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Article

The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress

by
Meiling Wu
1,
Gen Li
1,
Simiao Wang
1,
Ziteng Wang
1,
Longfei Li
1,2 and
Li Han
1,2,*
1
Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of Pharmaceutical Sciences, Hebei University, Baoding 071002, China
2
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(1), 208; https://doi.org/10.3390/agronomy15010208
Submission received: 10 December 2024 / Revised: 27 December 2024 / Accepted: 13 January 2025 / Published: 16 January 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

:
Dark septate endophytes (DSE) may facilitate plant growth and stress tolerance in stressful ecosystems. However, little is known about the response of medicinal plants to DSE, especially under heavy metal stress. This study aimed to investigate how DSE affects the growth of Dendranthema morifolium in medicinal plants under cadmium (Cd) stress. In this investigation, the sterile and non-sterile inoculations were carried out to evaluate the effect of three DSE strains on D. morifolium stressed with Cd. For the root, DSE15 sterile or non-sterile inoculation resulted in enhanced root biomass, root volume, the Cd content of roots, and the indoleacetic acid (IAA) levels in D. morifolium under Cd stress. DSE7 non-sterile inoculation significantly enhanced the Cd content of roots at 1 and 5 mg Cd/kg soil. Regarding impact stems and leaves, under sterile conditions, DSE7 and DSE15 effectively regulated the shoot biomass, plant height, chlorophyll level, and superoxide dismutase (SOD) content. Under sterile conditions, DSE15 positively influenced shoot biomass and plant height, while DSE7 had no significant effect on them when subjected to Cd stress. For effects on flowers under non-sterile conditions, DSE7 and DSE15 significantly increased the flower biomass under Cd stress, while DSE7 reduced the Cd transfer coefficient of flowers at 1 and 5 mg Cd/kg soil. Importantly, at 1 mg Cd/kg soil, DSE7 and DSE15 non-sterile inoculations promoted the 1, 5-dicaffeoylquinic acid content by 18.29% and 21.70%. The interaction between DSE and soil factors revealed that DSE species had significant effects on soil organic carbon and available nitrogen in D. morifolium non-sterile soil. The DSE15 inoculation enhanced soil organic carbon content, while the inoculation of DSE7 and DSE15 reduced soil available nitrogen content under Cd stress. These results contribute to a better understanding of DSE-plant interactions in habitats contaminated by heavy metals and demonstrate the potential utility of DSE strains for cultivating medicinal plants.

1. Introduction

As one of the main pollutants in soil, the accumulation of Cd soil poses a threat to agricultural cultivation worldwide [1]. Residual heavy metals in soil can cause abnormal physical and chemical properties of the soil, and even the morphological metabolism, as well as the physiology of plants [2]. Selectively employing beneficial microorganisms is an effective strategy for plants to combat heavy metal stress [3]. In recent years, the colonization of endophytic fungi, such as arbuscular mycorrhizal fungi (AMF) and dark septum endophytes (DSE), has been found in the roots of plants in various stress habitats, which can help host plants adapt to adverse environments by changing root morphology, changing plant biomass, and affecting plant metabolism [4,5].
Dark septate endophytes (DSE) are conidial or aseptic cyst fungi that colonize a wide range of hosts in a variety of habitats [6]. They can improve the tolerance of plants to abiotic stresses and play an important role in the survival of hosts in extreme conditions (such as drought, cold, and heavy metals, etc.) [7,8,9]. The interaction between plants and endophytic fungi in roots has always been the focus of researchers’ attention. Recently, more and more studies have demonstrated the coping strategies of DSE under heavy metal stress [10,11,12]. As Li et al. [13] suggested the colonization of DSE instigates a highly orchestrated shift in the accumulation patterns of organic acids within the root system to improve Pb resistance in plants. An important driver for endophytic fungi tolerance is the ability of this plant species to be associated with root symbionts and corresponds to a nutrient-uptake strategy trait, corresponding to various adaptations developed by plants to acquire phosphorus and nitrogen [14]. Moreover, endophytic fungi symbiosis frequently boosts heavy metal resistance through the facilitation of antioxidant enzyme actions and photosynthesis [15,16]. DSE inoculation can also affect the content of abscisic acid (ABA) and indoleacetic acid (IAA) in the root by regulating the expression of genes involved in the signal transduction and polar transport of phytohormone, which is also the main reason for promoting the growth of host plants [17]. DSE regulates the content of polysaccharide and insoluble phosphate Cd in the root cell wall, thereby increasing the retention of Cd in the roots [18,19]. In addition, the existence of the genes EpABC2.1, EpABC3.1, and EpABC4.1 in Exophiala pisciphila has been linked to the reduction of the Cd content and Cd transfer coefficient in the shoots of maize [20,21], making it relatively safe for above-ground portions of the plant.
Moreover, studies have contributed to our understanding that DSE inoculation can also promote the growth and quality of medical plants. For instance, He et al. [22] reported that compared with the non-inoculated control group, two DSE strains increased the plant biomass of Glycyrrhiza uralensis. Additionally, certain research demonstrated that utilizing fungi and bacteria to enhance the bioactive chemical content of medicinal plants represents a novel research avenue in the field of medicinal plant cultivation [23,24,25]. Nevertheless, little is known about the impact of beneficial fungi on medicinal parts and the precise mechanism of action remains inconclusive.
Dendranthema morifolium is a medicinal and edible cognate plant [26]. Modern studies showed that the medicinal ingredients of D. morifolium have many biological and pharmacological characteristics including antibacterial, anti-inflammatory, antioxidant, vasodilator, hypolipidemic, and anti-tumor characteristics, such as chlorogenic acid and 1, 5-dicaffeoylquinic acid [27,28,29]. The quality of the medication produced was also impacted by the accumulation of heavy metals in the soil that occurred during the planting of D. morifolium [30,31,32]. Therefore, the inoculation of medicinal plants with DSE has great significance in improving the quality of Chinese herbal medicines under heavy metal stress.
This study aimed to investigate how the DSE affects the performance and active ingredients accumulation of D. morifolium medicinal parts under Cd stress. Specifically, we addressed the following questions in this study: (1) What is the effect of DSE on the growth of D. morifolium under Cd stress? (2) How does the inoculation of DSE affect the active ingredients of D. morifolium under Cd stress? (3) How does DSE inoculation affect Cd accumulation in medicinal plants?

2. Materials and Methods

2.1. Fungal Isolates and Plant Materials

The three DSE strains, Paraphoma chlamydocopiosa (DSE7, isolated from D. morifolium root), Paraboeremia selaginellae (DSE15, isolated from Astragalus membranaceus root), and Paraphoma radicina (DSE20, isolated from D. morifolium root), were preserved in the culture collection of the Laboratory of Mycorrhizal Biology, Hebei University, China. The stress resistance of the three strains under cadmium stress in Astragalus membranaceus has been confirmed [33]. The DSE strains were cultured at 27 °C on PDA medium for 14 days before inoculation in the experiments.
Mature seeds of D. morifolium were collected from the medicinal herb planting base in Anguo, Hebei Province, China, and stored at 4 °C.

2.2. Inoculation Assay

The experiment consisted of two parts: sterile inoculation and non-sterile inoculation. Sterile conditions were designed to demonstrate the effect of a single strain on D. morifolium plants in the absence of other strains. Non-sterile conditions simulated the plant’s natural growth environment. The inoculation experiment was conducted using a completely randomized factorial design (4 DSE inoculations × 4 Cd concentrations), with three replicates. The four DSE inoculations were inoculated DSE7, DSE15, DSE20, and a non-inoculated control. The four Cd concentrations were 0, 1, 5, and 10 mg Cd/kg soil. There were three plants per pot, totaling 96 experimental pots.
Seed germination: D. morifolium seeds with a full shape were selected on a super-clean workbench, soaked in 5% sodium hypochlorite for 10 min, and rinsed with sterile water. The seeds were cultivated at 27 °C in the dark on a sterile petri dish. The seedlings with the same growth were randomly chosen for additional testing once the seeds germinated.
The soil substrate was prepared by mixing sand and soil (1:2 v/v), 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). The soil matrix was adjusted with CdCl2·2.5H2O to achieve 0, 1, 5, and 10 mg Cd/kg concentrations. For sterile inoculation, the soil was autoclaved at 121 °C for 90 min, while non-sterile inoculation used untreated soil.
Non-sterile inoculation: two 9 mm discs of pre-cultivated DSE colonies were placed in plastic pots (12 cm in height, 10 cm in bottom diameter, 17 cm in top diameter) containing 450 g of soil mixture and another 450 g of soil mixture was added to the pots. Meanwhile, the blank PDA medium was used as the control treatment. Randomly selected seedlings from each pot were planted on the soil surface, watered with 140 mL, and incubated under controlled conditions in a greenhouse: light intensity of 300 μmol m−2 s−1, relative humidity of 60%, and a photoperiod of 14 h light/10 h dark. The plants were harvested after five months of being cultivated.
Sterile Inoculation: a DSE disc (5 mm) was placed on the surface of a culture cup (15.5 cm in height, 5.5 cm in bottom diameter, 8.5 cm in top diameter) containing 200 g of sterile soil substrate, and covered with 300 g of sterile soil substrate under the sterile conditions. Randomly selected well-grown seedlings were planted on the soil substrate. A second cup the same size was inverted, the top was removed, and this was then sealed with air-permeable medical tape. In the control treatment, a blank PDA media of 5 mm diameter was used. 50 mL of sterile water was added following inoculation. All inoculation procedures were carried out in a completely sterile environment, and the pots were kept in a growth chamber (14 h photoperiod, 27 °C/22 °C (day/night), 60% mean relative humidity). The seedlings were harvested after three months.

2.3. Determination of Morphological Indicators in Plants

Following the harvest, the plant height, number of petals, flower diameter, branching number, and the number of leaves were recorded. Subsequently, the shoots and the roots from each pot were carefully separated. The root system was gently washed to remove adhering sand and soil, and the plant roots were scanned using an Epson V800 scanner (Epson, Nagano, Japan). The WinRHIZO image analysis system (Regent Instruments, Quebec, QC, Canada) was employed to assess the morphological traits such as root length, root surface area, and root volume. After scanning, the roots were collected for analysis of the DSE colonization rate. According to the method of Phillips and Hayman [34], DSE colonization was detected with clean, fresh root segments (0.5 cm). The fresh root segments were cleaned in 10% (w/v) potassium hydroxide and then stained in 0.5% (w/v) acid fuchsins. A microscopic examination was conducted on 30 randomly selected root segments from each sample at ×800 magnifications [35]. The DSE (total, mycelium, and microsclerotia) colonization rate (%) was expressed as the percentage of the number of infected root segments to the total number of root segments. DSE colonization intensity was determined by measuring the percentage of DSE presence in all observed intersections [36]. Plant biomass was assessed by measuring the dry weight of shoots and roots, which were dried at 70 °C for at least 48 h.

2.4. Measurement of Physiological and Biochemical Indicators

The chlorophyll value of each plant was determined using SPAD-502 (Konica Minolta Sensing, Osaka, Japan) and reported as SPAD values before harvesting. The chlorophyll content of a plant was determined by measuring and averaging the SPAD values of the leaves from the high, middle, and low parts of each plant. The SOD activity was extracted using the nitrotetrazolium blue chloride (NBT) photoreduction method [37].

2.5. Determination of Soil Factor Index

The soil pH value was determined using a precision pH meter (PHS-3C). The Cd mass fractions in different parts of the plants were determined using a flame atomic absorption spectrophotometer (TAS-990, Beijing Puxi Instrument Factory, Beijing, China) [16]. Soil organic carbon (SOC) was measured using the scorch mass method [38]. The soil available phosphorus (AP) was determined using the sodium bicarbonate extraction-molybdenum-antimony anti-colorimetric method [39]. The soil available nitrogen (AN) was determined using the alkaline hydrolysis-diffusion method [40].

2.6. Active Ingredient Content and IAA

The contents of chlorogenic acid (C16H18O9), 1, 5-dicaffeoylquinic acid (C25H24O12), and IAA were determined using high-performance liquid chromatography (HPLC) with the XBridge®C18 (250 mm × 4.6 mm, 5 μm, Waters Corp, Milford, MA, USA). The chlorogenic acid and 1, 5-dicaffeoylquinic acid were extracted from the flowers of D. morifolium, which were baked in an oven at 60 °C for 48 h, ground into a powder, and passed through a 40-mesh sieve. A 0.5 g dried flower sample was extracted with 25 mL of methanol using ultrasonic treatment for 40 min. The concentrated solution was obtained using a pressure rotary evaporator (Automatic Science, Shanghai, China), dissolved in 2.5 mL of methanol, and then filtered using a 0.22 μm microporous membrane to obtain the sample for testing. Chromatographic conditions: the mobile phase was a mixture of water with 0.1% phosphoric acid and acetonitrile in gradient elution. The percentage of acetonitrile was increased from 10% (v/v) at zero min to 18% at 11 min, and then increased to 20% at 30 min, and kept constant until 40 min. The detection wavelength was set at 328 nm (chlorogenic acid) and 348 nm (1, 5-dicaffeoylquinic acid). The sample size was 10 μL, the column temperature was set at 25 °C, and the flow rate was 1 mL·min−1 [41].
IAA was extracted from the plants according to the method described by Torelli et al. [42]. The extracting solution 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 PDA (photodiode array) detector. IAA was detected and quantified through the comparison of the peak retention time and area of the sample with those of the standard IAA (IAA, Sigma-Aldrich, St. Louis, MO, USA) [43].

2.7. Statistical Analysis

The growth indexes and physiological indexes of D. morifolium were analyzed using a one-way analysis of variance (ANOVA) with SPSS 21.0 software. A two-way ANOVA was performed to explore the effects of DSE inoculation treatment, heavy metal Cd stress treatment, and their interaction on biomass, morphological indexes, physiological indexes, and pharmaceutical ingredients (chlorogenic acid and 1, 5-dicaffeoylquinic acid) of D. morifolium. The biased ETA squared (ηp2) was used as a measure of the size of the share of the effect of each treatment. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Variance decomposition analysis (VPA) was performed using RStudio 1.2.5 to measure the contribution of DSE strains, different concentrations of Cd stress, and soil conditions on the growth of D. morifolium.

3. Results

3.1. Fungal Colonization

Following the harvest, the formation of DSE hyphae and micronucleus structures in the root cortex and vascular tissue was observed in all stained root segments that were sterile inoculated with DSE7, DSE15, and DSE20 (Figure S1). DSE colonization structures were not found in non-inoculated plants with DSE. The colonization rate of three DSE strains was measured under Cd stress in the roots of D. morifolium sterile-growth seedlings (Figure S2). For DSE7, the highest mycelial colonization (39.7%), microsclerotia colonization (30.2%), total colonization (39.7%), and colonization intensity (30.7%) were observed at 10 mg Cd/kg soil. The lowest mycelial colonization (19.9%), microsclerotia colonization (19.3%), and total colonization (19.9%) were observed at 1 mg Cd/kg soil. For DSE15, the highest mycelial colonization (70.1%), microsclerotia colonization (27.1%), and total colonization (70.1%) were observed at 10 mg Cd/kg soil. The lowest mycelial colonization (36.7%), total colonization (36.7%), and colonization intensity (17.8%) were observed at 0 mg Cd/kg soil, while the lowest microsclerotia colonization was 18.0% at 1 mg Cd/kg soil. For DSE20, the highest mycelial colonization (59.2%) and total colonization (59.2%) were observed at 5 mg Cd/kg soil, while the highest colonization intensity was 32.3% at 10 mg Cd/kg soil. The lowest mycelial colonization (20.2%), microsclerotia colonization (30.4%), total colonization (20.2%), and colonization intensity (13.5%) were observed at 0 mg Cd/kg soil. Maximal levels of mycelial, microsclerotia, and total colonization observed at 10 mg Cd/kg soil were significantly higher than those at 0 mg Cd/kg soil. However, the mycelial, microsclerotia, and total colonization showed no significant differences among 0 and 1 mg Cd/kg soil for DSE7 and DSE15.

3.2. Plant Morphological Parameter

The morphological parameter of D. morifolium was considerably affected by the interaction between fungal inoculation and Cd stress (Tables S1 and S2). Compared with CK, DSE15 exerted a considerable influence on shoot biomass, total biomass, and root biomass under Cd stress, regardless of sterile or non-sterile conditions (Figure 1 and Figure 2). The influence was alleviated by fungal non-sterile inoculation, although the addition of Cd had a negative influence on the biomass of the D. morifolium (Figure 2). Considering the flowers are the primary source of the bioactive compounds in the D. morifolium we tested, studying the flowers is essential. Figure S3 displays the morphology of the flowers. DSE7 and DSE15 non-sterile inoculation significantly increased the flower biomass under Cd stress.
At 1 and 5 mg Cd/kg soil, DSE15 significantly enhanced the plant height and root volume compared to CK under sterile and non-sterile application (Table 1, Figure 3). DSE20 treatment also enhanced the plant height compared to CK under sterile and non-sterile applications. The sterile application of DSE7 resulted in the enhancement of both plant height and root length under Cd stress. The difference is that the non-sterile inoculation of DSE7 conditions significantly promoted root length (Figure 3e). Moreover, DSE15 non-sterile treatment significantly increased petal number, and DSE7 non-sterile treatment also enhanced petal number and flower diameter under Cd stress (Figure 3c,d).

3.3. Plant Physiological Parameter and Active Ingredient

A two-way ANOVA revealed that both DSE inoculation and Cd stress, as well as their interactions, had a significant impact on SOD, and DSE inoculation had a significant impact on chlorophyll (Tables S1 and S2). Three DSE strains resulted in a marked enhancement of SPAD values under sterile conditions (Figure 4a). In the absence of Cd, DSE7 and DSE15 non-sterile inoculation significantly enhanced the SPAD values, with DSE7 showing no significant difference under sterile conditions. Moreover, the three DSE strains’ sterile inoculation resulted in increased SOD activity in D. morifolium seedlings, with the highest value observed in the 5 mg Cd/kg treatment (Figure 4d). Despite Cd stress, DSE15 sterile treatment significantly increased SOD content under sterile conditions.
DSE species, Cd stress, and their interaction had significant effects on the IAA concentration in the D. morifolium (Tables S1 and S2). Regardless of Cd stress, both DSE7 and DSE15 markedly elevated IAA levels in comparison to the uninoculated controls under sterile and non-sterile conditions (Figure 4b and Figure 5b). Importantly, chlorogenic acid was mainly affected by DSE species, while 1, 5-dicaffeoylquinic acid was significantly affected by Cd stress (Table S2). In the absence of Cd, the DSE15 non-sterile inoculation significantly enhanced chlorogenic acid. At 1 mg Cd/kg soil, DSE7 and DSE15 non-sterile inoculation promoted the content of 1, 5-dicaffeoylquinic acid by 18.29% and 21.70% (Figure 5d). At 5 mg Cd/kg soil, the DSE15 non-sterile inoculation significantly enhanced the chlorogenic acid content by 25.7% (Figure 5c).

3.4. Soil Properties and Cd Concentrations

The two-way ANOVA of the variance of soil factors revealed that DSE species had significant effects on soil organic carbon, available phosphorus, and available nitrogen in D. morifolium non-sterile soil. DSE species had a main effect on organic carbon content (p < 0.01, ηp2 = 0.339). Cd stress treatment expressed the greatest contribution rate to soil available phosphorus (p < 0.001, ηp2 = 0.647), and showed a significant effect on soil organic carbon, available nitrogen, organic carbon, and pH. The interaction between DSE species and Cd stress had a significant effect on the soil factors of D. morifolium (Table S2). Compared with the control group, the inoculation of DSE15 significantly reduced soil available nitrogen content without Cd stress. Under Cd stress, the DSE15 inoculation enhanced soil organic carbon content, while the inoculation of DSE7 and DSE15 reduced soil available nitrogen content (Figure S4).
As the concentration of Cd treatment increased, the cadmium levels at each site exhibited a consistent upward trend, irrespective of fungal inoculation (Figure 4c and Figure 6). DSE15 treatment significantly increased the Cd content of roots in both sterile and non-sterile conditions (Figure 4c and Figure 6a). Similarly, DSE15 non-sterile treatment also significantly enhanced the Cd content of stems and leaves, while DSE7 significantly increased the Cd content of roots 1 and 5 mg Cd/kg soil (Figure 6a). Conversely, the DSE20 non-sterile inoculation significantly decreased the Cd content of roots, while DSE20 increased the Cd transfer coefficient of flowers under the highest Cd concentration treatment (Figure 6a,c). In addition, in the DSE7 non-sterile inoculation into D. morifolium plants, the Cd transfer coefficient of flowers was reduced to 1 and 5 mg Cd/kg soil (Figure 6d).

3.5. Variance Decomposition of Different Factors on the Growth of D. morifolium Under Non-Sterile Inoculation

VPA was used to quantify the association between DSE species, Cd stress, soil nutrients, and plant growth (Figure 7). The growth indices of D. morifolium were 8.6%, 12.6%, and 6.6% for DSE species, Cd stress and soil factors, respectively. The co-explanatory amount of Cd stress and DSE species was 19.3%, and 6.5% with soil factors. DSE species and Cd stress explained 12.7% and 16.2% of the physiological indices of D. morifolium, respectively. DSE species and Cd stress together explained 8.3%, while soil factors together explained 7.6%, and Cd stress and soil factors together accounted for 4.8%. Hence, DSE species and Cd stress had major effects on plant growth.

4. Discussion

4.1. Effect of DSE Strains on Biomass

The root system, as the primary organ responsible for absorbing nutrients and water, is particularly susceptible to the impact of both beneficial and harmful environmental elements [44]. In both the sterile and non-sterile inoculation study of D. morifolium, it was observed that under different amounts of Cd stress, the root biomass of inoculated DSE15 was promoted more than that of the uninoculated. The root growth of host plants inoculated with DSE fungi indicates the result of improved nutrient absorption of the root [45,46]. Furthermore, DSE fungal infestation most likely alters the morphology and architecture of the roots, promoting the development of fibrous and adventitious roots [47]. For example, in our study, at 1 and 5 mg Cd/kg soil, DSE15 significantly enhanced the root volume compared to CK under sterile and non-sterile conditions. Similarly, Zhou et al. [48] discovered dark septate endophyte inoculation significantly increased root volume under Cd stress.
In the present study, when compared with the uninoculated, the non-sterile inoculation of DSE7 and DSE15 demonstrated promotive effects on the biomass of the medicinal parts (flowers). For instance, DSE7 and DSE15 non-sterile inoculation significantly increased the flower biomass under Cd stress. Li et al. [49] and Zhai et al. [50] found that microorganisms were used to promote the biomass of Isatis indigotica and Salvia miltiorrhiza. Consequently, employing DSE to enhance the biomass of medicinal parts may emerge as one of the effective strategies for improving the yield of Chinese medicinal materials.

4.2. Effect of DSE Inoculation on Physiological Parameters and Active Ingredients

Heavy metals are known for their detrimental effects on plant growth, leading to a decrease in transpiration and photosynthetic efficiency, thereby disrupting the photosynthetic process [51]. Likar and Regvar [52] reported that the inoculation of DSE into Salix caprea significantly can increase its chlorophyll content and transpiration rate. Our findings corroborated this observation, where DSE7 non-sterile inoculation significantly enhanced the SPAD values in the absence of Cd. However, DSE7 showed no significant difference under sterile conditions. One possible explanation is the synergistic effect of other bacterial species present in non-sterile soil with DSE7 in promoting chlorophyll growth. Furthermore, endophytic fungi can modulate reactive oxygen species and antioxidant activity in plants, thereby increasing photosynthesis in plant leaves [53]. In our study, under sterile conditions, Cd stress treatment inoculated with DSE15 significantly increased SOD content in D. morifolium compared to the non-inoculated treatment. We have reason to speculate that endophytic fungi in roots can assist plants in coping with and adapting to heavy metal pollution environments by regulating plant antioxidant enzyme activities [54]. When encountering adverse environmental stressors, plants can activate their superoxide dismutase (SOD) activity to catalyze the oxidation of ROS, thereby mitigating the deleterious impacts of free oxygen radicals, as documented by Collin-Hansen et al. [55] and Hamilton et al. [53]. Moreover, over-expressed Mn-SOD in GM Arabidopsis and tomato exhibited higher tolerance [56].
IAA is an essential endogenous growth regulator in plants and plays a crucial role in plant growth and development [57,58]. Coincidentally, DSE itself can secrete IAA and other plant hormones, directly stimulating the root growth, nutrient uptake, and plant growth of host plants under heavy metal stress [59,60,61]. In our study, at 1 and 5 mg Cd/kg soil, both DSE15 markedly elevated IAA levels while promoting the plant height and root volume under sterile and non-sterile conditions. Due to the consideration of the influence of other strains in non-sterile soil, the same promotive effects were produced under sterile and non-sterile conditions. One possible theory is that DSE15 plays a dominant role in promoting IAA hormones undisturbed by other strains or has a synergistic effect with other strains in non-sterile soil. Research has indicated that synergies between bacteria promote soybean growth, physiological and biochemical conditions, and reduce the transfer of nickel to the edible part of faba bean plants [62]. Consequently, using the synergistic effects of different strains of medicinal plants has emerged as a useful tactic.
As a special type of plant, medicinal species primarily serve as the raw materials for traditional Chinese herbal medicines and various therapeutic agents in the pharmaceutical sector [63]. To gain further insights into the application of DSE on medicinal plants, we analyzed the promotion ability of DSE in the contents of medicinal ingredients. In our study, DSE7 and DSE15 non-sterile inoculation promoted the content of 1, 5-dicaffeoylquinic acid by 18.29% and 21.70% in flowers at Cd content (1 mg/kg). Extant studies support the notion that the inoculation of DSE into the medicinal plant Epimedium significantly increased the biomass of the plant, and the total flavonoids and patchouli glycosides increased by 42.15% and 20.24%, respectively [64]. In addition, studies have indicated that the inoculated endophyte fungi can enhance the content of most geranium essential oil constituents and the secretion and accumulation of organic acids and mineral absorption in rice [13,25]. Xing et al. [65] reported that the live fungus and its mycelial extract of the endophytic fungus (Chaetomium globosum D38) significantly upregulated the production of tanshinone and the transcriptional activity of those key genes in the tanshinone biosynthetic pathway. The interaction between plants and endophytic fungi involves a cooperative relationship mediated by the chemical defense of plants [66]. The colonization process of fungi can trigger a series of defense responses in host plants, such as the enhancement of secondary metabolism like flavonoids [67]. Furthermore, Wu et al. [68] and Wang et al. [69] found that changes in secondary metabolites as a result of either the plant’s genetic regulation in response to unfavorable environments or the plant’s growth-promoting effect of extracellular metabolites secreted by DSE in soil.

4.3. Effect of DSE Strains on Cd Absorption

For our focus on medicinal plants, the accumulation of Cd in the medicinal parts is more worthy of attention. In our non-sterile inoculation study, DSE7 was inoculated into D. morifolium plants, the Cd transfer coefficient of flowers was reduced to 1 and 5 mg Cd/kg soil, and the transfer of Cd to medicinal parts was inhibited. This phenomenon is beneficial to our research on the medicinal plant D. morifolium. In general, DSE colonization can alter the translocation of heavy metals from the roots to shoots of host plants [70]. Similarly, Chen et al. [18] found that the Cd transport coefficient of maize significantly decreased by 26.3% under 20 mg·kg−1 of Cd stress with DSE inoculation treatment. Moreover, Li et al. [33] found Paraphoma chlamydocopiosa also can decrease the Cd translocation factor of Astragalus mongholicus. Studies have demonstrated that DSE colonization reduced the translocation of Cd to above ground by increasing the binding of Cd to pectins and proteins in the roots [71]. In addition, Xiao et al. [19] suggested that carboxyl, hydroxyl, and acidic groups were involved in the retention of Cd in the cell wall of roots. It has also been shown that host plants store large amounts of Cd in the cell wall and convert Cd into inactive or insoluble forms, which leads to a greater accumulation of inactive or insoluble forms of Cd in roots [18,71,72]. In addition, it has been shown that the endophytic fungus (Sphingomonas SaMR12) increases GSH by upregulating the expression of related genes, which increases Cd accumulation and tolerance in host plants [73]. Thus, the complex mechanisms related to DSE’s promotion of plant growth and tolerance to heavy metal stress need further clarification. Due to the wide application of medicinal plants in medicine, the accumulation of Cd in the medicinal parts should be taken into account when evaluating the growth promotion of DSE inoculation.

4.4. Effect of DSE Strains on Soil Factors

As a bridge between plants and the soil, DSE has been determined to convert soil organic carbon and nitrogen and insoluble phosphorus mineralization into effective forms, which can greatly expand plant organic nutrient pools [74,75]. Our findings were the opposite in that the DSE15 inoculation enhanced soil organic carbon content, while the inoculation of DSE7 and DSE15 reduced soil available nitrogen content under natural conditions and Cd stress treatments. Our research also revealed some variations in the growth-promoting properties of DSE in sterile and natural environments. A plausible explanation for this can be the influence exerted by other co-existing strains present in the soil [76]. Pu et al. [77] found that Rhizophagus irregularis significantly decreased the soil-dissolved SOC content of both types of MPs, whereas the combined inoculation of Rhizophagus irregularis and Bacillus velezens SC60 significantly increased it under Cd stress conditions. It can be seen that the effect of other strains in the soil should be considered in the practical application of strains.

5. Conclusions

This study investigated the interactions between D. morifolium in sterile or non-sterile inoculations and three dark septal endophytes, DSE7, DSE15, and DSE20, isolated from the roots of medicinal plants. In the present study, in both sterile and non-sterile inoculations, DSE15 showed positive effects on the biomass, plant height, root development, and IAA growth of D. morifolium under Cd stress compared with non-inoculated treatments. We conclude that DSE15 has a potent capacity to stimulate growth and is impervious to outside influences or a synergistic effect with other strains. However, the growth of DSE7 in sterile and non-sterile conditions showed some differences, which were probably related to the interaction with other microorganisms such as fungi and bacteria in non-sterile soils. Our study first showed the stimulating impact of DSE on the medicinal parts of D. morifolium under Cd stress. This finding provides a basis and strategy for cultivating medicinal plants in soils contaminated with heavy metals. Building upon this basis, further research will be conducted on the effects of DSE inoculation on microbial community dynamics or molecular pathways, which will be further studied.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15010208/s1. Table S1. Two-way ANOVA of the impacts of DSE inoculation, Cd stress, and their interactions on the growth of D. morifolium sterile seedlings. Table S2. Two-way ANOVA of the impacts of DSE inoculation, Cd stress, and their interactions on the growth and soil factors of D. morifolium non-sterile seedlings. Figure S1. Characteristics of DSE microsclerotia and mycelium structure of D. morifolium. H = DSE hyphae, M = DSE microsclerotia, bars = 50 μm. a–d was the DSE colonization structure in D. morifolium. Figure S2. Effect of DSE sterile colonization on mycelial colonization rate (a), microsclerotia colonization rate (b), total colonization rate (c), and colonization intensity (d) of three DSEs inoculation and Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 by Duncan’s test. Different lowercase letters indicate the significant difference among different Cd stress and the same DSE species. Figure S3. The morphology of the flowers of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). Figure S4. Effect of DSE colonization on soil organic carbon (a), soil available nitrogen (b), soil available phosphorus (c), and pH (d) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil).

Author Contributions

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

Funding

This research was supported by the National Natural Science Foundation of China (No. 22373030).

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.

Acknowledgments

We gratefully thank Yijin Wang and Jiawang He for their assistance in data curation.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 15 00208 g001
Figure 1. Effect of DSE colonization on shoot biomass (a), root biomass (b), total biomass (c), and root:shoot ratio (d) of D. morifolium sterile seedlings under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 1. Effect of DSE colonization on shoot biomass (a), root biomass (b), total biomass (c), and root:shoot ratio (d) of D. morifolium sterile seedlings under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 2. Effect of DSE colonization on shoot biomass (a), root biomass (b), total biomass (c), flower biomass (d), and root:shoot ratio (e) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 2. Effect of DSE colonization on shoot biomass (a), root biomass (b), total biomass (c), flower biomass (d), and root:shoot ratio (e) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 3. Effect of DSE colonization on plant height (a), number of leaves (b), number of petals (c), flower diameter (d), root length (e), and root volume (f) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 3. Effect of DSE colonization on plant height (a), number of leaves (b), number of petals (c), flower diameter (d), root length (e), and root volume (f) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 4. Effect of DSE colonization on SPAD value (a), IAA (b), Cd content of roots (c), and superoxide dismutase (SOD) (d) activities of D. morifolium sterile seedlings under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 4. Effect of DSE colonization on SPAD value (a), IAA (b), Cd content of roots (c), and superoxide dismutase (SOD) (d) activities of D. morifolium sterile seedlings under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 5. Effect of DSE colonization on SPAD value (a), IAA content of roots (b), chlorogenic acid (c), and 1, 5-dicaffeoylquinic (d) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 5. Effect of DSE colonization on SPAD value (a), IAA content of roots (b), chlorogenic acid (c), and 1, 5-dicaffeoylquinic (d) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 6. Effect of DSE colonization on Cd content of root (a), Cd content of stems and leaves (b), Cd content of flower (c), and Cd transfer coefficient of flower (d) (Cd content of flower/Cd content of root) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
Figure 6. Effect of DSE colonization on Cd content of root (a), Cd content of stems and leaves (b), Cd content of flower (c), and Cd transfer coefficient of flower (d) (Cd content of flower/Cd content of root) of D. morifolium non-sterile plants under Cd stress (0, 1, 5, and 10 mg Cd/kg soil). The error bars represent the standard error of the mean. Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Figure 7. Variation partitioning of different factors on the growth of D. morifolium non-sterile plants. Note: growth index of D. morifolium (a), physiological index of D. morifolium (b), DSE: DSE species; Cd: Cd stress; soil: soil factors.
Figure 7. Variation partitioning of different factors on the growth of D. morifolium non-sterile plants. Note: growth index of D. morifolium (a), physiological index of D. morifolium (b), DSE: DSE species; Cd: Cd stress; soil: soil factors.
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Table 1. The effects of DSE species and Cd stress treatment on the morphology of D. morifolium sterile seedlings.
Table 1. The effects of DSE species and Cd stress treatment on the morphology of D. morifolium sterile seedlings.
SpeciesDifferent TreatmentPlant Height
(cm)		Leaf NumberBranches NumberRoot Length
(mm)		Root Volume (cm3)
CK0 mg/kg20.33 ± 3.07 Aa30.33 ± 2.34 Aa1.00 ± 0.00 Aab673.22 ± 63.03 Aa0.37 ± 0.02 Ba
1 mg/kg20.33 ± 3.43 Ba30.00 ± 6.04 ABa1.00 ± 0.00 Aab593.37 ± 28.99 ABab0.36 ± 0.02 Ba
5 mg/kg19.30 ± 3.83 Ba34.33 ± 3.47 ABa1.33 ± 0.13 ABa503.28 ± 55.79 ABb0.32 ± 0.01 Bab
10 mg/kg16.00 ± 2.39 ABab22.00 ± 5.79 Bb1.00 ± 0.00 Aab493.25 ± 39.28 ABb0.28 ± 0.06 Bb
DSE70 mg/kg22.32 ± 2.01 Aab27.67 ± 3.11 Aa1.00 ± 0.00 Aa637.44 ± 24.38 ABb0.56 ± 0.03 Aa
1 mg/kg26.50 ± 1.25 Aa30.33 ± 3.74 ABa1.00 ± 0.00 Aa783.27 ± 39.53 Aa0.39 ± 0.08 Bab
5 mg/kg23.11 ± 3.47 ABab29.33 ± 2.07 Ba1.00 ± 0.00 ABa624.50 ± 35.02 Ab0.37 ± 0.04 Bab
10 mg/kg20.33 ± 4.91 Ab29.03 ± 3.77 Aa1.00 ± 0.00 Aa572.60 ± 39.47 Ab0.33 ± 0.06 Bb
DSE150 mg/kg21.43 ± 2.19 Aab33.33 ± 4.21 Ab1.00 ± 0.00 Aab703.70 ± 58.34 Aa0.41 ± 0.07 Bb
1 mg/kg25.89 ± 2.21 Aa37.66 ± 4.72 Aa1.00 ± 0.00 Aab634.44 ± 45.57 ABab0.89 ± 0.03 Aa
5 mg/kg28.20 ± 5.53 Aa42.00 ± 3.01 Aa1.33 ± 0.13 ABa535.50 ± 78.45 ABb0.79 ± 0.10 Aa
10 mg/kg19.49 ± 3.45 Ab20.00 ± 1.89 Bc1.00 ± 0.00 Aab504.84 ± 56.44 ABb0.57 ± 0.12 Ab
DSE200 mg/kg22.67 ± 3.53 Aab29.33 ± 4.22 Aa1.00 ± 0.00 Aa654.33 ± 48.33 Aa0.45 ± 0.04 ABb
1 mg/kg26.77 ± 3.88 Aa29.00 ± 5.90 Ba1.00 ± 0.00 Aa509.33 ± 59.33 Bb0.81 ± 0.11 Aa
5 mg/kg22.80 ± 4.94 Bab30.33 ± 4.45 Ba1.00 ± 0.00 ABa612.89 ± 58.34 Aab0.28 ± 0.06 Bc
10 mg/kg19.30 ± 2.19 Ab25.67 ± 4.33 ABab1.00 ± 0.00 Aa572.54 ± 44.33 Aab0.23 ± 0.04 Bc
Different letters above the error bars indicate a significant difference at p < 0.05 using a Duncan’s test. Different uppercase letters indicate significant differences between different strains of the same Cd treatment, and different lowercase letters indicate significant differences between different Cd treatments of the same strain.
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Wu, M.; Li, G.; Wang, S.; Wang, Z.; Li, L.; Han, L. The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress. Agronomy 2025, 15, 208. https://doi.org/10.3390/agronomy15010208

AMA Style

Wu M, Li G, Wang S, Wang Z, Li L, Han L. The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress. Agronomy. 2025; 15(1):208. https://doi.org/10.3390/agronomy15010208

Chicago/Turabian Style

Wu, Meiling, Gen Li, Simiao Wang, Ziteng Wang, Longfei Li, and Li Han. 2025. "The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress" Agronomy 15, no. 1: 208. https://doi.org/10.3390/agronomy15010208

APA Style

Wu, M., Li, G., Wang, S., Wang, Z., Li, L., & Han, L. (2025). The Promotion of Dark Septate Endophytes on the Performance and Active Ingredients Accumulation of Dendranthema morifolium Under Cd Stress. Agronomy, 15(1), 208. https://doi.org/10.3390/agronomy15010208

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