A cultivation regulation, female plant rate improvement and soil evaluation method based on differences in rhizosphere microbial community of different genders of sparganium stoloniferum

Abstract

This invention discloses a cultivation regulation method, soil evaluation method, and their application based on the characteristics of rhizosphere soil microorganisms of different sexes of *Calamus fragilis*, belonging to the field of medicinal plant cultivation and agricultural microecology technology. This method determines the soil microecological state by detecting the bacterial α-diversity index, relative abundance of dominant and specific bacterial groups in the rhizosphere soil of *Calamus fragilis*, and combining this with physicochemical indicators such as total carbon, total nitrogen, and total phosphorus. When the soil is determined to be in a male-type microecological state unfavorable to the formation of female plants, donor soil, microbial enrichment, or special microbial agents derived from the rhizosphere soil of female plants or mixed female-male plant rhizosphere soil are applied to reconstruct a rhizosphere microecological environment conducive to the formation and growth of female plants. This invention can be used to increase the female plant rate of *Calamus fragilis*, improve the yield of medicinal seeds, optimize crop rotation / intercropping systems, and restore the microecology of planting soil, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the fields of cultivation of Chinese and Tibetan medicinal materials, biotechnology, agricultural microecology and soil ecological regulation technology. Specifically, it relates to a method for identifying, regulating and optimizing the soil for cultivating *Herpetospermum pedunculosum* based on the differences in rhizosphere soil microbial community composition, bacterial diversity index and soil physicochemical indicators of different sexes, and the application of this method in increasing the proportion of female *Herpetospermum pedunculosum* plants, improving the yield of medicinal materials and optimizing the rotation / intercropping system. Background Technology

[0002] The Chinese gourd (Platycladus orientalis) is an important Tibetan medicinal herb, possessing medicinal value such as clearing heat and detoxifying, clearing the liver and promoting bile secretion. It is widely used in Tibetan medicine prescriptions. Because the Chinese gourd is a dioecious plant, with only female plants producing fruit and medicinal seeds, the low proportion of female plants in artificial cultivation is a significant factor limiting its large-scale production.

[0003] Previous studies have shown that there are differences in bacterial diversity, specific bacterial community composition, and total carbon, total nitrogen, and total phosphorus in the rhizosphere soil of different sexes of quince plants; in particular, the bacterial α diversity in the rhizosphere soil is significantly lower when only male plants are planted than when only female plants are planted or when male and female plants are planted together, while the difference in fungal α diversity is not significant.

[0004] The study also showed that there were identifiable differences in the rhizosphere bacterial communities of different sexes of quince at the phylum level. For example, Proteobacteria, Acidobacteria, Green Curvularia, Bacillus, Bdellovibrio, Cyanobacteria, and Thermomycosis can serve as indicator bacteria in response to the rhizosphere environment of plants of different sexes.

[0005] Current cultivation techniques for pine gourd mainly focus on seed breeding and field management, lacking systematic research and application of differences in rhizosphere soil microorganisms between different sexes. A cultivation regulation system based on rhizosphere microbial community characteristics and soil physicochemical indicators has not yet been established. Therefore, existing technologies struggle to effectively determine whether soil conditions are conducive to female plant formation, and there is a lack of technical means to improve the female plant rate, optimize the soil environment, and increase seed yield through microecological regulation. Summary of the Invention

[0006] The purpose of this invention is to provide a cultivation regulation method, a soil evaluation method, and their applications based on the differences in rhizosphere soil microbial communities between different sexes of quince.

[0007] To address the aforementioned issues, this invention determines the microecological state of the soil for growing pine gourd by detecting the characteristics of the rhizosphere soil microbial community and related physicochemical indicators. Based on this, targeted soil regulation is implemented to optimize the rhizosphere microecological environment, increase the female plant rate and seed yield of pine gourd, and provide technical support for the artificial cultivation, resource protection, and ecological planting of pine gourd.

[0008] Beneficial Effects: Compared with existing technologies, this invention constructs a cultivation regulation and soil evaluation method based on the differences in rhizosphere soil microbial communities between different sexes of *Calamus edulis*. This method can identify and improve the soil condition for *Calamus edulis* cultivation from the perspective of rhizosphere microecology, achieving targeted optimization of the cultivation environment. Through comprehensive analysis of rhizosphere bacterial diversity, dominant microbial communities, and related soil physicochemical indicators, it can provide a basis for female plant formation, beneficial microbial enrichment, and soil microecological reconstruction. This is beneficial for improving the female plant rate, seed yield, and artificial cultivation stability of *Calamus edulis*, and provides new technical approaches for crop rotation, intercropping, ecological restoration, and the protection of endangered Tibetan medicinal resources. Attached Figure Description

[0009] Figure 1 This is a flowchart of soil evaluation based on the differences in rhizosphere microorganisms between different sexes of wavy melon;

[0010] Figure 2 This is a flowchart of cultivation regulation to increase the female plant rate of pine melon;

[0011] Figure 3 This is a schematic diagram comparing the α-diversity index of rhizosphere soil bacteria in different sexes of wavy melon.

[0012] Figure 4 This is a schematic diagram comparing the α-diversity index of rhizosphere soil fungi in different sexes of Cucurbita pulverata.

[0013] Figure 5 This is a bar chart showing the relative abundance of dominant bacterial phyla in the rhizosphere soil of different sexes of *Melon rotundifolia*. Detailed Implementation

[0014] This invention provides a cultivation regulation and soil evaluation method based on the differences in rhizosphere soil microorganisms between male and female pineapple plants. This method is based on the differences in rhizosphere microecology formed by pineapple plants of different sexes. By collecting rhizosphere soil samples, detecting the soil microbial community structure and related physicochemical indicators, the soil microecological state is determined, and corresponding regulatory measures are taken to optimize the soil environment for pineapple cultivation, thereby increasing the percentage of female pineapple plants and seed yield.

[0015] In this embodiment, *Calabash melon* was planted in the Nyingchi region of Tibet, with three biological replicates in each group: male-only group M, female-only group F, and mixed male-female group FM. Rhizosphere soil samples were collected at the initial flowering stage. Some samples were cryopreserved for microbial sequencing, while others were air-dried for the determination of total carbon, total nitrogen, and total phosphorus in the soil. After impurity removal and sieving, some of the collected soil samples were used for soil physicochemical index detection, and the other part was used for microbial community detection. The preferred soil physicochemical indexes included total carbon, total nitrogen, and total phosphorus, and the preferred microbial community detection included bacterial 16S rRNA amplification sequencing and fungal ITS amplification sequencing, with bacterial community detection results serving as the primary evaluation criterion.

[0016] After high-throughput sequencing of the collected soil samples, the raw data underwent quality control, noise reduction, splicing, chimera removal, and species annotation. Soil microbial diversity indices were then calculated. For example... Figure 3-5 As shown, the ACE index, Chao1 index, Shannon index, and Simpson index were calculated for the soil bacterial community to reflect its richness and diversity. Simultaneously, the relative abundance changes of dominant and specific bacterial groups were combined to comprehensively evaluate the rhizosphere microecological status under different planting conditions. The test results indicate that the rhizosphere soil bacterial diversity was relatively low when only male plants were planted, while it was relatively high under conditions of only female plants or mixed male-female planting, suggesting that different sexes of wavy gourd plants have different effects on the rhizosphere bacterial community.

[0017] After obtaining the soil microecological status evaluation results, further regulation can be implemented on the planting plots. Select rhizosphere soil from the female plant group or mixed planting group as donor soil, sieve it, and add it to the soil to be cultivated at a volume ratio of 5%–20%. Mix well and let stand for 3–15 days before sowing the pine gourd. After sowing, perform a root irrigation during the seedling stage. The irrigation solution can be an extract of the donor soil, fermented bacterial solution, or a combination thereof. After one growth cycle, if the bacterial diversity of the soil to be cultivated increases and tends to converge with the female plant type / mixed planting type microbial community structure, then continue to maintain this regulation plan.

[0018] Furthermore, bacterial communities are isolated from the rhizosphere soil of female plants or mixed-plant rhizosphere soil, and a special microbial inoculant for pine melon is prepared through enrichment culture, compounding, and carrier adsorption. The inoculant can be formulated as powder, granules, or suspension. Application methods include:

[0019] (1) Seed dressing: Use 10-500 grams of inoculant per kilogram of seeds;

[0020] (2) Root irrigation: according to 10 5 ~10 9 After dilution to a CFU / mL concentration, administer;

[0021] (3) Substrate addition: Mix with seedling substrate at a mass ratio of 0.1% to 10%.

[0022] (4) After the application of the microbial agent, the increase in bacterial α diversity index or the increase in the abundance of the target bacterial community is used as an effective criterion.

[0023] This invention can also use the soil evaluation results for screening planting sites for pine nut cultivation, optimizing crop rotation layouts, and constructing intercropping systems. In practical applications, soil samples can be taken from target sites intended for pine nut cultivation, and their microbial community structure and related physicochemical indicators can be tested using the methods described above. When the bacterial diversity of the soil to be tested is low, the composition of the dominant bacterial community is unfavorable to the formation of female pine nut plants, or the overall microecological state is male-type, the site can be given priority for microecological restoration before planting. The restoration methods may include applying one or more of the following: rhizosphere donor soil from female plants, rhizosphere donor soil from mixed female-male plants, donor soil extract, enriched bacterial solution, or special microbial agents. The restored site can be tested again. When its bacterial community structure is close to the female-type or mixed female-male plant microecological state, it is more suitable as a site for pine nut cultivation, seed propagation, or germplasm resource protection and utilization. In crop rotation, suitable crop types for successive planting with pine gourd can be selected based on the impact of the previous crop on the soil microecology. In intercropping, companion plants that can maintain or promote the enrichment of beneficial bacterial communities can be prioritized for planting to improve the ecological stability of the soil. These methods can optimize the pine gourd cultivation environment from the perspective of rhizosphere microecological regulation, increasing the female plant rate, fruit setting capacity, and seed yield under artificial cultivation conditions, and providing technical support for the ecological cultivation of pine gourd and the protection of endangered Tibetan medicinal resources.

[0024] Furthermore, a soil microecological status reference model can be established based on the detection results of rhizosphere soil samples from different sexes of *Calamus edulis*. This reference model may include the bacterial diversity index range, dominant bacterial community composition characteristics, specific bacterial community distribution characteristics, and relevant soil physicochemical index ranges for soils planted with male plants alone, female plants alone, and mixed male-female plants. In actual evaluation, bacterial community indicators can be prioritized as the primary criterion, with fungal community indicators serving as a secondary reference. When the bacterial diversity level of the tested soil sample is close to the reference range for male plants and simultaneously exhibits bacterial community composition characteristics unfavorable to female plant formation, the soil can be identified as a plot requiring priority microecological regulation. When the bacterial diversity level of the tested soil sample is close to the reference range for female plants or mixed male-female plants, and the composition of dominant and specific bacterial communities is consistent with favorable soil types, the plot can be determined as suitable for planting, propagation, or continuous cultivation of *Calamus edulis*. By combining soil evaluation with subsequent regulation measures, a complete technical path from soil collection, index detection, status determination, microecological regulation to cultivation application can be achieved.

[0025] Those skilled in the art should understand that any equivalent substitutions or conventional changes made to the detection methods, types of indicators, control materials, application methods, and parameter ranges described in the embodiments without departing from the spirit and essence of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for regulating the cultivation of pine gourd based on the characteristics of rhizosphere soil microorganisms of different sexes, characterized in that: (1) Collect rhizosphere soil samples from the planting plots of pine melon; (2) Detect the bacterial community diversity index and / or relative abundance of dominant and specific bacterial groups in the rhizosphere soil samples; (3) Determine whether the soil is in a male-type microecological state, a female-type microecological state, or a mixed male-female planting microecological state based on the test results. (4) When the soil is in a microecological state that is not conducive to the formation of male plants from female plants, female plant rhizosphere donor soil, female-male mixed plant donor soil, microbial enrichment from the donor soil or a combination thereof are applied to the soil to be cultivated in order to regulate the soil bacterial community structure. (5) Sow or transplant pine melons, and continue to adjust the amount and number of applications according to the changes in bacterial community during the growth process, so as to improve the proportion of female plants, seed yield and / or plant growth uniformity.

2. The method according to claim 1, characterized in that, The bacterial community diversity index includes at least two of the ACE index, Chao1 index, Shannon index, and Simpson index; the donor soil is derived from the rhizosphere soil of female monoculture melons and / or mixed female-male melon rhizosphere soil; the proportion of the donor soil added to the soil to be cultivated is 1% to 40% (volume ratio); the microbial enrichment is a complex bacterial community isolated, enriched, or amplified from the rhizosphere soil of female monocultures or mixed female-male melons; the soil condition is determined by detecting at least one of total carbon, total nitrogen, and total phosphorus in the soil, and by combining the physicochemical and microbiological indicators.

3. A method for evaluating soil for the cultivation of pine gourd, comprising collecting soil samples to be tested; detecting its bacterial α diversity index, bacterial community composition and soil physicochemical indicators; comparing the test results with a preset reference model; and outputting the evaluation results of whether the soil is suitable for the cultivation of female pine gourd plants, mixed male and female planting, or soil microecological restoration.

4. The method according to claim 3, characterized in that, The preset reference model is established using a database of microbial characteristics of soil from single female plants, single male plants, and mixed female-male plants.

5. The method according to claim 3, characterized in that, The evaluation results shall include at least one of the following: (1) Whether it is necessary to apply female-type donor soil; (2) Whether it is necessary to apply microbial agents; (3) Whether it is suitable as a breeding ground; (4) Whether it is suitable to carry out crop rotation or intercropping.

6. A microbial inoculant for the cultivation of pine gourd, characterized in that, The microbial agent contains a complex bacterial community derived from the rhizosphere soil of female pine melon plants and / or the rhizosphere soil of mixed male and female pine melon plants. After the agent is applied to the rhizosphere of pine melon plants, it can enhance soil bacterial diversity and cause the microbial community structure to evolve towards a female plant type or mixed plant type microecology.

7. The microbial agent according to claim 6, characterized in that, The microbial agent is prepared as any one of the following: powder, granules, liquid, suspension, or carrier adsorbent; the application methods include one or more of the following: seed dressing, root irrigation, furrow application, hole application, drip irrigation, or mixing with seedling substrate.

8. The application of the method or microbial agent according to any one of claims 1-7 in improving the female plant rate of pine melon, improving the seed yield of pine melon, optimizing the pine melon rotation / intercropping system, or restoring the soil microecology of pine melon cultivation.

Images