Method for determining rhizosphere microecological regulation effect of amorphophallus konjac under exogenous selenium application condition

Abstract

This invention belongs to the field of plant rhizosphere research and discloses a method for determining the regulatory effect of exogenous selenium application on the rhizosphere microecology of Amorphophallus bulbifera. The method includes: experimental design and sample collection; soil microbial DNA extraction and amplicon sequencing; and quality control and analysis of the microbiome data. This invention utilizes Illumina MiSeq high-throughput sequencing technology to analyze the changes in the composition and diversity of rhizosphere soil bacteria communities in Amorphophallus bulbifera under exogenous selenium treatment, and further analyzes the functions of related communities through PICRUSt functional prediction analysis. The conclusions are as follows: exogenous selenium treatment induces significant changes in the rhizosphere soil bacterial community of Amorphophallus bulbifera, which is beneficial for creating a stable and more diverse rhizosphere microbial community structure.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of plant rhizosphere research technology, and particularly relates to a method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera. Background Technology

[0002] Konjac is a perennial herbaceous plant belonging to the genus *Amorphophallus* in the family Araceae. It is the only plant species in the world rich in glucomannan (KGM). KGM is a water-soluble polysaccharide (dietary fiber) with unique physical and chemical properties, and is widely used in food, medicine, health care, and chemical industries. *Amorphophallus muelleri* is an important cultivated variety of konjac, exhibiting strong resistance to soft rot and abundant KGM content. In recent years, with the continuous development of konjac products, the demand for konjac bulbs has increased year by year. However, problems such as continuous cropping obstacles in the konjac cultivation process seriously restrict the development of my country's konjac industry. Therefore, exploring the impact of different planting or fertilization methods on the growth of *Amorphophallus muelleri* and soil health is of great significance for selecting appropriate planting methods to maximize konjac yield and achieve the healthy and robust development of my country's konjac industry.

[0003] The plant rhizosphere is considered one of the most complex ecosystems on Earth and a hotspot for various microorganisms. Rhizosphere microbial communities play a crucial role in promoting soil nutrient cycling, improving soil structure, promoting plant growth and development, and enhancing plant defense mechanisms, thus significantly contributing to plant productivity and health in the natural environment. Studies by Mendes and Dong et al. have indicated that most disease resistance and developmental mechanisms in plants are directly related to the diversity of rhizosphere microorganisms. However, the composition of the rhizosphere microbial community is influenced by multiple factors. Plant genotype and soil type are two major factors affecting rhizosphere microbial community composition. Changes in tillage practices and cultivation management often alter the soil environment, leading to changes in the structure, composition, and diversity of the soil microbial community. Liu Ruowei and Ke Lingjie et al. investigated the effects of in-situ or exogenous selenium treatments (different types or amounts of selenium fertilizer) on the structure, diversity, and crop quality of the plant rhizosphere soil microbial community, clarifying that appropriate concentrations of selenium fertilizer help enhance soil enzyme activity, enrich beneficial rhizosphere microorganisms, and improve the richness and diversity of the soil microbial community. However, little is currently known about the effects of exogenous selenium fertilizer on the rhizosphere soil microbial community of Amorphophallus bulbifera.

[0004] Bacteria constitute the largest proportion of soil microorganisms, are the most active factor in the soil, and are an important indicator of soil health, playing a key role in promoting crop growth. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera.

[0006] This invention is implemented as follows: a method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera, characterized in that the method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera specifically includes:

[0007] S1: Experimental design and sample collection;

[0008] S2: Soil microbial DNA extraction and amplicon sequencing;

[0009] S3: Quality control and analysis of microbiome data.

[0010] Furthermore, in S1, the experiment included two treatments: a selenium treatment group (TSe): 150 g / mu of selenium fertilizer was applied to the soil exogenously; and a control group (TCK): no selenium fertilizer was applied. A randomized block design was used, with three replicates per treatment and a plot area of ​​75 m². 2 Konjac was planted in single ridges with a ridge width of 80cm, a ridge height of 20cm-30cm, a furrow width of 25cm-30cm between ridges, and a plant spacing of 40cm. The tested konjac bulbs were planted uniformly on April 28, 2023, and the tested selenium fertilizer was applied as a base fertilizer mixed with soil during konjac planting.

[0011] Furthermore, in S1, the sample collection is as follows:

[0012] During the tuber enlargement stage of konjac (October 2023), konjac plants were selected using a five-point sampling method. After removing the topsoil, the entire konjac plant was pulled up, and the loose soil was shaken off. Tiny soil particles attached to the roots were carefully collected with a brush as rhizosphere soil. The rhizosphere soil samples were named TSe (rhizosphere soil of konjac plants in the selenium treatment group) and TCK (rhizosphere soil of konjac plants in the control group). After sampling, the rhizosphere soil samples taken from 5 konjac plants were thoroughly mixed to form one sample. Each treatment contained 3 replicates, for a total of 6 samples. The collected konjac rhizosphere soil samples were flash-frozen in liquid nitrogen and then immediately stored in a -80°C freezer for later use.

[0013] Further, in step S2, DNA was extracted from six rhizosphere soil samples following the steps of the FastDNA® SPIN Kit for Soil (MP, USA) soil genomic DNA extraction kit. DNA concentration and purity were detected using NanoDrop2000, and DNA extraction quality was assessed using 1% agarose gel electrophoresis. Using the extracted DNA as a template, the V3-V4 variable region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The recovered product was purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing using the Illumina Miseq PE300 platform.

[0014] Furthermore, in S3, the paired-end raw sequencing sequences were quality controlled using FASTP, and paired-end sequences were assembled using FLASH software. UPARSE software was used to cluster the quality-controlled assembled sequences into Operational Taxonomic Units (OTUs) based on 97% similarity and to remove chimeras. RDP classifier was used to perform OTU taxonomic annotation against the SILVA 16S rRNA gene database (v138), with a confidence threshold of 70%. Mothur software was used to calculate Alpha diversity indices such as Chao 1 and Shannon index, and Wilxocon rank-sum test was used to analyze inter-group differences in Alpha diversity. PCoA analysis (principal coordinate analysis) based on the Bray-Curtis distance algorithm was used to examine the overall changes in the microbial community structure among samples, and PERMANOVA nonparametric test was used to analyze whether the differences in microbial community structure among sample groups were significant.

[0015] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0016] Exogenous selenium treatment affects the diversity, composition, and metabolic functions of the rhizosphere bacterial community in Amorphophallus bulbifera. Exogenous selenium treatment helps increase the abundance and diversity of bacterial communities in the rhizosphere soil of Amorphophallus bulbifera, and also helps increase the relative abundance of beneficial microorganisms such as Actinobacteria, Chloramphenicol, Acidobacteria, and Sphingomyces, Streptomyces, and Bacillus. By reshaping a stable and more diverse rhizosphere microbial community structure through the exogenous addition of selenium fertilizer, a healthier rhizosphere microecological environment can be created. The results of this invention provide a scientific basis for the rational application of selenium fertilizer to improve the soil microecological environment, optimize the planting benefits of konjac, and maintain the sustainable development of the konjac industry.

[0017] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are:

[0018] In agricultural production: This technology can guide the precise application of selenium fertilizer in the cultivation of bulbils and konjac, improving disease resistance and yield by optimizing the rhizosphere microecology and reducing losses caused by continuous cropping obstacles. Based on calculations of a 10%–15% increase in yield and an additional 500–800 yuan per mu (approximately 0.16 acres), large-scale application can significantly improve the economic benefits for konjac growers. Simultaneously, the rational application of selenium fertilizer can reduce the use of chemical fertilizers, lower agricultural non-point source pollution, and achieve both ecological and economic benefits.

[0019] Industrial applications: Selenium-enriched konjac products have a significantly increased added value due to their combination of high glucomannan content and selenium nutritional characteristics, which can be expanded into high-end markets such as health foods and functional foods, thus extending the konjac industry chain.

[0020] Technical service side: Based on the rhizosphere microecological detection and selenium fertilizer application guidance technology of this invention, a standardized detection service solution can be formed to provide technical consultation and field guidance for konjac production areas, creating commercial value for technical services.

[0021] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad.

[0022] Currently, there is limited research both domestically and internationally on the effects of selenium fertilizer on the rhizosphere microorganisms of konjac, and a method for determining the correlation between exogenous selenium application and the structure and function of the rhizosphere bacterial community in Amorphophallus bulbiferus has not yet been established. This invention, for the first time, through high-throughput sequencing and bioinformatics analysis, clarifies the enrichment pattern of beneficial microorganisms in the rhizosphere of Amorphophallus bulbiferus by selenium fertilizer and its regulatory mechanism on metabolic function, filling the gap in the technology of selenium regulation of the rhizosphere microecology of Amorphophallus bulbiferus.

[0023] Existing technologies mostly focus on the impact of selenium fertilizer on crop yield or selenium content, lacking a technical system to analyze the regulatory role of selenium fertilizer from the perspective of rhizosphere microorganisms. The "sampling-sequencing-data analysis-effect determination" full-process method constructed in this invention fills the gap in microecological-based fertilization decision-making technology in the konjac industry and provides a referable technical paradigm for the application of selenium fertilizer in other tuber crops.

[0024] (3) The technical solution of the present invention solves a technical problem that people have long wanted to solve but have never been able to solve successfully.

[0025] This invention addresses the challenge of microecological regulation in konjac continuous cropping: Continuous cropping of konjac is a key bottleneck restricting the industry's development, and effective methods for improving the rhizosphere microecology have long been lacking. This invention regulates the rhizosphere bacterial community through selenium fertilizer, enriching beneficial microorganisms such as Actinobacteria and Streptomyces, enhancing soil nutrient cycling and plant disease resistance, thus providing a practical and feasible technical approach to overcoming continuous cropping obstacles.

[0026] This invention solves the problem of accurately determining the effect of selenium fertilizer application: traditional selenium fertilizer application relies on experience and cannot quantify its impact on the rhizosphere microecology. It establishes a determination system based on bacterial community diversity index, species composition, and functional prediction, enabling visualized and quantitative evaluation of the selenium fertilizer's regulatory effect, thus addressing the technical pain point of "how much selenium fertilizer to apply and what the effect will be."

[0027] (4) The technical solution of the present invention overcomes technical bias.

[0028] This invention overcomes the technical bias that "selenium fertilizer is only used to increase the selenium content of crops": the industry generally believes that the core function of selenium fertilizer is to increase selenium accumulation in crops, neglecting its regulatory value on the rhizosphere microecology. This invention confirms that appropriate concentrations of selenium fertilizer can reshape the rhizosphere bacterial community structure of Amorphophallus bulbifera, enhance community diversity and metabolic function, and clarify the dual role of selenium fertilizer in "nutrient supply + microecological regulation".

[0029] This invention overcomes the technical bias that "rhizosphere microbial detection is difficult to guide field fertilization": Traditional views hold that microbial sequencing technology is complex and costly, making it difficult to translate into practical field applications. This invention simplifies the sampling and analysis process, establishes standardized judgment methods, and directly links laboratory data with fertilization decisions, demonstrating that microbial detection technology can efficiently serve agricultural production practices. Attached Figure Description

[0030] Figure 1 This is a flowchart of a method for determining the effect of exogenous selenium application on the rhizosphere microecology of Amorphophallus bulbifera under the conditions provided in this embodiment of the invention.

[0031] Figure 2This is a Venn diagram showing the distribution of OTUs in the rhizosphere soil of Amorphophallus bulbifera in different treatment groups according to embodiments of the present invention.

[0032] Figure 3 PCoA analysis of the rhizosphere soil bacterial community composition of Amorphophallus bulbifera in different treatment groups provided in the embodiments of the present invention;

[0033] Figure 4 This refers to the relative abundance of bacterial phyla in the rhizosphere soil of Amorphophallus bulbifera in different treatment groups provided in the embodiments of the present invention.

[0034] Figure 5 This refers to the species composition of the rhizosphere soil bacterial community at the genus level in different treatment groups of Amorphophallus bulbifera provided in the embodiments of the present invention;

[0035] Figure 6 This is an analysis of the significant differences in bacterial community species at the genus level among different treatment groups provided in the embodiments of the present invention;

[0036] Figure 7 This invention provides a predictive analysis of the PICRUSt function of rhizosphere soil bacterial communities in different treatment groups of Amorphophallus bulbifera, based on embodiments of the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0038] like Figure 1 As shown, this embodiment of the invention provides a method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera. The method specifically includes:

[0039] S1: Experimental design and sample collection;

[0040] S2: Soil microbial DNA extraction and amplicon sequencing;

[0041] S3: Quality control and analysis of microbiome data.

[0042] S1, the experiment included two treatments: a selenium treatment group (TSe): 150 g / mu of selenium fertilizer was applied to the soil exogenously; and a control group (TCK): no selenium fertilizer was applied. A randomized block design was used, with three replicates per treatment and a plot area of ​​75 m². 2 Konjac was planted in single ridges, with a ridge width of 80cm, a ridge height of 20cm-30cm, and a furrow width of 25cm-30cm between ridges. The plant spacing was 40cm. The tested konjac bulbs were planted uniformly on April 28, 2023. The tested selenium fertilizer was applied as a base fertilizer mixed into the soil during konjac planting.

[0043] Sample Collection: Konjac plants were selected using a five-point sampling method during the tuber enlargement stage (October 2023). After removing the topsoil, the entire plant was uprooted, and the loose soil was shaken off. Tiny soil particles attached to the roots were carefully collected with a brush as rhizosphere soil. These rhizosphere soil samples were named TSe (rhizosphere soil from the selenium-treated group) and TCK (rhizosphere soil from the control group). After collection, the rhizosphere soil samples from five plants were thoroughly mixed to form one sample. Each treatment included three replicates, for a total of six samples. The collected rhizosphere soil samples were flash-frozen in liquid nitrogen and immediately stored at −80 °C for later use.

[0044] In step S2, DNA was extracted from six rhizosphere soil samples following the procedures outlined in the FastDNA® SPIN Kit for Soil (MP, USA). DNA concentration and purity were assessed using a NanoDrop 2000, and DNA extraction quality was determined by 1% agarose gel electrophoresis. Using the extracted DNA as a template, the V3-V4 variable region of the bacterial 16S rRNA gene was amplified using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The recovered product was purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing using an Illumina Miseq PE300 platform.

[0045] In step S3, FASTP was used to perform quality control on the raw paired-end sequencing sequences, and FLASH software was used to assemble the paired-end sequences. UPARSE software was used to cluster the quality-controlled assembled sequences into Operational Taxonomic Units (OTUs) based on 97% similarity and to remove chimeras. RDP classifier was used to perform OTU taxonomic annotation against the SILVA16S rRNA gene database (v138), with a confidence threshold of 70%. Mothur software was used to calculate Alpha diversity indices such as Chao 1 and Shannon index, and Wilxocon rank-sum test was used to analyze inter-group differences in Alpha diversity. Principal coordinate analysis (PCoA) based on the Bray-Curtis distance algorithm was used to examine the overall changes in microbial community structure among samples, and the PermanoVA nonparametric test was used to analyze whether the differences in microbial community structure among sample groups were significant.

[0046] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0047] (a) OTU abundance and Alpha and Beta diversity of bacteria

[0048] 396,435 valid bacterial sequences were obtained from the rhizosphere soil of six Amorphophallus bulbifera samples using Illumina MiSeq high-throughput sequencing, with an average sequence length of 415 bp. Classification and annotation of the valid reads were performed at a 97% sequence similarity level, resulting in 6659 bacterial operational taxonomic units (OTUs) annotated from the six Amorphophallus bulbifera rhizosphere soil samples. The selenium-treated group and the control group contained 5036 and 4098 bacterial OTUs, respectively, with the bacterial abundance in the selenium-treated group being 1.23 times that of the control group. The Venn diagram shows the number of shared and unique bacterial OTUs in the rhizosphere soil of Amorphophallus bulbifera in the selenium-treated and control groups. The two treatment groups shared 3285 bacterial OTUs, accounting for 49.33% of the total bacterial OTUs in the Amorphophallus bulbifera rhizosphere soil. Figure 2 Comparing the two treatments, the number of unique OTUs in the selenium-treated group (1751) was 1.08 times that in the control group (1623). These results indicate that exogenous selenium treatment simultaneously increased the abundance of bacterial communities and the number of unique bacterial OTUs in the rhizosphere soil of Amorphophallus bulbifera.

[0049] The effects of exogenous selenium treatment on the diversity and richness of bacterial communities in the rhizosphere soil of *Amorphophallus bulbifera* were further analyzed using comparative analysis of Alpha diversity indices. The results are shown in Table 1. The Shannon index of the bacterial community in the rhizosphere soil of *Amorphophallus bulbifera* in the selenium-treated group increased by 0.74% compared to the control group, while the Simpson index decreased by 13.64%. However, the ACE and Chao1 indices in the selenium-treated group decreased by 0.63% and 1.19% respectively compared to the control group, although the differences were not statistically significant. This indicates that exogenous selenium treatment can, to some extent, increase the diversity of bacterial communities in the rhizosphere soil of *Amorphophallus bulbifera* while decreasing its richness.

[0050]

[0051] Note: Different lowercase letters indicate significant differences (P<0.05).

[0052] Principal coordinate analysis (PCoA) based on the Bray-Curtis distance algorithm was used to evaluate the effects of exogenous selenium treatment on the rhizosphere soil bacterial community structure (Beta diversity) of Amorphophallus bulbifera. Figure 3 Principal component 1 (PC1) and principal component 2 (PC2) explained 56.87% and 18.04% of the sample variance, respectively, totaling 74.91%. The selenium-treated and control samples showed significant separation on the first principal component, with the samples from the selenium-treated and control groups located on opposite sides of the PC1 axis and spaced far apart, indicating a significant difference in the bacterial community structure of the rhizosphere soil of *Amorphophallus bulbifera* between the two groups.

[0053] (ii) Species composition and relative abundance of bacterial communities at different taxonomic levels

[0054] To further analyze the effects of exogenous selenium treatment on the composition of rhizosphere soil bacteria in Amorphophallus bulbifera, we analyzed the species composition at different taxonomic levels. Firstly, at the phylum level, the rhizosphere soil bacterial communities of Amorphophallus bulbifera plants under different treatments were mainly composed of Proteobacteria, Actinobacteriota, Chloroflexi, Acidobacteriota, Firmicutes, and Bacteroidota, with the relative abundance of these six phyla accounting for over 80% of the total soil bacteria. Figure 4When comparing the two treatment groups, the relative abundance of Proteobacteria, Firmicutes, and Bacteroidetes in the selenium-treated group was lower than that in the control group, decreasing by 15.30%, 16.99%, and 42.39%, respectively. Conversely, the relative abundance of Actinobacteria, Chlorophyta, and Acidobacteria was higher in the selenium-treated group than in the control group, increasing by 21.13%, 9.46%, and 35.56%, respectively.

[0055] The Circos diagram illustrates the species composition of bacterial communities at the genus level in samples from different treatment groups. Figure 5 Bacillus, Acidobacteriales, Gaiellales, Vicinamibacterales, Xanthobacteraceae, and Mycobacterium are the main dominant bacterial communities in the rhizosphere soil of Amorphophallus bulbifera. To further clarify the changes in the relative abundance of bacterial communities in the rhizosphere soil of Amorphophallus bulbifera after exogenous selenium treatment, we conducted a significant difference analysis between groups. A bar chart using the species difference test was used to show the relative abundance differences of the top 20 species between the selenium-treated group and the control group. Figure 6First, among the accurately classified and named species, the relative abundance of many bacterial communities showed a significant increasing trend in the selenium-treated groups. Specifically, the relative abundance of *Sphingomonas* (1.76%), *Gaiella* (1.45%), *Streptomyces* (1.006%), and *Gemmatimonas* (0.67%) in the selenium-treated groups increased by 112.05%, 229.55%, 26.58%, and 116.13%, respectively, compared to the control group (P < 0.05). Conversely, the relative abundance of *Devosia* and *Rhodanobacter* was significantly lower in the selenium-treated groups than in the control group, decreasing by 46.05% and 67.53%, respectively, compared to the control group (P < 0.01). Furthermore, the relative abundance of many unclassified species, including *Subgroup 7*, *Gemmatimonadaceae*, *RBG-13-54-9*, *Rokubacteriales*, *Vicinamibacteraceae*, and *C0119*, was significantly higher in the selenium-treated group than in the control group. These results indicate that exogenous selenium treatment had a significant impact on the dominant species composition and relative abundance of the rhizosphere soil bacterial community of *Amorphophallus bulbifera*.

[0056] (III) Predictive analysis of rhizosphere soil bacterial community function in different treatment groups of Amorphophallus bulbifera

[0057] To better investigate the microecological function of bacterial communities in the rhizosphere soil of Amorphophallus bulbifera in the selenium-treated and control groups, functional prediction analysis was performed using PICRUSt software. Figure 7According to PICRUSt, the KEGG pathway's Level 1 functions can be categorized into functional groups such as metabolism, environmental information processing, genetic information processing, and cellular processes. At the Level 2 functional group, bacterial communities from different treatment groups exhibited similar gene functions, primarily metabolic, including global and overview pathways, carbohydrate metabolism, amino acid metabolism, energy metabolism, and nucleotide metabolism. They also involved functions related to membrane transport, signal transduction, translation, and prokaryotic cell community sensing. Heatmaps were used to illustrate the differences in the relative abundance of the top 20 genes at pathway Level 3 between the two treatment groups. The results showed that exogenous selenium treatment increased bacterial community metabolism and genetic information processing-related functions in the rhizosphere soil of *Amorphophallus villosa*. Among the top 20 functions, the average relative abundance of genes related to secondary metabolite biosynthesis, microbial metabolism in different environments, amino acid biosynthesis, carbon metabolism, purine metabolism, oxidative phosphorylation, glyoxylate and dicarboxylate metabolism, pyruvate metabolism, glycolysis / glucose production, prokaryotic carbon fixation pathways, glycine, serine and threonine metabolism, fatty acid metabolism, and amino sugar and nucleotide sugar metabolism were all higher in the selenium-treated group than in the control group. Conversely, the average relative abundance of ABC transporters and two-component systems, which are related to environmental information processing, was lower in the selenium-treated group than in the control group.

[0058] Soil microbial communities are influenced by various factors, such as plant type, climate, soil properties, and agricultural practices. This invention uses 16S amplicon sequencing to analyze the effects of exogenous selenium treatment on the composition and diversity of bacterial communities in the rhizosphere soil of *Amorphophallus bulbifera*. First, PCoA principal coordinate analysis based on the Bray-Curtis distance algorithm revealed that samples from the selenium-treated group and the control group were distributed on opposite sides of the PC1 axis with significant distances between them, indicating a marked difference in the bacterial community structure of the rhizosphere soil between the two groups. Furthermore, we found that exogenous selenium treatment simultaneously increased the abundance, diversity, and number of unique bacterial OTUs in the rhizosphere soil of *Amorphophallus bulbifera*. The Shannon index of the bacterial community in the rhizosphere soil of the selenium-treated group increased by 0.74% compared to the control group, while the Simpson index decreased by 13.64%. These findings are consistent with previous studies, suggesting that changes in the rhizosphere bacterial community of *Amorphophallus bulbifera* are influenced to some extent by exogenous selenium treatment, as soil microbial communities typically change dynamically under the combined influence of host and environmental factors. Soil microbial community diversity is crucial for the integrity, stability, and sustainability of soil ecosystems. High microbial diversity and activity promote plant growth, enhance plant defenses, and suppress soil-borne diseases. The increased abundance and diversity of bacterial communities after exogenous selenium treatment in this invention suggest a beneficial transformation of the rhizosphere microecological environment of Amorphophallus bulbifera.

[0059] Further analysis of the differences in bacterial community composition revealed that the relative abundance of Proteobacteria, Firmicutes, and Bacteroidetes in the selenium-treated group decreased compared to the control group, while the relative abundance of Actinobacteria, Chlorconoids, and Acidobacteria increased. Studies have shown that actinomycetes are important decomposers in the carbon cycle, utilizing persistent carbon sources by secreting hydrolytic enzymes to degrade cellulose, lignin, and lignocellulose; actinomycetes can also produce antibiotics that inhibit the growth and development of various soil plant pathogens. Chlorconoids include anaerobic thermophiles and anaerobic organohalogen-respiring bacteria, which utilize organohalogen compounds. Acidobacteria mainly participate in iron cycling and single-carbon compound metabolism, playing a crucial role in degrading cellulose and lignin in plant residues. In this invention, the high abundance of Actinomycetes, Chlorconoids, and Acidobacteria in the rhizosphere soil of Amorphophallus bulbifera after exogenous selenium treatment enhances the cycling of essential nutrients, thereby improving soil fertility and sustainable utilization. Simultaneously, a higher abundance of actinomycetes also enhances plant defense mechanisms and inhibits the growth of soil-borne pathogens.

[0060] Similarly, at the genus level, we observed a significant increase in the relative abundance of several bacterial communities in the selenium-treated group. Specifically, the relative abundance of *Sphingomonas*, *Gaiella*, *Streptomyces*, and *Gemmatimonas* in the selenium-treated group increased by 112.05%, 229.55%, 26.58%, and 116.13%, respectively, compared to the control group (P < 0.05). Some strains of *Sphingomonas* have been reported to be closely related to nitrogen fixation; many species of *Streptomyces* have been reported as plant growth-promoting and biocontrol bacteria, capable of producing substances that inhibit plant pathogens. Overall, exogenous selenium fertilizer, to some extent, increased the relative abundance of beneficial bacteria in the rhizosphere soil of *Amorphophallus bulbifera*.

[0061] PICRUSt functional prediction showed that the rhizosphere bacterial community of Amorphophallus bulbifera exhibited significantly improved metabolic functions compared to the control group after exogenous selenium treatment, including amino acid metabolism, carbohydrate metabolism, and energy metabolism. Soil microorganisms primarily participate in soil nutrient cycling and transformation through their metabolic activities, regulating the metabolic processes of organisms. Higher metabolic functions may promote the turnover and metabolism of nutrients in the rhizosphere bacterial community, providing plants and bacteria with the energy and metabolic products needed for growth and development, thus contributing to enhanced stability of the rhizosphere soil microecology.

[0062] Exogenous selenium treatment affected the diversity, composition, and metabolic functions of the rhizosphere bacterial community in Amorphophallus bulbifera. Exogenous selenium treatment helped increase the abundance and diversity of bacterial communities in the rhizosphere soil of Amorphophallus bulbifera, while also increasing the relative abundance of beneficial microorganisms such as Actinobacteria, Chloramphenicol, Acidobacteria, and Sphingomyces, Streptomyces, and Bacillus. Reshaping a stable and more diverse rhizosphere microbial community structure through exogenous selenium fertilizer application helps create a healthier rhizosphere microecological environment. These results provide a scientific basis for the rational application of selenium fertilizer to improve the soil microecological environment, optimize the planting benefits of konjac, and maintain the sustainable development of the konjac industry.

[0063] Example 1

[0064] In a typical Amorphophallus bulbifera growing area (soil type: red soil, texture: sandy loam), experimental plots with consistent soil pH, organic matter content, total nitrogen content, and other physicochemical properties were selected, with an area of ​​450 m². The experiment included two groups: a selenium-treated group (TSe) and a non-selenium-treated control group (TCK). Each group was replicated three times, with each replicate plot measuring 75 m², using a randomized block design.

[0065] The planting specifications were single-ridge planting, with a ridge width of 80 cm, a ridge height of 25 cm, a furrow width of 28 cm, and a plant spacing of 40 cm between konjac bulbs. "Zhuma Jin No. 1" bulbs were uniformly planted on April 28, 2023. The selenium-treated group received selenium fertilizer as basal fertilizer at planting time, at a rate of 150 g / mu, with a selenium content of 10 g / kg. The control group did not receive selenium fertilizer, but other field management practices remained the same.

[0066] During the tuber enlargement stage of *Amorphophallus konjac* in October 2023, five healthy *Amorphophallus konjac* plants were selected from each replicate plot using a five-point sampling method. After removing the top 2 cm of soil, the entire plant was uprooted, and loose soil from the root surface was shaken off. Soil particles tightly attached to the roots were collected using a sterile brush as rhizosphere soil. The rhizosphere soil from each of the five plants in each plot was thoroughly mixed to prepare one pooled sample, resulting in a total of six samples across two groups. The samples were flash-frozen in liquid nitrogen and then stored at -80°C for later use.

[0067] Soil microbial DNA was extracted using the FastDNA® SPIN Kit for Soil. DNA concentration and purity were assessed using a NanoDrop 2000, and DNA integrity was verified by 1% agarose gel electrophoresis. PCR amplification was performed using the V3-V4 variable region of the bacterial 16S rRNA gene as the target fragment, employing primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The amplified products were purified using the AxyPrep DNAGel Extraction Kit and then analyzed on an Illumina MiSeq PE300 platform.

[0068] After the data was subjected to FASTP quality control and FLASH stitching, OTUs were obtained by clustering with 97% similarity using Uparse software, and species annotation was completed using the SILVA database. Alpha diversity indices for Shannon, Simpson, ACE, and Chao1 were calculated using Mothur software, and Beta diversity was analyzed using PCoA based on Bray-Curtis distance.

[0069] The results showed that the abundance of bacterial OTUs in the selenium-treated group was 5036, which was 1.23 times that of the control group (4098); the number of unique OTUs was 1751, which was 1.08 times that of the control group (1623). The Shannon index increased by 0.74% and the Simpson index decreased by 13.64% compared with the control group. In the PCoA analysis, the two groups were clearly separated in the first principal component (56.87% explanatory power). Based on the above differences in diversity indices and community structure, it was determined that exogenous selenium application can significantly regulate the rhizosphere microecological environment of Amorphophallus bulbifera.

[0070] Example 2

[0071] Based on the experimental design and operation procedure of Example 1, rhizosphere soil samples from the three replicate plots of each treatment group were extracted, sequenced, and analyzed independently without sample mixing, resulting in three groups of selenium treatment data (TSe-1, TSe-2, TSe-3) and three groups of control data (TCK-1, TCK-2, TCK-3).

[0072] Statistical analysis of the Alpha diversity index of the six samples showed that the mean Shannon index of the three replicates in the selenium-treated group was 6.81±0.049, with an intra-group coefficient of variation of only 0.72%; the mean Shannon index of the control group was 6.76±0.200, with an intra-group coefficient of variation of 2.96%. The intra-group coefficients of variation for ACE and Chao1 indices in the selenium-treated group were 2.03% and 2.19%, respectively, both lower than those in the control group (1.28% and 0.81%).

[0073] In PCoA analysis, the three replicates from the same treatment group clustered on the ordination plot, with the distance between samples within a group being less than the distance between samples in the treatment group. The PERMANOVA test showed that the p-value for significant differences between treatment groups was <0.05, while the p-value for significant differences within treatment groups was >0.05.

[0074] Consistency verification of replicated data confirmed the stability and reproducibility of changes in rhizosphere bacterial communities induced by selenium application, eliminating the interference of single-sample randomness on the judgment results. This implementation method closely integrates laboratory analysis results with actual field conditions, improving the reliability of the judgment method in agricultural production, and enabling the conclusions on selenium fertilizer's regulation of rhizosphere microecology to directly guide fertilization decisions in konjac cultivation.

[0075] Example 3

[0076] While maintaining the same selenium application rate (150 g / mu), planting specifications, field management and other conditions as in Example 1, the time for collecting rhizosphere soil samples was precisely limited to the rapid expansion stage of underground bulbs (mid-October 2023). During this stage, the konjac root system secretion is strongest and the activity of rhizosphere microorganisms reaches its peak, which can maximize the interaction effect between exogenous selenium and microorganisms.

[0077] Two sampling time points were set up concurrently: the pre-expansion period (mid-September 2023) and the post-expansion period (mid-November 2023) as controls. Three samples were collected from the selenium-treated group and three from the control group at each time point, and the same sequencing and analysis procedures were carried out.

[0078] The results showed that in the samples collected during the rapid expansion period, the relative abundance of Actinobacteria, Chlorobacteria, and Acidobacteria in the selenium-treated group increased by 21.13%, 9.46%, and 35.56% respectively compared with the control group; the relative abundance of beneficial bacteria such as Sphingosporobacter, Gastrodia, Streptomyces, and Bacillus increased by 112.05%, 229.55%, 26.58%, and 116.13% respectively, which were significantly higher than those in the early expansion period (increase range 3.21%~89.74%) and the late expansion period (increase range 5.12%~95.36%).

[0079] PCoA analysis showed that the selenium-treated group during the rapid expansion period had the highest degree of separation from the control group, with the first principal component contributing 56.87%, while the first principal component contributions in the early and late expansion periods were 42.15% and 38.69%, respectively.

[0080] The above results indicate that sampling the rhizosphere soil during the rapid expansion period of underground bulbs can more accurately reflect the regulatory intensity of selenium fertilizer on the rhizosphere microecology. This implementation method clarifies the optimal sampling period, providing technical support for the accurate determination of the regulatory effect of selenium fertilizer, and also providing a scientific basis for selecting the timing of selenium fertilizer application in the field.

[0081] Example 4

[0082] Based on the classification and abundance data of six OTU samples obtained in Example 1, a Bray-Curtis distance matrix was constructed using the vegan package in R. This matrix quantifies the degree of difference in bacterial community structure between any two samples. The distance matrix values ​​range from 0 to 1, with values ​​closer to 1 indicating greater differences in community structure.

[0083] The analysis results showed that the average distance between samples within the selenium treatment group was 0.12, the average distance between samples within the control group was 0.15, and the average distance between samples between the selenium treatment group and the control group was 0.48. The distance between groups was significantly higher than the distance within groups.

[0084] PCoA analysis based on the distance matrix showed that the cumulative explanatory power of the first principal component (PC1) and the second principal component (PC2) reached 74.91%. All three samples in the selenium treatment group were clustered to the left of the PC1 axis, while all three samples in the control group were clustered to the right of the PC1 axis. The two groups of samples formed obvious segregated clusters in the ordination space, with no overlap.

[0085] The significance of the differences between groups was further verified by the PERMANOVA nonparametric test. The results showed that R²=0.72 and P=0.001, indicating that selenium application was the dominant factor leading to changes in the rhizosphere bacterial community structure, explaining 72% of the community structure variation.

[0086] This implementation method verifies the regulatory effect of exogenous selenium on rhizosphere microecology from the perspective of community structure spatial distribution, clarifies that microbial community structure can be used as the core criterion for the regulatory effect of selenium fertilizer, and proves the feasibility and effectiveness of the determination method of the present invention at the system level.

[0087] Example 5

[0088] Five gradient selenium fertilizer application schemes were set up: 0 g / mu (CK), 75 g / mu (T1), 150 g / mu (T2), 225 g / mu (T3), and 300 g / mu (T4). Each scheme was replicated three times, and the plot area, planting specifications, and other conditions were consistent with those in Example 1. Rhizosphere soil samples were collected in October 2023 during the rapid expansion period of underground bulbs, and the same DNA extraction, sequencing, and bioinformatics analysis procedures were performed.

[0089] The rhizosphere bacterial communities of the five schemes were comprehensively evaluated, and the evaluation indicators included: ① Alpha diversity index (Shannon, Simpson); ② relative abundance of beneficial microorganisms (Actinomycetes, Chlorophytes, Acidobacteria, and Sphingosporobacter spp., etc.); ③ community structure stability (average distance between samples within the group).

[0090] The results showed that the T2 scheme (150 g / mu) had the highest Shannon index (6.81) and the lowest Simpson index (0.0038); the relative abundance of beneficial microorganisms reached its peak, with Actinobacteria accounting for 18.25% and Streptomyces accounting for 1.006%; the average distance between samples within the group was the smallest (0.12), and the community structure stability was the best.

[0091] The abundance of beneficial microorganisms in the T1 scheme (75 g / mu) was lower than that in the T2 scheme, while the Shannon index of the T3 (225 g / mu) and T4 (300 g / mu) schemes decreased to 6.52 and 6.38, respectively. The abundance of some beneficial microorganisms decreased, indicating that high concentrations of selenium fertilizer had an inhibitory effect on the microbial community.

[0092] Through comprehensive scoring, scheme T2 (150 g / mu) was selected as the optimal selenium fertilizer application scheme. This implementation method demonstrates that the determination method of the present invention can not only determine whether selenium fertilizer has a regulatory effect, but also screen the merits of different selenium application schemes, achieving refined regulation of agricultural inputs and providing customized selenium fertilizer application strategies for Amorphophallus bulbifera cultivation in different regions and with different soil types.

[0093] Example 6

[0094] Three consecutive seasons of location-based monitoring experiments were conducted on the same experimental plot. The experimental protocol for the first season was the same as in Example 1, with a selenium application group (150 g / mu) and a control group. After the first harvest, the rhizosphere bacterial community structure was measured, and the results showed that the abundance of beneficial microorganisms in the selenium application group was significantly higher than that in the control group.

[0095] In the second season of the trial, based on the monitoring results of the first season, the selenium application rate was adjusted to 120 g / mu to further optimize the community structure. Monitoring after the second harvest revealed that the abundance of beneficial microorganisms increased by 5.23% compared to the first season, and the stability of the community structure was further enhanced.

[0096] In the third season of the experiment, the selenium application rate was maintained at the level of the second season. Monitoring results showed that the rhizosphere bacterial community structure remained stable, and the coefficients of variation of indicators such as Shannon index and abundance of beneficial microorganisms were all less than 3%.

[0097] Through multi-season data feedback and dynamic adjustments to the selenium application plan, a closed-loop control system of "monitoring-analysis-adjustment-re-monitoring" has been formed. Monitoring results from three consecutive seasons indicate that the determination method of this invention can be applied long-term to field selenium fertilizer management, continuously maintaining the stability of the rhizosphere microecology.

[0098] This implementation demonstrates the long-term application value of the technical solution of this invention, proving that it can effectively solve the problem of continuous cropping obstacles in konjac, providing technical support for the sustainable development of the konjac industry, and has significant industrial promotion significance. As can be seen from the above embodiments, this technical solution does not stop at the level of simple detection or analysis, but establishes a stable, determinable, and feedback-able technical correlation between exogenous selenium application behavior and changes in rhizosphere bacterial community structure. It provides a novel implementation path based on a microecological regulation mechanism for fertilization decisions in selenium-enriched konjac production, meeting the requirements of full disclosure and implementation methods supporting the technical solution.

[0099] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for determining the effect of exogenous selenium application on the rhizosphere microecological regulation of Amorphophallus bulbifera, characterized in that, Rhizosphere soil samples were collected during the tuber enlargement stage of konjac underground stems under both selenium-treated and selenium-free treatments. Amplicon sequencing was performed on the bacterial community genetic information in the samples. Based on the combined criterion of changes in bacterial community diversity index and differences in community structure, the regulatory effect of exogenous selenium application on the stability of the rhizosphere microecology of konjac was determined, and the determination results were output to guide the decision-making on selenium fertilizer application.

2. The method according to claim 1, characterized in that, The selenium-treated and non-selenium-treated treatments were randomly assigned to the field, with each treatment replicated three times.

3. The method according to claim 1, characterized in that, The rhizosphere soil sample was obtained by removing the topsoil, and the soil was found to be tightly attached to the surface of the konjac root system.

4. The method according to claim 1, characterized in that, When the bacterial community diversity index and community structure under selenium treatment conditions changed significantly compared to the untreated condition, it was determined that exogenous selenium had a regulatory effect on the rhizosphere microecology.

5. A rhizosphere microecological analysis system for determining the ecological regulation effect of exogenous selenium application, characterized in that, It includes a sample acquisition module, a microbial genetic information processing module, and a regulatory effect assessment module. in, The sample acquisition module is used to acquire konjac rhizosphere soil samples under selenium-treated and non-selenium-treated conditions; The microbial genetic information processing module is used to generate rhizosphere bacterial community structure data; The regulation effect judgment module is used to output the ecological regulation judgment results of selenium fertilizer application based on the changes in diversity index and differences in community structure.

6. The system according to claim 5, characterized in that, The microbial genetic information processing module includes sequence quality control, sequence assembly, and abnormal sequence removal functions.

7. The system according to claim 5, characterized in that, The regulation effect determination module determines the differences in community structure by using a principal coordinate analysis method based on the distance between samples.

8. A method for optimizing exogenous selenium application schemes based on rhizosphere bacterial community structure, characterized in that, After obtaining data on rhizosphere bacterial community structure under different selenium application conditions, By jointly evaluating community diversity indices and differences in community structure. We screened out the selenium application schemes that had a better impact on the stability of the rhizosphere microecology, and used the schemes as the basis for selenium application in konjac production.

9. The method according to claim 8, characterized in that, The rhizosphere bacterial community structure data were constructed based on operational taxonomic units.

10. The method according to claim 8, characterized in that, By combining the results of changes in community diversity index with the results of differences in community structure, the exogenous selenium application plan can be dynamically revised.

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