A ginseng high-yield planting method based on short stick branch lecanora material optimization and ginseng stress resistance activation

Abstract

The application discloses a high-yield planting method of Gastrodia elata based on short stick branch leucopaxillus material optimization and Gastrodia elata stress resistance activation, and realizes yield increase and quality improvement through composite strengthening treatment of the material and composite strain inoculation technology. The method comprises the following steps: 1) the short stick branch leucopaxillus substrate is soaked in a high-activity material formed by fermentation of lactobacillus plantarum and nano-composite nutrient solution; 2) nitrogen-fixing bacteria, disease-resistant actinomycetes and armillaria mellea are mixed and inoculated at a ratio of 3:1:1 to improve nitrogen supply and reduce the incidence of black rot; 3) the Gastrodia elata is activated by a pulse electric field (5 kV / cm, 10 μs) to activate GASA stress resistance genes, and combined with calcium signal pathway strengthening technology, so that the germination rate reaches 88.5%; and 4) the azoxystrobin is loaded on chitosan-silicon dioxide nanoparticles to realize long-acting prevention and control of diseases. The application makes the per mu yield of Gastrodia elata increase by 30%-40%, the content of Gastrodia elata element increase by more than 30%, the chemical pesticide dosage decrease by 55%, the strain passage purity is stable, is suitable for large-scale planting in multiple climate zones, and provides an innovative solution for green production of traditional Chinese medicinal materials.

Description

Technical Field

[0001] This invention belongs to the field of Gastrodia elata cultivation technology, specifically, it relates to a high-yield cultivation method for Gastrodia elata based on the optimization of short-stem linden fungal material and the activation of stress resistance in seed Gastrodia elata. Background Technology

[0002] As a precious traditional Chinese medicine, the core of Gastrodia elata's cultivation lies in the highly efficient symbiotic system between Armillaria mellea and the substrate. Traditional techniques for preparing the substrate present the following key challenges: Current substrate preparation techniques mostly use coarse wood (diameter > 5 cm) as the substrate, requiring 3-4 months of natural fermentation before inoculation with Armillaria mellea. This process has the following drawbacks: (1) Traditional fungal materials rely solely on a simple mixture of sawdust and wheat bran. After colonization, the mycelial growth rate of Armillaria mellea is only 0.3-0.5 cm / d, and repeated subculturing (>3 times) easily leads to mycelial degeneration and a 40%-60% decrease in fluorescence intensity (RFU value). The preparation of fungal materials and the planting of Gastrodia elata need to be carried out in stages, and synchronous sowing cannot be achieved, resulting in a limited number of planting batches per year.

[0003] (2) The nutrient supply is limited and the nitrogen fixation capacity of Armillaria mellea is limited. Additional chemical nitrogen fertilizer (such as urea) needs to be applied, which increases the planting cost. Summary of the Invention

[0004] To overcome the problems existing in the prior art, this invention proposes a high-yield cultivation method for Gastrodia elata based on the optimization of short-stem linden mycelium and activation of stress resistance in seed tubers, comprising the following steps: S1. Pretreatment and activity enhancement of fungal materials S1.1 Mix short linden branches (0.5-1cm in diameter, 2-3cm in length) with sawdust and wheat bran in a mass ratio of 42%:42%:16%, add 1% sugar and 0.2% nitrogen-fixing fertilizer, and sterilize by high-pressure steam at 121℃ for 30 minutes; S1.2 The sterilized substrate was soaked in Lactobacillus plantarum solution (OD600=0.8) for 24 hours, and then inoculated with Armillaria mellea mother culture "Yifeng No. 4" and cultured at 20-25℃ in the dark for 30-45 days to form active mycelial material; S2. Composite reinforcement treatment of fungal substrate S2.1 Make fish-scale-like cuts with a depth of 2-3 mm and a spacing of 1 cm on the surface of the fungal material obtained in step S1.2, and dry it until the moisture content is 25%-30%; S2.2 was soaked in a nanocomposite nutrient solution (chitosan-silica particles loaded with azoxystrobin, particle size 80±20nm, drug loading 15%) containing 2% glucose, 0.01% folic acid, 0.05% caffeine, and 0.1% natamycin for 2 hours. S3. Co-inoculation of compound strains: Mix nitrogen-fixing bacteria, disease-resistant actinomycetes and Armillaria mellea at a live count ratio of 3:1:1, and inoculate the fungal material treated in step S2. Incubate at 25°C in the dark for 7 days. S4. Synchronous sowing of hemp seeds and bed construction S4.1 The fungal material obtained in step S3 and the seed of "Yihongyou No. 1" Gastrodia elata, which had been treated with a pulsed electric field (field strength 5kV / cm, pulse width 10μs, treatment for 10 minutes) and 0.5% trehalose-50mCaCl2 solution (molar ratio 1:2), were simultaneously sown in sandy loam soil with a pH of 5.8-6.0. S4.2 The gaps in the mushroom bed are filled with 0.5-1mm quartz sand (3-5cm thick). A permeable non-woven fabric (80g / m²) is laid under the quartz sand layer. An ultrasonic insect repellent device (frequency 25kHz, power 10W, working for 5 minutes at 30-minute intervals) is buried per square meter.

[0005] Furthermore, the nanocomposite nutrient solution is prepared by the following method: 2.1 Preparation of chitosan-silica nanoparticles by ionic crosslinking: Chitosan (3wt%) and tetraethyl orthosilicate (TEOS, 1wt%, molar ratio of chitosan to 1:2) were dissolved in acetic acid solution, sodium tripolyphosphate was added dropwise for crosslinking, the pH was adjusted to 5.0, and nanoparticles with a particle size of 80±20nm were obtained after centrifugation and washing. 2.2 Drug loading: Azoxystrobin (15 mg / mL) was dissolved in ethanol and mixed with the nanoparticle solution at a volume ratio of 1:5. After stirring for 24 hours, the mixture was centrifuged and dried, and the drug loading reached 15%. 2.3 Slow-release performance: The release rate of azoxystrobin was detected by HPLC, and a first-order kinetic equation was fitted, with R²≥0.98 (simulated soil solution: pH6.0, 25℃).

[0006] Further, the seed treatment includes: 3.1 Gene expression induction: Gastrodia elata GASA family genes were activated by pulsed electric field (5kV / cm, 10μs pulse width); 3.2 Activation of calcium signaling pathway: Hemp cells were loaded with Fluo-4AM probe (5 μM concentration), and the peak concentration of cytoplasmic calcium ions was increased by 40% and the germination rate reached 85%-90% by laser confocal microscopy (excitation wavelength 488 nm).

[0007] Furthermore, the disease prevention and control includes: 4.1 Pretreatment of the mushroom bed: Apply 500g / m² of lime powder before sowing to adjust the soil pH to 6.0-6.5; 4.2 Biological control agent combination: Alternate spraying of Trichoderma agent (1×10 8 CFU / mL) and Bacillus subtilis agent (5×10)8 Mix CFU / mL at a volume ratio of 1:5 and spray once every 15 days. This method improves the control efficiency by 15%-20% compared to the conventional chemical control group (pyraclostrobin spray).

[0008] Furthermore, the method for controlling the propagation of the Armillaria mellea mother culture is as follows: 5.1 Fluorescent labeling detection: The purity of the hyphae was verified by fluorescence microscopy using a GFP-labeled Armillaria mellea-specific promoter (glyceraldehyde-3-phosphate dehydrogenase gene promoter). 5.2 Purity standards: mycelial growth rate 0.5-0.8 cm / d, fluorescence intensity RFU > 3000, and contamination rate ≤ 0.1%.

[0009] Compared with the prior art, the present invention achieves at least the following beneficial effects: 1. This invention uses Lactobacillus plantarum fermentation and nano-composite nutrient solution soaking to shorten the colonization time of Armillaria mellea mycelium to 3-5 days (7-10 days in the traditional method), increase the mycelial growth rate to 0.5-0.8 cm / d (0.3-0.5 cm / d in the traditional method), shorten the preparation cycle of fungal material by 40%, and realize the simultaneous sowing of "fungal material-seed hemp".

[0010] 2. This invention mixes nitrogen-fixing bacteria, disease-resistant actinomycetes, and Armillaria mellea in a 3:1:1 ratio to form a synergistic system. The nitrogen-fixing bacteria increase nitrogen supply by 30% and reduce chemical fertilizer usage by 20%-30%, while the disease-resistant actinomycetes reduce the incidence of black rot by 40% and reduce chemical pesticide usage by 55%.

[0011] 3. This invention activates the Gastrodia elata GASA family genes through a pulsed electric field (5 kV / cm, 10 μs pulse width), and combined with calcium signaling pathway activation technology, the germination rate reaches 85%-90% (70% with traditional methods), the tuber enlargement period is extended by 7-10 days, and the fresh weight of a single plant increases by 15%-20%. Detailed Implementation

[0012] Unless otherwise stated, all materials and reagents used in this invention are commercially available.

[0013] 1. Experimental Materials and Equipment Mushroom substrate materials: short linden branches (0.5-1 cm in diameter, 2-3 cm in length), sawdust (pine wood), wheat bran (commercially available); Microbial strains: "Yifeng No. 4" Armillaria mellea, nitrogen-fixing bacteria, and disease-resistant actinomycetes; Seedlings: "Yihong You 1" Gastrodia elata zero-generation seedlings (diameter 1-2 cm); Equipment: Autoclave, pulsed electric field generator (5 kV / cm), laser confocal microscope (Olympus FV3000), ultrasonic insect repellent device (25 kHz, 10 W).

[0014] 2. Implementation Steps 2.1 Pretreatment and activity enhancement of fungal materials Preparation of mixed matrix (S1.1): Mix short sticks, linden branches, sawdust, and wheat bran in a ratio of 42%:42%:16%, add 1% sucrose and 0.2% nitrogen-fixing fertilizer (urea), and pack into sterilization bags; sterilize at 121℃ with high-pressure steam for 30 minutes, and cool to room temperature.

[0015] Activation of fungal material (S1.2): After sterilization, the substrate was soaked in Lactobacillus plantarum solution (OD600=0.8) for 24 hours, drained, and then inoculated with Armillaria mellea mother culture "Yifeng No. 4" (inoculation amount 5% w / w). After being cultured at 25℃ in the dark for 40 days, the mycelial coverage reached 95%, and the fluorescence intensity RFU=5200±300.

[0016] 2.2 Composite reinforcement treatment of fungal substrate (S2.1-S2.2) Surface treatment: Use a special knife to make fish-scale-like cuts 2.5 mm deep and 1 cm apart on the surface of the fungus; Dry at 45℃ until the moisture content is 28%.

[0017] Soaking in nano-nutrient solution: Prepare a nanocomposite nutrient solution containing 2% glucose, 0.01% folic acid, 0.05% caffeine, and 0.1% natamycin; After soaking the fungal material for 2 hours, it is drained and a nano-drug film is formed on the surface.

[0018] 2.3 Preparation of Nanocomposite Nutrient Solution Nanoparticle synthesis: Dissolve 3 wt% chitosan and 1 wt% tetraethyl orthosilicate (TEOS, molar ratio 1:2) in a 2% acetic acid solution; Add 5% sodium tripolyphosphate (pH=5.0) dropwise, centrifuge (8000 rpm, 15 min) to obtain particles with a diameter of 80±20 nm.

[0019] Drug loading and sustained release validation: Mix 15 mg / mL azoxystrobin ethanol solution with nanoparticle solution at a ratio of 1:5 and stir for 24 hours. HPLC analysis showed a cumulative release rate of 82.3% over 60 days, with a first-order kinetic fit R² = 0.983.

[0020] 2.4 Co-inoculation with mixed bacterial strains (S3) Mix nitrogen-fixing bacteria, disease-resistant actinomycetes and Armillaria mellea at a live count ratio of 3:1:1 (total inoculum 10% w / w). After inoculation, the mycelial fluorescence intensity was 4800±250 after 7 days of incubation at 25°C in the dark.

[0021] 2.5 Simultaneous sowing of hemp seeds and construction of seedbeds (S4.1-S4.2) Hemp seed treatment: Pulsed electric field treatment: field strength 5 kV / cm, pulse width 10 μs, treatment time 10 minutes; Soaking solution: 0.5% trehalose + 50 mM CaCl2 (molar ratio 1:2), soaking for 48 hours; Laser confocal microscopy showed that the peak calcium ion concentration increased by 42%, and the germination rate was 88.5% (compared to 70.2% in the control group).

[0022] Mushroom bed construction: Sow the fungal material and seed hemp in sandy loam soil with a pH of 5.9 at a ratio of 5:1 (by weight); Fill with 0.5-1 mm quartz sand (4 cm thick), and lay a bottom layer of 80 g / m² permeable non-woven fabric; An ultrasonic insect repellent device is installed per square meter (working cycle: 30-minute interval, 5-minute operation).

[0023] 2.6 Disease Prevention and Control Pretreatment of the mushroom bed: Apply 500 g / m² of lime powder and adjust the pH to 6.2; Biological control: Alternately spray Trichoderma agent (1×10) 8 CFU / mL) and Bacillus subtilis agent (5×10) 8 CFU / mL), volume ratio 1:5, sprayed at 15-day intervals; The incidence rate of black rot was 3.8%.

[0024] 2.7 Control of strain passage The Armillaria mellea-specific promoter (glyceraldehyde-3-phosphate dehydrogenase gene promoter) labeled with GFP was used for detection after 3 passages. The mycelial growth rate was 0.7 cm / d, the fluorescence intensity RFU was 3100±150, and the contamination rate of other microorganisms was 0.08%.

[0025] 3. Control experiment setup 1. Control group setup A total of 4 groups of experiments were set up, each group Example: Implement all steps of claims 1-5 to verify the synergistic effect of the entire technical solution; Control 1: The substrate used for the fungal culture was traditional coarse linden wood (5-7 cm in diameter, naturally fermented for 4 months). All other steps were exactly the same as in the example, verifying the advantages of short linden wood substrate + rapid fermentation technology. Control 2: Step S2.2 (soaking in nanocomposite nutrient solution) is omitted, and the other steps are exactly the same as in the example, to verify the contribution of nano slow-release nutrient solution to mycelial activity and disease control; Control 3: In step S3, only Armillaria mellea was inoculated (live bacteria ratio 0:0:1), and the other steps were exactly the same as in the example, to verify the enhancement of nitrogen fixation and disease resistance of the compound strain.

[0026] 2. Comparison of Experimental Data Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A high-yield cultivation method for Gastrodia elata based on optimization of short-stem linden mycelium and activation of stress resistance in seed tubers, characterized in that, Includes the following steps: S1. Pretreatment and activity enhancement of fungal materials S1.1 Mix short linden branches with a diameter of 0.5-1cm and a length of 2-3cm with sawdust and wheat bran in a mass ratio of 42%:42%:16%, then add 1% sugar and 0.2% nitrogen-fixing fertilizer, and sterilize by high-pressure steam at 121℃ for 30 minutes. S1.2 The sterilized substrate was soaked in a solution of Lactobacillus plantarum with an OD600 value of 0.8 for 24 hours, and then inoculated with Armillaria mellea mother culture "Yifeng No. 4" and cultured at 20-25℃ in the dark for 30-45 days to form active mycelial material; S2. Composite reinforcement treatment of fungal substrate S2.1 Make fish-scale-like cuts with a depth of 2-3 mm and a spacing of 1 cm on the surface of the fungal material obtained in step S1.2, and dry it until the moisture content is 25%-30%; S2.2 The dried fungal material was soaked in a nanocomposite nutrient solution for 2 hours. The nanocomposite nutrient solution contained 2% glucose, 0.01% folic acid, 0.05% caffeine and 0.1% natamycin, and was loaded with azoxystrobin encapsulated in chitosan-silica particles with a particle size of 80±20 nm and a drug loading of 15%. S3. Co-inoculation with compound bacterial strains Nitrogen-fixing bacteria, disease-resistant actinomycetes and Armillaria mellea were mixed at a live count ratio of 3:1:1 and inoculated onto the fungal material treated in step S2. The mixture was then incubated at 25°C in the dark for 7 days. S4. Simultaneous sowing of hemp seeds and construction of seedbeds S4.1 The fungal material obtained in step S3 and the "Yihongyou No. 1" Gastrodia elata seed hemp treated with pulsed electric field and seed soaking were simultaneously sown in sandy loam soil with a pH of 5.8-6.

0. The pulsed electric field strength was 5kV / cm, the pulse width was 10μs, and the treatment time was 10 minutes. The seed soaking treatment used a mixed solution of 0.5% trehalose and 50mM CaCl2, wherein the molar ratio of trehalose to CaCl2 was 1:

2. S4.2 Fill the gaps in the mushroom bed with quartz sand with a particle size of 0.5-1mm and a filling thickness of 3-5cm. Lay a permeable non-woven fabric with a weight of 80g / m² under the quartz sand layer, and bury an ultrasonic insect repellent device in each square meter of mushroom bed. The device has a frequency of 25kHz, a power of 10W, and works for 5 minutes every 30 minutes.

2. The method for high-yield cultivation of Gastrodia elata based on optimization of short-stemmed linden mycelium and activation of stress resistance in seed tubers, as described in claim 1, is characterized in that... The nanocomposite nutrient solution is prepared by the following method: 2.1 Preparation of chitosan-silica nanoparticles by ionic crosslinking: 3% by mass of chitosan and 1% by mass of tetraethyl orthosilicate were dissolved in acetic acid solution, wherein the molar ratio of tetraethyl orthosilicate to chitosan was 1:

2. Sodium tripolyphosphate was added dropwise for crosslinking, the pH was adjusted to 5.0, and nanoparticles with a particle size of 80±20 nm were obtained after centrifugation and washing. 2.2 Drug loading: Azoxystrobin was dissolved in ethanol to prepare a 15 mg / mL solution. This solution was mixed with the nanoparticle solution at a volume ratio of 1:

5. After stirring for 24 hours, the mixture was centrifuged and dried to achieve a drug loading of 15%. 2.3 The release performance of azoxystrobin in the obtained nanocomposite nutrient solution meets the following requirements: in a simulated soil solution with pH 6.0 and temperature 25℃, the release rate was detected by HPLC and a first-order kinetic equation was fitted, and the fitting correlation coefficient R² ≥ 0.

98.

3. The method for high-yield cultivation of Gastrodia elata based on optimization of short-stemmed linden mycelium and activation of stress resistance in seed tubers, as described in claim 1, is characterized in that... The pulsed electric field described in step S4.1 is also used to activate the Gastrodia elata GASA family genes.

4. The method for high-yield cultivation of Gastrodia elata based on optimization of short-stem linden mycelium and activation of stress resistance in seed tubers, as described in claim 1, is characterized in that... It also includes disease control, which includes: 4.1 Before sowing, sprinkle lime powder on the surface of the mushroom bed at a rate of 500g / m² and adjust the soil pH to 6.0-6.5; 4.2 During the growth period, alternately spray with Trichoderma agent and Bacillus subtilis agent, wherein the viable bacterial concentration of the Trichoderma agent is 1×10⁻⁶. 8 The viable bacterial concentration of the Bacillus subtilis agent is 5 × 10 CFU / mL. 8 CFU / mL, the two are mixed at a volume ratio of 1:5 and sprayed once every 15 days.

5. The method for high-yield cultivation of Gastrodia elata based on optimization of short-stemmed linden mycelium and activation of stress resistance in seed tubers, as described in claim 1, is characterized in that... The Armillaria mellea mother culture meets the following purity and activity quality control standards: 5.1 The purity of the mycelium was verified by a fluorescent labeling detection method, which utilizes a GFP-labeled Armillaria mellea-specific promoter, namely the glyceraldehyde-3-phosphate dehydrogenase gene promoter, and the purity of the mycelium was verified by fluorescence microscopy. 5.2 The quality control standards are as follows: mycelial growth rate is 0.5-0.8 cm / d, fluorescence intensity RFU value is greater than 3000, and contamination rate of miscellaneous bacteria does not exceed 0.1%.

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