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Article

The Effect of Intercropping with Eucommia ulmoides on the Growth and Quality of Abelmoschus manihot and Its Rhizosphere Microbial Community

by
Minghao Han
1,2,3,†,
Ze Zhang
1,2,3,†,
Han Yang
1,2,3,
Jiyu Du
1,2,3,
Xue Wu
1,2,3 and
Yujie Fu
4,5,*
1
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China
2
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, China
3
College of Chemistry, Chemical Engineering and Resource Utilization, Northeast Forestry University, Harbin 150040, China
4
The College of Forestry, Beijing Forestry University, Beijing 100083, China
5
National Engineering Research Center of Tree Breeding and Ecological Restoration, Beijing 100083, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(4), 863; https://doi.org/10.3390/agronomy15040863
Submission received: 24 February 2025 / Revised: 25 March 2025 / Accepted: 27 March 2025 / Published: 30 March 2025
(This article belongs to the Section Innovative Cropping Systems)
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Abstract

:
Intercropping is a specific agricultural practice where multiple crops are alternately planted in the same field, focusing on optimizing crop interactions and resource use. The key advantages of this approach encompass the complementary ecological niches of crops, which facilitate efficient resource utilization, promote soil microbial diversity, and ultimately lead to enhanced crop yield and quality. Within this context, rhizosphere microorganisms play a pivotal role in plant growth, not only maintaining crop health but also augmenting resistance to various stressors through intricate mechanisms, such as colonizing the plant rhizosphere to produce phytohormones that stimulate plant growth, activating plant defense systems, and competitively excluding soil pathogens. Abelmoschus manihot (A. manihot) is widely distributed and possesses medicinal value; thus, it is utilized to treat a variety of diseases. When cultivating A. manihot, we hope to make rational use of limited planting space, while ensuring the yield of A. manihot and enhancing its medicinal quality. Therefore, a field experiment was conducted in which two planting patterns for A. manihot were designed. Monocropping plots featured A. manihot planted at 0.3 m intervals with a row spacing of 0.5 m. In the intercropping plots, A. manihot was also planted at 0.3 m intervals, maintaining a row spacing of 0.5 m. Furthermore, Eucommia ulmoides (E. ulmoides) was planted at 0.3 m intervals, with a row spacing of 0.25 m between A. manihot and E. ulmoides. Through the field experiment, we evaluated the effects of monocropping and intercropping with E. ulmoides on the quality and biomass of A. manihot, as well as the rhizosphere microbial community structure. The results showed that intercropping can promote the growth of A.manihot, especially by increasing the number of flowers and fruits, but the quality of the medicinal properties is not affected. Specifically, in 2024, the number of flowers in the intercropping plants was 20 ± 2, compared to 13 ± 2 in the monocropping; in 2023, the number of fruits in the intercropping plants was 19 ± 2, compared to 13 ± 2 in the monocropping; and in 2024, the number of fruits in the intercropping plants was 20 ± 2, compared to 13 ± 2 in the monocropping. This effect is due to the self-regulation of A. manihot in response to the biological stress from E. ulmoides. The composition and function of the A. manihot rhizosphere fungal community in the intercropping system changed significantly, which may be the reason for the growth and development of A. manihot. This discovery reveals the potential of intercropping as an agricultural practice in promoting plant growth and increasing yield. Intercropping with E. ulmoides significantly promoted the growth of A. manihot, increasing the number of its flowers and fruits without compromising the quality of its medicinal properties. This finding offers valuable insights for agricultural production: by employing rational intercropping configurations, crop yields can be increased without compromising crop quality.

1. Introduction

Intercropping refers to planting two or more plants in the same field for most of the crop growth period [1]. Intercropping, a time-honored agricultural technique, has been utilized in China for over two millennia. The advantages of the intercropping system include the following: First, the ecological niche complementation between crops can effectively use resources, improve the growth environment, and enhance their stress resistance and adaptability [2]. This reduces competition and promotes synergistic growth, thus increasing crop yields [3]. Second, the interaction between crops can regulate the secondary metabolism of plants and improve the components in medicinal plants, thus enhancing the survival ability and functionality of plants [4,5]. Third, a variety of crops can provide a rich source of nutrients for rhizosphere microorganisms, thereby promoting the diversity and activity of these microorganisms [6]. This helps to increase the number of beneficial microorganisms, which can enhance nutrient availability by competitively excluding soil pathogens (e.g., through resource competition or by secreting antibiotics) [7,8] and by increasing the dissolution of phosphorus [9] and the fixation of atmospheric nitrogen [10]. By optimizing the soil microbial community structure, these crops positively impact crop growth and flowering [11].
Rhizosphere microorganisms have extremely important effects on plant growth and health. In monocropping systems, the long-term cultivation of the same crop often leads to soil structure degradation, nutrient imbalance, a tendency towards simplification of the soil microbial community structure, and a reduction in soil microbial diversity [12]. Different crop roots in an intercropping system can directly interact, thereby altering the nutrient environment in the soil. This change is beneficial for the growth and reproduction of rhizosphere microorganisms, which in turn promotes the recycling of nutrients in the soil, including the transformation and release of key nutrients, such as carbon, nitrogen, and phosphorus [13]. The effective utilization of these nutrients can enhance the growth potential and stress resistance of crops, thereby increasing the overall productivity of the intercropping system [14]. Root exudates and residues provide rich and diverse living environments and nutrient sources for microorganisms, thereby promoting the development of microbial diversity [15]. In turn, these microorganisms facilitate crop growth by decomposition organic matter, releasing nutrients, and promoting nutrient circulation, thus affecting the yield and quality of crops in intercropping systems [10,16]. At the same time, rhizosphere microorganisms also provide effective protection for crops by producing antibiotics, inhibiting pathogens, and significantly enhancing the stress resistance of crops [8,17]. The role of these microorganisms in the growth process of crops cannot be ignored, and complex and subtle interaction mechanisms are formed between them and crops. Therefore, the deep study of the interaction mechanism between microorganisms and crops in the rhizosphere is important for optimizing agricultural production and improving crop yield and quality.
Nontargeted metabolomics is a comprehensive analytical method for studying metabolites within biological systems. It aims to conduct integrated detection and quantification of all detectable metabolites without the need to predefine target metabolites. In intercropping systems, nontargeted metabolomics can be used to analyze the effects of intercropping on crop metabolites, changes in the root exudates of different crops [18], and variations in soil microbial metabolites [19]. By detecting and analyzing these metabolites, the impact of intercropping on crop quality and the mechanisms of interaction between crops, such as allelopathy, nutrient competition and promotion, and root interactions, can be revealed [20,21,22]. 16S rRNA gene sequencing [23] and ITS sequencing [24], with their high throughput, accuracy, and low cost, can be utilized in intercropping systems. These technologies enable the analysis of the structure of soil microbial communities and the diversity of microbial communities in crop roots [25]. By comparing the differences in microbial communities under different intercropping patterns, the impact mechanisms of intercropping on soil microbial communities can be revealed. Therefore, in this study, we will employ untargeted metabolomics to explore the changes in plant secondary metabolites in the intercropping system and use 16S rRNA gene sequencing and ITS sequencing to analyze the evolution of microbial diversity and community structure in the rhizosphere.
Abelmoschus manihot is a robust erect annual or perennial herb. A. manihot flowers have been used as a conventional Chinese medicine for clearing dampness and heat, reducing swelling, and detoxifying [26]. Modern pharmacological research results suggest that A. manihot flowers are rich in flavonoids, polyphenols, and other active substances and exhibit a variety of biological properties, such as the treatment of diabetic nephropathy [27] as well as anti-inflammatory [28], antioxidant [29], anticonvulsant [30], and anti-tumor effects [31]. Although studies on the cultivation of A. manihot are rare, research on the cultivation of its congener, okra (Abelmoschus esculentus), indicates that intercropping okra with cassava (Manihot esculenta) does not affect the yield of either crop but can achieve results such as weed suppression [32]. However, there is little record of its effects on quality and the mechanisms of action on soil microorganisms. In this study, we will construct a monocropping system and intercropping system with Eucommia ulmoides seedlings. The reason for choosing E. ulmoides as a symbiotic plant lies in the various ecological and economic advantages that this woody plant possesses. Firstly, E. ulmoides has a deep and extensive root system, which can effectively stabilize the soil and avoid excessive competition with the shallow root system of A. manihot. Moreover, E. ulmoides is rich in various medicinal components and possesses high medicinal value. Its bark, leaves, and fruits can all be utilized as medicinal materials for treating a variety of ailments [33], thus offering additional economic benefits. The aim of constructing these two systems is to conduct a metabolomic analysis using liquid chromatography–mass spectrometry (LC-MS) technology to determine the compositional differences in the metabolites of the flowers between the monoculture and intercropping systems. Furthermore, 16S rRNA gene sequencing and ITS sequencing technology will be used to explore the composition and diversity differences in the bacteria and fungi in this monocropping system and intercropping system with E. ulmoides seedlings. In this study, our objective is to gain an in-depth understanding of the quality and microbial mechanisms in the intercropping systems of A. manihot. More specifically, we hypothesize two points: firstly, intercropping can enhance the medicinal quality of A. manihot without affecting its yield, and secondly, changes in the rhizosphere microbial community structure and composition drive the changes in its quality characteristics.

2. Materials and Methods

2.1. Experimental Site and Environmental Conditions

Field experiments were conducted in Fushun, Liaoning Province (41°41′29.14″ N, 124°47′49.75″ E, elevation 324 m), from May 2022 to October 2023. All plot soil types were determined as phaeozems according to the World Reference Base for Soil Resources (WRB).

2.2. Experimental Design

Three plots of 10 m × 3 m were selected as three biological replicates, and each plot was set with 2 treatments, which were (1) monocropping of A. manihot (control) and (2) intercropping of A. manihot and E. ulmoides.
A. manihot is a large herbaceous plant. Based on preliminary experiments, it has been observed that if the planting density is too high, the plants have fewer branches, fewer flowers per plant, and smaller flowers, whereas if the planting density is too low, although the flowers are larger and there are more branches per plant, the number of flowers per unit area is also lower. The field planting pattern was designed considering these observations, as depicted in Figure 1. The monocropping plots featured A. manihot planted at 0.3 m intervals with a row spacing of 0.5 m. In the intercropping plots, A. manihot was also planted at 0.3 m intervals, maintaining a row spacing of 0.5 m. Furthermore, E. ulmoides was planted at 0.3 m intervals, with a row spacing of 0.25 m between A. manihot and E. ulmoides.

2.3. Plant Materials

The experiment required seedlings of A. manihot and E. ulmoides, which were sourced from the Key Laboratory of Forest Plant Ecology at Northeast Forestry University. The seedlings were transplanted in May 2022, and throughout that period, they received meticulous care and were harvested in October of the same year. Given that A. manihot is an annual herb, it was replanted in May 2023 and subsequently harvested in October 2023. Five plants of A. manihot were collected from each of the 3 replicates in both the monocropping and intercropping plots for biomass and morphological index measurements. A. manihot flowers were collected from the monocropping and intercropping plots during the flowering period of A. manihot and dried in the shade for future use. The A. manihot flowers were pulverized and sieved with a 60-mesh sieve. Then, 100 mg of the sample powder was accurately weighed and dissolved in 20 mL of iced ethanol–water (30:70, v/v), followed by ultrasonic treatment at 4 °C and 300 W for 30 min and centrifugation at 12,000 rpm for 5 min. Finally, the supernatant was collected for later use.

2.4. Soil Sampling

During the harvest of A. manihot, a spade was used to carefully uproot the plants, ensuring they were completely extracted. Any large clumps of soil were shaken off, and any residual soil was brushed away from the roots. Then, soil samples were collected from 5 different locations, mixed together, and sifted through a 2 mm mesh to remove plant roots and other impurities. The sifted soil represents the rhizosphere samples of the A. manihot, collected in 3 replicates for both the monocropping and intercropping systems. Throughout the sampling process, any residues on the spade were carefully wiped off with sterile paper. The collected rhizosphere soil was placed into sterile test tubes, each clearly labeled for identification, and immediately frozen with liquid nitrogen for the determination of rhizosphere microorganisms.

2.5. Reagents

Rutin and gallic acid were purchased from Weikeqi Biological Technology Co., Ltd. (Chengdu, China). UPLC-MS grade acetonitrile, methanol, and formic acid were sourced from Fisher Scientific (Geel, Belgium). The pure water utilized for analysis with the UPLC-Q-Exactive Orbitrap MS was sourced from the Wahaha Group Co., Ltd. (Hangzhou, China). All samples were filtered through a 0.22 μm polytetrafluoroethylene membrane (Millipore, MA, USA).

2.6. Data Collection

2.6.1. Observations Recorded

During the harvest period, we measured the growth characteristics and biomass of A. manihot. The main root length (cm), plant height (cm), and ground diameter (mm) were measured using a tape measure and a caliper. The above-ground and below-ground biomass of A. manihot was measured using a balance. In addition, we manually counted and recorded the number of branches, leaves, and fruits for each plant. The number of flowers was observed and recorded daily.

2.6.2. Determination of Total Flavonoid Content (TFC)

The determination of the total flavonoid content (TFC) followed the method of Xu et al. [34] with moderate modifications. In brief, 1 mL of the diluted sample solution was added to 3.5 mL of an aqueous solution, followed by the injection of 0.3 mL of a 5% NaNO2 solution for a 6 min reaction. Then, 0.3 mL of a 10% AlCl3 solution was added. Finally, 1 mL of a 4% NaOH solution was added and mixed evenly, after which the aqueous solution was brought up to a total volume of 10 mL, and the absorbance of the blank sample at 510 nm was recorded (UV-1800, MAPADA, Shanghai, China). Using catechin as the standard (y = 0.4836x − 0.0431, R2 = 0.997), the TFC content results were expressed as mg of catechin equivalent (CE) per gram dry weight (mg CE/g DW) of A. manihot flowers.

2.6.3. Determination of Total Polyphenol Content (TPC)

The determination of the total polyphenol content (TPC) was based on the method of Bai et al. [35] with the following modifications. In brief, 40 mL of the sample solution was accurately diluted with 1.8 mL of a 20-fold diluted Folin–Ciocalteu reagent and kept in the dark for 5 min. Then, 1.2 mL of a 7.5% (w/v) Na2CO3 solution was added and reacted in the dark at 25 °C for 2h to determine the absorbance at 760 nm. A blank was used as the control. Gallic acid was used as the standard (y = 2.7882x − 0.0109, R2 = 0.996). The results are expressed as mg of gallic acid equivalent (GAE) per gram dry weight (mg GAE/g DW) of A. manihot flowers.

2.6.4. UPLC-Q-Exactive Orbitrap MS Analysis

This experiment utilized an ultra-high-performance liquid chromatography system coupled with a quadrupole-Orbitrap high-resolution mass spectrometer (UPLC-Q-Exactive Orbitrap MS, Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation was performed on a Hypersil Gold C18 column (2.1 × 100 mm, 1.9 μm). The liquid chromatography conditions and mass spectrometry conditions were set based on Xu et al. [34]. Under this mode, the data acquisition software (Compound Discoverer 3.3, Thermo Fisher Scientific, USA) continuously evaluated the full scan mass spectrometry data based on preset criteria and collected and triggered the acquisition of MS/MS spectra. Thermo Compound Discoverer™ 3.0 software (Thermo Fisher Scientific) was utilized to process raw data from 12 A. manihot flowers (6 monocropping and 6 intercropping) analyzed by UPLC-Q-Exactive Orbitrap MS. We used spectral matching with Compound Discoverer™ 3.0 software against the mzCloud and ChemSpider databases. Only those metabolites with a match score ≥ 85 and supported by both accurate mass and MS/MS fragmentation patterns were retained for further analysis.

2.6.5. Soil Microbial Determination

The sequencing stage was conducted by Beijing Allwegene Technologies Co., Ltd. (Beijing, China). Soil microbial DNA was extracted using an E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Inc., Norcross, GA, USA). The V3–V4 region of the 16S rRNA gene was used to sequence soil bacteria for absolute and relative quantitative analysis. Relative quantitative sequencing analysis of soil fungi was conducted using the ITS1 region. The primer sequences are listed in detail in Table 1 [25]. The Illumina Miseq/NovaSeq 6000 platform was used for paired-end sequencing of the collected A. manihot rhizosphere soil samples at 2 × 250 bp. Strict quality control measures were taken on the data, and then bioinformatics analysis was carried out. This resulted in bar graphs representing the composition of the microbial community based on OTU analysis. In addition, alpha and beta diversity analyses were performed.

2.7. Statistical Analysis

Data organization was carried out using Excel 2019 software. Prior to conducting statistical analysis, normality tests were performed on all data sets. The data were then analyzed using SPSS software (version 27.0) for analysis of variance (ANOVA), and normality tests were conducted on all data sets. Independent sample t-tests were employed to evaluate the same indicators for the monocropping and intercropping systems. The differences were found to be statistically significant with a threshold of p < 0.05. Data graphs were drawn using Origin 2024 software and the Metware Cloud platform.

3. Results

3.1. Response of Biomass and Morphological Characteristics of A. manihot to Intercropping

The morphological characteristics and biomass of each part of A. manihot are shown in Table 2 and Table 3. Comparing the intercropping A. manihot and monocropping A. manihot, the regularity was basically the same in the two years, and the number of fruits of A. manihot increased significantly (p = 0.009 and p = 0.011). In the second year, the number of flowers per plant was counted. The number of flowers per plant of A. manihot (p = 0.011) increased significantly. In terms of biomass, there was no significant difference between above-ground and underground biomass during the two years.

3.2. Analysis of Secondary Metabolites and Differences in A. manihot Flowers

3.2.1. Total Flavonoid Content and Total Polyphenol Content of A. manihot Flowers

Figure 2 shows the TFC and TPC of A. manihot flowers in the two planting methods. As can be seen from the figure, there is no significant difference in the content of TFC and TPC under the two planting modes. It is evident that intercropping does not diminish the total amount of secondary metabolites in the flowers of A. manihot.

3.2.2. Difference Analysis of Secondary Metabolites in A. manihot Flowers

Next, the A. manihot flowers were analyzed using LC−MS technology, detecting 16,196 substances. After spectral matching annotation, 73 characteristic peaks with the highest similarity to the reference mass spectra were retained for further statistical analysis (Table S1). Metabolite classification revealed that the main metabolites in A. manihot flowers were flavonoids, phenolic acids, amino acids and derivatives, nucleotides and derivatives, lignans and coumarins, alkaloids, and other organic compounds (Figure 2B). Among them, myricetin, rutin, hyperoside, quercetin, isoquercitrin, chlorogenic acid, and caffeic acid are known as important compounds.
The principal component analysis (PCA) metabolomics score plot shows the comparison between intercropping and monocropping A. manihot flowers, as depicted in Figure 2C. There were significant differences in the content of six metabolites between the two treatment groups. The relative content of three metabolites in the intercropping A. manihot flowers was higher than that of the monocropping flowers (Figure 2D). These metabolites include L-valine, L-threonine, and 3,5-dicaffeoylquinic acid. Conversely, the relative content of three metabolites in the monocropping A. manihot flowers was higher than that of the intercropping flowers. These metabolites included 4-hydroxybenzaldehyde, isochlorogenic acid B, and isochlorogenic acid C (Table S2).

3.3. The Soil Physical and Chemical Properties of the Intercropping and Monocropping Systems of A. manihot

The results shown in Table 4 indicate that the total N g·kg−1 in the monocropping soil, i.e., 0.79 ± 0.11 g·kg−1, was significantly higher than that in the intercropping soil, which was 0.26 ± 0.05 mg·kg−1 (p < 0.01). The nitrate N mg·kg−1 in the intercropping soil, i.e., 122.74 ± 23.58 mg·kg−1, was significantly higher than that in the monocropping soil, which was 48.64 ± 6.97 mg·kg−1 (p < 0.01). The available P in the intercropping soil, i.e., 25.89 ± 3.25 mg·kg−1, was significantly higher than that in the monocropping soil, which was 17.22 ± 2.39 mg·kg−1 (p < 0.05). Although there were differences in soil SOC, Ammoniume N, and Available K, they did not reach a statistically significant level.

3.4. The Difference in Alpha and Beta Diversity Indices of Rhizosphere Bacteria and Fungi Between the Intercropping and Monocropping Systems of A. manihot

The results indicate, as depicted in Figure 3A,B, that in both the monocropping and intercropping systems, the Chao1 index, Observed species index, PD_Whole tree index, and Shannon index of rhizosphere bacteria in the monocropping A. manihot were higher than those in the intercropping. However, these differences were not statistically significant (Figure 3A). In contrast, the Chao1 index, Observed species index, PD_Whole tree index, and Shannon index of the rhizosphere fungi in the intercropping A. manihot were higher than those in the monocropping, with the Chao1 index (p = 0.038), Observed species index (p = 0.008), and PD Whole tree index (p = 0.020) achieving statistical significance (Figure 3B). It is clear that intercropping exerts a more pronounced effect on the rhizosphere fungi of A. manihot.
Beta diversity is a key indicator used to assess the differences between microbial communities by comparing their structural characteristics. Figure 3C,D illustrate the differences in the composition of rhizosphere bacterial and fungal communities in the monocropping and intercropping A. manihot. In the PCoA plot for bacteria (Figure 3C), there is not a clear separation between the samples from the monocropping and intercropping. However, in the PCoA plot for fungi, there is a clear separation between the samples from monoculture and intercropping (Figure 3D), indicating that intercropping has a more significant effect on the rhizosphere fungal community than on the bacterial community in A. manihot.

3.5. Venn Diagram of Bacterial and Fungal Communities Between Intercropping and Monocropping

As depicted in Figure 4, the outcomes suggest that the unique OTUs of the rhizosphere bacteria in the monocropping A. manihot amounted to 1398, while in the intercropping A. manihot, the unique OTUs of the rhizosphere bacteria were 1003 (Figure 4A). As for fungi, the unique OTUs of the rhizosphere fungi in the monocropping A. manihot were 310, while in the intercropping A. manihot, the unique OTUs of the rhizosphere fungi were 869 (Figure 4B). This shows that intercropping significantly affects the characteristics of the rhizosphere soil microbial community of A. manihot, with fungi playing a greater role than bacteria.

3.6. Phylum-Level Community Composition of Rhizosphere Bacteria and Fungi Between Intercropping and Monocropping

In the bacterial community, at the phylum level, the major microbial communities comprised Proteobacteria, Actinobacteriota, Bacteroidota, Acidobacteriota, Gemmationadetes, and Verrucomicrobia (Figure 5A). Collectively, these phyla constitute the major bacterial phyla, accounting for over 80% of the bacterial community. Although there were variations in the proportion of each phylum in the rhizosphere of the monocropping and intercropping A. manihot, no significant differences were observed between the monocropping and intercropping at the phylum level (Figure 5C).
In the fungal community, at the phylum level, the rhizosphere fungi of A. manihot were mainly composed of Ascomycota, Basidiomycota, and Mucoromycota (Figure 5B). Notably, the relative abundance of Ascomycota in the rhizosphere of the monocropping A. manihot was 43.32%, while that of the intercropping A. manihot was 60.22%, which was significantly higher than that of the monocropping (p < 0.01) (Figure 5D). The relative abundance of Basidiomycota in the rhizosphere of the monocropping A. manihot was 34.57%, while that of the intercropping A. manihot was 14.23%, which was significantly higher than that of the intercropping (p < 0.05) (Figure 5D). This indicates that intercropping exerts a more substantial influence on the phylum-level fungal community composition of the rhizosphere than on the bacterial community in A. manihot.

3.7. Genus-Level Community Composition of Rhizosphere Bacteria and Fungi Between Intercropping and Monocropping

In the bacterial community, as shown in Figure 6A, the rhizosphere bacteria of A. manihot mainly included Paludibaculum, Vicinamibacter, Limisphaera, Brevitalea, Gemmatimonas, Chryseolinea, and Thermomarinilinea. We conducted a relative abundance analysis of 986 genera of the rhizosphere bacteria (Figure 6B), and the results showed that the relative abundance of 14 genera significantly increased, with 1 genus being unique to the intercropping. The relative abundance of 20 genera significantly decreased, with 4 genera being unique to the monoculture. Among the top 20 genera in terms of bacterial relative abundance, the relative abundance of Vicinamibacter in the rhizosphere of the monocropping A. manihot was 6.69%, which was significantly higher than that of the intercropping at 3.76% (p < 0.05). The relative abundance of Thermomarinilinea in the rhizosphere of the monocropping A. manihot was 1.58%, which was significantly higher than that of the intercropping at 0.72% (p < 0.05) (Table S3).
In the analysis of the fungal community, as shown in Figure 6C, the rhizosphere bacteria of A. manihot primarily consisted of Entoloma, Linnemannia, Plectosphaerella, Unidentified, and Leohumicola. We conducted a relative abundance analysis of 604 genera of the rhizosphere fungi (Figure 6C), and the results showed that the relative abundance of 54 genera significantly increased, among which 11 genera were unique to the intercropping. The relative abundance of 28 genera significantly decreased, among which 3 genera were unique to the monocropping. Among the top 20 genera in terms of fungal relative abundance, Entoloma and Tausonia in the rhizosphere of the monocropping A. manihot were 23.12% and 3.72%, respectively, which were significantly higher than 0.79% and 1.28% in the rhizosphere of the intercropping A. manihot. In the rhizosphere of the intercropping A. manihot, Mortierella, Collembolispora, Pseudogymnoascus, Gibellula, and Arthropsis were 8.88%, 4.34%, 3.24%, 2.10%, 1.78% and 2.56%, respectively, which were significantly higher than those of the monocropping A. manihot (Table S4).
A comprehensive comparison of the differences between fungi and bacteria at the genus level reveals that intercropping exerts a more substantial influence on the fungi-level fungal community composition of the rhizosphere than on the bacterial community in A. manihot.

3.8. Predictive Analysis of Rhizosphere Bacterial and Fungal Functions Between Intercropping and Monocropping

The bacterial functional predictions generated by PICRUSt2 (Figure 7A) indicate that in the rhizosphere of A. manihot, the predominant functions were associated with amino acid metabolism, biosynthesis of other secondary metabolites, carbohydrate metabolism, cell growth and death, cell motility, cellular community—prokaryotes, energy metabolism, folding, sorting and degradation, glycan biosynthesis and metabolism, lipid metabolism, membrane transport, metabolism of cofactors and vitamins, metabolism of other amino acids, metabolism of terpenoids and polyketides, nucleotide metabolism, replication and repair, signal transduction, signaling molecules and interaction, transcription, transport and catabolism, translation, xenobiotics biodegradation and metabolism, and function unknown. The result indicates that the intercropping of A. manihot had a minimal impact on bacterial functional predictions.
The FUNGuild fungal function prediction results (Figure 7B) indicate that the fungal functions in the rhizosphere of A. manihot primarily include unknown, animal-associated biotroph–plant pathogen, animal pathogen, animal pathogen–dung saprotroph–endophyte–lichen parasite–plant pathogen–undefined saprotroph, animal pathogen–endophyte–fungal parasite–lichen parasite–plant pathogen–wood saprotroph, animal pathogen–endophyte–fungal parasite–plant pathogen–wood saprotroph, animal pathogen–endophyte–lichen parasite–plant pathogen–soil saprotroph–wood saprotroph, animal pathogen–endophyte–plant pathogen–wood saprotroph, animal pathogen–plant pathogen–undefined saprotroph, animal pathogen–undefined saprotroph, bryophyte parasite–ectomycorrhizal–ericoid mycorrhizal–undefined saprotroph, bryophyte parasite–litter saprotroph–wood saprotroph, dung saprotroph–endophyte–undefined saprotroph, ectomycorrhizal, ectomycorrhizal–fungal parasite–soil saprotroph–undefined saprotroph, ectomycorrhizal–fungal pathogen–undefined saprotroph, endomycorrhizal–plant pathogen–undefined saprotroph, endophyte–epiphyte–fungal parasite–insect parasite, endophyte–litter saprotroph–soil saprotroph–undefined saprotroph, endophyte–undefined saprotroph, fungal parasite, plant pathogen, plant pathogen–wood saprotroph, plant saprotroph–wood saprotroph, soil saprotroph, soil saprotroph–undefined saprotroph, undefined saprotroph, and others. Intercropping consistently led to a reduction in the functional abundance of plant pathogens and ectomycorrhizal fungi parasites–soil saprotrophs–undefined saprotrophs, whereas it caused an increase in the functional abundance of bryophyte parasites-litter saprotrophs–wood saprotrophs, unknown, and others.

4. Discussion

4.1. The Effect of Intercropping on the Growth of A. manihot

In the first year of our experiments, we found a significant increase in the number of A. manihot fruits in the intercropping system, so in the second year, we not only focused on the quantity of A. manihot fruits but also counted the number of flowers during the growth process of A. manihot. As expected, the number of flowers also increased significantly. This is different from our first hypothesis. Flowers and fruits are important reproductive organs of plants; therefore, we hypothesize that when there is interspecific competition, A. manihot could adjust its survival strategies to cope with the competition and turn more resources to reproductive growth. Studies have shown that plants adjust their biomass allocation to the environment under biotic and abiotic stresses [36,37,38]. For instance, excess nutrition helps to promote the vegetative growth of a plant [39], which reduces the nutrient distribution to the reproductive organs [38]. In the case of insufficient nutritional growth, plants will preferentially meet their basic nutritional growth needs to ensure survival [40]. Therefore, balancing the relationship between vegetative growth and reproductive growth under stress conditions is crucial for plant health and yield. By moderately controlling vegetative growth and ensuring the smooth progress of reproductive growth, the yield and quality of crops can be maximized. In the intercropping system, the growth of E. ulmoides also needs certain space and nutrients, which brings biological stress to the growth of A. manihot to a certain extent. Therefore, A. manihot adjusts its biomass allocation, resulting in more flowering quantity than that of A. manihot. It can be seen that in the intercropping system, A. manihot gives priority to reproductive growth under the condition of ensuring the basic needs of reproductive growth. This phenomenon has been extensively studied in plant ecology, revealing how plants adapt their growth strategies to different environmental stresses to maximize survival and reproduction opportunities [37].

4.2. The Effect of Intercropping on Secondary Metabolites in A. manihot Flowers

A. manihot not only has ornamental value, but its medicinal ingredients also attract much attention. As a traditional Chinese medicine, its flowers are rich in flavonoids, polyphenols, and other active ingredients, which give a variety of pharmacological activities, such as anti-inflammatory, anti-oxidation, etc. [26]. In the intercropping system, the medicinal components of A. manihot may be influenced by interactions with other crops. Therefore, we determined the content of TFC and TPC in monocropping and intercropping modes, and the results showed that the difference between TFC and TPC in A. manihot was not significant between the two cultivation modes. We next obtained the results of untargeted metabolite analysis on the flowers of the major medicinal sites in the monocropping and intercropping mode using LC-MS, which showed no significant differences in the content of important active components such as myricetin, rutin, hyperoside, quercetin, isoquercitrin, chlorogenic acid, and caffeic acid. It can be seen that the medicinal components of the intercropping species are not affected, which also suggests that A. manihot can maintain the main components of flowers under the different cultivation modes, indicating the resilience of the process that determines the viability of reproductive tissues [41]. Although this is different from our first hypothesis, it is also an acceptable result.

4.3. The Effect of Intercropping on the Rhizosphere Microorganisms of A. manihot

In this study, we analyzed the impact of intercropping on the rhizosphere microbial community of A. manihot through high-throughput sequencing technology and predicted its functions. The results revealed that the response of the rhizosphere fungi of A. manihot to intercropping was more significant compared to the rhizosphere bacteria. The reason for this phenomenon may lie in the interactions between the crop roots and changes in the soil environment within the intercropping system. Specifically, intercropping may lead to the reallocation of key nutrients such as soil nutrients, thereby affecting the growth and reproduction of fungi. Studies have shown that soil alkali-hydrolyzable nitrogen, available phosphorus, and available potassium significantly affect the composition of bacterial communities, while soil total nitrogen, alkali-hydrolyzable nitrogen, available phosphorus, and available potassium significantly affect the composition of fungal communities [42]. The content of soil nitrate N is the main environmental factor determining the soil fungal community [43]. In turn, an increase in the abundance of Mortierella may accelerate nitrogen turnover in the rhizosphere, converting the nitrogen pool from total nitrogen to more mobile forms, such as nitrate [44,45,46]. Significant changes in the soil total nitrogen, ammonium nitrogen, and available phosphorus in this experiment may be one of the reasons for the changes in the rhizosphere fungal community. In addition, the chemical substances secreted by the roots of different crops may also selectively affect the fungal community, promoting the growth of certain fungal groups while inhibiting the development of others [47]. These factors collectively contribute to a more significant response of the rhizosphere fungal community of A. manihot to intercropping.
Under the two planting patterns, the PCoA plot of fungi shows a clear separation between the intercropping and monoculture samples. This indicates that intercropping reshaped the rhizosphere fungal community structure. Specifically, in the intercropping system, Ascomycota significantly increased, which may be related to their key role in organic matter decomposition and nutrient cycling. Studies have shown that fungi of the Ascomycota phylum play a significant role in soil ecosystems [48]. They are capable of decomposing complex organic materials, such as cellulose [49] and lignin [50], thereby releasing nutrients that are available to plants. Moreover, the arbuscular mycorrhiza fungi (AMF) of the Ascomycota phylum form symbiotic relationships with plant roots, such as mycorrhizae, which help plants absorb water and minerals and enhance their resistance to adverse conditions [51]. Therefore, an increase in Ascomycota fungi under intercropping systems may have a positive impact on the growth and health of A. manihot.
Furthermore, at the genus level, we also observed an increase in Pseudogymnoascus and Mortierella and a decrease in Tausonia in the intercropping system. Pseudogymnoascus is widely present in the soil, playing a significant role in the decomposition of soil organic matter and nutrient cycling [52]. Studies have revealed that specific species in the genus Pseudogymnoascus can colonize plants, which can help plants better adapt to the ecological environment [53]. Certain species of Pseudogymnoascus may positively influence plant growth and reproduction by altering the soil microbial community structure and its diversity and complexity [54]. Similarly, studies have revealed that some fungi of the genus Mortierella can colonize plant roots and promote the growth of both above-ground and below-ground biomass by secreting auxin IAA through their hyphae [55,56,57]. Moreover, certain fungi of the genus Mortierella secrete total lipids and arachidonic acid that directly inhibit the growth of Fusarium spp. and Aspergillus sp. while also promoting the growth of beneficial microorganisms such as Pseudomonas geniculata, Trichoderma velutinum, and Bacillus velezensis [58]. However, Tausonia is recorded as a pathogen, and its population decreases in the case of the continuous cropping of Atractylodes lancea, while it increases with the continuous cropping of other crops [59]. It is worth noting that the chemical substances secreted by A. lancea during its growth have an inhibitory effect on pathogens [60]. Consequently, the increase in these beneficial fungi in the intercropping system may help build a healthier soil microbial environment, further enhancing the stress resistance and productivity of A. manihot. The reduction in pathogenic fungi like Tausonia may also reduce the occurrence of diseases, which is beneficial for the healthy growth of A. manihot. This was further confirmed in the FUNGuild fungal function prediction analysis. The function prediction analysis revealed that these increased fungi may positively impact the growth and health of A. manihot by increasing ectomycorrhizal fungi to promote nutrient uptake and inhibit the growth of pathogenic fungi.

5. Conclusions

The intercropping of A. manihot with E. ulmoides can, to some extent, promote the growth of A. manihot, especially by significantly increasing the number of flowers and fruits of A. manihot. This increase is a self-regulatory response of A. manihot to the biological stress caused by E. ulmoides. In the intercropping system, despite a significant increase in A. manihot flowers, this does not affect the quality of A. manihot flowers as a medicinal herb. The content of TFC, TPC, and important compounds did not undergo significant changes. In the A. manihot intercropping system, there were significant changes in the composition and functional diversity of the rhizosphere fungal community of A. manihot. Specifically, there were notable alterations in alpha diversity indices, beta diversity indices, OTUs, phylum-level community composition, and genus-level community composition of the rhizosphere fungal community of A. manihot. These changes probably promoted the growth of A. manihot. Therefore, a well-designed intercropping system of A. manihot and E. ulmoides is expected to promote the transformation of beneficial rhizosphere microbial communities in the future. Farmers can leverage this advantage to alleviate disease pressure and enhance crop yields in sustainable agriculture.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15040863/s1: Table S1: Compounds with 73 characteristic peaks matching in A. manihot flowers; Table S2: Comparison of the relative contents of 73 compounds under two planting patterns; Table S3: Comparison of the bacterial genus levels composition in the rhizosphere of A. manihot; Table S4: Comparison of the fungal genus level composition in the rhizosphere of A. manihot.

Author Contributions

Conceptualization, M.H. and Y.F.; methodology, H.Y.; software, M.H. and H.Y.; validation, M.H., J.D. and Z.Z.; formal analysis, M.H.; investigation, M.H. and J.D.; resources, Y.F.; data curation, M.H. and J.D.; writing—original draft preparation, M.H. and Z.Z.; writing—review and editing, M.H. and X.W.; visualization, M.H.; supervision, M.H.; project administration, M.H.; funding acquisition, Y.F. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key R&D Program of China (2022YFD2200602), the National Key Natural Science Foundation of China (31930076), the Natural Science Foundation of China (32271805), the National XA Science and Technology innovation project (2022XACX1100), the 111 Project (B20088), and the Heilongjiang Touyan Innovation Team Program (Tree Genetics and Breeding Innovation Team).

Data Availability Statement

Data will be made available on request.

Acknowledgments

The authors would like to thank all the reviewers who participated in the review.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AMFarbuscular mycorrhiza fungi
LC-MSliquid chromatography–mass spectrometry
TFCtotal flavonoid content
TPCtotal polyphenol content
CEcatechin equivalent
GAEgallic acid equivalent
PCAprincipal component analysis
PCoAprincipal co-ordinatest analysis

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Agronomy 15 00863 g001
Figure 1. Schematic diagram of the monocropping and intercropping of A. manihot and E. ulmoides.
Figure 1. Schematic diagram of the monocropping and intercropping of A. manihot and E. ulmoides.
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Figure 2. Composition analysis diagram of A. manihot flowers. (A) TFC and TPC of A. manihot flowers. (B) Classification of annotated and identified metabolites in A. manihot flowers. (C) Scatter plot of the principal component analysis (PCA) scores for A. manihot flowers in the monocropping and intercropping systems. (D) Volcano plot of A. manihot flowers: intercropping vs. monocropping.
Figure 2. Composition analysis diagram of A. manihot flowers. (A) TFC and TPC of A. manihot flowers. (B) Classification of annotated and identified metabolites in A. manihot flowers. (C) Scatter plot of the principal component analysis (PCA) scores for A. manihot flowers in the monocropping and intercropping systems. (D) Volcano plot of A. manihot flowers: intercropping vs. monocropping.
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Figure 3. Alpha and beta diversity of rhizosphere microorganisms in the monocropping and intercropping systems of A. manihot. (A) Alpha diversity indices of rhizosphere bacteria in the monocropping and intercropping A. manihot. (B) Alpha diversity indices of rhizosphere fungi in the moncropping and intercropping A. manihot. (C) Scatter plot of the principal co-ordinatest analysis (PCoA) scores of the rhizosphere bacteria in the monocropping and intercropping A. manihot. (D) Scatter plot of the principal co-ordinatest analysis (PCoA) scores of the rhizosphere fungi in the monocropping and intercropping A. manihot.
Figure 3. Alpha and beta diversity of rhizosphere microorganisms in the monocropping and intercropping systems of A. manihot. (A) Alpha diversity indices of rhizosphere bacteria in the monocropping and intercropping A. manihot. (B) Alpha diversity indices of rhizosphere fungi in the moncropping and intercropping A. manihot. (C) Scatter plot of the principal co-ordinatest analysis (PCoA) scores of the rhizosphere bacteria in the monocropping and intercropping A. manihot. (D) Scatter plot of the principal co-ordinatest analysis (PCoA) scores of the rhizosphere fungi in the monocropping and intercropping A. manihot.
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Figure 4. The effect of monocropping and intercropping on the number of bacterial and fungal OTUs in the rhizosphere of A. manihot. (A) The effect of monocropping and intercropping on the number of bacterial OTUs in the rhizosphere of A. manihot. (B) The effect of monocropping and intercropping on the number of fungal OTUs in the rhizosphere of A. manihot.
Figure 4. The effect of monocropping and intercropping on the number of bacterial and fungal OTUs in the rhizosphere of A. manihot. (A) The effect of monocropping and intercropping on the number of bacterial OTUs in the rhizosphere of A. manihot. (B) The effect of monocropping and intercropping on the number of fungal OTUs in the rhizosphere of A. manihot.
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Figure 5. Composition of the rhizosphere microbial community of A. manihot at the phylum level. (A) Composition of the rhizosphere bacterial community of A. manihot at the phylum level. (B) Composition of the rhizosphere fungal community of A. manihot at the phylum level. (C) Relative abundance of A. manihot rhizosphere bacteria at the phylum level. (D) Relative abundance of A. manihot rhizosphere fungi at the phylum level.
Figure 5. Composition of the rhizosphere microbial community of A. manihot at the phylum level. (A) Composition of the rhizosphere bacterial community of A. manihot at the phylum level. (B) Composition of the rhizosphere fungal community of A. manihot at the phylum level. (C) Relative abundance of A. manihot rhizosphere bacteria at the phylum level. (D) Relative abundance of A. manihot rhizosphere fungi at the phylum level.
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Figure 6. Composition of the rhizosphere microbial community of A. manihot at the genus level. (A) Composition of the rhizosphere bacterial community of A. manihot at the genus level. (B) Volcano plot of rhizosphere bacterial relative abundance of A. manihot at the genus level: intercropping vs. monocropping. (C) Composition of the rhizosphere fungal community of A. manihot at the genus level. (D) Volcano plot of rhizosphere fungal relative abundance of A. manihot at the genus level: intercropping vs. monocropping.
Figure 6. Composition of the rhizosphere microbial community of A. manihot at the genus level. (A) Composition of the rhizosphere bacterial community of A. manihot at the genus level. (B) Volcano plot of rhizosphere bacterial relative abundance of A. manihot at the genus level: intercropping vs. monocropping. (C) Composition of the rhizosphere fungal community of A. manihot at the genus level. (D) Volcano plot of rhizosphere fungal relative abundance of A. manihot at the genus level: intercropping vs. monocropping.
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Figure 7. Effect of intercropping A. manihot on the prediction and analysis of bacterial and fungal functions in the rhizosphere. (A) Variations in the composition of bacterial functional groups inferred by PICRUSt2. (B) Variations in the composition of fungal functional groups inferred by FUNGuild.
Figure 7. Effect of intercropping A. manihot on the prediction and analysis of bacterial and fungal functions in the rhizosphere. (A) Variations in the composition of bacterial functional groups inferred by PICRUSt2. (B) Variations in the composition of fungal functional groups inferred by FUNGuild.
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Table 1. Primer sequences.
Table 1. Primer sequences.
PrimersPrimer TypeF-End SequenceR-End SequenceLength
338F_806Bacteria 16S rRNAACTCCTACGGGAGGCAGCAGGGACTACHVGGGTWTCTAAT468
ITS1F_ITS2Rfungus ITSCTTGGTCATTTAGAGGAAGTAAGCTGCGTTCTTCATCGATGC350
Table 2. Morphological characteristics of each part and biomass per plant of A. manihot in 2022.
Table 2. Morphological characteristics of each part and biomass per plant of A. manihot in 2022.
Abelmoschus manihotBranchesLeaf NumbersGround Diameter (mm)Height (cm)Fruit NumbersAbove-Ground Biomass (g)Underground Biomass (g)Total Biomass (g)
Monocropping4 ± 320 ± 1217.38 ± 1.671.56 ± 0.2313 ± 2 **64.68 ± 21.1318.90 ± 5.8783.58 ± 27.00
Intercropping1 ± 124 ± 620.12 ± 1.781.77 ± 0.1419 ± 282.62 ± 7.3321.25 ± 4.16103.87 ± 11.49
** p < 0.01.
Table 3. Morphological characteristics of each part and biomass per plant of A. manihot in 2023.
Table 3. Morphological characteristics of each part and biomass per plant of A. manihot in 2023.
Abelmoschus manihotBranchesLeaf NumbersGround Diameter (mm)Height (m)Fruit NumbersFlowers NumbersAbove-Ground Biomass (g)Underground Biomass (g)Total Biomass (g)
Monocropping0 ± 117 ± 420.09 ± 2.112.29 ± 0.089 ± 2 **13 ± 2 **64.56 ± 7.0110.65 ± 2.0975.21 ± 8.33
Intercropping1 ± 117 ± 419.55 ± 1.532.12 ± 0.0814 ± 220 ± 259.82 ± 8.4612.02 ± 2.4171.84 ± 10.86
** p < 0.01.
Table 4. Physical and chemical properties of soil in the monocropping and intercropping systems of A. manihot.
Table 4. Physical and chemical properties of soil in the monocropping and intercropping systems of A. manihot.
Soil CharacteristicsMonocropping A. manihotIntercropping A. manihot
Total N (g·kg−1)0.79 ± 0.11 **0.26 ± 0.05
Ammoniume N (mg·kg−1)12.99 ± 1.21 10.79 ± 1.39
Nitrate N (mg·kg−1)48.64 ± 6.97122.74 ± 23.58 **
Available P (mg·kg−1)17.22 ± 2.39 25.89 ± 3.25 *
Available K (mg·kg−1)155.44 ± 9.26 161.81 ± 18.23
SOC (g·kg−1)118.54 ± 29.20 94.31 ± 31.59
** p < 0.01, * p < 0.05.
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MDPI and ACS Style

Han, M.; Zhang, Z.; Yang, H.; Du, J.; Wu, X.; Fu, Y. The Effect of Intercropping with Eucommia ulmoides on the Growth and Quality of Abelmoschus manihot and Its Rhizosphere Microbial Community. Agronomy 2025, 15, 863. https://doi.org/10.3390/agronomy15040863

AMA Style

Han M, Zhang Z, Yang H, Du J, Wu X, Fu Y. The Effect of Intercropping with Eucommia ulmoides on the Growth and Quality of Abelmoschus manihot and Its Rhizosphere Microbial Community. Agronomy. 2025; 15(4):863. https://doi.org/10.3390/agronomy15040863

Chicago/Turabian Style

Han, Minghao, Ze Zhang, Han Yang, Jiyu Du, Xue Wu, and Yujie Fu. 2025. "The Effect of Intercropping with Eucommia ulmoides on the Growth and Quality of Abelmoschus manihot and Its Rhizosphere Microbial Community" Agronomy 15, no. 4: 863. https://doi.org/10.3390/agronomy15040863

APA Style

Han, M., Zhang, Z., Yang, H., Du, J., Wu, X., & Fu, Y. (2025). The Effect of Intercropping with Eucommia ulmoides on the Growth and Quality of Abelmoschus manihot and Its Rhizosphere Microbial Community. Agronomy, 15(4), 863. https://doi.org/10.3390/agronomy15040863

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