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Article

The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Astragalus mongholicus under Cadmium Stress

by
Min Li
1,
Li Han
2,
Chao He
3,*,
Xia Li
1 and
Xueli He
1,*
1
College of Life Sciences, Hebei University, No. 180, Wusidong Rd., Baoding 071002, China
2
College of Pharmaceutical Sciences, Hebei University, No. 180, Wusidong Rd., Baoding 071002, China
3
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(8), 1801; https://doi.org/10.3390/agronomy14081801
Submission received: 10 July 2024 / Revised: 1 August 2024 / Accepted: 14 August 2024 / Published: 15 August 2024
(This article belongs to the Section Soil and Plant Nutrition)
Browse Figures
Versions Notes

Abstract

:
Dark septate endophytes (DSE) commonly inhabit the root systems of plants that flourish in heavy metal-contaminated soils. Nevertheless, there is limited understanding regarding the overall response of medicinal plants to DSE under heavy metal stress. The present study utilized a non-sterile pot experiment to evaluate the beneficial impacts of DSE (Paraphoma chlamydocopiosa, Paraboeremia selaginellae, and Paraphoma radicina) inoculation on Astragalus mongholicus under cadmium (Cd) stress. At 0 mg Cd/kg soil, inoculation with DSE led to a significant increase in the total biomass of the host plant by up to 34.0%. Although plant biomass decreased at 5 mg Cd/kg soil and 10 mg Cd/kg soil, the total biomass of the host plant increased by up to 72.3% after DSE inoculation. The plant height, branch number, soil and plant analyzer development (SPAD) value, and biomass were not affected by Cd concentration after inoculation with P. chlamydocopiosa and P. selaginellae. DSE emerged as the most prominent explanatory variable (44.2%) for plant growth at 10 mg Cd/kg soil. Inoculation with P. chlamydocopiosa increased root length by 47.9% and root biomass by 74.1%, and inoculation with P. chlamydocopiosa and P. selaginellae increased the indole-3-acetic acid (IAA) content by 103.6% and 105.8%, respectively, at 10 mg Cd/kg soil. Additionally, P. radicina inoculation was preferred to increase the content of calycosin-7-O-glucoside, while P. chlamydocopiosa increased the content of formononetin. DSE enhanced the accumulation of Cd in the shoot, whereas P. radicina exhibited an inhibitory effect on Cd accumulation in the root system. P. chlamydocopiosa decreased the Cd translocation factor, while P. radicina increased the Cd translocation factor, which exceeded one. The interaction between DSE and soil factors significantly contributed to the host plant growth. DSE inoculation improved soil organic carbon content and inoculation with P. chlamydocopiosa, and P. selaginellae reduced available nitrogen content, regardless of the Cd stress. DSE inoculation reduced available phosphorus content at 10 mg Cd/kg soil. Importantly, P. radicina promote the performance and calycosin-7-O-glucoside accumulation of the host plant, and reduced Cd content in the roots at 5 mg Cd/kg soil. These data enhanced comprehension of the ecological function of DSE in heavy metal-contaminated soils and demonstrated the potential utility of DSE strains for cultivating medicinal plants.

1. Introduction

Cadmium (Cd) is classified as a non-essential element for plant development and is recognized as one of the most hazardous heavy metal contaminants. Cd content in the soil exceeds the standard of 7.0%, ranking first among heavy metal pollutants [1]. Soil Cd pollution has become a limiting factor for cultivating medicinal plants in agricultural systems [2,3]. Selectively employing beneficial microorganisms is an effective strategy for plants to combat heavy metal stress [4,5]. The microbiomes linked to plants, such as symbiotic microorganisms, have been extensively studied for their positive effects on host plants, including enhancing growth performance, nutrient absorption, and stress resistance [6,7].
Dark septate endophytes (DSE), a diverse group of ascomycetes, have the ability to develop characteristic dark septate hyphae and microsclerotia within plant roots [8]. They are recognized for their significant role in improving plant tolerance to heavy metal stress [5]. And DSE are found in various habitats, especially in arid, salinity, and heavy metal-polluted areas [9,10]. They can colonize the root cortex cells and intercellular spaces, as well as penetrate plant vascular tissues [11,12]. Research findings have shown that DSE can improve the absorption of water and nutrients by host plants through the formation of a fungal network in the root system, which is particularly evident in stressful environments [13]. In addition, DSE can also effectively regulate plant growth by synthesizing auxin [14]. For example, under Cd stress, DSE produces indole-3-acetic acid (IAA) to promote the root development, nutrient absorption, and overall plant growth of the host plant [15]. Moreover, DSE possesses the ability to convert complex organic substances found in the soil into forms that are easily assimilated and utilized by plants, thereby improving the effectiveness of plants in utilizing carbon, nitrogen, and phosphorus [16]. Wu et al. [17] demonstrated that DSE has the capacity to promote the growth of the endangered species Saussurea involucrata and elevate its rutin concentration. Similarly, Zhu et al. [18] indicated that DSE can enhance the biomass, flavonoids and icariin contents of Epimedium wushanense. Tan et al. [19] also found that DSE significantly increased the glycyrrhizic acid content of Glycyrrhiza glabra. Plant-associated microbiomes are highly sensitive to changes in plant and environmental participation, and their community structure and ecological functions may be deeply influenced by plant characteristics and environmental behavior, which in turn affect soil fertility and plant life activities [20,21]. Moreover, the symbiosis formed by plants and microorganisms can significantly enhance the development of plant root systems and the production of secretions in stressful environments, regulate soil nutrient structure, and thereby affect microbial ecological functions [22,23]. Therefore, a comprehensive understanding of the growth-promoting and tolerance mechanisms of DSE will help to explore its application potential, especially in enhancing the efficiency of medicinal plants and remediation of polluted soil [24].
Astragalus mongholicus (Leguminosae) is a perennial herbaceous plant extensively cultivated in China, with roots used both as medicine and food. The roots of this plant contain several active ingredients, including calycosin-7-glucopyranoside and formononetin, which are acknowledged for their efficacy in the treatment of conditions related to qi and blood deficiency, cancers, neurological diseases, allergic diseases, metabolic diseases, cardiovascular diseases, and gastrointestinal diseases [25,26,27,28,29]. Currently, due to the threatened condition of wild Astragalus resources, the severity of habitats, and high market demand, the supply of Astragalus medicinal materials in the market is mainly artificial cultivation, and the quality of medicinal plants mainly depends on a suitable natural environment for growth [23]. Therefore, a pressing objective is to enhance the Astragalus cultivation soil environment and the yield and quality of Astragalus cultivated products.
In the current study, we posited that DSE (Paraphoma chlamydocopiosa, Paraboeremia selaginellae, and Paraphoma radicina, isolated from Astragalus mongholicus) could improve the performance and Cd tolerance of A. mongholicus under Cd stress conditions. Hence, we conducted a pot experiment that involved a two-factor interaction of DSE inoculation and Cd concentration treatment under nonsterile soil conditions, with the aim to (1) determine the effects of DSE on plant performance and active ingredient contents, (2) determine the effect of DSE on Cd content in different parts of the host plant, (3) determine the effect of DSE on the physicochemical properties of the soil.

2. Materials and Methods

2.1. Biological Materials and Culture Substrate

The three DSE strains, Paraphoma chlamydocopiosa (MT723851), Paraboeremia selaginellae (MT723853), and Paraphoma radicina (MT723859), were obtained from the roots of A. mongholicus and identified by Han et al. [30]. Before inoculation, three DSE strains were activated in potato dextrose agar (PDA) for 14 d, respectively, and then used for the experiment. The mature A. mongholicus seeds were collected from the medicinal herb planting base in Anguo, Hebei Province, China, and stored in the laboratory at 4 °C.
The culture substrate was the soil matrix, which was a 2:1 (w/w) mixture of soil and sand (with particles smaller than 2 mm), containing 20.32 mg/g of soil organic carbon (SOC), 121.34 μg/g of soil-available nitrogen (SAN), and 18.63 μg/g of soil-available phosphorus (SAP).
According to the soil pollution-risk screening criteria established in China [31], the permissible upper limit for Cd content is established at 1 mg/kg. Wu et al. [32] reported that the highest provincial-level average Cd content was measured at 10.03 mg/kg. Additionally, our investigation revealed that the cadmium content in the soil of the mining area in the Hebei Province ranges from 5.4 to 11.1 mg/kg [33], while in the Baiyangdian wetland, it was 9.97 mg/kg [34]. Consequently, the cadmium stress range established for this study was 0–10 mg Cd/kg.

2.2. Inoculation Assay

The inoculation trial was carried out utilizing a fully randomized factorial design, incorporating four distinct DSE inoculations and four different Cd conditions, with a total of five replicates for each experimental condition. The four DSE inoculations were P. chlamydocopiosa, P. selaginellae, P. radicina, and a non-inoculated control. The four distinct Cd treatment conditions were 0, 1, 5 and 10 mg Cd/kg soil. Each pot contains two plants, making a total of 80 experimental pots (Figure 1).
The seeds were soaked in 50% concentrated sulfuric acid (v/v) for a duration of 10 min, followed by a subsequent immersion in 5% sodium hypochlorite (v/v) for an additional 10 min, and finally rinsed with sterile water. The sterile seeds were cultured in the dark at 27 °C for germination. The A. mongholicus seedlings were transferred to sterile plastic pots measuring 12 cm in height and 17 cm in diameter. Each pot contained 900 g of a soil matrix. CdCl2·2.5H2O was used to treat soil with heavy metal contents of 0, 1, 5, and 10 mg Cd/kg soil, respectively. To initiate DSE inoculation, a 9 mm disc was excised and placed 1 cm away from the seedling roots. As a control treatment, sterile PDA medium devoid of fungal inoculation was utilized. The seedlings were cultivated in a greenhouse under light conditions of 300 μmol m−2 s−1, with a relative humidity of 60% and a photoperiod of 14 h/10 h (light/darkness). The plants were harvested after five months of being cultivated.

2.3. DSE Colonization of Plant Roots

The potted Astragalus roots were sectioned into segments measuring 0.5 cm in length and then treated with 10% potassium hydroxide (w/v) for clarification [35]. The colonization structure of DSE was observed using a biomicroscope, and the colonization rate of DSE was examined following the method outlined by Biermann and Linderman [36].

2.4. Plant Growth Parameters and Cd Contents

The plant height, leaf number, and branch number of each seedling were documented before harvesting. The measurement of the SPAD value was conducted utilizing the SPAD-502 instrument (Konica Minolta Sensing, Osaka, Japan).
The root sediment was rinsed with water, after which the plant roots were digitized utilizing an Epson V800 scanner (Epson, Nagano, Japan). The morphological indices of the roots were subsequently analyzed through the WinRHIZO image analysis system (Regent Instruments, Quebec, QC, Canada) to ascertain various morphological parameters, including total length, average diameter, surface area, and volume.
To determine the Cd content in roots and shoots, 0.2 g of dried plant samples was digested with a digestion solution containing HClO4/HNO3 (1:3, v/v) at 200–250 °C. The resulting transparent solutions were subsequently diluted with 0.2% HNO3 in a 50 mL volumetric flask. A flame atomic absorption spectrometer (TAS-990, Beijing, China) was employed for the quantification of Cd mass fractions in the plant tissues [5].

2.5. IAA and Active Ingredient Content

The contents of IAA, calycosin-7-glucoside, and formononetin in roots were determined using high-performance liquid chromatography (HPLC) using a reversed-phase C18 symmetry column (Waters Corp., Milford, MA, USA) (Table S1). IAA was extracted from the roots utilizing the methodology outlined by Torelli et al. [37]. The HPLC run was carried out using the equal elution mode (80% methanol: 20% water) at 30 °C, and a flow rate of 1.0 mL/min. The eluted compounds were detected spectrophotometrically at 269 nm using a 2998 photodiode array detector. The detection and quantification of IAA were achieved by comparing the retention time and peak area of the sample with those of a standard IAA solution (IAA, Sigma-Aldrich, St. Louis, MI, USA).
To determine the content of calycosin-7-glucopyranoside and formononetin, dried root specimens were pulverized into a fine powder and subsequently sieved through a 40-mesh sieve. A 0.5 g sample of the root was subjected to extraction in 25 mL of methanol for a duration of 3 h utilizing an ultrasonic bath [38]. The concentrated solution was obtained using a pressure rotary evaporator (Automatic Science, Shanghai, China), reconstituted in 2.5 mL of methanol. The mobile phase employed for the analysis comprised 0.2% formic acid (A) and acetonitrile (B) in a gradient elution format (0–20 min, transitioning from 80% to 60% A; 20–30 min, maintaining 60% A) at a temperature of 30 °C, with a flow rate set at 1.0 mL/min and detection wavelength at 260 nm. The standards for calycosin-7-glucopyranoside and formononetin were obtained from the China National Institutes for Food and Drug Control.

2.6. Soil Physicochemical Properties

The precision pH meter (PHS-3C) was utilized for determining soil pH values. The soil organic carbon content (SOC) was assessed utilizing the loss-on-ignition technique [39]. The determination of soil-available nitrogen (SAN) was conducted through the alkaline hydrolysis diffusion method [40]. Additionally, soil-available phosphorus (SAP) was quantified employing the sodium bicarbonate leaching-molybdenum antimony colorimetric method [41].

2.7. Statistical Analyses

Two-way ANOVA was employed to assess the impacts of DSE inoculation, Cd stress, and their interactions on the growth and soil factors of the host plant. One-way ANOVA was utilized to compare the mean values of various plant growth parameters, active ingredients, and soil nutrient contents of each DSE species or Cd stress treatments. To compare mean values, Tukey’s tests and T-tests were conducted, with a significance level set at p < 0.05. The statistical analyses were performed using SPSS version 21.0. Additionally, principal component analysis (PCA) was used to visualize differences in plant growth across DSE species and Cd treatment. Variation partitioning analysis (VPA) was conducted to examine the interactive effects of DSE inoculation and soil nutrients on plant growth. Random forest model was used to assess factors affecting plant growth. All statistical analyses were executed using R version 4.0.0. The correlation among the different variables was analyzed using the Pearson coefficient with Origin 2021 software. The values presented in the figures represent the averages of at least three replicates.

3. Results

3.1. Quantification of DSE Colonization

Three DSE colonizations in the roots of A. mongholicus were measured at different levels of Cd/kg soil (Table S2). For P. chlamydocopiosa, the highest hyphal colonization (77.8 ± 6.2%), microsclerotia colonization (43.3 ± 3.2%), and total colonization (77.8 ± 3.9%) appeared at 10 mg Cd/kg soil. The lowest hyphal colonization was 43.2% (±2.3%) at 0 mg Cd/kg soil, while the lowest microsclerotia colonization (19.3 ± 3.1%) and total colonization (48.1 ± 4.5%) appeared at 1 mg Cd/kg soil. For P. selaginellae, the highest hyphal colonization (69.7 ± 1.9%), microsclerotia colonization (40.9 ± 4.3%), and total colonization (69.7 ± 4.3%) appeared at 10 mg Cd/kg soil. The lowest hyphal colonization (38.2 ± 4.4%) and total colonization (38.2 ± 2.3%) were recorded at 0 mg Cd/kg soil, while the lowest microsclerotia colonization was 20.2% (±4.3%) at 1 mg Cd/kg soil. For P. radicina, the highest hyphal colonization (45.6 ± 5.1%), microsclerotia colonization (26.3 ± 1.3%), and total colonization (53.6 ± 4.3%) were recorded at 10 mg Cd/kg soil, while the lowest hyphal colonization (30.8 ± 2.3%), microsclerotia colonization (13.3 ± 2.1%), and total colonization (30.8 ± 3.3%) were observed at 0 mg Cd/kg soil. The highest levels of hyphal and microsclerotia colonization were recorded at a concentration of 10 mg Cd/kg soil, which was significantly greater than the levels observed at 0 mg Cd/kg soil. However, the hyphal and microsclerotia colonization rates of DSE showed no significant differences among 0–5 mg Cd/kg soil.

3.2. Plant Growth Parameters

At 0 mg Cd/kg soil, P. radicina increased the plant height, and P. selaginellae and P. radicina increased the number of leaves and branches, respectively, in comparison to the control plants (Figure 2a–c). At 1 mg Cd/kg soil, P. radicina increased plant height and branch count, and P. selaginellae and P. radicina increased leaf numbers, respectively, compared with the control plants. At 5 mg Cd/kg soil, P. selaginellae and P. radicina increased the plant height, inoculation with P. chlamydocopiosa, P. selaginellae, and P. radicina increased the number of leaves, and inoculation with P. radicina increased the number of branches, respectively, compared with the control plants. At 10 mg Cd/kg soil, DSE exerted a considerable influence on both plant height and leaf number, and P. radicina alone increased the number of branches. For plants not inoculated with DSE, the morphological indices exhibited significant differences at 5 mg Cd/kg soil and 10 mg Cd/kg soil. For DSE inoculation, the difference caused by Cd stress treatment gradually decreased. The plant height and branch number inoculated with P. chlamydocopiosa and P. selaginellae were not affected by Cd concentration (Figure 2).
The SPAD value is indicative of the relative chlorophyll content in leaves. At 0 mg Cd/kg soil, P. selaginellae increased the SPAD value by 26.8% (Figure 2d) compared with the control plant. At 1 mg Cd/kg soil, P. chlamydocopiosa increased the SPAD value by 50.4% compared with the control plant. At 5 mg Cd/kg soil, inoculation with P. chlamydocopiosa, P. selaginellae, and P. radicina increased SPAD values compared with the control plant. At 10 mg Cd/kg soil, DSE showed varying degrees of growth-promoting effects on SPAD values.
Two-way ANOVA revealed that both DSE inoculation and Cd stress, as well as their interactions, had a significant impact on plant biomass (Table 1). At 0 mg Cd/kg soil, DSE contributed to an enhancement in both shoot biomass and total biomass (Figure 2e,g), while P. selaginellae resulted in a 34.8% increase in root biomass and a 28.6% increase in the root–shoot ratio (Figure 2f,h) compared with the control plants. At 1 mg Cd/kg soil, P. selaginellae and P. radicina increased the shoot biomass and total biomass, and P. selaginellae alone increased the root biomass by 36.5%, respectively, compared with the control plants. At 5 mg Cd/kg soil, the application of three DSE resulted in an enhancement of both shoot biomass and total biomass. Furthermore, the species P. chlamydocopiosa and P. selaginellae exhibited a notable growth-promoting influence on root biomass, increasing it by 32.0% and 33.8%, respectively, compared with the control plants. At 10 mg Cd/kg soil, DSE showed positive growth-promoting effects on shoot biomass and total biomass, and P. chlamydocopiosa alone had a significant growth-promoting effect on 74.1% of the root biomass, respectively, in comparison to the control plants. Despite a reduction in the biomass observed under moderate and severe Cd stress conditions, the influence of DSE on biomass enhancement was quite significant, increasing by up to 82.8%. Inoculation with P. chlamydocopiosa prevented the root biomass from being affected by Cd stress (Figure 2).
At 0 mg Cd/kg soil, P. chlamydocopiosa and P. selaginellae increased the root length by 10.0% and 25.7% (Figure 3a), and P. selaginellae increased the root volume by 32.0% (Figure 3b), in comparison to control plants. At 1 mg Cd/kg soil, P. chlamydocopiosa and P. radicina increased root length by 30.3% and 11.8%, and P. chlamydocopiosa increased the root volume by 22.3%, in comparison to control plants. At 5 mg Cd/kg soil, P. chlamydocopiosa and P. radicina increased root length by 4.13% and 5.1%, respectively, while P. selaginellae increased root volume by 30.7%, in comparison to control plants. At 10 mg Cd/kg soil, P. chlamydocopiosa and P. selaginellae increased root length by 47.9% and 15.7%, respectively, while P. selaginellae increased the root volume by 23.1%, compared to control plants.

3.3. IAA, Active Ingredients and Cd Content

Two-way ANOVA revealed that both DSE inoculation and Cd stress, as well as their interactions significantly influenced IAA content. DSE inoculation had a greater impact on IAA content (Table 1). At 0 mg Cd/kg soil, P. chlamydocopiosa and P. selaginellae increased the IAA content by 25.9% and 11.0%, respectively (Figure 3c). At 1 mg Cd/kg soil, P. selaginellae boosted the IAA content by 30.2%. At 5 mg Cd/kg soil, P. selaginellae boosted the IAA content by 58.6% in comparison to control plants. At 10 mg Cd/kg soil, P. chlamydocopiosa and P. selaginellae increased the IAA content by 103.6% and 105.8%, respectively. Furthermore, P. selaginellae rendered the IAA content unaffected by Cd stress (Figure 3c).
Two-way ANOVA revealed that DSE inoculation had a greater impact on calycosin-7-glucopyranoside content (Table 1). At 0 mg Cd/kg soil, P. radicina increased the calycosin-7-glucopyranoside content by 31.7% (Figure 3d), and P. chlamydocopiosa and P. selaginellae increased the content of formononetin by 2.8% and 26.4% (Figure 3e), respectively, compared with the control plants. At 1 mg Cd/kg soil, three DSE increased the content of calycosin-7-glucopyranoside by 20.1%, 15.4%, and 29.9%, and formononetin content by 6.4%, 49.6%, and 8.0%, respectively, in comparison to the control plants. At 5 mg Cd/kg soil, P. radicina increased the calycosin-7-glucopyranoside content by 32.2%, and P. chlamydocopiosa increased the formononetin content by 7.5%, respectively, compared with the control plants. At 10 mg Cd/kg soil, P. chlamydocopiosa and P. radicina increased the calycosin-7-glucopyranoside content by 31.0% and 39.9%, and P. chlamydocopiosa and P. selaginellae increased the formononetin content by 24.8% and 9.5%, respectively. Despite the concentration of Cd stress, inoculation with P. radicina had a stronger growth-promoting effect on calycosin-7-glucopyranoside content, while P. chlamydocopiosa inoculation had a stronger effect on formononetin content (Figure 3).
Two-way ANOVA revealed that DSE inoculation had a greater impact on the translocation factor (Table 1). An increase in the Cd content observed in both the shoots and roots was associated with a proportional elevation in Cd concentration (Figure 4a,b). At 1 and 5 mg Cd/kg soil, inoculation with the three DSE significantly increased the accumulation of Cd in shoots. At 10 mg Cd/kg soil, inoculation with P. selaginellae and P. radicina resulted in a marked enhancement of Cd accumulation in the shoots when compared to the control plants (Figure 4a). Inoculation with P. chlamydocopiosa and P. selaginellae at Cd concentrations of 1 and 10 mg Cd/kg soil significantly enhanced the accumulation of Cd in the roots. Conversely, P. radicina exhibited an inhibitory effect on Cd accumulation in the roots, leading to no difference when compared to the control group. At 5 mg of Cd/kg soil, inoculation with P. chlamydocopiosa and P. selaginellae resulted in a significant enhancement of Cd accumulation in the roots. In contrast, P. radicina inoculation led to a significant reduction in Cd accumulation in roots (Figure 4b). Inoculation with P. chlamydocopiosa reduced the translocation factor, while inoculation with P. radicina increased the translocation factor, with the coefficient exceeding 1 (Figure 4c).

3.4. Soil Physicochemical Properties

Inoculation with DSE improved SOC content, regardless of the Cd stress (Figure 5a). Compared with uninoculated plants, inoculation with P. chlamydocopiosa and P. selaginellae reduced SAN content regardless of the Cd stress. Compared to no Cd stress, inoculation with P. chlamydocopiosa and P. radicina significantly increased the SAN content under moderate and severe Cd stress (Figure 5b). Regardless of DSE inoculation, the highest SAP content was found at 10 mg Cd/kg soil (Figure 5c). At 0 mg Cd/kg soil, inoculation with P. selaginellae reduced SAP content. At 1 mg Cd/kg soil, P. radicina reduced SAP content. At 5 mg Cd/kg soil, P. chlamydocopiosa reduced SAP content. At 10 mg Cd/kg soil, DSE inoculation reduced SAP content compared with uninoculated plants.

3.5. Factors Affecting the Growth of the Host Plant

The VPA revealed that, irrespective of Cd stress, the explanatory variables of DSE were higher than those of soil factors (Figure 6). The explanatory variables of the interaction between soil and DSE had the most significant impact on plant growth. At 10 mg Cd/kg soil, the independent variable of DSE had the highest impact on plant growth, accounting for 44.2% of the variance (Figure 6D). Therefore, as the stress levels increase, the beneficial impact of DSE on the growth of the host plant becomes more pronounced.
PCA revealed that the impact of P. radicina on plant growth differed from that of P. chlamydocopiosa and P. selaginellae. P. chlamydocopiosa and P. selaginellae exhibited similar growth promotion mechanisms at 1 and 5 mg Cd/kg soil, while each DSE treatment had its unique way of responding to Cd stress at 10 mg Cd/kg soil (Figure 7).
Correlation analysis revealed that the SPAD value, the content of IAA and active ingredient, leaf number and biomass of A. mongholicus uninoculated with DSE were negatively correlated with the Cd content (Figure 8A). While the number of leaves inoculated with P. chlamydocopiosa was not correlated with the Cd content. The root–shoot ratio exhibited a positive correlation with the Cd content (Figure 8B). However, the root–shoot ratio and Cd concentration demonstrated an inverse relationship following inoculation with P. selaginellae and P. radicina (Figure 8C,D). After inoculation with P. selaginellae, the root length and root surface area exhibited a positive correlation with Cd content. There was no significant correlation between the SPAD value and the root Cd and shoot Cd content inoculated with P. selaginellae and P. radicina. The SPAD value inoculated with P. selaginellae exhibited a positive correlation with the root–shoot ratio. The IAA concentration in roots inoculated with P. selaginellae exhibited a significant positive correlation with the Cd concentrations in both the shoots and roots (Figure 8C). The IAA content after inoculation with P. radicina was positively correlated with active ingredients and biomass (Figure 8D). The IAA content increased after inoculation with P. chlamydocopiosa and P. selaginellae, showing a positive correlation with the SAP content. The shoot and root Cd contents exhibited a significant negative correlation with SOC, while demonstrating a positive correlation with SAP. Plant growth index increased after inoculation with P. selaginellae and P. radicina, showing a negative correlation with SAP (Figure 8).
The random forest model indicated that SOC and SAP were the most significant factors contributing to plant biomass (49.5%, 44.0%). Plant height, IAA content, leaf number, and root length explained 30.7%, 30.0%, 26.6%, and 23.1% of plant biomass, respectively. The shoot Cd content had little effect on plant biomass (Figure 9, Table S3).

4. Discussion

4.1. Effect of DSE Strains on the Growth of the Host Plant

Although increasing evidence suggests the diversity and distribution of DSE strains [4,42], their impacts on plant growth in stressful environments, especially on medicinal plant growth under heavy metal stress, are still poorly studied. The current study revealed that the inoculation of DSE significantly enhanced both the growth and Cd tolerance of the host plant. The enhancement of the host plant growth following inoculation with DSE may be attributed to an increase in nutrient uptake efficiency [43,44]. Liu et al. [45] demonstrated that inoculation with DSE resulted in a significant enhancement of the biomass of orchid seedlings. Additionally, Mikheev et al. [46] reported that Phialocephala fortinii inoculation resulted in an increase in the biomass of Vaccinium macrocarpon. Here, under Cd stress, the SPAD value, plant height, branch number, and biomass were not affected by Cd concentration after inoculation with P. chlamydocopiosa and P. selaginellae. Under moderate and high Cd stress, the number of leaves and biomass increased by up to 58.6% and 82.8%, respectively, compared with uninoculated plants. It is evident that DSE inoculation can markedly enhance Cd tolerance and reduce the phytotoxicity of Cd [47,48]. Xiao et al. [49] found that high concentrations of Cd are toxic to maize, leading to a serious decrease in biomass and plant height, while inoculation with DSE can alleviate these negative effects. Ban et al. [50] indicated that exposure to Pb stress resulted in a notable increase in both the height and biomass of maize seedlings when treated with Gaeumannomyces cylindrosporus. This enhancement was attributed to improved photosynthetic efficiency and modifications in the transport and accumulation of Pb within the plant. Our research results indicated that plant biomass is more prominent, sometimes even reaching its maximum value at 1 mg Cd/kg soil, which is consistent with previous research results [51]. In addition, the three DSE strains tested respond to plant growth differently under Cd stress. Berthelot et al. [52] observed that the impact of DSE inoculation on plant growth varied under Cd stress conditions. Therefore, it can be inferred that the nature of the interaction between DSE and plants is likely to be primarily determined by the particular species of DSE present [53].
Despite Cd stress, inoculation with DSE exhibited a beneficial impact on the root development of the host plant, especially at 10 mg Cd/kg soil, and P. chlamydocopiosa increased root length by 47.9%, indicating that plants inoculated with DSE allocated more resources to root production in response to high-concentration Cd stress. Hou et al. [5] reported that DSE significantly increased both the root length and root surface area of Artemisia ordosica, while also enhancing the plant’s resilience to Cd stress. Huot et al. [54] discovered that longer root length could be more advantageous for plant growth. Consequently, as the volume and biomass of roots expand, the development of a deep and extensive root network increases the surface area in contact with the soil, thereby improving the efficiency of water and nutrient absorption by the roots. Therefore, the greater root volume and length of host plants may confer advantages in their adaptability to environments contaminated with heavy metals [55].

4.2. Effect of DSE Inoculation on Active Ingredients

At 0 mg Cd/kg soil, inoculation with P. chlamydocopiosa and P. selaginellae significantly elevated the IAA content compared to control plants. This may be due to the fact that DSE can manipulate plant growth either through its own production of IAA or by stimulating IAA production in the host plant [56]. In addition to promoting plant growth, IAA is a key factor in plant adaptation to heavy metal-induced stress [57]. Shah et al. [58] found that IAA not only promotes the biomass production of the host plant but also effectively alleviates Cu stress in plants. This study indicated that the influence of DSE on IAA was amplified in response to elevated Cd stress. Consequently, it is plausible that one of the mechanisms through which DSE assists host plants in managing Cd stress involves the regulation of the plant hormone equilibrium, thereby improving the overall productivity of the host plants. And all three DSE demonstrated a pro-growth effect on the active ingredients. Inoculation with P. chlamydocopiosa exhibited a more pronounced influence on the content of formononetin, irrespective of Cd stress. This finding suggests that DSE can enhance both the growth and the concentration of active compounds in medicinal plants [17,19]. Inoculating medicinal plant Epimedium brevicornu with DSE not only significantly increased plant biomass, but also elevated the concentration of total flavonoids and icariin by 42.15% and 20.24%, respectively [18]. On the other hand, DSE can also significantly increase the content of medicinal components under stressful environments [19]. The interaction between plants and endophytic fungi involves a cooperative relationship mediated by the chemical defense of plants [59]. The colonization process of fungi can trigger a series of defense responses in host plants, such as the enhancement of secondary metabolism like flavonoids [17,59]. Wu et al. [60] discovered that DSE colonization can lead to various changes in plant biosynthetic pathways, including phenylpropanoid biosynthesis and flavonoid biosynthesis, as well as plant hormone signaling pathways. And changes in plant hormones can regulate the production of flavonoids [61]. Similar metabolic regulation has been observed in symbiotic associations of Phialocephala fortinii–Pinus massoniana and Suillus bovinus–P. massoniana [62]. Furthermore, DSE can enhance the surface area of plant roots, facilitate the absorption of various nutrients [45,63], and increase the bioaccumulation of medicinal ingredients.

4.3. Effect of DSE Strains on Cd Absorption in Medicinal Plants

Generally, DSE colonization influences the transportation of heavy metals from the root system to aerial parts of the host plant [64]. Inoculation with P. chlamydocopiosa reduced the translocation factor, while P. radicina inhibited Cd accumulation in the roots and increased the Cd translocation factor of the host plant. Xiao et al. [49] found that the accumulation of Cd in maize was positively correlated with elevated Cd concentrations, with the Cd levels in the roots surpassing those in the stems. Li et al. [65] found that DSE inhibited the movement of Cd from the roots to the aerial portions of the plant.
Cd was segregated in the roots, leading to a reduction in the ratio of Cd content between shoot and root (i.e., translocation factor). Studies have found that the cell wall functions as the principal location for Cd binding, acting as the initial defense mechanism against heavy metals. This function is essential for facilitating Cd tolerance and accumulation within the organism [66,67]. The association of metal ions with cell walls mitigates the toxicity of excess metal ions on organelles [68]. It was concluded that DSE colonization increased the accumulation of Cd within the root cell wall and converted Cd to inactive forms, thus significantly reducing the transfer of Cd to the shoot [69]. Moreover, inoculation with DSE increased root length and root volume, facilitating more efficient metal uptake by the root system due to the increased root–soil contact area. It has been proposed that endophytic fungi possess the capability of absorbing Cd into their spores and hyphae, using vacuole isolation as an adaptive strategy [48]. Moreover, melanin in DSE hyphae is considered the most crucial component of the cell wall and possesses a strong biosorption ability for metal ions. This ability can reduce the harmful effect of Cd on root cells [70]. However, Zhu et al. [6] reported that DSE reduced Cd accumulation in roots, thereby enhancing the growth and resilience of tomato plants exposed to heavy metal stress. He et al. [64] observed that the colonization of DSE significantly reduced Cd concentration in maize roots under 50 and 100 mg Cd/kg soil. Thus, the accumulation and transfer of Cd in host plants may also be related to DSE strains. Meanwhile, when medicinal plants are inoculated with DSE, the accumulation of Cd in plant parts should be considered. Therefore, it is worth considering how to find a balance between the promoting impact of DSE on medicinal plant development and Cd accumulation.

4.4. Effect of DSE Strains on Soil Factors

The VPA revealed that the interaction between soil and DSE exerted the most significant influence on host plant, highlighting the crucial role of DSE in nutrient exchange during plant growth. Among them, soil organic C and available P dominated. Research has indicated that DSE can significantly increase plant organic nutrient pools by converting soil-insoluble carbon and phosphorus into bioavailable forms. DSE also increases the contact area between plant roots and soil, thereby enhancing nutrient absorption by the host plant. The Cd content in plant root and shoot exhibited a positive association with soil-available P content while showing a negative relationship with soil organic C content. Research has demonstrated that P is crucial in the uptake and transport of Cd and can reduce Cd toxicity by forming Cd–phosphate complexes on the hyphal cell wall [71,72]. Li and Wei [73] found that soils with high soil organic matter content exhibit an increased adsorption capacity, thereby promoting the stable formation of cadmium complexes. Chen et al. [74] indicated that an elevation in soil carbon could significantly diminish the concentration of heavy metals within the soil matrix. Consequently, phosphorus and carbon are essential in the processes of fixation and detoxification of Cd and may serve as a significant mechanism for DSE to assist in plant detoxification.

5. Conclusions

The present study revealed that DSE inoculation has the potential to mitigate the negative impacts of Cd stress on the growth of A. mongholicus. DSE inoculation promoted the performance and active ingredient content of the host plant and altered the redistribution of Cd in roots and stems under Cd stress; this positive effect depends on the DSE strain. In particular, inoculation with P. radicina not only enhanced plant growth and active ingredient accumulation but also inhibited the accumulation of Cd in roots. Moreover, in more stressful conditions, DSE has a greater assisting effect on the growth of the host plant. Our study has demonstrated, for the first time, that DSE positively promotes the growth and Cd tolerance of A. mongholicus. This finding establishes a basis and approach for the cultivation of medicinal plants in soils that are contaminated with heavy metals. Building upon this basis, further research will be conducted to examine the impact of DSE inoculation on the rhizosphere microbial communities and the chemical forms of Cd present in medicinal plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14081801/s1. Table S1. Linear relationships of IAA, calycosin-7-glucopyranoside, and formononetin. Table S2: DSE colonization rate in A. membranaceus roots under different Cd stress. Distinct lowercase letters signify a statistically significant difference among various Cd stress (p < 0.05); Table S3 Factor coefficients affecting the growth of Astragalus membranaceus.

Author Contributions

Conceptualization, M.L. and X.H.; methodology, M.L., L.H. and X.L.; formal analysis, M.L. and C.H.; data curation, M.L., L.H. and X.L.; writing—original draft preparation, M.L. writing—review and editing X.H. and C.H.; visualization, M.L. and L.H.; project administration, X.H.; funding acquisition, X.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Natural Science Foundation of Hebei Province (grant number H2022201056) and Central Guidance for Local Scientific and Technological Development Funding Projects (grant number 236Z2904G).

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Inoculation assay of Astragalus mongholicus cultivated in pot experiments. The designation “Control” refers to the non-inoculated control group, “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
Figure 1. Inoculation assay of Astragalus mongholicus cultivated in pot experiments. The designation “Control” refers to the non-inoculated control group, “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
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Figure 2. The effects of DSE inoculation and Cd stress on the plant height (a), leaf number (b), branch number (c), SPAD value (d), shoot biomass (e), root biomass (f), total biomass (g), and root–shoot ratio (h) of Astragalus mongholicus. The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
Figure 2. The effects of DSE inoculation and Cd stress on the plant height (a), leaf number (b), branch number (c), SPAD value (d), shoot biomass (e), root biomass (f), total biomass (g), and root–shoot ratio (h) of Astragalus mongholicus. The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
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Figure 3. The effects of DSE inoculation and Cd stress on the root length (a), root volume (b), IAA content (c), calycosin-7-glucopyranoside content (d), and formononetin content (e) of Astragalus mongholicus. The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
Figure 3. The effects of DSE inoculation and Cd stress on the root length (a), root volume (b), IAA content (c), calycosin-7-glucopyranoside content (d), and formononetin content (e) of Astragalus mongholicus. The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
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Figure 4. The effects of DSE inoculation and Cd stress on the shoot Cd content (a), root Cd content (b), and translocation factor (c). The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
Figure 4. The effects of DSE inoculation and Cd stress on the shoot Cd content (a), root Cd content (b), and translocation factor (c). The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
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Figure 5. The effects of DSE inoculation and Cd stress on the content of soil organic carbon (a), soil-available nitrogen (b), and soil-available phosphorus (c). The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
Figure 5. The effects of DSE inoculation and Cd stress on the content of soil organic carbon (a), soil-available nitrogen (b), and soil-available phosphorus (c). The error bars illustrate the standard error of the mean. The lowercase letters assigned to the columns denote statistically significant differences (p < 0.05) within the same DSE strain across various levels of Cd stress. Conversely, the uppercase letters signify significant differences (p < 0.05) among the different DSE strains under the same Cd treatment. The designation “C” refers to the non-inoculated control group, while “PC”, “PS”, and “PR” represent plants inoculated with Paraphoma chlamydocopiosa, Paraboeremia selaginellae, Paraphoma radicina, respectively.
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Figure 6. The variation partitioning analysis (VPA) of soil factors and DSE inoculation on growth indicators of Astragalus mongholicus at 0 mg Cd/kg soil (A), 1 mg Cd/kg soil (B), 5 mg Cd/kg soil (C), and 10 mg Cd/kg soil (D).
Figure 6. The variation partitioning analysis (VPA) of soil factors and DSE inoculation on growth indicators of Astragalus mongholicus at 0 mg Cd/kg soil (A), 1 mg Cd/kg soil (B), 5 mg Cd/kg soil (C), and 10 mg Cd/kg soil (D).
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Figure 7. The principal component analysis (PCA) showing growth indicators of Astragalus mongholicus with different DSE species and Cd treatments.
Figure 7. The principal component analysis (PCA) showing growth indicators of Astragalus mongholicus with different DSE species and Cd treatments.
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Figure 8. Correlation analysis of the growth indicators and soil properties of Astragalus mongholicus inoculated with DSE species. (A): un-inoculated control, (B): Paraphoma chlamydocopiosa inoculation, (C): Paraboeremia selaginellae inoculation, (D): Paraphoma radicina inoculation. RCd: root Cd content, SCd: shoot Cd content, TF: translocation factor, CG: calycosin-7-glucopyranoside, For: formononetin, RL: root length, RB: root biomass, SB: shoot biomass, R/S: root–shoot ratio. (An asterisk (*) denotes a statistically significant difference at a probability level of p < 0.05, while two asterisks (**) indicate a statistically significant difference at a probability level of p < 0.01).
Figure 8. Correlation analysis of the growth indicators and soil properties of Astragalus mongholicus inoculated with DSE species. (A): un-inoculated control, (B): Paraphoma chlamydocopiosa inoculation, (C): Paraboeremia selaginellae inoculation, (D): Paraphoma radicina inoculation. RCd: root Cd content, SCd: shoot Cd content, TF: translocation factor, CG: calycosin-7-glucopyranoside, For: formononetin, RL: root length, RB: root biomass, SB: shoot biomass, R/S: root–shoot ratio. (An asterisk (*) denotes a statistically significant difference at a probability level of p < 0.05, while two asterisks (**) indicate a statistically significant difference at a probability level of p < 0.01).
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Figure 9. Random forest model showing main factors affecting the growth of Astragalus mongholicus. RV: root volume, SCd: shoot Cd content, RL: root length, RCd: root Cd content.
Figure 9. Random forest model showing main factors affecting the growth of Astragalus mongholicus. RV: root volume, SCd: shoot Cd content, RL: root length, RCd: root Cd content.
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Table 1. Two-way ANOVA of the impacts of DSE inoculation, cadmium stress and their interactions on the growth and soil factors of Astragalus mongholicus.
Table 1. Two-way ANOVA of the impacts of DSE inoculation, cadmium stress and their interactions on the growth and soil factors of Astragalus mongholicus.
DSECdDSE × Cd
Fηp2Fηp2Fηp2
Height60.34 ***0.8531.21 ***0.756.44 ***0.64
Leaf number151.10 ***0.9335.11 ***0.7721.15 ***0.86
Branch61.46 ***0.8513.46 ***0.563.05 ***0.46
SPAD56.29 ***0.8448.74 ***0.8222.85 ***0.87
Shoot biomass126.73 ***0.92143.78 ***0.9317.10 ***0.83
Root biomass93.06 ***0.90144.15 ***0.9322.38 ***0.86
Total biomass146.31 ***0.93221.79 ***0.9515.45 ***0.81
Root–shoot ratio45.73 ***0.8117.03 ***0.6120.54 ***0.85
IAA56.32 ***0.843.97 *0.2713.45 ***0.79
Root length270.15 ***0.9691.23 ***0.9085.37 ***0.96
Root volume80.27 ***0.8883.87 ***0.8939.49 ***0.92
Calycosin-7-O-glucoside304.85 ***0.97139.96 ***0.9319.96 ***0.85
Formononetin614.60 ***0.98955.70 ***0.99156.82 ***0.98
Shoot Cd277.98 ***0.961037.17 ***0.9928.91 ***0.89
Root Cd271.20 ***0.961782.58 ***0.9951.45 ***0.94
Translocation factor282.29 ***0.9617.59 ***0.6219.25 ***0.84
SOC205.64 ***0.95203.76 ***0.9513.72 ***0.79
SAN142.78 ***0.9337.64 ***0.7811.95 ***0.77
SAP147.34 ***0.931185.99 ***0.9983.49 ***0.96
An asterisk (*) denotes a statistically significant difference at a probability level of p < 0.05, three asterisks (***) indicate a statistically significant difference at a probability level of p < 0.001.
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Li, M.; Han, L.; He, C.; Li, X.; He, X. The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Astragalus mongholicus under Cadmium Stress. Agronomy 2024, 14, 1801. https://doi.org/10.3390/agronomy14081801

AMA Style

Li M, Han L, He C, Li X, He X. The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Astragalus mongholicus under Cadmium Stress. Agronomy. 2024; 14(8):1801. https://doi.org/10.3390/agronomy14081801

Chicago/Turabian Style

Li, Min, Li Han, Chao He, Xia Li, and Xueli He. 2024. "The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Astragalus mongholicus under Cadmium Stress" Agronomy 14, no. 8: 1801. https://doi.org/10.3390/agronomy14081801

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