A mycorrhizal cultivation method for improving stress resistance of pinus kesiya var. lasiocarpa seedlings

Abstract

The application relates to a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. thurberi seedlings, and belongs to the technical field of agricultural planting. The cultivation method comprises the following steps: (1) compounding Rhizopogon fulvum, Boletus and Russula variabilis to form a fungicide, and preparing microcapsules after culture; (2) taking grass carbon, vermiculite, humus soil and perlite as a matrix, and adding the microcapsule fungicide and a water-retaining agent; (3) disinfecting and sowing seeds, and domesticating seedlings through light intensity gradient, water stress and temperature fluctuation; and (4) treating the root system with abscisic acid when transplanting, pre-filling organic fertilizer and pouring mycorrhizal promoting activation liquid. The application significantly improves the stress resistance, survival rate and afforestation adaptability of Pinus kesiya var. thurberi seedlings through the synergistic effect of composite mycorrhizal fungi and stress domestication.

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 kesiya var. langbianensis seedlings. BACKGROUND

[0002] Pinus kesiya var. 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 and Guangxi. 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 kesiya var. 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, which uses sodium alginate to wrap fungi, 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 kesiya var. 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 kesiya var. langbianensis seedlings, which comprises the following steps: (1) mixing the Rhizopogon fulvum, Boletus complex fungicide, and green and red Russula fungicide, and then culturing them in PDMY solid medium, mixing with sodium alginate solution, and high-pressure homogenization to prepare micro-mycelia fragments; mixing with chitooligosaccharide solution, humic acid slow-release particles, potassium dihydrogen phosphate, and magnesium sulfate, adding calcium chloride solution for solidification, and then soaking in salicylic acid solution to prepare microcapsules; (2) mixing and sterilizing grass charcoal, vermiculite, humus soil, and perlite, adding microcapsule fungicide and polyacrylamide to prepare the substrate; (3) disinfecting the Pinus kesiya var. langbianensis seeds and then 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 hole with organic fertilizer and sulfur powder, perfusing the rhizosphere with a promoting liquid containing methyl jasmonate and chitooligosaccharide, and covering the seedlings with a mixture of pine needles and rice husks to keep them moist and suppress weeds.

[0006] Further, the Rhizopogon velleius, the Tricholoma matsutake and the Russula virescens are mixed in a weight ratio of (55-65):(20-30):(10-20).

[0007] Further, the grass carbon, the vermiculite, the humus soil and the perlite are mixed in 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.

[0008] Further, 45-55 g / L of the microcapsule microbial agent and 2.5-3.5 g / L of the polyacrylamide are added.

[0009] Further, before transplanting, the seedlings are acclimated to light intensity gradient, the light intensity is increased by 5000 lux to 35000 lux per day, the seedlings are acclimated to water stress, the water content of the substrate is decreased to 30% in stages, and the seedlings are acclimated to temperature fluctuation, the temperature is 35 DEG C during the day and 12 DEG C at night.

[0010] 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.

[0011] Further, the high-pressure homogenization condition is 80 MPa pressure for 3 cycles.

[0012] Further, the microcapsule solidification condition includes 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 DEG C high-pressure steam sterilization for 60 min; the seed treatment in step (3) is 0.5% potassium permanganate soaking for 30 min and 25 DEG C warm water soaking for 12 hours; and the light intensity acclimation period in step (3) is 7 days, and the light intensity is increased by 5000 lux per day.

[0013] Further, the rhizosphere promoting 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 raising 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 raising environment control in step (3) is 25 DEG C during the day and 18 DEG C 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.

[0014] Compared with the prior art, the mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings has the following beneficial effects:

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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

[0020] 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

[0021] 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.

[0022] In addition, the description such as "first", "second", etc. in the present application is only for the purpose of description, and is not intended to particularly indicate the order or sequence, nor to limit the present application, which is 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 with "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.

[0023] 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.

[0024] Pine ectomycorrhizal fungi complex preparation is Rhizopogon spp. fungicide, MycoApply® Endo / Ecto Mix; boletus complex fungicide is Boletus edulis complex fungicide, Plant Success® Granular Mycorrhizae; Russula general fungicide is MycoBloom® Russula Blend; yeast extract is BD Difco™ 212750 of BDBiosciences, USA; malt extract powder is Merck™ 1.05398.0500 of Merck Group, Germany; agar is Sigma-Aldrich™ A1296 of Sigma-Aldrich, USA; sodium alginate is Sigma-Aldrich™ W201502; chitosan oligosaccharide is Golden-Shell® Oligochitosan; humic acid slow-release particles are Humiforte™ 10-0-0; salicylic acid is Sigma-Aldrich™ S5922; Sigma-Aldrich™ S5922 is Sigma-Aldrich™ 392707; abscisic acid is S-elicitor; grass carbon is northeast sphagnum peat (0-10mm) of Heilongjiang Xing'an Peat Factory; vermiculite is horticultural grade (2-4mm) of Lingshou County Mineral Products Company; humus soil is Inner Mongolia Mengcao Ecological; perlite is expanded type (3-6mm) of Xinyang Perlite Base; polyacrylamide is PAM-12.

[0025] Embodiment 1: The embodiment discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings, and comprises the following steps.

[0026] 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 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 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 12h / 12h, 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 1:3 (w / v), and subjected to high-pressure homogenization circulation for 3 times under 80 MPa to prepare micro-mycelia fragments.

[0027] 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 potassium dihydrogen phosphate and 0.3 g / L magnesium sulfate are added, and then added into 1.5% calcium chloride solution, and then solidified for 20 min by using a peristaltic pump (flow rate 5 ml / min, needle diameter 0.7 mm) to prepare microcapsules, the microcapsules are washed by using sterile water, soaked in 0.01% salicylic acid solution for 4 hours, and stored at 4 DEG C for standby.

[0028] Step 3: 40% grass charcoal, 30% vermiculite, 20% humus soil 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, and then 50 g / L of the micro-encapsulated microbial agent and 3 g / L of water-retaining agent polyacrylamide are added and stirred for 15 min to prepare a substrate.

[0029] Step 4: The seeds of Pinus kesiya var. lasiocarpa were soaked in 0.5% potassium permanganate for 30 min, washed with clean water, and then soaked in warm water at 25°C for 12 hours. Two seeds were sown in each 300 ml black nutrient cup containing the substrate, with a soil depth of 1.5 cm. The temperature was 25°C during the day and 18°C at night, the humidity was 70%, and the light intensity was 5000 lux. The water content of the substrate was checked every 3 days to maintain 60% of the field water holding capacity. Seven days before transplanting, the light intensity was increased to 35000 lux. Five days before transplanting, the water content of the substrate was reduced from 60% to 30% in a stepwise manner. Three days before transplanting, the temperature was increased to 35°C during the day and decreased to 12°C at night. The seedlings were transplanted when they reached a height of 20 cm. The nutrient cups were immersed in a 0.001% abscisic acid (ABA) solution for 10 seconds to form a root system protective film. A 40x40x40 cm planting hole was prepared by pre-filling 200 g of decomposed organic fertilizer and 100 g of sulfur powder. The rhizosphere was irrigated with a mycorrhizal activation liquid containing 0.1 mmol / L methyl jasmonate and 0.05% chitinous oligosaccharide, 100 ml per plant. A 5 cm thick mixture of pine needles and rice husk was used to cover the rhizosphere, and the rhizosphere humidity was maintained and weeds were inhibited.

[0030] In this embodiment, the light intensity gradient acclimation was achieved by increasing the light intensity to 35000 lux for 7 days before transplanting, simulating full sunlight in the afforestation area. The water stress acclimation was achieved by reducing the water content of the substrate from 60% to 30% in a stepwise manner for 5 days before transplanting. The temperature fluctuation acclimation was achieved by increasing the temperature to 35°C during the day and decreasing it to 12°C at night for 3 days before transplanting, simulating the diurnal temperature difference in a dry and hot valley.

[0031] Example 2: This embodiment discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings. The root-hair cystidium agent, boletus complex agent, and greenish-red agaricus agent 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 45 g / L of micro-encapsulated bacteria agent and 2.5 g / L of water-retaining agent polyacrylamide are added. The mixture is stirred for 10 min to prepare the substrate. The other contents are consistent with those of Example 1.

[0032] Example 3: This embodiment discloses a mycorrhizal cultivation method for improving the stress resistance of Pinus kesiya var. lasiocarpa seedlings. The root-hair cystidium agent, boletus complex agent, and greenish-red agaricus agent 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 55 g / L of micro-encapsulated bacteria agent and 3.5 g / L of water-retaining agent polyacrylamide are added. The mixture is stirred for 20 min to prepare the substrate. The other contents are consistent with those of Example 1.

[0033] Comparative Example 1

[0034] 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.

[0035] Comparative Example 2

[0036] 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, and 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 increased to 35℃ and the nighttime temperature is decreased to 12℃, and then the seedlings are directly transplanted, and the rest is the same.

[0037] Comparative Example 3

[0038] 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.

[0039] Performance test

[0040] 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 count the number of surviving plants (n=100); the disease resistance detection method is to inoculate the rhizosphere with Rhizoctonia solani fungus cake (diameter 5mm), and count the incidence rate after 7 days; the root activity detection is by TTC (chloride triphenyl tetrazolium) reduction method, Fresh Pinus kesiya 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 segments, and placed in a 10mL test tube for TTC (chloride triphenyl tetrazolium) reduction method detection, and the results are shown in Table 1.

[0041] Table 1

[0042]

[0043] 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.

[0044] 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 to enhance the stress resistance of Pinus kesiao seedlings, characterized in that, Includes the following steps: (1) Mix the root fibrous fungal agent, Boletus compound fungal agent, and green red mushroom fungal agent, culture them on PDMY solid medium, mix them with sodium alginate solution, and homogenize them under high pressure to make micro mycelial fragments; mix them with chitin oligosaccharide solution, humic acid slow-release granules, potassium dihydrogen phosphate and magnesium sulfate, add calcium chloride solution to solidify, and soak them in salicylic acid solution to make microcapsule fungal agent; (2) Sterilize the mixture of peat moss, vermiculite, humic soil and perlite, add microcapsule fungal agent and polyacrylamide to make a substrate; (3) Disinfect the seeds of Pinus sylvestris and sow them in the substrate, and control the temperature, humidity and light; before transplanting, carry out light intensity gradient acclimatization, water stress acclimatization and temperature fluctuation acclimatization; (4) Transplanting treatment: when the seedlings reach 20cm, immerse the nutrient cup in abscisic acid solution, prefill the planting hole with organic fertilizer and sulfur powder, irrigate the root zone with a activating solution containing methyl jasmonate and chitin oligosaccharide, and cover with a mixture of pine needles and rice husks to keep moist and suppress weeds; Before transplanting, light intensity gradient acclimatization was carried out, with daily light increase from 5000 lux to 35000 lux. Water stress acclimatization was also carried out, with substrate moisture content gradually reduced to 30%. Temperature fluctuation acclimatization was carried out at 35℃ during the day and 12℃ at night.

2. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, Mix the root-root fungal agent, the Boletus compound fungal agent, and the green-green red mushroom fungal agent in a weight ratio of (55-65):(20-30):(10-20).

3. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, Mix peat moss, vermiculite, humus and perlite in a volume ratio of (30-45):(25-35):(15-25):(5-15) and sterilize. Add microcapsule agent and polyacrylamide to make a matrix.

4. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to any one of claims 1 or 3, characterized in that, Add 45-55 g / L of microcapsule agent and 2.5-3.5 g / L of polyacrylamide.

5. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, PDMY solid medium contains 500 ml / L potato juice, 1 g / L yeast extract, 2.1 g / L malt extract, 5 g / L glucose and 16 g / L agar.

6. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, The high-pressure homogenization condition is 3 cycles at a pressure of 80 MPa.

7. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, The microencapsulated bacterial agent solidification time is 20 min; the sterilization conditions are 115-130℃ high-pressure steam sterilization for 60 min; in step (3), the seeds are soaked in 0.5% potassium permanganate for 30 min and soaked in 25℃ warm water for 12 hours; in step (3), the light intensity gradient acclimatization period is 7 days, with an increase of 5000 lux per day.

8. The mycorrhizal cultivation method for enhancing the stress resistance of Pinus kesiao seedlings according to claim 1, characterized in that, In step (4), the activating solution contains 0.1 mmol / L methyl jasmonate and 0.05% chitin oligosaccharide; 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 substrate is placed in a seedling container, which is a 300 ml black nutrient cup, with 2 seeds sown in each cup and a soil covering depth of 1.5 cm; the seedling environment is controlled at 25℃ during the day and 18℃ at night, with a humidity of 70%, a light intensity of 5000 lux, and the substrate moisture content is maintained at 60% field capacity.

Images