Method for promoting growth and development and increasing yield and quality of traditional Chinese medicinal materials by using photosynthetic microorganisms

Abstract

The application provides a method for promoting growth and development and yield and quality improvement of traditional Chinese medicinal materials by using photosynthetic microalgae, and belongs to the technical field of microalgae application. The application provides application of microalgae capable of photosynthesis in cultivation of traditional Chinese medicinal materials, wherein the microalgae are planted on leaves of the traditional Chinese medicinal materials by foliar spraying. The microalgae capable of photosynthesis can be planted on leaves of the traditional Chinese medicinal materials, thereby promoting growth and development of angelica sinensis; endogenous hormones and microbial community composition of the leaves are changed, thereby regulating rhizosphere soil microbial community structure and function, changing soil physical and chemical properties, thereby significantly promoting yield increase of the traditional Chinese medicinal materials, reducing diseases, improving effective component content, and effectively improving stress resistance. The application further provides a cultivation method of the traditional Chinese medicinal materials, wherein the microalgae fungicide is sprayed on leaves after true leaves grow or after transplanting; the method is not affected by environmental factors, soil types, farm operations and indigenous microorganisms, and has stable effect and saves time and labor.

Description

Technical Field

[0001] This invention belongs to the field of microalgae application technology, specifically relating to a method for promoting the growth, development, yield increase, and quality improvement of Chinese medicinal materials using photosynthetic microorganisms. Background Technology

[0002] Angelica sinensis ranks first among the authentic medicinal herbs of Gansu Province. As a product with both medicinal and edible uses, its main bioactive components include flavonoids, ferulic acid, volatile oils, polysaccharides, amino acids, and various trace elements. The main cultivation areas of Angelica sinensis are located in Gansu, Qinghai, and Yunnan provinces of my country. In traditional producing areas, yield and quality have significantly declined due to continuous cropping obstacles and changing climate conditions. Traditional cultivation methods heavily rely on chemical fertilizers and pesticides, which do not align with the current pursuit of reducing chemical inputs. Therefore, the development of new alternative inputs is imperative.

[0003] Cyanobacteria are a type of prokaryotic photosynthetic microorganism widely distributed in terrestrial environments. In recent years, their agricultural applications as renewable biofertilizers and soil conditioners have received widespread attention, effectively improving soil fertility and plant productivity, and replacing chemical fertilizers and pesticides. As important primary producers, cyanobacteria can assimilate atmospheric carbon dioxide into organic matter, thereby significantly increasing the soil organic carbon (C) content in agricultural ecosystems. Secondly, cyanobacteria have nitrogen-fixing capabilities; for example, nitrogen-fixing algae (such as those in the genus *Anabaena*)... Anabaena azotica It can improve soil fertility. Furthermore, different algae may contain plant growth regulators such as auxins, gibberellins, and cytokinins; they may also contain chemical substances such as algal toxins that inhibit plant growth and development and interfere with bacterial community structure. The cultivation of medicinal plants follows the principle of prioritizing quality while considering yield, and the effects of different cyanobacteria on medicinal plants are rarely reported. Summary of the Invention

[0004] This invention provides a method for promoting the growth, development, yield, and quality improvement of Chinese medicinal herbs by utilizing photosynthetic microorganisms. This method can be achieved through foliar spraying, which promotes the growth and development of Chinese medicinal herbs and increases the content of effective components.

[0005] This invention provides the application of photosynthetic microalgae in the cultivation of Chinese medicinal herbs, wherein the microalgae are established on the leaves of the Chinese medicinal herbs by foliar spraying.

[0006] In one specific embodiment of the present invention, the type of microalgae includes cyanobacteria.

[0007] In one specific embodiment of the present invention, the microalgae include *Microsheatha* (a type of algae). Microcoleus vaginatus ).

[0008] In one specific embodiment of the present invention, the medicinal material includes Angelica sinensis.

[0009] The present invention also provides the application of photosynthetic microalgae in promoting the growth of Chinese medicinal materials, reducing diseases and improving quality, wherein the microalgae are established on the leaves of the Chinese medicinal materials by foliar spraying.

[0010] In one specific embodiment of the present invention, the promotion of growth, reduction of diseases, and improvement of quality of Chinese medicinal materials includes at least one of the following: (1) Significantly promotes increased yield, reduces disease, and increases the content of effective components in traditional Chinese medicine; (2) Significantly reduces the content of 1-aminocyclopropanecarboxylic acid; increases the content of abscisic acid and jasmonic acid, effectively improving stress resistance; (3) Significantly altered the leaf microbial community; (4) Significantly promotes soil carbon fixation, nitrogen assimilation and potassium activation capacity; (5) Significant enrichment of soil-specific probiotics; (6) Significantly reduced abundance of marker pathogens.

[0011] The present invention also provides a method for cultivating Chinese medicinal materials, including spraying microalgae capable of photosynthesis onto the leaves of the Chinese medicinal materials during the cultivation period.

[0012] In one specific embodiment of the present invention, the foliar spraying includes spraying a microalgae agent capable of photosynthesis; The method for preparing the microalgae inoculant includes inoculating the photosynthetic microalgae into BG-11 culture medium for cultivation to obtain the microalgae inoculant.

[0013] In one specific embodiment of the present invention, the effective bacteria in the microalgae agent are *Micrococcus sheathingii* (…). Microcoleus vaginatus When the effective viable count is (1~3)×10⁻⁶, the effective viable count is (1~3)×10⁻⁶. 5 per mL.

[0014] In one specific embodiment of the present invention, the spraying is carried out after the true leaves have grown or after transplanting.

[0015] In one specific embodiment of the present invention, the spraying is performed 3 to 5 times, and the time interval between two consecutive sprayings is 3 to 5 weeks.

[0016] Beneficial Effects: This invention provides the application of photosynthetic microalgae in the cultivation of Chinese medicinal herbs. The microalgae are established on the leaves of the medicinal herbs through foliar spraying. The photosynthetic microalgae described in this invention can establish themselves on the leaves of Chinese medicinal herbs, thereby promoting the growth and development of Angelica sinensis and increasing the content of its active ingredients.

[0017] The microalgae described in this invention can be applied to leaves and colonize them, thereby altering the composition of endogenous hormones and microbial communities in the leaves. This, in turn, regulates the structure and function of the rhizosphere soil microbial community, changes the soil's physicochemical properties, and thus significantly promotes the yield of medicinal herbs, reduces diseases, and increases the content of effective components. It also significantly reduces the content of 1-aminocyclopropanecarboxylic acid; increases the content of abscisic acid and jasmonic acid, effectively improving stress resistance, especially under high temperature and drought stress; and significantly alters the leaf microbial community, increasing the number of photosynthetic cyanobacteria, thereby improving leaf nitrogen fixation, nitrate reduction, urea decomposition, and oxidative photoautotrophy. Fermentation produces acid, promotes the accumulation of photosynthetic sugars in leaves, the conversion of nitrate nitrogen to ammonium nitrogen, and the scavenging of reactive oxygen species; foliar spraying significantly promotes soil carbon fixation, nitrogen assimilation, potassium activation, and the conversion of ammonium nitrogen to nitrate nitrogen; foliar spraying significantly enriches soil-specific beneficial bacteria *Pseudomonas putida* and beneficial fungi *Rhizopus irregularis*; foliar spraying significantly reduces the abundance of the specific pathogenic bacteria *Stenotrophomonas maltophilia* and pathogenic fungi *Fusarium oxysporum*, *Fusarium vanetenianum*, *Fusarium solani*, and *Cladosporium*; foliar spraying mainly affects the content of JA and ABA and the abundance of Nocardia spp. strain S5 (…). Nocardioides sp The abundance of S5 affects the average weight of a single plant and the content of ferulic acid in medicinal materials.

[0018] This invention provides the application of *Micrococcus sheathingans* in the cultivation of *Angelica sinensis*. The examples demonstrate that foliar spraying with *Micrococcus sheathingans* significantly promotes the accumulation of photosynthetic sugars in *Angelica sinensis* leaves compared to the control, accelerates the conversion of nitrate nitrogen to ammonium nitrogen, and significantly enhances the scavenging capacity of reactive oxygen species. Under the planting year and environmental conditions of the examples, the absolute value of 1-aminocyclopropanecarboxylic acid content in leaves was very high, which was significantly reduced after *Micrococcus sheathingans* treatment; the absolute value of abscisic acid content was low, which was significantly but slightly increased after T treatment; jasmonic acid content was significantly increased; and DPPH free radical scavenging capacity, total phenol content, and glycolate oxidase activity were significantly improved, indicating that *Micrococcus sheathingans* has a significant effect on improving stress resistance, especially under high temperature and drought stress. After foliar spraying with *Micrococcus sheathingans*, there was a significant correlation between leaf hormones and bacterial community structure. The treatment significantly altered the leaf microbial community, increased the number of photosynthetic cyanobacteria, thereby improving nitrogen metabolism (nitrogen fixation, nitrate reduction, urea decomposition, etc.), oxidative photoautotrophic function, fermentation acid production, sucrose content, and nitrite reductase activity. Rhizosphere soil functional annotation, enrichment difference analysis, and physicochemical index detection showed that the main functional differences in rhizosphere soil treated with *Micrococcus sheathus* were in carbon and nitrogen metabolism, promoting soil carbon fixation, nitrogen assimilation, potassium activation, conversion of ammonium nitrogen to nitrate nitrogen, and degradation of phenolic acid autotoxic substances. Energy was provided centered on sugar degradation metabolism, producing different short-chain organic acid molecules, such as naphthoic acid and malic acid, activating potassium nutrition, and ultimately promoting the growth and yield of Angelica sinensis. The treatment with *Micrococcus sheathingus* significantly reduced the diversity of bacteria and fungi in the rhizosphere soil, increased the abundance of the characteristic beneficial bacteria *Pseudomonas putida* and the beneficial fungus *Rhizopus irregularis*, and decreased the abundance of the characteristic pathogenic bacteria *Stenotrophomonas maltophilia* and the pathogenic fungi *Fusarium oxysporum*, *Fusarium vanetenianum*, *Fusarium solani*, and *Cladosporium*. In addition, the soil was significantly enriched with *Pseudomonas*, which promoted soil nutrient transformation; *Trichophyton*, which participated in the degradation of organic aromatic pollutants and nitrogen cycling; and *Nocardia*, which enhanced the decomposition of organic matter and the cycling of substances.

[0019] This invention also provides a method for cultivating Chinese medicinal herbs, which involves foliar spraying of microalgae inoculants after the true leaves have emerged or after transplanting. This solves the problems of unstable effects and time-consuming and labor-intensive processes associated with current solid root application microbial products on the market, which are affected by environmental factors, soil type, agricultural operations, and indigenous microorganisms. Attached Figure Description

[0020] Figure 1 Figure showing the results of Prouker analysis of differential bacterial genera and differential hormones in leaves; Figure 2 A KEGG enrichment analysis plot of differentially expressed genes; Figure 3 Mantel-test heatmap of leaf hormones and agronomic traits; Figure 4Cluster heatmap showing the correlation between leaf hormones and agronomic traits; Figure 5 Mantel-test heatmap of rhizosphere soil microorganisms (species) and agronomic traits; Figure 6 A heatmap showing the correlation between rhizosphere soil microorganisms (species) and agronomic traits. Detailed Implementation

[0021] This invention provides the application of photosynthetic microalgae in the cultivation of Chinese medicinal herbs, wherein the microalgae are established on the leaves of the Chinese medicinal herbs by foliar spraying.

[0022] The microalgae described in this invention contain chloroplasts or chloroplast-like structures and can autonomously perform photosynthesis. These microalgae can be prokaryotic or eukaryotic. In one embodiment of this invention, cyanobacteria among prokaryotic microalgae are used as an example, particularly *Micrococcus sheathingans* (*Micrococcus spp.*). Microcoleus vaginatus The examples are provided for illustration, but should not be construed as representing the entire scope of protection of this invention.

[0023] The microalgae described in this invention can be established on the leaves of medicinal herbs through foliar spraying, thereby promoting the growth of the herbs, reducing diseases, and improving their quality. One embodiment uses Angelica sinensis as an example, but this should not be considered as representing the entire scope of protection of this invention.

[0024] The present invention also provides the application of photosynthetic microalgae in promoting the growth of Chinese medicinal materials, reducing diseases and improving quality, wherein the microalgae are established on the leaves of the Chinese medicinal materials by foliar spraying.

[0025] The methods for promoting the growth, reducing diseases, and improving the quality of Chinese medicinal herbs as described in this invention include at least one of the following: (1) Significantly promotes increased yield, reduces disease, and increases the content of effective components in traditional Chinese medicine; (2) Significantly reduces the content of 1-aminocyclopropanecarboxylic acid; increases the content of abscisic acid and jasmonic acid, effectively improving stress resistance, especially the resistance to high temperature stress and drought stress; (3) Significantly alters the leaf microbial community, increases the number of photosynthetic cyanobacteria, thereby enhancing the leaf's nitrogen fixation, nitrate reduction, urea decomposition, oxidative photoautotrophy, and fermentation acid production functions, promoting the accumulation of photosynthetic sugars in the leaves, the conversion of nitrate nitrogen to ammonium nitrogen, and the removal of reactive oxygen species; (4) Significantly promotes soil carbon fixation, nitrogen assimilation and potassium activation capacity, as well as promotes the conversion of ammonium nitrogen to nitrate nitrogen; (5) Significant enrichment of soil-marking probiotics; for example, enrichment of soil-marking probiotic bacteria Pseudomonas putida and soil-marking probiotic fungi Rhizopus irregularis; (6) Significantly reduce the abundance of marker pathogens; the pathogens include pathogenic bacteria and pathogenic fungi, the marker pathogenic bacteria include Stenotrophomonas maltophilia, and the marker pathogenic fungi include Fusarium oxysporum, Fusarium van estiensis, Fusarium solani, and Cladosporium.

[0026] The present invention also provides a method for cultivating Chinese medicinal materials, which includes spraying microalgae capable of photosynthesis onto the leaves of the Chinese medicinal materials during the cultivation period.

[0027] The spraying method described in this invention includes spraying a microalgae inoculant; the preparation method of the microalgae inoculant includes inoculating the microalgae into BG-11 culture medium for cultivation to obtain the microalgae inoculant. The effective viable count of the microalgae inoculant of this invention is (1~3)×10⁻⁶. 5 The spraying method described in this invention is carried out after true leaves have emerged or after transplanting, and the number of sprayings is 3 to 5, with an interval of 3 to 5 weeks between two consecutive sprayings. When performing foliar spraying, the amount of spraying can be 20 to 40 L / mu per acre, depending on the size of the crop canopy.

[0028] Experiments in the examples have confirmed that when the microbial agent of the present invention is applied to the cultivation of Angelica sinensis, foliar spraying mainly affects the content of JA, ABA and Nocardia strain S5 (…). Nocardioides sp The abundance of S5 affects the average weight of a single Angelica plant and the content of ferulic acid, thereby increasing yield and improving quality.

[0029] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for promoting the growth, development, yield, and quality improvement of Chinese medicinal materials using photosynthetic microorganisms, should not be construed as limiting the scope of protection of the present invention.

[0030] In this embodiment of the invention, the experimental field was located in an organic medicinal herb planting base in Tianzhu County, Gansu Province. The initial physicochemical properties of the soil were as follows: pH=7.70, organic matter 3.54%. The experiment adopted a single-factor randomized design, with each treatment replicated 3 times, and each plot area was 30m². 2 The planting spacing for Angelica sinensis is 10cm × 20cm.

[0031] All reagents and culture media used in the experiment were chemically pure. The activity assay kits for cellulase, laccase, xylanase, peroxidase, ACC deaminase, glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, and phenylalanine ammonia-lyase, as well as the assay kits for ABTS, DPPH, ferrophosphate, malondialdehyde, ammonium nitrogen, nitrate nitrogen, amino nitrogen, soluble sugars, chlorophyll, flavonoids, nitrite nitrogen, and nitrate nitrogen were purchased from Beijing Box Biotechnology Co., Ltd.

[0032] The composition of the culture medium used in the embodiments of the present invention is as follows: BG-11 medium: Sodium nitrate (NaNO3) 1500 mg / L, dipotassium hydrogen phosphate (K2HPO4) 40 mg / L, magnesium sulfate (MgSO4) 36.6 mg / L, calcium chloride (CaCl2) 27.2 mg / L, citric acid 6 mg / L, ammonium ferric citrate. The following are the concentrations of various sodium chlorides: 6 mg / L of EDTA (6 mg / L), 1 mg / L of disodium ethylenediaminetetraacetate (Na2EDTA), 20 mg / L of sodium carbonate (Na2CO3), 2.86 mg / L of boric acid (H3BO3), 1.81 mg / L of manganese chloride (MnCl2·4H2O), 0.22 mg / L of zinc sulfate (ZnSO4), 0.39 mg / L of sodium molybdate (Na2MoO4·2H2O), 0.08 mg / L of copper sulfate (CuSO4·5H2O), and 0.0409 mg / L of cobalt chloride (CoCl2·6H2O).

[0033] Example 1 Microsheathing algae (Pterygota spp.) purchased from the Freshwater Algae Bank (FACHB-collection, Wuhan), Institute of Hydrobiology, Chinese Academy of Sciences Microcoleus vaginatus The culture medium was placed in a sterile Erlenmeyer flask containing BG-11 medium and incubated on a light shaker under the following conditions: temperature 25℃, light-dark cycle 14:10h, light intensity 100 μmol·m⁻¹. -2 ·s -1 140 r / min, cultured for 7 days, microscopic concentration was 3 × 10 6 per mL.

[0034] Example 2 The experimental groups were set up as follows: Treatment group (T): The culture medium of *Micrococcus sheathingus* cultured in BG-11 medium at 25°C was diluted 10-fold with sterile water until the effective viable bacterial count was 3 × 10⁻⁶. 5 The dosage is 2L per application area.

[0035] Control group (CK): BG-11 culture medium alone, diluted by the same factor as the treatment group T, with a spraying volume of 2L / plot each time.

[0036] The steps are as follows: After the angelica seedlings emerge, foliar spraying will be carried out from May 15 to August 25, 2025, once every 4 weeks, for a total of 4 sprays. Conventional agronomic practices will be adopted for field management.

[0037] Seven days after the fourth treatment, 100 Angelica plants were randomly selected from each plot, and leaves from the same part were picked. The corresponding rhizosphere soil was collected, mixed separately, and brought back to the laboratory for later use.

[0038] Angelica sinensis was harvested at the end of October. Disease incidence was assessed, and the topsoil and mud were washed away with running water. The plants were then dried in the shade and weighed to calculate the average weight per plant. The contents of ferulic acid and ligustilide were determined according to the methods described in the pharmacopoeia and literature (Determination of ferulic acid and ligustilide content in 30 batches of Angelica sinensis, Chinese Journal of Experimental Traditional Medical Formulae, 2011). Relevant indicators are shown in Table 1 (Student T-test analysis, FC value is the corresponding fold change).

[0039] Table 1. Yield, disease rate, and quality of Angelica sinensis under different treatments ( - (x±se, n=3)

[0040] In Table 1, different lowercase letters indicate significant differences (P < 0.05) (the same applies below). Table 1 shows that, compared to the control group CK, the average weight of a single Angelica plant treated with the *Micrococephala stenoptera* T group described in this invention was 1.38 times that of the control, the ferulic acid content was 1.36 times that of the control, and the disease incidence was 21% of the control, indicating that the *Micrococephala stenoptera* described in this invention effectively promoted the yield and quality of Angelica.

[0041] Example 3 Effects of treatment group T (Microcoleiae sheathii) on physiological and biochemical parameters of Angelica sinensis leaves The test setup and test plan are the same as in Example 2.

[0042] Leaf physiological and biochemical parameters were determined by spectrophotometry according to the kit instructions. The equivalent values ​​of ABTS scavenging capacity and DPPH scavenging capacity, as well as the contents of total phenols, malondialdehyde, ammonium nitrogen, nitrate nitrogen, carotenoids, sucrose, and chlorophyll, and the activities of glutamine synthase, glutamate synthase, nitrite reductase, nitrate reductase, peroxidase, phenylalanine ammonia-lyase, and glycolate oxidase were measured. Each treatment was repeated three times. The Student's T test was used to analyze the statistically significant differences in the parameters, as shown in Table 2 (FC value is the corresponding fold change).

[0043] Table 2 Detection of physiological and biochemical indicators of leaves ( - (x±se, n=3)

[0044] Table 2 shows that in the T treatment group, the DPPH free radical scavenging capacity, total phenol content, and glycolate oxidase activity were significantly increased, indicating that *Microsporum spp.* significantly improved the reactive oxygen species scavenging capacity. The sucrose content and nitrite reductase activity were also significantly increased, indicating that *Microsporum spp.* promoted the accumulation of photosynthetic sugars and nitrogen metabolism, especially the conversion of nitrate nitrogen to ammonium nitrogen.

[0045] Thirty-four hormones in leaves were detected by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). HPLC conditions: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm); mobile phase A was ultrapure water with 0.04% acetic acid added, and mobile phase B was acetonitrile with 0.04% acetic acid added; gradient elution program: 0 min A / B 95:5 (V / V), 1.0 min 95:5 (V / V), 8.0 min 5:95 (V / V), 9.0 min 5:95 (V / V), 9.1 min 95:5 (V / V), 12.0 min 95:5 (V / V); flow rate 0.35 mL / min; column temperature 40℃; injection volume 2 μL.

[0046] The main mass spectrometry conditions included: electrospray ionization (ESI) temperature of 550℃, mass spectrometry voltage of 5500V in positive ion mode, mass spectrometry voltage of -4500V in negative ion mode, and curtain gas (CUR) of 35psi. A Metal Database (MWDB) was constructed based on standards for qualitative analysis of the mass spectrometry data. Quantitative analysis was performed using multiple reaction monitoring (MRM) mode of triple quadrupole mass spectrometry. OPLS-DA analysis was used to screen for indicators with a VIP > 1, and the degree of difference was analyzed by ANOVA and fold change (FC). Indicators with significant differences are shown in Table 3.

[0047] Table 3. Leaf hormone index detection (ng / g) - (x±se, n=3)

[0048] The OPLS-DA analysis showed an R²Y of 0.999 and a Q² of 0.995, indicating excellent model differentiation and fit. Table 3 shows that the T treatment significantly reduced the levels of three major growth-related hormones: auxin, cytokinin, and gibberellin; it also reduced salicylic acid content; the absolute value of 1-aminocyclopropanecarboxylic acid was very high, but significantly decreased after T treatment, indicating that *Micrococcus sheathingii* has a role in alleviating high-temperature stress; the absolute value of abscisic acid was low, but significantly and slightly increased after T treatment, indicating that *Micrococcus sheathingii* has a role in alleviating drought stress; and the jasmonic acid content significantly increased, indicating that *Micrococcus sheathingii* has a role in enhancing stress resistance.

[0049] Example 4 The effects of foliar spraying of *Micrococcus sheathingus* on soil nutrients and enzyme activity in Angelica sinensis were investigated. The experimental setup and scheme were the same as in Example 2.

[0050] The activities of sucrase, urease, β-glucosidase, xylanase, alkaline phosphatase, catalase, and polyphenol oxidase in soil, as well as the contents of ammonium nitrogen and nitrate nitrogen, were determined by spectrophotometry using a kit. Alkaline available nitrogen was determined by alkaline hydrolysis-diffusion method. Available potassium was determined by flame photometry after ammonium acetate dissolution extraction. Total iron was determined by tetraacid digestion-inductively coupled plasma atomic emission spectrometry. Available phosphorus in soil was determined by sodium bicarbonate solution extraction and molybdenum-antimony colorimetric method. Total phenolic acid content was detected using methods described in the reference (Determination of Total Phenolic Acid and Total Flavonoid Content in Dandelion Flowers and Its Antioxidant Properties, Food Science, 2011). Analysis of variance and fold change (FC) analysis were used to analyze the degree of difference. Indicators of significant differences are shown in Table 4.

[0051] Table 4 Soil physicochemical indicators ( - (x±se, n=3)

[0052] Table 4 shows that treatment T significantly increased the activity of soil alkaline xylanase, promoting the degradation of organic matter; it also significantly increased the content of ammonium nitrogen, nitrate nitrogen, and available potassium, as well as the nitrate nitrogen / ammonium nitrogen ratio, indicating that treatment T can promote nitrogen assimilation, potassium activation, and the conversion of ammonium nitrogen to nitrate nitrogen; the increased polyphenol oxidase activity promoted the degradation of phenolic acid autotoxic substances.

[0053] Example 5 The effects of foliar spraying of *Micrococcus sheathatum* on bacterial diversity and function in *Angelica sinensis* leaves. The experimental setup and protocol were the same as in Example 2.

[0054] Leaves under different treatments were subjected to amplicon sequencing by a third party to analyze bacterial diversity and function. The bacterial α diversity index of the leaves is shown in Table 5.

[0055] Table 5. Leaf bacterial microbial α-diversity ( - (x±se, n=3)

[0056] The Chao1 index measures species richness, i.e., the number of species, while the Shannon index measures species diversity. A higher index value indicates a higher number and diversity of species in the sample. The T treatment significantly promoted the number and diversity of leaf species, resulting in a more complex, homogeneous, and stable community structure.

[0057] The LEfSe method is a combination of nonparametric tests and linear discriminant analysis, and is a screening tool for characteristic microorganisms. The nonparametric statistical analysis of the characteristic bacterial species Kruskal Wallis in T treatment and CK leaf bacteria LEfSe (LDA threshold of 4) is shown in Table 6 (FC value is the corresponding fold of difference).

[0058] Table 6. LEfSe analysis of differences in bacterial genera abundance in leaves ( - (x±se, n=3)

[0059] The T-leaf marker bacteria for the treatment of *Micrococcus sheathingus* were annotated to six genera: *Tetracoccus* ( Quadrisphaera ), Spirals ( Spirosoma ), Methylobacterium ( Methylobacterium ), *Mesophytes* genus ( Mesorhizobium ), Sphingosine Box Bacteria ( Sphingopyxis ), Enterobacteriaceae ( Enterobacter ); CK treatment identified three genera of phyllographia marker bacteria: *Aureomyces* (…). Aureimonas ), Sphingosomalmonella ( Sphingomonas ), Pseudomonas spp. Pseudomonas ).

[0060] Kruskal Wallis nonparametric statistical analysis of leaf photosynthetic bacteria abundance is shown in Table 7 (FC value is the corresponding fold of difference).

[0061] Table 7 Effects of Micrococcus sheathingus treatment on the photosynthetic bacterial community of Angelica folia ( - (x±se, n=3)

[0062] The treatment T with *Microcoleus sheathingus* significantly increased the relative abundance of cyanobacteria (Cyanobacteriota), microcoleus, and the photosynthetic bacterium *Pseudorhodoplanes*, indicating that foliar spraying of *Microcoleus sheathingus* can not only effectively colonize it, but also promote the enrichment of other photosynthetic bacteria.

[0063] FAPROTAX analysis is applicable to functional annotation of chemical cycling processes. Using FAPROTAX software, the functional abundance table of the sample is predicted based on the sample microbial abundance table. Kruskal Wallis nonparametric statistical analysis is shown in Table 8 (FC values ​​are the corresponding fold differences).

[0064] Table 8 Prediction of FAPROTAX function in leaf bacteria ( - (x±se, n=3)

[0065] The T treatment of *Micrococcus sheathingus* significantly improved leaf nitrogen metabolism, including nitrogen fixation, nitrate reduction, and urea decomposition; significantly increased the number of photosynthetic cyanobacteria; and significantly improved oxidative photoautotrophy and fermentation acid production.

[0066] Procrustes analysis was conducted to reveal the consistency between the hormone metabolomics and 16S amplicon sequencing data. M² was the sum of squared residuals; a smaller value indicated better consistency between the two sets of data. Permutation tests were used to calculate M² and p-values. Procrustes analysis of differentially expressed bacteria and hormones at the leaf genus level is described below. Figure 1 The results showed that, compared with the control CK, the treatment T (labeled JQ) exhibited significant consistency in the ordination configuration of leaf-level differentially expressed bacteria and leaf hormone data at the genus level (M... 2 =0.413, p =0.026), indicating a significant consistency between community structure and hormone levels. From the sample distribution, the line segment lengths in the T treatment group were shorter than those in the CK group, indicating smaller sample residuals and a stronger correlation; while the CK group had longer sample line segment lengths, significantly larger residuals, and a weaker correlation.

[0067] Example 6 The effects of *Micrococcus sheathingii* on the diversity and function of rhizosphere microorganisms in *Angelica sinensis*. The experimental setup and protocol were the same as in Example 2.

[0068] After rhizosphere soil samples were collected and kept cold with dry ice, they were sent to a third party for metagenomic sequencing analysis, as well as microbial diversity and functional analysis. Table 9 shows the rhizosphere soil microbial α-diversity indices. The T treatment with *Micrococcus sheathatus* had no significant effect on bacterial and fungal species richness; however, it significantly reduced bacterial and fungal diversity.

[0069] Table 9. Rhizosphere soil microbial α-diversity ( - (x±se, n=3)

[0070] The LEfSe method is a combination of nonparametric tests and linear discriminant analysis, and is a screening tool for characteristic microorganisms. Table 10 shows the nonparametric statistical analysis of the abundance of characteristic bacteria Kruskal Wallis at the LEfSe (LDA threshold of 4) at the rhizosphere soil level for T treatment and CK treatment, and Table 11 shows the abundance of fungi (FC value is the corresponding fold difference).

[0071] Table 10 Rhizosphere soil bacteria LEfSe species ( - (x±se, n=3)

[0072] Table 10 shows that the T treatment of *Micrococcus sheathingii* significantly increased the growth of *Pseudomonas putida* (…). Pseudomonas putida ), Trichomonas vaginalis ( Comamonas testosteroni ), Nocardia spp. Nocardioides sp The abundance of strain S5 was significantly reduced; it also significantly reduced the abundance of hydrogen-eating bacteria (Strain S5). Hydrogenophaga sp.) strain PBC, Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia The abundance of ) . Among them, *Pseudomonas putida* is a typical probiotic, and *Stenotrophomonas maltophilia* is a typical opportunistic pathogen.

[0073] Table 11 Rhizosphere soil fungi LEfSe species ( - (x±se, n=3)

[0074] Table 11 shows that the T treatment of *Micrococcus sheathingus* significantly increased the mycorrhizal fungus *Gymnospermum irregularis* (…). Rhizophagus irregularis The abundance of Beauveria bassiana ( ) was reduced; Beauveria bassiana ), pathogen Fusarium oxysporum ( Fusarium oxysporum ), Fusarium van Eyten Fusarium vanettenii Fusarium solani () Fusarium solani ), Cladosporium ( Cladosporium cladosporioides Abundance. T treatment can prevent and control root rot, leaf spot, and leaf blight.

[0075] Using HUMAnN3, the quality control and host-de-hosted sequences were compared with the protein database (UniProt database UniRef90) based on DIAMOND. According to the correspondence between the UniRef90 ID and the MetaCyc functional database ID, the abundance of genes (UniRef90) belonging to the same function was accumulated to obtain the relative abundance of the corresponding function in the functional database. LEfSe analysis was used to identify the functional marker pathways of MetaCyc in rhizosphere soil microorganisms. Kruskal-Wallis nonparametric statistical analysis and Log2FC analysis were used to analyze the degree of difference. As shown in Table 12, the marker pathways of rhizosphere soil treated with *Micrococcus sheathingus* were: glucose and glucose-1-phosphate degradation, tricarboxylic acid cycle IV (2-ketoglutarate decarboxylase type), reductive tricarboxylic acid cycle I, incomplete reductive tricarboxylic acid cycle, 1,4-dihydroxy-6-naphthoic acid biosynthesis II, and phosphatidylglycerol biosynthesis II (non-plastotic pathway), indicating that the main function of its rhizosphere microorganisms was carbon fixation; energy was provided by sugar degradation metabolism, producing different small organic acid molecules (such as naphthoic acid, malic acid, etc.), and promoting potassium activation. The marker pathways of rhizosphere soil treated with CK were: saturated fatty acid elongation, stearic acid biosynthesis II (bacteria and plants), and (5Z)-dodecenoic acid biosynthesis II, indicating that its rhizosphere soil mainly participated in the synthesis and metabolism of long-chain fatty acids, which are not easily utilized by plants.

[0076] Table 12 Functions of MetaCyc, a microbial community in rhizosphere soil ( - (x±se, n=3)

[0077] Using the online analytical tool DESeq2 from the Life Science Cloud, differentially expressed genes / proteins were selected based on the UniRef90 protein abundance table from the Humann analysis results. The screening threshold was a fold change |log2FC| > 1.0, with a corrected P-value < 0.05. Further KEGG ORA enrichment analysis was used to infer the biological processes involved by these differentially expressed proteins. Results are as follows: Figure 2 As shown, the KEGG enrichment pathways of differentially expressed genes (proteins) in the T treatment compared to the CK treatment mainly include: carbon metabolism, ribosomes, cofactor biosynthesis, oxidative phosphorylation, pyruvate metabolism, amino acid biosynthesis, photosynthetic carbon fixation, tricarboxylic acid cycle, glycolysis / gluconeogenesis, glyoxylate and dicarboxylic acid metabolism, propionic acid metabolism, pyrimidine metabolism, nucleotide metabolism, lipoic acid metabolism, and Calvin cycle carbon fixation. This indicates that the main differential function between the T-treated and control *Micrococcus spp.* treatments is the carbon and nitrogen cycle.

[0078] Based on the KO abundance table for different samples, the carbon, nitrogen, phosphorus, and sulfur cycling pathways were analyzed using DiTing software. LEfSe analysis was performed on pathways and marker genes. Kruskal-Wallis nonparametric statistical analysis and Log2FC analysis were used to assess differences. The nitrogen metabolism pathway (KO00910) and LEfSe marker gene analysis results are shown in Table 13. The T treatment in *Micrococephalum spp.* promoted nitrification and nitrate reduction assimilation, while inhibiting denitrification, consistent with the soil's different forms of nitrogen content and the nitrate / ammonium nitrogen ratio. The LEfSe analysis results for carbon cycling pathways are shown in Table 14. The T treatment in *Micrococephalum spp.* promoted photosystem II, cytochrome b6 / f complex cellular components, reverse tricarboxylic acid cycle, and formic acid fermentation pathways, indicating that the T treatment promoted carbon fixation and the production of soluble small-molecule organic acids, consistent with MetaCyc functional statistical analysis.

[0079] Table 13. Analysis of marker genes for the LEfSe nitrogen metabolism pathway (x̄±se, n=3) (kw test)

[0080] Table 14 Analysis of LEfSe pathways in the carbon cycle (x̄±se, n=3) (kw test)

[0081] Example 7 The associations between yield and quality agronomic traits (NYXZ) and plant leaf hormones and rhizosphere soil differential microorganisms (species level) were studied using Mantel test and Pearson correlation analysis. The analysis and plotting were performed using the Metware Cloud Platform (https: / / cloud.metware.cn).

[0082] The Mantel test results for agronomic traits (NYXZ) and leaf hormones are shown in the figure. Figure 3 Agronomic traits (NYXZ) were significantly associated with indolecarboxylic acid (ICA), cis-zeatin (TZ), jasmonic acid (JA), and abscisic acid (ABA). Pearson correlation hierarchical clustering heatmap analysis is shown below. Figure 4 The average single plant weight (DZZ) and ferulic acid (AWS) content were significantly positively correlated with JA and ABA, respectively. p <0.01).

[0083] Mantel test for the relationship between agronomic traits (NYXZ) and differentially expressed microorganisms (species level) is shown in [reference needed]. Figure 5 Agronomic traits (NYXZ) and Nocardia strain S5 ( Nocardioides sp S5), Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ), Beauveria bassiana ( Beauveria bassiana ) and Fusarium solani ( Fusarium solani There is a significant correlation. See the Pearson correlation hierarchical clustering heatmap analysis. Figure 6 The average single plant weight (DZZ) and ferulic acid (AWS) content were respectively compared with Nocardia strain S5 ( Nocardioides sp. S5) showed a positive correlation.

[0084] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of photosynthetic microalgae in the cultivation of Chinese medicinal herbs, characterized in that, The microalgae are established on the leaves of the Chinese medicinal herbs through foliar spraying.

2. The application according to claim 1, characterized in that, The types of microalgae include cyanobacteria.

3. The application according to claim 1 or 2, characterized in that, The microalgae include Microsheatha scabra ( Microcoleus vaginatus ).

4. The application according to claim 1, characterized in that, The medicinal materials mentioned include Angelica sinensis.

5. The application of photosynthetic microalgae in promoting the growth of Chinese medicinal herbs, reducing diseases, and improving quality, characterized in that... The microalgae are established on the leaves of the Chinese medicinal herbs through foliar spraying.

6. A method for cultivating Chinese medicinal herbs, characterized in that, This includes foliar spraying of microalgae capable of photosynthesis during the cultivation period of Chinese medicinal herbs.

7. The cultivation method according to claim 6, characterized in that, The foliar spraying includes spraying microalgae agents that can perform photosynthesis; The method for preparing the microalgae inoculant includes inoculating the photosynthetic microalgae into BG-11 medium for cultivation to obtain the microalgae inoculant.

8. The cultivation method according to claim 7, characterized in that, The effective bacteria in the microalgae inoculant are *Microsheatha spp.* (… Microcoleus vaginatus When the effective viable count is (1~3)×10⁻⁶, the effective viable count is (1~3)×10⁻⁶. 5 per mL.

9. The cultivation method according to claim 7 or 8, characterized in that, The foliar spraying is carried out after the true leaves have grown or after transplanting.

10. The cultivation method according to claim 9, characterized in that, The spraying is performed 3 to 5 times, with a time interval of 3 to 5 weeks between two consecutive sprayings.

Images