Effects of the Radicle Sheath on the Rhizosphere Microbial Community Structure of Seedlings in Early Spring Desert Species Leontice incerta

Abstract

Most research on plant–microbe interactions emphasize the effects of micronutrients on the rhizosphere microbial community structure. However, the influence of seed structures, particularly the radicle sheath, on microbial diversity at the seedling root tips under varying temperatures and humidity has been less explored. This study conducted controlled indoor experiments in the northern desert of Xinjiang to assess the radicle sheath’s impact on microbial community composition, diversity, and function. The results indicated no significant changes in the Chao1 index for bacteria and fungi, but notable differences were observed in the Shannon and Simpson indices (p < 0.05). Under drought conditions, the radicle sheath significantly reduced bacterial infections without affecting fungi. Genus-level analysis showed an increased abundance of specific dominant bacterial groups when the radicle sheath was retained. NMDS analysis confirmed its significant effect on both bacterial and fungal community structures. LEfSe analysis identified 34 bacterial and 15 fungal biomarkers, highlighting the treatment’s impacts on microbial taxonomic composition. Functional predictions using PICRUSt 2 revealed that the radicle sheath facilitated the conversion of CH₄ to CH₃OH and various nitrogen cycle processes under drought. Overall, the radicle sheath plays a crucial role in maintaining rhizosphere microbial community stability and enhancing the functions of both bacteria and fungi under drought conditions.

1. Introduction

The middle desert ecosystem in northwest China is a typical representative ecosystem characterized by a dry climate, sparse vegetation, severe salinization, poor drainage, and diverse terrain [1]. Xinjiang is situated deep inland, experiencing scarce precipitation and limited water resources. Coupled with windy conditions and sparse vegetation, salinization and desertification have become the most significant environmental challenges [2]. Therefore, desert vegetation is dominated by drought-resistant plants and low shrubs. In addition, desert ecosystems contain a large number of microbial resources [3]. Studies have shown that microbial communities in deserts are similar to those in temperate soils at the phylum level but are highly specialized at the species level and are significantly affected by physical and chemical factors [4,5].

Natural actinomycete strains in the Monte Desert serve as natural promoters of vegetation growth and can improve the survival and growth of desert plants, such as Atriplex patens and Capsicum annuum seedlings, by enhancing their nutritional status [6]. Frankia and Alpova diplophloeus bacteria can decompose rock mineral nutrients by producing organic acids and transporting them to young roots to promote the growth of cactus seedlings [7]. Firmicutes and Glomeromycota enhance the resistance of Artemisia annua seedlings to drought and pathogenic bacteria by degrading seed mucus [8]. Heterotrophic bacterial communities in desert environments use atmospheric trace gases as their main energy source to maintain community diversity [9]. Therefore, in-depth research on the interactions between desert plants and microorganisms can provide a theoretical foundation for the restoration and protection of desert ecosystems.

Temperature and humidity are two major environmental factors in the soil. They directly affect aspects such as soil moisture, oxygen levels, and microbial activity and may significantly impact the structure and function of rhizosphere microbial communities [10,11]. The effects of rhizosphere microorganisms on plant growth and development are multifaceted and complex [12]. First, rhizosphere microorganisms can form symbiotic relationships with plants, providing essential nutrients and organic matter for growth by mediating carbon and nitrogen cycles, biological nitrogen fixation, producing plant growth hormones, and dissolving minerals in the soil [6,13,14,15]. This promotes root growth and enhances the absorption capacity of plants [16]. Second, rhizosphere microorganisms can inhibit the growth of soil pathogens, reducing the damage caused by soil-borne pathogens to plants and thus increasing plant resistance to environmental stress [12]. In addition, rhizosphere microorganisms can participate in regulating the root architecture and development patterns of plants, affecting the root surface area and root hair formation, which, in turn, influences the plants’ ability to absorb and utilize water and nutrients in the soil [17,18]. Therefore, rhizosphere microorganisms play a vital role in the growth and development of plants, significantly impacting their growth processes and population establishment. Although the importance of rhizosphere microorganisms is widely recognized, the effect of the unique structure of the radicle sheath in the desert plant Leontice incerta on the structure of the root tip microbial community has not been fully studied.

This study selected Leontice incerta Pall., a perennial short-lived plant of the genus Leontice of the Berberidaceae family that is only distributed in Kazakhstan and Xinjiang, China, as the research material. In addition to the seed coat, endosperm, and embryo, the seeds also develop a special structure called the radicle sheath. This structure typically forms as the radicle breaks through the seed coat during germination, wrapping around the young root in the early stages of seedling development. Therefore, we hypothesized that the presence of the radicle sheath increases the dominance of bacterial communities in the rhizosphere of seedlings, thereby improving their survival ability.

This study aims to systematically explore the mechanism by which the presence or absence of radicle sheaths affects the structure of the rhizosphere microbial community in early spring desert plant Leontice incerta seedlings in a windy and rainless environment. By analyzing the effects of the presence or absence of radicle sheaths on the composition, diversity, and function of the rhizosphere microbial community, we aim to answer the following scientific questions: (1) What is the effect of the radicle sheath on the structure and function of the microbial community at the root tip of Leontice incerta seedlings? (2) Do radicle sheaths promote beneficial microorganisms that positively impact the growth of Leontice incerta seedlings? (3) What role does the presence of the radicle sheath play in the adaptation of seedlings to the dry and windy desert environment?

2. Materials and Methods

2.1. Overview of the Study Area

The study area is located in the low mountain desert belt of the southern mountains in Anjihai Town, Shawan County, Xinjiang Uygur Autonomous Region, China (Figure 1). The geographical coordinates are 44°36′08.6″ N latitude and 84°90′04.6″ E longitude, with an elevation ranging between 400 and 800 m. This region’s geographical environment exhibits typical desert ecological characteristics, providing a unique natural laboratory for studying desert ecosystems [19]. The climate conditions of the area include an average annual temperature of approximately 6.5 °C, with extreme temperatures ranging from as low as −28 °C in January to as high as 37 °C in July. The average annual precipitation is around 131.8 mm, with about 65% of rainfall occurring during the spring and summer months. The annual evaporation ranges from 1237 to 1770 mm, and the area receives between 2200 and 3200 h of sunshine annually [20,21].

2.2. Experimental Design

Mature seeds were collected from natural populations in May 2023 and brought back to the laboratory for further use. We sterilized the seeds in 70% anhydrous ethanol for 5 min. Then, we soaked the sterilized seeds in 500 mL sterile water at room temperature for 4 to 6 h and transferred them to a flowerpot in an original habitat (25 cm × 15 cm × 10 cm) with 30 plants per pot and 5 replicates. Each pot contained 30 seedlings, with five replicates per treatment. The experiment included four treatments: removal of the radicle sheath under drought conditions (DTCF), retention of the radicle sheath under drought conditions (DTCL), removal of the radicle sheath under moist conditions (WTCF), and retention of the radicle sheath under moist conditions (WTCL). The seedlings were placed in a flowerpot and cultivated for 40 days, and a soil temperature and humidity meter was used to monitor the temperature and humidity every 8 h every day. After the treatment period, 10 uniform seedlings were selected and combined into one replicate (four treatments and three replicates). The root tips were stored in 50 mL sterile centrifuge tubes at −80 °C for subsequent DNA extraction, library construction, and NovaSeq PE250 high-throughput sequencing.

2.3. Determination Method

For the pretreated samples, nucleic acids were extracted using the Mag Beads Fast DNA Kit for Soil (116564384) (MP Biomedicals, San Diego, CA, USA). The extracted DNA was assessed for molecular size using 0.8% agarose gel electrophoresis and quantified with a Nanodrop spectrophotometer for subsequent experiments.

The V3V4 hypervariable region of the bacterial 16S rRNA gene was amplified using primers 338F and 806R [22,23], while the ITS1 region of the fungal genome was amplified using primers ITS1F and ITS2 [24]. PCR was performed using ABclonal DNA polymerase with the following setup: 0.25 μL of ABclonal DNA polymerase, approximately 2 μL of template DNA, and 10 μm of forward and reverse primers. The PCR conditions were as follows: initial denaturation at 98 °C for 3 min, followed by 30 s of denaturation at 98 °C, 30 s of annealing at 53 °C, and 45 s of extension at 72 °C. This cycle was repeated 27 times for bacteria and 30 times for fungi, with a final extension at 72 °C for 5 min. The amplified products were stored at 12 °C. The amplification results were checked using 2% agarose gel electrophoresis, and the target fragments were excised and purified using a magnetic bead recovery method. The PCR products were quantified on a Microplate reader (BioTek, FLx800, Shanghai, China) using the Quant-iT Pico Green dsDNA Assay Kit (Invitrogen, Shanghai, China), and samples were pooled according to the required data amount for each sample [25]. Library construction was performed using the Tru Seq Nano DNA LT Library Prep Kit (Illumina, San Diego, CA, USA). Library quality was assessed, and qualified libraries were subjected to 2 × 250 bp paired-end sequencing on an Illumina Nova Seq machine (San Diego, CA, USA) using the NovaSeq6000 SP Reagent Kit (Shanghai, China) (500 cycles). Libraries (with unique indices) were diluted to 2 nM, pooled according to the required data amount, and denatured into single strands with 0.1 N NaOH for sequencing. The final library concentration was controlled between 15–18 pM based on the actual situation [26,27,28,29,30].

2.4. Data Quality Control and Analysis

One-way ANOVA was used to analyze the differences in microbial communities between treatments. Before data analysis, the data were tested for normal distribution and homogeneity of variances. If consistent, the least significant difference (LSD, least significant difference) method was used for multiple comparisons. If it does not meet the requirements, the logarithmic or square root method is used to transform the data. If the data still does not meet the requirements after transformation, the Kruskal-Wallis non-parametric test is performed on the data. All the above analyzes are performed in SPSS 23.0 (SPSS Inc., Chicago, IL, USA) software.

Using R software (v4.1.2 version) and QIIME2 software (2022.11 version) using the UniFrac distance metric to investigate changes in microbial community structure between samples, and visualized using the non-metric multidimensional scaling (NMDS) method. The composition profile of the species at the genus level was also analyzed. Using QME2 software, the composition and abundance tables of each sample at the six classification levels of phylum, class, order, family, genus, and species were obtained, and the analysis results were presented in a bar graph. The linear discriminant analysis effect size (LES) method was used to detect differentially abundant taxa between groups [25].

3. Results

3.1. Analysis of Rhizosphere Microbial Community Taxonomic Composition

At the genus level, the effects of different treatments on the rhizosphere microbial community structure were analyzed by exploring changes in the relative abundance of dominant bacterial and fungal groups (Figure 2). In the bacterial community, the relative abundance of Rhizobium under DTCF, DTCL, WTCL, and WTCF treatments was 41.26%, 55.25%, 9.87%, and 8.59%, respectively. Among them, the relative abundance of Pseudomonas and Sphingomonas in the DTCF treatment was 11.23% and 13.16% lower than that in the DTCL treatment; the relative abundance of Enterobacter in WTCL treatment was 19.46% lower than that in the WTCF treatment (Figure 2A). In the fungal community, the relative abundance of Alternaria under DTCF, DTCL, WTCL, and WTCF treatments was 3.91%, 52.49%, 92.62%, and 99.43%, respectively. The relative abundance of Fusarium and Nectriella under DTCF, DTCL, and WTCL treatments were 40.7%, 3.03%, and 7.11%, respectively; the relative abundance of Nectriella under DTCF treatment was 41.01% (Figure 2B).

3.2. α and β Biodiversity

Shannon, Simpson, and Chao1 indices were used to evaluate the effects of the four treatments on the α diversity of the rhizosphere microbial community of seedlings. The Shannon and Simpson indices represent the α diversity index of the microbial community, and the Chao1 index represents the richness index (Figure 3). Chao1 index values under different treatments highlight the lack of significant differences between groups, and error bars represent standard deviations. The results show that the Chao1 index of the bacterial and fungal communities in the rhizosphere of seedlings was not significantly affected by the different treatments, while the Shannon and Simpson indices of the bacterial and fungal communities in the rhizosphere of seedlings were significantly different (p < 0.05). Among them, the α diversity index of the bacteria in the radicle sheaths retained under drought conditions was significantly lower than that of the radicle sheaths removed under the same conditions (p < 0.05), while there was no significant effect on the fungi; however, the α diversity index of the fungi in DTCF was significantly higher than that in WTCF.

Figure 4 shows the non-metric multidimensional scaling (NMDS) analysis of the microbial communities under the four treatments, all of which showed good representation (S < 0.2). In the bacterial community structure, there was no significant difference between the WTCL and WTCF functional groups, but there was a significant difference between the DTCL and DTCF functional groups (Figure 4A). In the fungal community structure, the DTCF functional group was significantly different from the other treatments (Figure 4B).

3.3. Differences in the Rhizosphere Microbial Communities of Leontice incerta Seedlings

The sequencing results showed that under the four treatments, the number of common bacterial OUTs in DTCF and DTCL treatments was 92, the number of unique OUTs in DTCF treatment was 1478, and the number of unique OUTs in DTCL treatment was 677 (Figure 5A); the number of common bacterial OUTs in WTCL and WTCF treatments was 104, the number of unique OUTs in WTCL treatment was 689, and the number of unique OUTs in WTCF treatment was 872 (Figure 5A); and under the four treatments, the number of common fungal OUTs was 18, the number of unique OUTs in DTCF treatment was 37, and the number of unique OUTs in DTCL treatment was 42 (Figure 5B). The number of common fungal OUTs was 17, the number of unique OUTs in WTCL treatment was 29, and the number of unique OUTs in WTCF treatment was 42 (Figure 5B).

In further analysis between treatments using LEfSe, significant changes in the abundance of root-associated microbiota were identified (Figure 6). At the genus level, significant differences (LDA score > 2) were observed in bacterial and fungal communities. With an LDA threshold of 2, LEfSe results revealed 34 bacterial biomarkers in the communities (Figure 6A): 11 in DTCF (including Streptomycetales, Sphingomonadales, Burkholderiales, Pseudomonadales, Streptomycetaceae, Sphingomonadaceae, Oxalobacteraceae, Pseudomonadaceae, Streptomyces, Massilia, Pseudomonas), 9 in DTCL (including Alphaproteobacteria, Caulobacterales, Rhizobiales, Comamonadaceae, Caulobacteraceae, Rhizobiaceae, Brevundimonas, Rhizobium, Sphingobium), 6 in WTCL (including Actinobacteriota, Actinobacteria, Alcaligenaceae, Nocardiopsaceae, Nocardiopsis, Streptosporangiales, Enterobacter), and 8 in WTCF (including Proteobacteria, Devosiaceae, Devosia, Enterobacteriaceae, Gammaproteobacteria, Novosphingobium, Enterobacterales).

LEfSe identified 15 fungal biomarkers in the communities (Figure 6B): 6 in DTCF (including Sordariomycetes, Hypocreales, Bionectriaceae, Nectriaceae, Nectriella, Fusarium), 1 in DTCL (Sordariales), 1 in WTCL (Ascomycota), and 7 in WTCF (Dothideomycetes, Pleosporales, Pleosporaceae, Alternaria, etc.).

3.4. Predicted Functions of Rhizospheric Microbial Communities in Leontice incerta Seedlings

The functions of bacterial communities in different treatments were annotated based on the Cluster of Homologous Proteins (COG) database using PICRUSt 2 database 7.1. The functions of bacterial communities were mainly manifested in the occurrence of the cell wall/membrane/envelope, post-translational modification, protein tumors and chaperones, signal transduction mechanisms, translation, ribosome structure and biogenesis, transcription, amino acid transport and metabolism, carbohydrate transport and metabolism, etc. (Figure 7). The top two metabolic pathways in abundance were the following: amino acid transport and metabolism and carbohydrate transport and metabolism (Figure 7).

In the KEGG database, a detailed analysis of its metabolic pathways showed that the presence of radicle sheath bacteria in the bacterial community under drought conditions could significantly promote the conversion of CH₄ to CH₃OH (Figure 8A). In the N cycle (Figure 8B), the abundance of genes K10535, K10944, K10945, and K10946 in the rhizosphere retained in the radicle sheath under drought conditions was significantly higher than that in other treatments (p < 0.05), and they were mainly involved in the conversion of NH₃ to NH₂OH in the nitrification reaction and the production of NO₂.

4. Discussion

During the growth of seedlings, the composition of the rhizosphere microbial community is a dynamic and complex process [31]. Plant rhizosphere microorganisms include bacteria, fungi, actinomycetes, and other microorganisms, which form a close interactive relationship with plant roots [6,32,33]. This study found that the radicle sheath plays an important role in regulating the rhizosphere microbial community of seedlings. The Chao1 index did not show significant differences, indicating that the treatments did not have a significant effect on the overall richness of the microbial community. However, the analysis of the Shannon and Simpson index revealed that the removal of the radicle sheath significantly increased the diversity of bacteria under drought conditions, indicating that the presence of the radicle sheath can effectively reduce the risk of infection by other bacteria and increase the relative abundance of dominant bacterial communities (Figure 3). Dawson et al. (2017) studied the specificity of soil bacterial communities of 19 perennial herbs such as Asteraceae, Lamiaceae, and Leguminosae, indicating that a few rhizosphere bacterial OTUs are mainly dominated by members that maintain relatively low abundance populations [34]. For the fungal community, the removal of the radicle sheath treatment increased the richness of fungi under drought conditions, which indirectly highlights the key role of the radicle sheath in maintaining the structure and functional stability of the rhizosphere microbial community (Figure 2).

The results of this study showed that the presence of the radicle sheath can significantly promote the conversion of CH₄ to CH₃OH; in the N cycle, it significantly promotes the conversion of NH₃ to NH₂OH and the generation of NO₂ and the conversion of NO₃ (Figure 8). This is consistent with the research results demonstrating that arbuscular mycorrhizal can form a symbiotic relationship with plant roots and provide plants with absorbable carbon and nitrogen sources and other nutrients by fixing nitrogen in the atmosphere [18]. In addition, studies have shown that some bacteria can produce growth hormones. For example, 12 endophytic bacteria isolated from Cymbidium eburneum can promote the production of indoleacetic acid (IAA), thereby improving plant growth and development [35,36]. Some probiotics have disease resistance, which inhibits the growth of plant pathogens [37]. Mycorrhizal fungi form a mycorrhizal symbiotic relationship with plants, providing plants with additional water and mineral nutrients. For example, the endophytic fungi Neotyphodium coenophialum infects Festuca arundinacea and accelerates the closure of stomata; the water content of plants infected with endophytic fungi is higher than that of uninfected plants [38]. Pathogenic fungi may cause harm to plants and cause diseases such as root rot. Some actinomycetes can produce antibiotics, which have a protective effect on inhibiting plant pathogens [7]. This study’s results are consistent with previous findings, indicating that, under drought conditions, retaining rhizobia can significantly enhance water retention at the root tip and reduce infection from other pathogenic microorganisms (Figure 2).

The diversity and functional diversity of the rhizosphere microbial community are essential for plant growth and health. Plants interact with rhizosphere microorganisms to form symbiotic relationships, which enhance nutrient absorption efficiency, improve stress resistance, and regulate plant growth and development [10,39,40,41,42,43,44].

5. Conclusions

This study is the first to focus on the impact of the radicle sheath on rhizosphere microbial communities during seedling growth, exploring the significant role of the radicle sheath in regulating plant–microbe interactions. The results indicate that the removal of the radicle sheath under drought conditions significantly increased the diversity of rhizosphere bacteria, while retaining the radicle sheath effectively reduced the risk of infection by other bacteria. Additionally, the removal of the radicle sheath also significantly increased the richness of rhizosphere fungi, particularly under drought stress. These findings provide a foundation for further research into the ecological functions of rhizosphere microbial communities in L. incerta seedlings, highlighting the importance of microbial community diversity and functionality for plant growth and health. Future studies could further explore the ecological functions of these microbial communities under different environmental conditions and their interactions with plant growth mechanisms.