Cultivation method for increasing yield and improving quality of anoectochilus formosanus

Abstract

The invention provides a cultivation method for increasing yield and improving quality of anoectochilus formosanus, which is characterized in that three-element synergistic interaction of plants, soil and microorganisms is driven by inoculating a pseudomonas agent, the problems of low yield, large fluctuation of active ingredients and the like in the traditional cultivation of anoectochilus formosanus are solved, and the specific method comprises the following steps: (1) substrate cultivation; (2) microbial agent construction and activation; according to the cultivation method, while the growth amount and medicinal active ingredients of the anoectochilus formosanus are remarkably increased, the rhizosphere microbial community structure is optimized, double improvement of the yield and quality of the anoectochilus formosanus is synergistically promoted, theoretical support is provided for efficient cultivation and precise regulation and control of the anoectochilus formosanus, and the cultivation method has important scientific significance and application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of roxburghii cultivation, and in particular to a cultivation method for increasing the yield and improving the quality of roxburghii. Background Art

[0002] As an endangered orchid with both ecological and medicinal scarcity, Anoectochilus roxburghii faces the dual dilemma of innovating cultivation techniques and elucidating its secondary metabolic regulation mechanisms. This rare species, known as "pharmaceutical gold," has demonstrated unique value in anti-tumor, hypoglycemic, and immune-modulating activities. Its edible and medicinal properties have been explored in recent years, leading to a surge in market attention and demand. However, Anoectochilus roxburghii production also faces technical bottlenecks, including the unclear mechanisms of action of its active ingredients and a backward industrial breeding and cultivation system. Summary of the Invention

[0003] The object of the present invention is to provide a cultivation method for increasing the yield and improving the quality of Anoectochilus roxburghii, so as to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for increasing the yield and improving the quality of Anoectochilus roxburghii, the method comprising the following steps: Step S1: Material preparation: a. Select disease- and insect-free golden thread vine seedlings that are 20-30 cm tall; b. Prepare a cultivation medium by mixing peat soil and perlite in a volume ratio of 3:1, and plant the seedlings in the cultivation medium; Step S2, preparation of bacterial agent: a. Activate and culture Pseudomonas on LB solid medium at 28 ± 5°C for 24 h. b. Inoculate the activated strain into LB liquid medium and culture at 28°C with shaking at 200 rpm for 24 h. c. Adjust the concentration of bacterial suspension; Step S3, inoculation process: a. Water the roots of Anoectochilus roxburghii seedlings with 20 mL of bacterial suspension. Culture conditions: temperature 28 ± 5°C, humidity 70%, and photoperiod 12 h / d. b. Replenish the bacterial suspension regularly after inoculation.

[0005] Furthermore, the peat soil in step S1 is German peat No. 933, and the seedling pre-cultivation stage is carried out in an artificial growth chamber under the following conditions: temperature 20-25°C, humidity 70-80%, light duration 12h / d, and light intensity 3000-5000lux.

[0006] Furthermore, the pH value of the LB solid medium is 7.0, and its components include 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, and 20 g agar; the pH value of the LB liquid medium is 7.0, and its components include 10 g / L peptone, 10 g / L NaCl, and 5 g / L yeast powder; the concentration of the bacterial suspension is adjusted to OD600 of 1.0.

[0007] Furthermore, in step S3, the bacterial suspension is replenished every other day, and the total replenishment amount is not less than 50% of the initial inoculum amount.

[0008] Furthermore, the Pseudomonas in step S2 needs to be inactivated before activation culture. The inactivation method is as follows: in a sterile environment, the Pseudomonas dilution is spread on an LB plate, and after culturing at 25-28°C for 3-4 days, a single colony is picked and purified by multiple plate streaking until a single strain with uniform morphology is obtained. Compared with the prior art, the present invention has the following beneficial effects: The present invention significantly optimizes the physiological state and metabolic activity of Anoectochilus roxburghii by inoculating Pseudomonas agents. On the one hand, it comprehensively promotes plant morphological development and biomass accumulation: key agronomic traits such as plant height, stem thickness, number of leaves and root length are significantly improved, the root system expansion enhances nutrient absorption capacity, and the fresh weight of a single plant is greatly increased, laying the foundation for high yield. On the other hand, it directionally regulates the synthesis of medicinal active ingredients: the content of flavonoids is significantly increased, the antioxidant and medicinal effects are enhanced, and the stability of basic metabolic substances such as polysaccharides and amino acids is maintained. This shows that the bacterial agent accurately acts on the secondary metabolic pathway and improves quality without interfering with basic physiology.

[0009] This approach systematically optimizes the "plant-soil-microbe" interaction system through microbial community intervention. Improved soil conditions manifest as significant increases in available phosphorus, nitrate nitrogen, and microbial biomass carbon, enhancing fertility supply efficiency. Microbial community reorganization enriches beneficial bacteria such as Pseudomonas and Trichoderma (which possess phosphate solubilizing, hormone-producing, and pathogen-fighting capabilities), inhibits pathogens such as Fusarium, and forms a disease-resistant microecological network. This coordinated regulation of endogenous and exogenous microbiota simultaneously achieves efficient nutrient utilization, reduces disease risk, and enhances ecological adaptability, providing a closed-loop solution for green, high-yield cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The treated seedlings (left) and the control seedlings (right) grown for three months; Figure 2 This is a graph showing the agronomic traits of Anoectochilus roxburghii. (Ai) in the figure shows the comparative changes in fresh weight per plant, plant height, stem diameter, number of leaves, root length, leaf length, leaf width, and leaf weight. Figure 3This is a graph showing the content index of the main active ingredients of Anoectochilus roxburghii. (af) in the figure show the comparative content of flavonoids, total polysaccharide, polysaccharide monosaccharide, amino acids, total phenols and vitamin C. Figure 4 The effects of inoculation with Pseudomonas aureus on the physical and chemical properties of the soil. (Af) Comparison of available phosphorus, available potassium, ammonium nitrogen, nitrate nitrogen, microbial biomass carbon, and soluble organic carbon. Figure 5 The figure shows the index of endophytic bacterial community in the root system of Anoectochilus roxburghii; (ad) in the figure shows the bacterial community composition map, bacterial community composition heat map, PCoA analysis map, and LDA score map; Figure 6 The figure shows the index map of endophytic fungal community in the root system of Anoectochilus roxburghii; (ad) in the figure shows the fungal community composition map, fungal community composition heat map, PCoA analysis map, and LDA score map; In the figure, CK is the control group and OF is the treatment group. DETAILED DESCRIPTION

[0011] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0012] The present invention provides a method for increasing the yield and improving the quality of roxburghii roxburghii, which specifically comprises the following steps: Step S1: Material preparation: a. Select disease-free and insect-free golden thread lotus tissue culture seedlings with a height of 20-30 cm; b. Plant the seedlings in a culture medium prepared by mixing German peat moss No. 933 and perlite in a volume ratio of 3:1. The seedling pre-cultivation phase was conducted in an artificial growth chamber at a temperature of 20-25°C, a humidity of 70-80%, a photoperiod of 12 hours per day, and a light intensity of 3000-5000 lux.

[0013] Step S2, bacterial culture: a. Aseptically spread the diluted Pseudomonas bacteria onto LB plates and incubate at 25-28°C for 3-4 days. b. Pick a single colony and repeatedly streak it onto a plate to purify it until a single strain with uniform morphology is obtained.

[0014] Step S3, preparation of bacterial agent: a. Prepare LB solid medium with a pH of 7.0 using 10 g of peptone, 10 g of NaCl, 5 g of yeast powder, and 20 g of agar. Activate Pseudomonas sp. on this solid medium and incubate at 28°C for 24 h. b. Prepare LB liquid medium with a pH of 7.0 by mixing 10 g of peptone, 10 g of NaCl, and 5 g of yeast powder. Inoculate the activated strain into the LB liquid medium and incubate in a shaker at 28°C and 200 rpm for 24 h. c. After the bacteria have fully grown, measure the OD600 value of the bacterial suspension using a UV spectrophotometer and adjust the bacterial concentration to OD600 of 1.0 using sterile LB liquid medium; Step S4, inoculation process: a. Divide tissue culture seedlings of A. roxburghii into treatment and control groups. Water the roots of the inoculated A. roxburghii seedlings with 20 mL of bacterial suspension and replenish the suspension regularly every other day. The control group was supplemented with an equal amount of sterile water. Both treatment and control groups were placed in a light incubator at 28°C, with a relative humidity of approximately 70% and a photoperiod of 12 hours per day.

[0015] (1) After 150 days of growth, complete plants of the treatment group and the control group were collected. Morphological indicators such as plant height, stem diameter, number of leaves, number of roots, root length, leaf length and leaf width were measured using a vernier caliper and a soft ruler, respectively. Biomass parameters such as fresh weight of a single plant and the ratio of leaf weight to fresh weight were determined using an electronic balance.

[0016] For comparison results, please see Figure 1 and Figure 2 As shown in the results, compared with the control group, the fresh weight of each plant of Anoectochilus roxburghii inoculated with Pseudomonas increased significantly by 83.2%, plant height increased by 56.7%, stem diameter increased by 26.9%, leaf number increased by 23.3%, root length increased by 89.0%, and leaf length and leaf width increased by 16.1% and 5.7%, respectively. However, the ratios of root number and leaf weight to fresh weight did not differ significantly among the treatments, indicating that the addition of Pseudomonas significantly improved the growth performance and agronomic traits of Anoectochilus roxburghii by enhancing the plant's ability to absorb and utilize nutrients.

[0017] (2) Determination of the content of bioactive substances in Anoectochilus roxburghii: Complete plants of Anoectochilus roxburghii that were 150 days old were selected. After removing the mud and sand from the roots, the leaves and stems were quick-frozen in liquid nitrogen, freeze-dried for 48 hours to constant weight, and crushed through an 80-mesh sieve. The total polysaccharide and monopolysaccharide content were determined using the phenol-sulfuric acid method, the amino acids were determined using high-performance liquid chromatography, and the total phenols, flavonoids, and vitamin C were determined using ultraviolet spectrophotometry.

[0018] The results of the measurement can be found in Figure 3As shown in the results, compared with the control group, inoculation with Pseudomonas australis significantly affected the content of some active ingredients in Anoectochilus roxburghii. Among them, the flavonoid content increased significantly by 34.3%, and the total phenolic content decreased significantly by 23.7%. The polysaccharide, amino acid, and vitamin C contents were slightly lower than those in the control group, but the differences were not significant. This suggests that the beneficial bacteria may regulate the plant's secondary metabolic pathways, promote flavonoid synthesis, and interfere with phenolic accumulation, while having little effect on basal metabolism.

[0019] (3) Determination of physical and chemical properties of soil with golden thread lotus: A certain amount of soil from the treatment group and the control group was taken to test the soil physical and chemical properties: Ammonium nitrogen: indigo blue colorimetry (UV-5500 spectrophotometer, 625 nm); Nitrate nitrogen: UV spectrophotometry (UV-5500, 220 / 275 nm dual wavelength calibration); Soluble organic carbon (DOC): water-soil oscillation method (5:1 water-to-soil ratio, 180 rpm × 12 h, centrifugation at 3500 rpm for 25 min, and determination by TOC instrument); Microbial biomass carbon: chloroform fumigation-potassium sulfate extraction method (fumigation for 24 h, 0.5 mol / L K2SO4 extraction, TOC instrument detection).

[0020] The results of the measurement can be found in Figure 4 As shown, the addition of beneficial bacteria to roxburghii soil significantly impacted its physical and chemical properties. Specifically, the effects on available phosphorus, ammonium nitrogen, nitrate nitrogen, and microbial biomass carbon were significant, with some impact on soluble organic carbon. There was no significant difference in available potassium content. Compared with the control group, the available phosphorus content in the roxburghii treatment group increased by 84.8%, ammonium nitrogen content decreased by 23.3%, nitrate nitrogen content increased by 64.3%, microbial biomass carbon content increased by 23.8%, and soluble organic carbon content decreased by 57.2%. Available potassium content was essentially the same in the control and treatment groups, with the treatment group slightly higher than the control.

[0021] (4) Determination of endophytic microorganisms in the root system of Anoectochilus roxburghii. The determination method includes the following steps: 1. Sequencing Process 1. Sample processing: Take the intact root system of Anoectochilus roxburghii, freeze it in liquid nitrogen and grind it into powder. Use CTAB method to extract total DNA. The purity requirement is A260 / A280=1.8-2.0.

[0022] 2. Amplicon selection: Fungi: ITS1-ITS2 region (primers ITS1F / ITS2R); Bacteria: 16S rRNA gene V5-V7 region (primers 799F / 1193R, containing specific barcode).

[0023] 2. PCR Amplification Conditions (1) Fungal ITS amplification Reaction system (30 µL): 15 µL Phusion® High-Fidelity PCR Master Mix (NEB), 0.5 U AccuPrimer™ Taq (Life Technologies), 0.2 µM primers, and 10 ng template DNA.

[0024] Thermal cycling parameters: Pre-denaturation at 98°C for 1 min; 30 cycles (98°C 10 s→50°C 30 s→72°C 60 s); Final extension was performed at 72°C for 5 min.

[0025] (2) Bacterial 16S amplification Reaction system (20 µL): 2 µL 10× PCR Buffer, 2 µL 2.5 mmol / L dNTPs, 0.8 µL 5 µmol / L primers, 0.2 µL rTaq enzyme, 0.2 µL BSA, 10 ng template DNA.

[0026] Thermal cycling parameters: Pre-denaturation at 95°C for 3 min; 35 cycles (95°C 30 s→55°C 30 s→72°C 45 s); Final extension was performed at 72°C for 10 min and the cells were stored at 10°C.

[0027] 3. Sequencing and Analysis Platform: Illumina Novaseq 6000 (PE250 mode); Data quality control: remove low-quality sequences (Q20 < 90%) and chimeras (UCHIME algorithm); Species annotation: fungi are based on the UNITE v10 database, bacteria are based on Silva v138; Statistical tools: Alpha diversity (Shannon / Chao1 index) was calculated using Qiime2, and differences between groups were analyzed using Lexar Element Analysis (LDA > 3.0).

[0028] The results of the measurement can be found in Figure 5 and Figure 6 As shown in Figure 2, the effects of inoculation with Pseudomonas on the root endophytes and soil microorganisms of Anoectochilus roxburghii are as follows: the composition of the top 20 bacterial communities ranked by abundance at the genus level shows that ( Figure 5a) The abundance of the genera Asticcacaulis, Enterobacter, Novosphingobium, Acidothemus, Altererythrobacter, Acidicaldus, Roseiarcus, and Pseudolabrys in the inoculated treatment was higher than that in the CK. Among them, compared with the CK, the proportion of the genus Asticcacaulis in the bacterial community increased by 1.47%, the proportion of the genus Enterobacter increased by 3.65%, the proportion of the genus Novosphingobium increased by 2.54%, the proportion of the genus Acidothemus increased by 0.45%, the proportion of the genus Altererythrobacter increased by 0.04%, the proportion of the genus Acidicaldus increased by 0.58%, the proportion of the genus Roseiarcus increased by 0.15%, and the proportion of the genus Pseudolabrys increased by 0.13%.

[0029] From the heat map of the top 20 bacterial communities ranked by abundance at the genus level, we can see that ( Figure 5 b). The abundances of Pseudomonas, Asticcacaulis, Enterobacter, Altererythrobacter, Novosphingobium, Acidicaldus, Acidothemus and Roseiarcus in the inoculated treatment were significantly higher than those in the CK, while the abundances of Bradyrhizobium, Bordetella, Sphingomonas, Bacillus, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Reyranella, Paenibacillus, Burkholderia-Caballeronia-Paraburkholderia, Sphingobium and Caulobacter were significantly lower than those in the CK.

[0030] Depend on Figure 5 c shows that the first two axes of the PCoA analysis of the bacterial community of Anoectochilus roxburghii can explain 36.9% of the results. On the PCo1 axis, there is no intersection between the inoculated treatment and the CK samples.

[0031] According to the LDA score map of LEfSe ( Figure 5d) As can be seen, the LEfSe treatment detected more significantly different clades (10 clades, 2 phyla, 2 classes, 2 orders, 3 families, and 1 genus) than the CK treatment (6 clades, 2 phyla, 2 classes, 1 order, and 1 family). Among them, Pseudomonas was the most dominant genus in the inoculated treatment.

[0032] The composition of the top 20 fungal communities at the genus level shows that ( Figure 6 a) The abundance of the genera Trichoderma, Longitudinalis, Talaromyces, Cyphellophora, and Aspergillus in the inoculated treatment was higher than that in the CK. Compared with the CK, the proportion of Trichoderma in the fungal community increased by 10.03%, Longitudinalis by 0.82%, Talaromyces by 0.28%, Cyphellophora by 0.45%, and Aspergillus by 0.24%.

[0033] From the heat map of the top 20 fungal communities ranked by abundance at the genus level, we can see that ( Figure 6 b) The abundance of Penicillium, Cyphellophora, Aspergillus, Humicola, Hypomyces, Talaromyces, Trichoderma, Longitudinalis, Pyrenochaetopsis and Exophiala in the inoculated treatment was significantly higher than that in the CK, while the abundance of Neopyrenochaeta, Fusarium, Saitozyma, Auricularia and Colletotrichum was significantly lower than that in the CK.

[0034] Depend on Figure 6 c shows that the first two axes of the PCoA analysis of the fungal community of Anoectochilus roxburghii can explain 60.6% of the results. On the PCo1 axis, there is no intersection between the inoculated treatment and the CK samples.

[0035] According to the LDA score graph of LEfSe ( Figure 6 d) As can be seen, the number of significantly different clades detected in the inoculated LEfSe treatment (15 clades, 2 orders, 6 families, and 7 genera) was less than that in the CK treatment (4 clades, 2 families, and 2 genera). Among them, Trichoderma, Gelasinospora, and Acremonium were the significantly dominant genera in the inoculated treatment.

[0036] The inoculation treatment significantly altered the root endophytes and soil microbial communities of A. roxburghii. The abundance of beneficial bacterial genera, particularly Pseudomonas and Trichoderma, increased significantly, while the abundance of potential pathogenic genera, such as Penicillium and Fusarium, decreased. These changes in the microbial community contribute to enhanced disease resistance and nutrient uptake efficiency in A. roxburghii. The increased abundance of Pseudomonas, a bacterial community capable of solubilizing insoluble phosphates in the soil, significantly altered the endophytic microbial community structure in A. roxburghii, thereby affecting its nutrient supply.

[0037] Trichoderma, a common soil and rhizosphere fungus, has been widely studied and applied in agricultural production. It produces hormonally active metabolites, such as indoleacetic acid (IAA), which promote root growth and increase root surface area, thereby improving the plant's ability to absorb nutrients and minerals. This has a certain positive effect on the nutrient absorption and growth of A. roxburghii. Furthermore, Trichoderma is known for its potent antagonistic and parasitic abilities, inhibiting soil-borne plant pathogens by producing a variety of antibiotics and parasitizing other fungi. This suggests that inoculation with Pseudomonas significantly alters the microbial environment in the rhizosphere of A. roxburghii. Such changes in the abundance of root endophytes and soil microorganisms contribute to enhanced disease resistance and growth performance of A. roxburghii. These changes have a certain impact on the agronomic traits, soil physical and chemical properties, active substances, and quality of A. roxburghii after inoculation.

[0038] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for increasing the yield and improving the quality of Anoectochilus roxburghii, characterized in that: The method comprises the following steps: Step S1: Material preparation: a. Select disease- and insect-free golden thread vine seedlings that are 20-30 cm tall; b. Prepare a cultivation medium by mixing peat soil and perlite in a volume ratio of 3:1, and plant the seedlings in the cultivation medium; Step S2, preparation of bacterial agent: a. Activate and culture Pseudomonas on LB solid medium at 28 ± 5°C for 24 h. b. Inoculate the activated strain into LB liquid medium and culture at 28°C with shaking at 200 rpm for 24 h. c. Adjust the concentration of bacterial suspension; Step S3, inoculation process: a. Water the roots of Anoectochilus roxburghii seedlings with 20 mL of bacterial suspension. Culture conditions: temperature 28 ± 5°C, humidity 70%, and photoperiod 12 h / d. b. Replenish the bacterial suspension regularly after inoculation.

2. The method for increasing yield and improving quality of Anoectochilus roxburghii according to claim 1, wherein: In step S1, the peat soil is German peat No. 933, and the seedling pre-cultivation stage is carried out in an artificial growth chamber under the following conditions: temperature of 20-25° C., humidity of 70-80%, light duration of 12 h / d, and light intensity of 3000-5000 lux.

3. The method for increasing yield and improving quality of Anoectochilus roxburghii according to claim 1, wherein: The pH value of the LB solid medium is 7.0, and its components include 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast powder, and 20 g agar; The pH value of the LB liquid medium is 7.0, whose components include peptone 10 g / L, NaCl 10 g / L, and yeast powder 5 g / L; the concentration of the bacterial suspension was adjusted to an OD600 of 1.

0.

4. The method for increasing yield and improving quality of Anoectochilus roxburghii according to claim 1, wherein: In step S3, the bacterial suspension is replenished every other day, and the total replenishment amount is not less than 50% of the initial inoculum amount.

5. The method for increasing yield and improving quality of Anoectochilus roxburghii according to claim 1, wherein: The Pseudomonas in step S2 must be inactivated before activation culture. The inactivation method is as follows: in a sterile environment, the Pseudomonas dilution is spread on an LB plate, and after culturing at 25-28°C for 3-4 days, a single colony is picked and purified by multiple plate streaking until a single strain with uniform morphology is obtained.

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