Gansu cyst and growth promoting effect of Gansu cyst on radix bupleuri

Abstract

The invention provides a method for realizing ecological planting of radix bupleuri based on arbuscular mycorrhizal fungi. According to the present invention, the morphological characteristics and the molecular identification results show that the new arbuscular mycorrhizal fungi, i.e., Dominikia gansuensis, are found and named as the new arbuscular mycorrhizal fungi, i.e., Dominikia gansuensis; a new species of Gansu cyst is subjected to propagation culture and inoculated to the rhizosphere of radix bupleuri, so that the growth promoting function of the new species of Gansu cyst is studied. According to the present invention, it is proved that the new species of Gansu cyst significantly promotes the growth of bupleurum chinense, reshapes the root structure, enhances the nutrient absorption, is helpful for reducing the excessive dependence of bupleurum chinense on chemical fertilizers, and provides a new approach for the ecological planting of bupleurum chinense by using a biotechnology method.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a newly discovered arbuscular mycorrhizal fungus and its growth-promoting effect on the traditional Chinese medicine Bupleurum chinense, aiming to provide a method for achieving ecological cultivation of Bupleurum chinense. Background Technology

[0002] Bupleurum chinense is an important traditional medicinal plant in my country with a long history of cultivation and clinical application. It is commonly used to treat colds, fever, and liver stagnation, playing a vital role in traditional Chinese medicine and ethnic minority medicine. In recent years, with the continuous development of traditional Chinese medicine and its increasing international recognition, Bupleurum chinense has not only been widely cultivated in major medicinal plant producing areas in my country but has also gradually spread to many countries and regions, becoming one of the internationally influential medicinal resources. However, with the expansion of Bupleurum chinense cultivation and the increase in market demand, problems such as soil degradation, continuous cropping obstacles, and excessive use of pesticides and fertilizers have emerged, seriously affecting the yield and quality of the medicinal material. Therefore, exploring green, scientific, and efficient cultivation strategies, especially utilizing functional microorganisms to improve the growth and quality of Bupleurum chinense, has become a key scientific issue that urgently needs to be addressed. This has significant practical implications and application value for promoting the sustainable development of the Bupleurum chinense industry.

[0003] Traditional Chinese medicine (TCM) ecological agriculture, as an important development model for the sustainable production of TCM herbs, aims to achieve the dual goals of coordinated development of TCM herb production and the ecological environment by fully utilizing natural resources and ecosystem services while protecting and improving the ecological environment. In this model, microbial communities, especially functional microorganisms that closely interact with plant roots, play a crucial role in maintaining ecosystem stability and improving the production performance of medicinal herbs. Arbuscular mycorrhizal (AM) fungi are among the most widely distributed and ecologically significant symbiotic fungi in soil. They can significantly promote the absorption of nitrogen, phosphorus, and various trace elements from the soil by medicinal plants, enhance their resistance to drought stress and pests and diseases, and induce the synthesis and accumulation of various bioactive components such as terpenes, flavonoids, and alkaloids within medicinal plants, ultimately improving the quality of medicinal herbs. Therefore, AM fungi show broad application prospects in the green cultivation of TCM herbs and the development of microbial fertilizers, and are an important microbial resource for achieving green and sustainable agricultural development.

[0004] AM fungi belong to the phylum Glomeromycota and are obligate symbiotic fungi that rely on plant roots to establish a symbiotic relationship to complete their life cycle. This biological characteristic makes the isolation, purification, and propagation of AM fungi extremely difficult. As of May 25, 2025, only 355 AM fungal germplasm resources have been discovered and clearly recorded globally (data from https: / / amf-phylogeny.jimdofree.com / species-list / ). This limited germplasm diversity significantly restricts their in-depth application and promotion in fields such as ecological agriculture, bio-fertilizer development, and plant function enhancement.

[0005] The Qinghai-Tibet Plateau, known as the "Roof of the World" and the "Third Pole," boasts diverse ecological environments and abundant germplasm resources, yet research on the discovery and cultivation of agronomica mycorrhizae (AM) germplasm resources remains lacking. Therefore, we have dedicated ourselves to the isolation and purification of AM fungi in this region. Through identification, we discovered two new AM fungi, and the relevant findings have been published in the journal *Mycological Progress*, titled "Glomus chinense and Dominikia gansuensis, two new Glomeraceae species of arbuscular mycorrhizalfungi from high altitude in the Tibetan Plateau." Given that AM fungi typically possess important nutritional and ecological functions, but different AM fungal strains exhibit distinct functional characteristics, the growth-promoting effects of the new species *Dominikia gansuensis* on plants have not yet been studied. This patent will primarily introduce the effects of the new *Dominikia gansuensis* strain on the growth, root traits, and nutrient absorption of *Bupleurum chinense*, aiming to provide highly efficient inoculants and technical support for the ecological cultivation of *Bupleurum chinense*. Summary of the Invention

[0006] The main content of this invention is to improve the cultivation method of Bupleurum chinense by using microbial fertilizer technology, reduce the use of chemical fertilizers, and realize the ecological cultivation of Bupleurum chinense.

[0007] This invention provides a novel *Agromycium gansuense* fungus and verifies its growth-promoting effect on *Bupleurum chinense*. Studies show that the *Agromycium gansuense* fungus exhibits a positive promoting effect on the growth of *Bupleurum chinense*. Inoculation with the *Agromycium gansuense* fungus increased the aboveground and underground biomass of *Bupleurum chinense* by 3 times and 29.2 times, respectively, with a particularly significant increase in root biomass and a significant increase in the average diameter of the roots. Regarding nutrient absorption, inoculation with the *Agromycium gansuense* fungus increased the nitrogen content of the tissue by 89.5% and the phosphorus content by 74.6%.

[0008] The applicant's research group successfully isolated a new AM fungus from the rhizosphere soil of plants mainly composed of birch (Betula platyphylla), euonymus (Euonymus alatus), and some understory herbaceous plants in the understory grassland ecosystem of Hezuo City, Gannan Tibetan Autonomous Prefecture, Gansu Province (35°06′27.77″N, 102°51′18.78″E, altitude 2816 meters). After morphological and molecular identification, it was named *Gansu multi-nest fungus*.

[0009] The morphological identification method used in this invention involves observing and measuring the morphological characteristics of AM fungal spores under a microscope, specifically including spore size, shape, color, wall structure and thickness, and the presence or absence of accessory structures. Molecular identification is performed through AM fungal spore DNA extraction, PCR amplification, cloning and sequencing, and sequence alignment to construct a phylogenetic tree to determine its phylogenetic position, thus proving that this AM fungus is a new species of the genus *Dominikia*.

[0010] The AM fungal inoculant used in this invention is obtained through propagation and culture technology. Specifically, spores of *Polycystis gansuensis* are inoculated into flowerpots containing an equal volume mixture of sterilized sand and zeolite. Sterilized sorghum and alfalfa seeds that have sprouted are sown, and propagation and culture are carried out under greenhouse conditions. During this period, distilled water is applied every three to four days, and low-phosphorus Hoagland nutrient solution is added every two weeks. After four months of cultivation, watering is stopped, and after two weeks of drying, the above-ground parts are cut off. The plant roots and potting substrate are harvested, containing AM fungal spores, infected roots, and ectomycorrhizae, which constitute the *Polycystis gansuensis* inoculant.

[0011] The new species of *Polycystis suis* provided by this invention has the following effects:

[0012] 1) Significantly promotes the growth of Bupleurum plants.

[0013] 2) Significantly increases the aboveground and underground biomass of Bupleurum.

[0014] 3) Significantly increases the nitrogen and phosphorus content of Bupleurum tissue.

[0015] 4) It provides an efficient microbial inoculant to enhance the growth of Bupleurum chinense, which helps to improve the yield and quality of Bupleurum chinense and reduce excessive reliance on chemical fertilizers. Attached Figure Description

[0016] Figure 1 This describes the morphological characteristics of *Polycystis gansuensis*. In this diagram, a represents the morphology of the aquatic sporocarps; b represents the sporocarp wall structure in PVLG; c represents the structure and color reaction of the sporocarps and hyphal walls in PVLG + Melzer's stain; and d represents the arbuscular structure formed by this strain infecting the root system.

[0017] Figure 2 This is the molecular phylogenetic tree of *Polycystis gansuensis*.

[0018] Figure 3 This refers to the effect of AM fungi on the growth of Bupleurum plants.

[0019] Figure 4 This describes the effect of AM fungi on the biomass of Bupleurum chinense. Here, a represents the total biomass of each Bupleurum chinense plant; b represents the ratio of root biomass to aboveground biomass.

[0020] Figure 5 This describes the effect of AM fungi on the root characteristics of Bupleurum chinense. Where a represents the root length of Bupleurum chinense; b represents the average diameter of Bupleurum chinense roots.

[0021] Figure 6 This study investigated the effects of AM fungi on the nitrogen and phosphorus content of Bupleurum chinense tissue. Where a represents the nitrogen content of Bupleurum chinense tissue, and b represents the phosphorus content. Detailed Implementation

[0022] The techniques and methods used in this patent will be described in further detail below.

[0023] In the following embodiments, some of the experimental materials were sourced from the following sources:

[0024] The new strain of *Polycystis gansuensis* was isolated by our research group and preserved in the Mycorrhizal Research Laboratory of Lanzhou University. All other materials and reagents were purchased from the market.

[0025] Example 1

[0026] Isolation, purification and identification of *Polycystis multifiliis* strains from Gansu

[0027] The applicant's research group isolated an AM fungus from the rhizosphere soil of plants in the understory grassland ecosystem of Hezuo City, Gannan Tibetan Autonomous Prefecture, Gansu Province (35°06′27.77″N, 102°51′18.78″E, altitude 2816 meters). Based on the combined identification methods of morphological squashing and molecular sequencing, it was identified as a new AM fungus and named *Gansu multilocularis*.

[0028] result

[0029] The AM fungal spores described in this invention have the following morphological characteristics: spores are rarely solitary in the soil, and usually exist in the form of sporocarps (…). Figure 1a). The sporocarps are dark yellow (8-40-83-0) to yellowish-brown (0-51-76-36), measuring 99-(198)-354×188-(434)-463μm, and contain dozens to hundreds of spores randomly. The spores are pale yellow (0-4-29-0) to yellowish-brown (0-51-76-36), spherical to nearly spherical, with a diameter of (20-)47(-86)μm, rarely ovoid, measuring 18-42×30-60μm, and have one opposite hyphae. The spore wall has three layers: the first layer forms the spore surface, is ephemeral, transparent, and (0.4–)0.8(–1.4) μm thick; the second layer is 0.5 μm thick and usually adheres tightly to the third layer, even in violently broken spores; the third layer is laminated, dark yellow (8-40-83-0) to yellowish-brown (0-51-76-36) in color, and (0.3–)0.7(–1.2) μm thick. In Melzer's reagent, only the first layer is stained pale purple (0-40-0-0) to pinkish-purple (0-80-65-0). Opposite hyphae are straight, curved, or cylindrical in shape, with a base width of (4.4–)6.6(–9.9) μm. The hyphal pores are (1.0–)2.5(–4.5) μm wide and are continuously closed at intervals. Germination tubes arise from the septum at the base of the spore and emerge through the lumen of adjacent hyphae. Figure 1 b, c). When AM fungi establish a symbiotic relationship with plants, typical arborescent and hyphal structures can be observed in the plant roots. Figure 1 d).

[0030] The molecular identification results of the AM fungus described in this invention are as follows: Sequencing results show that the SSU-ITS-LSU segment of the AM fungus is 1533-1538 bp in length. Phylogenetic analysis confirms that this AM fungus is a new species of the genus *Dominikia*, and its sequence similarity to its nearest relative, *Dominikia glomerocarpica*, is less than 97%. Figure 2 ).

[0031] Example 2

[0032] Study on the growth-promoting effect of *Polycystis multifiliis* on Bupleurum chinense in Gansu

[0033] In this embodiment, two inoculation treatments were set up: a control group and an AM fungal inoculation group. The inoculated strain was *Polycystis gansuensis*, with six replicates for each treatment. The test soil was forest soil, which was sterilized twice by high-pressure steam at 121℃ for 75 minutes, and then used as the potting substrate, with 1.3 kg of substrate per pot. 15 sterilized *Bupleurum chinense* seeds with white sprouts were sown in each pot. Two weeks after emergence, 10 seedlings were transplanted from each pot.

[0034] AM fungal inoculant was prepared using propagation culture technology. Spores of *Polycystis gansuensis* were inoculated into flowerpots containing an equal volume mixture of sterilized sand and zeolite. Sterilized sorghum and alfalfa seeds that had begun to sprout were sown, and propagation culture was carried out under greenhouse conditions. After four months of cultivation, watering was stopped, and the above-ground parts were cut off after two weeks of drying. The plant roots and potting substrate were harvested, containing AM fungal spores, infected roots, and ectomycorrhizal mycelia, thus yielding the *Polycystis gansuensis* inoculant.

[0035] The AM fungal inoculation treatment involved directly inoculating the flowerpots with AM fungal inoculum at 4 / 5 of their depth, using 70g of *Polygonum multiflorum* agent (approximately 1000 AM fungal spores) per pot. The control group received an equal amount of autoclaved AM fungal inoculum, along with 50mL of inoculum filtrate passed through a 38μm sieve. Additionally, to supplement other soil microorganisms, all treatments received 100mL of fresh soil microbial filtrate passed through a 38μm sieve.

[0036] After 120 days of growth, the growth indicators of Bupleurum chinense were measured, including plant height, aboveground and underground biomass, root traits such as root length, root specific surface area, root tissue density and average root diameter, and nutritional indicators such as nitrogen and phosphorus content in Bupleurum chinense tissue.

[0037] result

[0038] AM fungi significantly promote the growth of Bupleurum plants. Figure 3 Compared with the control, inoculation with AM fungi increased the aboveground and underground biomass of Bupleurum chinense by 3 times and 29.2 times, respectively, and the total biomass of Bupleurum chinense by 9 times. Figure 4 a). Furthermore, AM fungi significantly increased the root-to-shoot ratio of Bupleurum chinense (a). Figure 4 b) This directs more resources toward root growth, significantly promoting the accumulation of underground biomass in Bupleurum chinense, which helps improve the yield and quality of Bupleurum chinense.

[0039] AM fungi significantly affected the root traits of Bupleurum chinense. Compared with the control, inoculation with AM fungi significantly reduced the specific root length of Bupleurum chinense roots. Figure 5 a) significantly increased the average diameter of Bupleurum root system ( Figure 5 b) indicates that after inoculation with AM fungi, the root characteristics of Bupleurum chinense became shorter and thicker, reducing the plasticity of the root system.

[0040] AM fungi can help plants absorb mineral nutrients such as phosphorus, nitrogen, and potassium from the soil, improving their nutritional status. Compared with the control, inoculation with AM fungi increased the nitrogen content of Bupleurum chinense tissue by 89.5%. Figure 6 a) The tissue phosphorus content increased by 74.6% ( Figure 6 b) indicates that inoculation with AM fungi significantly increases the nitrogen and phosphorus content of Bupleurum tissue, improves the nutritional level of Bupleurum, and promotes its growth.

[0041] In summary, inoculation with the new species *Polycystis gansuensis* significantly promoted the growth of *Bupleurum chinense*, reshaped the root structure, and enhanced its ability to absorb nitrogen and phosphorus, providing a new technical approach for improving the growth of *Bupleurum chinense* using biotechnology.

[0042] The specific examples described above are for illustrating the technical methods and objectives of the present invention. It should be noted that the above descriptions are merely specific examples of the present invention and may be modified.

Claims

1. A new species of AM fungus, *Dominikia gansuensis*, characterized by: The nucleotide sequences are shown in Sequence 1-5.

2. The application of the Gansu Polycystic Mycorrhizal Agent as described in claim 1 in the growth of the medicinal plant Bupleurum chinense, so as to realize the ecological cultivation of Bupleurum chinense.

3. The application of Gansu Polycystic Mycorrhizal Agent in the growth of Bupleurum chinense according to claim 2, characterized in that, AM fungal inoculation treatment involved inoculating the flowerpots with *Polycystis aureus* inoculum from Gansu Province, filling each pot to 4 / 5 full. Each pot contained 70g of *Polycystis aureus* inoculum (approximately 1000 AM fungal spores). The control group was inoculated with an equal amount of *Polycystis aureus* inoculum from Gansu Province, sterilized in an autoclave, and 50mL of AM-free inoculum filtrate was added to each pot.

4. The fungal inoculation treatment according to claim 3AM, characterized in that, The preparation method of *Polycystis gansuensis* inoculant includes the following steps: *Polycystis gansuensis* spores are inoculated into sterilized flowerpots containing an equal volume of sand and zeolite. Sterilized sorghum, alfalfa, and clover seeds that have sprouted white leaves are sown, and the mixture is propagated in a greenhouse. After four months of cultivation, watering is stopped, and after two weeks of drying, the above-ground parts are cut off. The plant roots and potting substrate are harvested, containing *AM* fungal spores, infected roots, and extra-root mycelia, thus obtaining a pure *AM* fungal inoculant.

5. The matrix sterilization treatment containing a mixture of sand, zeolite, and other materials in equal volumes as described in claim 4, characterized in that... Sterilize using a high-pressure steam sterilizer at 121℃ for 75 minutes, and repeat sterilization after 24 hours to ensure thorough sterilization.

6. The method for disinfecting sorghum, alfalfa, and clover seeds according to claim 4, characterized in that, First, disinfect with 75% alcohol for 1 minute, then with 0.5% sodium hypochlorite for 3 minutes, and finally with 75% alcohol for 30 seconds. Rinse several times with sterile water to avoid disinfectant residue inhibiting seed germination. Spread the disinfected seeds evenly in a clean tray and germinate in the dark and humid environment.

7. The watering management during the propagation and cultivation of the microbial agent according to claim 4, characterized in that, Water with distilled water every three to four days, and add low-phosphorus Hoagland nutrient solution every two weeks.

8. The spore density of the final *Polycystis suis* inoculant obtained according to claim 4, characterized in that, Each 25 grams of substrate contains approximately 350-390 *Polycystis gansuensis* spores.

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