can be combined with each other, but it must be based on the realization of ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the protection scope required by the present application.

[0024] Unless otherwise specified, the examples and comparative examples are parallel tests with the same components, component contents, preparation steps and preparation parameters. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are analytical reagents (A.R.) unless otherwise specified, which are purchased from commercial channels.

[0025] The needle tree ectomycorrhizal fungus complex preparation is Rhizopogon spp. agent, Endo / Ecto Mix; the Boletus edulis complex agent is Boletus edulis complex agent, Plant Granular Mycorrhizae; the Russula general agent is Russula general agent, Russula Blend; the yeast extract is BD Difco TM 212750; the malt extract powder is Merck of Germany Merck Group TM 1.05398.0500; the agar is Sigma-Aldrich of USA Sigma-Aldrich TM A1296; the sodium alginate is Sigma-Aldrich TM W201502; the chitooligosaccharide is Oligochitosan; the humic acid slow-release granule is Humiforte TM 10-0-0; the salicylic acid is Sigma-Aldrich TM S5922; Sigma-Aldrich TM S5922 is Sigma-Aldrich TM392707; abscisic acid is S-elicitor; grass carbon is northeast moss grass carbon (0-10 mm) of Heilongjiang Xing'an grass carbon factory; vermiculite is horticultural grade (2-4 mm) of Lingshou County Mineral Products Company; humus is Inner Mongolia Mengcao Ecological; perlite is expanded type (3-6 mm) of Xinyang Perlite Base; polyacrylamide is PAM-12.

[0026] Example 1

[0027] The embodiment discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings, comprising the following steps:

[0028] Step 1: a composite microbial agent is prepared by using 60 parts of needle leaf tree ectomycorrhizal fungus composite preparation, 25 parts of boletus composite microbial agent and 15 parts of Russula general microbial agent according to weight parts, PDMY solid culture medium containing 500 ml / L of potato juice, 1 g / L of yeast extract, 2.1 g / L of malt extract powder, 5 g / L of glucose and 16 g / L of agar is used for dark culture at 25 DEG C for 15 days, then the culture is transferred to a light culture room, the light intensity of the light culture room is 2000 lux, the light period is 12 h / 12 h, and the culture is cultured at 25 DEG C for 5 days, the extension mycelium differentiation is induced, the colonies are scraped by using a sterile spatula, mixed with 0.1% sodium alginate solution at a ratio of 1:3 (w / v), and subjected to high-pressure homogenization circulation for 3 times under 80 MPa to prepare micro-mycelia fragments.

[0029] Step 2: the micro-mycelia fragments are mixed with 0.5% chitinous oligosaccharide solution, stirred at 300 rpm for 30 min, humic acid slow-release particles, 0.3 g / L of potassium dihydrogen phosphate and 0.3 g / L of magnesium sulfate are added, and then added into 1.5% calcium chloride solution, and the microcapsules are prepared by using a peristaltic pump (flow rate 5 ml / min, needle diameter 0.7 mm) solidification for 20 min, then washed with sterile water, soaked in 0.01% salicylic acid solution for 4 hours, and stored at 4 DEG C for standby.

[0030] Step 3: 40% grass carbon, 30% vermiculite, 20% humus and 10% perlite are mixed according to volume percentage, sterilized by high-pressure steam at 121 DEG C for 60 min, cooled to room temperature, 50 g / L of microencapsulated microbial agent and 3 g / L of water-retaining agent polyacrylamide are added, and stirred for 15 min to prepare a substrate.

[0031] Step 4: The seeds of Pinus kesiya var. lasiocarpa are soaked in 0.5% potassium permanganate for 30 min, rinsed with water, and then soaked in warm water at 25°C for 12 hours. Two seeds are planted in each 300 ml black nutrient cup containing the substrate, with a soil depth of 1.5 cm. The temperature is 25°C during the day and 18°C at night, the humidity is 70%, and the light intensity is 5000 lux. The substrate water content is checked every 3 days to maintain 60% of the field water holding capacity. Seven days before transplanting, the light intensity is increased to 35000 lux. Five days before transplanting, the substrate water content is reduced from 60% to 30% in stages. Three days before transplanting, the daytime temperature is increased to 35°C and the nighttime temperature is decreased to 12°C. The seedlings are transplanted when they are 20 cm tall. The nutrient cups are immersed in a 0.001% abscisic acid (ABA) solution for 10 seconds to form a root system protective film. A 40x40x40 cm planting hole is pre-filled with 200g of decomposed organic fertilizer and 100g of sulfur powder. The rhizosphere is irrigated with a mycorrhizal activation liquid containing 0.1 mmol / L methyl jasmonate and 0.05% chitinous oligosaccharide, 100ml per plant. A 5cm thick cover of a mixture of pine needles and rice husks is applied to maintain the rhizosphere humidity and suppress weeds.

[0032] In this example, the light intensity gradient acclimation is achieved by increasing the light intensity to 35000 lux daily for 7 days before transplanting, simulating full sunlight in a reforestation area. The water stress acclimation is achieved by reducing the substrate water content from 60% to 30% in stages 5 days before transplanting. The temperature fluctuation acclimation is achieved by increasing the daytime temperature to 35°C and decreasing the nighttime temperature to 12°C 3 days before transplanting, simulating the diurnal temperature difference in a dry and hot valley.

[0033] Example 2

[0034] This example discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings. The Rhizopogon fulvum, Boletus complex, and Russula variabilis agents are mixed in a weight ratio of 55:20:10. 35% grass carbon, 25% vermiculite, 25% humus soil, and 15% perlite are mixed in a volume percentage. The mixture is sterilized at 115°C for 60 min under high-pressure steam, cooled to room temperature, and then 45g / L of micro-encapsulated bacteria and 2.5g / L of water-retaining agent polyacrylamide are added. The mixture is stirred for 10 min to form the substrate. The other contents are consistent with Example 1.

[0035] Example 3

[0036] This example discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings. The Rhizopogon fulvum, Boletus complex, and Russula variabilis agents are mixed in a weight ratio of 65:30:20. 45% grass carbon, 35% vermiculite, 15% humus soil, and 5% perlite are mixed in a volume percentage. The mixture is sterilized at 130°C for 60 min under high-pressure steam, cooled to room temperature, and then 55g / L of micro-encapsulated bacteria and 3.5g / L of water-retaining agent polyacrylamide are added. The mixture is stirred for 20 min to form the substrate. The other contents are consistent with Example 1.

[0037] Comparative Example 1

[0038] The difference between Example 1 and Comparative Example 1 is that only the conifer ectomycorrhizal fungus complex preparation is used, and the rest is the same.

[0039] Comparative Example 2

[0040] The difference between Example 1 and Comparative Example 2 is that the light intensity / moisture / temperature acclimation step is missing, i.e. 7 days before transplanting, the light intensity is increased by 5000 lux per day, finally to 35000 lux, 5 days before transplanting, the water content of the substrate is reduced from 60% to 30% in steps; 3 days before transplanting, the daytime temperature is raised to 35℃ and the nighttime temperature is lowered to 12℃, then the seedlings are directly transplanted, and the rest is the same.

[0041] Comparative Example 3

[0042] The difference between Example 1 and Comparative Example 3 is that the complex mycelium fragments are directly mixed into the substrate without being made into microcapsules, and the rest is the same.

[0043] Performance Test

[0044] The performance of the examples and comparative examples is tested, the mycorrhizal infection rate detection method is to randomly take 30 seedling main roots, slice and observe the Hartig net structure under a microscope; the transplant survival rate detection method is to stop watering for 15 days after transplanting, and the number of surviving plants (n=100) is counted; the disease resistance detection method is to inoculate the rhizosphere with Rhizoctonia solani fungus cake (diameter 5mm), and the incidence rate is counted after 7 days; the root activity detection is by TTC (triphenyltetrazolium chloride) reduction method, Fresh Pinus kesiya var. langbianensis seedling root tips 5cm are washed with deionized water for 3 times, filter paper is used to absorb the water, 0.5g sample is accurately weighed, cut into 2mm fragments, and placed in a 10mL test tube for TTC (triphenyltetrazolium chloride) reduction method detection, and the results are shown in Table 1.

[0045] Table 1

[0046]

[0047] As can be seen from Table 1, the comprehensive performance of Example 1 is the best, and Example 1 improves stress resistance through the synergistic effect of complex mycorrhizal fungi, stress acclimation and microcapsule embedding.

[0048] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. If these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A method for mycorrhizal cultivation of Pinus massoniana seedlings to improve their stress resistance, characterized in that, It comprises the following steps: (1) mixing the Rhizopogon licheniformans agent, the Boletus complex agent and the Russula virescens agent, culturing them in PDMY solid medium, mixing them with sodium alginate solution, and preparing micro-mycelium fragments by high-pressure homogenization; mixing them with chitooligosaccharide solution, humic acid slow-release particles, potassium dihydrogen phosphate and magnesium sulfate, adding calcium chloride solution for solidification, and preparing micro-capsules by soaking in salicylic acid solution; (2) mixing peat, vermiculite, humus soil and perlite, sterilizing them, adding micro-capsule agent and polyacrylamide to prepare the substrate; (3) disinfecting the Pinus kesiya var. thurberi seeds, sowing them in the substrate, and controlling the temperature, humidity and light; before transplanting, performing light intensity gradient acclimation, water stress acclimation and temperature fluctuation acclimation; (4) transplanting treatment: when the seedlings reach 20 cm, immersing the nutrient cups in abscisic acid solution, pre-filling the planting holes with organic fertilizer and sulfur powder, pouring the rhizosphere with activation liquid containing methyl jasmonate and chitooligosaccharide, and covering the mixture of pine needles and rice husks to keep the soil moist and suppress weeds.

2. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, The nutrient agent comprises biochemical potassium fulvic acid, molasses fermentation powder, algal powder and rare earth nitrate.

3. The method for preparing a microbial fertilizer for citrus disease control according to claim 1, characterized in that, The Rhizopogon licheniformans agent, the Boletus complex agent and the Russula virescens agent are mixed at a weight ratio of (55-65):(20-30):(10-20).

4. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, The peat, vermiculite, humus soil and perlite are mixed at a volume ratio of (30-45):(25-35):(15-25):(5-15), sterilized, and added with micro-capsule agent and polyacrylamide to prepare the substrate.

5. The method for preparing a microbial fertilizer for citrus disease control according to any one of claims 1 or 4, characterized in that, 45-55 g / L of micro-capsule agent and 2.5-3.5 g / L of polyacrylamide are added.

6. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, Before transplanting, light intensity gradient acclimation is performed, the light is increased by 5000 lux to 35000 lux per day, water stress acclimation is performed, the water content of the substrate is decreased by steps to 30%, and temperature fluctuation acclimation is performed with a day temperature of 35℃ and a night temperature of 12℃.

7. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, The PDMY solid medium contains potato juice 500 ml / L, yeast extract 1 g / L, malt extract powder 2.1 g / L, glucose 5 g / L and agar 16 g / L.

8. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, The high-pressure homogenization condition is 80 MPa pressure for 3 cycles.

9. The microbial fertilizer preparation method for citrus disease control according to claim 1, characterized in that, The micro-capsule solidification time is 20 min; the sterilization condition is 115-130℃ high-pressure steam sterilization for 60 min; in step (3), the seed treatment is 0.5% potassium permanganate soaking for 30 min and 25℃ warm water soaking for 12 hours; in step (3), the light intensity acclimation period is 7 days, and the light is increased by 5000 lux per day.

10. The method for preparing a microbial fertilizer for citrus disease control according to claim 1, characterized in that, In step (4), the rhizosphere activation liquid contains 0.1 mmol / L methyl jasmonate and 0.05% chitooligosaccharide; in step (4), the planting hole is pre-filled with 200 g of decomposed organic fertilizer and 100 g of sulfur powder; in step (3), the seedling raising container is a 300 ml black nutrient cup, 2 seeds are sown in each cup, and the soil depth is 1.5 cm; in step (3), the seedling raising environment control is a day temperature of 25℃ and a night temperature of 18℃, a humidity of 70%, a light intensity of 5000 lux, and a substrate water content of 60% of the field water holding capacity.

Images