Concentration-Dependent Effects of Penicillium oxalicum on Leaf Physiology and Secondary Metabolite Accumulation in Eleutherococcus senticosus

Abstract

Objective

This study investigates the physiological and biochemical responses of E. senticosus leaves to varying concentrations of P. oxalicum.

Methods

Spore suspensions of P. oxalicum at 10³ (CL), 10⁴ (CM), and 10⁵ (CH) CFU/mL were applied via foliar spraying of E. senticosus leaves. Growth indices (fresh weight, dry weight, and Leaf drying rate), photosynthetic pigments (chlorophyll a, chlorophyll b, and carotenoids), carbohydrate metabolites (soluble sugars and starch), antioxidant enzyme activities, and chlorophyll fluorescence parameters were quantified. Additionally, total phenolic and flavonoid contents were measured.

Results

CM treatment significantly increased leaf biomass, whereas CL treatment increased pigment accumulation, photosynthetic efficiency, and antioxidant enzyme activities. The contents of soluble sugars, starch, proteins, chlorophyll fluorescence, phenolics, and flavonoids also increased. In contrast, CH treatment suppressed several parameters.

Conclusion

P. oxalicum induced a concentration-dependent biphasic response in E. senticosus. Lower concentrations promoted growth, photosynthetic function, and antioxidant capacity, whereas higher concentrations led to physiological inhibition. These findings emphasize the critical ability of spore concentration to modulate physiological processes and offer insights into strategies that can help improve the quality of medicinal plants.

Introduction

Endophytic fungus–plant interactions are now a major focus of ecological and biotechnological research.^1,2 Endophytic fungi can colonize internal plant tissues asymptomatically, forming stable symbiotic associations.^3,4 These fungi effectively regulate the plant life cycle by altering developmental pathways and enhancing stress adaptation.^5,6 Endophytes secrete hormones, enzymes and metabolites that modulate host physiological processes.^7,8 Endophytic fungi also improve nutrient uptake and stress tolerance, and they modulate specialized metabolic pathways, thereby promoting plant growth.^9,10 Thus, endophytic fungi are promising bioinoculants that can increase medicinal plant growth and bioactive metabolite production.

Penicillium oxalicum (P. oxalicum), a member of the order Eurotiales, family Aspergillaceae, and genus Penicillium, is an endophytic fungus isolated from the roots, stems, and leaves of Eleutherococcus senticosus (E. senticosus). ¹¹ The internal transcribed spacer (ITS) sequence has been submitted to GenBank under accession number KX674635.1. The fungal strain is preserved in the Department of Traditional Chinese Medicine Resources and Development, Heilongjiang University of Chinese Medicine. Numerous studies have confirmed the ability of this fungus to stimulate root proliferation, increase shoot vigor, increase biomass accumulation, and improve nutrient uptake efficiency, all of which can help increase host plant development. ¹² In addition to its growth-promoting effects, P. oxalicum has been reported to upregulate chlorophyll biosynthesis in plants, thereby enhancing photosynthetic efficiency. ¹³ It can also activate key antioxidant enzymes in the host, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), which collectively enhance plant defense systems against oxidative stress by scavenging reactive oxygen species (ROS). ¹⁴ P. oxalicum also induces phenolic/flavonoid biosynthesis, improving host medicinal value. ¹⁵ However, its effects on the leaves of E. senticosus remain poorly understood. Therefore, targeted studies are needed to elucidate the role played by P. oxalicum in regulating growth dynamics, photosynthetic performance, antioxidant responses, and secondary metabolism in this pharmacologically important species.

E. senticosus, commonly known as Siberian ginseng or Ciwujia, is a traditional Chinese medicinal herb renowned for its broad spectrum of pharmacological activities.^16,17 Its leaves are rich in pharmacologically active constituents, including eleutherosides, ¹⁸ triterpenoid saponins, ¹⁹ flavonoids, and phenolic compounds.^20,21 These bioactive metabolites confer diverse pharmacological properties to E. senticosus, including cardioprotective and neuroprotective effects, ²² anticancer activity, ²³ sedative and hypnotic effects, ²⁴ and strong antioxidant and anti-inflammatory activities. ²⁵ Collectively, these activities render this herb a promising therapeutic agent for managing conditions such as ischemic stroke, chronic fatigue syndrome, and oxidative stress-related disorders.^26,27 Given the global demand for natural health products, evidence-based strategies are needed to improve E. senticosus cultivation.

To elucidate the complex bidirectional dynamics of plant–fungus symbiosis, we conducted a systematic investigation of the morphological and physiological responses of E. senticosus leaves to foliar applications of various concentrations of P. oxalicum spore suspensions. This work advances our foundational knowledge of plant–endophyte interactions and offers translational insights that have potential applications in sustainable agriculture and medicinal plant biotechnology. Importantly, the findings of this study may facilitate the development of biostimulant-based cultivation strategies to increase leaf biomass and enhance the pharmacological quality of E. senticosus.

Materials and Methods

Plant Materials and Growth Conditions

Two-year-old E. senticosus seedlings were obtained from Yichun city, Heilongjiang Province, China. Voucher specimens were taxonomically authenticated by Professor Shenglei Guo from the Department of Traditional Chinese Medicine Resources, Heilongjiang University of Chinese Medicine. All the experiments were conducted in a climate-controlled greenhouse located at the Medicinal Botanical Garden of Heilongjiang University of Chinese Medicine. In early April 2023, the seedlings employed in this research were transplanted into plastic pots (20 cm in diameter) filled with 3 kg of field-collected soil. A single seedling was placed in each pot to ensure uniform growth conditions and minimize interference between plants.

Preparation of the Endophytic Fungal Spore Suspension

The P. oxalicum strain was aseptically inoculated onto potato dextrose agar (PDA) slants and incubated at 28 °C for three days. After incubation, 5 mL of sterile double-distilled water was added to each slant tube, and the spore-laden mycelia were dislodged using a sterile inoculation loop. The suspension was mixed and subsequently filtered four times through sterile paper to clarify the spores. Preliminary tests revealed that 10² CFU/mL had no effect, whereas 10⁶ CFU/mL caused phytotoxicity (chlorosis/necrosis in 15 days). On the basis of these findings, three effective concentrations (10³, 10⁴, and 10⁵ CFU/mL) were prepared via aseptic serial dilution. These concentrations were designated low-dose (CL: 10³ CFU/mL), medium-dose (CM: 10⁴ CFU/mL), and high-dose (CH: 10⁵ CFU/mL) in subsequent experiments. ²⁸

Coculture Experiment

In early May 2023, Sterilized soil, which was prepared via dry heat treatment at 121 °C for 2 h, ²⁹ was mixed with perlite at a 3:1 (v/v) ratio and transferred into polyethylene pots. Uniform two-year-old E. senticosus seedlings were transplanted into the pots once the soil reached field capacity. To prevent contamination, a 1-cm-thick layer of sterile soil was added to the surface of each pot.

The experiment consisted of four treatment groups, each of which consisted of six biological replicates, including three technical replicates per biological replicate. The experiment thus included a negative control group (CK) and three fungal treatment groups corresponding to different P. oxalicum spore concentrations: CL, CM, and CH. One week after transplantation, 125 mL of the indicated spore suspension was applied to each treatment group at 8:30 am, whereas the control group received 125 mL of sterile distilled water. The treatments were repeated once per week. Leaf samples were collected for 30 days after treatment, starting from the third node downward, and were subsequently used in the physiological and biochemical analyses.

Measurement of Growth and Physiological Indices

Leaf area was measured with the assistance of Adobe Photoshop software, in line with the methods described in a previous study. ³⁰ The fresh and dry weights of the leaves were measured using an electronic analytical balance. The leaf drying rate was calculated as follows:

$$\mathbf{L}\mathbf{e}\mathbf{a}\mathbf{f}\mathbf{d}\mathbf{r}\mathbf{y}\mathbf{i}\mathbf{n}\mathbf{g}\mathbf{r}\mathbf{a}\mathbf{t}\mathbf{e}(\text{\%})=(\mathbf{L}\mathbf{e}\mathbf{a}\mathbf{f}\mathbf{d}\mathbf{r}\mathbf{y}\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}/\mathbf{L}\mathbf{e}\mathbf{a}\mathbf{f}\mathbf{f}\mathbf{r}\mathbf{e}\mathbf{s}\mathbf{h}\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t})\times \mathbf{100}\text{\%}.$$

A LI-6400 portable photosynthesis system (LI-COR, USA) was used to measure the photosynthetic parameters. ³¹ Chlorophyll fluorescence parameters were assessed using a PAM-2500 portable chlorophyll fluorescence analyzer (Walz, Germany) following a 30-min dark adaptation period. ³² The starch and soluble sugar contents were quantified using the anthrone method, ³³ whereas the soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method. ³⁴ Enzyme activity assays were performed according to the manufacturers’ protocols with the assistance of commercial kits (Wuhan Eddichem Biotechnology Co., Ltd, China).^35–37 The following physiological indices were measured: acid phosphatase (ACP), SOD, CAT, POD, ascorbate peroxidase (APX), glutathione reductase (GR), malate dehydrogenase (MDH), nitrate reductase (NR), and malondialdehyde (MDA).

Data Processing

A single-factor completely randomized experimental design was employed in this study, which focused on four treatment groups (CK, CL, CM, and CH), each of which was associated with six biological replicates (individual plants) and three technical replicates per biological replicate. Data analysis was performed via SPSS 22.0 software (IBM Corp., USA). Data normality was assessed on the basis of the Shapiro–Wilk test, and homogeneity of variance was evaluated using Levene's test (P > 0.05). A one-way analysis of variance (ANOVA) was used for post hoc comparisons, followed by Duncan's multiple range test. All the results are expressed as the means ± standard errors (SEs). Graphs were generated with the assistance of GraphPad Prism 7.0 software (GraphPad Software, USA).

Results

Effects on the Growth of E. senticosus Leaves

To evaluate the concentration-dependent effects of P. oxalicum, we first examined the fundamental growth parameters. Compared with the CK treatment, all the fungal treatments increased the leaf fresh/dry weight and leaf drying rate (P < 0.01; Table 1). The CM group exhibited the highest values for leaf fresh weight (6.7270 ± 0.7473 g), dry weight (2.5683 ± 0.2373 g), and Leaf drying rate (38.18 ± 0.97), indicating that this concentration might be optimal for biomass accumulation. However, the leaf area indicated a different trend, such that the highest value was observed in the CK group (224.8229 ± 11.6137 mm²), whereas the lowest value was observed in the CH group (142.5508 ± 3.3292 mm²). These results suggested that the fungus-induced increase in biomass might favor dry matter accumulation over leaf expansion, thus providing a basis for understanding how P. oxalicum influences resource allocation in E. senticosus.

Table 1.

Effects on Growth Parameters (x ± s, n = 6).

Groups	Leaf fresh weight (g)	Leaf dry weight (g)	Leaf drying rate (%)	Leaf area (mm2)
CK	3.3530 ± 0.3871	0.7048 ± 0.0458	21.01 ± 0.53	238.2287 ± 4.2875
CL	6.1940 ± 0.3748**	2.1053 ± 0.2029**	33.99 ± 2.10**	224.8229 ± 11.6137**
CM	6.7270 ± 0.7473**	2.5683 ± 0.2373**	38.18 ± 0.97**	196.8717 ± 4.5199**
CH	4.4083 ± 0.1164**	1.2678 ± 0.1250**	28.76 ± 2.76**	142.5508 ± 3.3292**

Note: CK: distilled water; CL: P. oxalicum (10³CFU/mL); CM: P. oxalicum spore suspension (10⁴CFU/mL); CH: P. oxalicum spore suspension (10⁵CFU/mL). Group comparisons were performed on the basis of a one-way ANOVA with Duncan's HSD test for multiple comparisons. Data: mean ± SD (n = 6). *P < 0.05, **P < 0.01.

Effects on the Photosynthetic Pigment Content

An analysis of photosynthetic pigments revealed potential mechanisms through which P. oxalicum might increase carbon assimilation (Figure 1A-E). The chlorophyll a and b contents were highest in the CL group (3.77 ± 0.12 mg/g (Figure 1A) and 1.40 ± 0.02 mg/g (Figure 1B), respectively), which exhibited a 68% increase in total chlorophyll content in comparison with the CK group (Figure 1D). The highest chlorophyll a/b ratio (2.69 ± 0.04) was detected in the CL group, suggesting better PSII light-harvesting efficiency in this context (Figure 1C). The carotenoid content, which plays a crucial photoprotective role, peaked in the CM group (0.48 ± 0.006 mg/g, Figure 1E), suggesting a balance between light utilization and oxidative stress mitigation at moderate fungal concentrations. These pigment changes may drive improvements in functional photosynthesis.

Figure 1.

Effects on the Photosynthetic Pigments, Photosynthetic Physiology and Chlorophyll Fluorescence Parameters. (a-e) Photosynthetic Pigments; (f-i) Photosynthetic Physiology Parameters; (j-m) Chlorophyll Fluorescence Parameters. CK: Distilled Water; CL: P. oxalicum (10³ CFU/mL); CM: P. oxalicum (10⁴ CFU/mL); CH: P. oxalicum (10⁵ CFU/mL). Group Comparisons Were Performed on the Basis of a One-Way ANOVA with Duncan's HSD Test for Multiple Comparisons. Data: Mean ± SD (n = 6). *P < .05, **P < .01.

Effects on Photosynthetic Parameters

Photosynthetic performance exhibited a biphasic response to the fungal concentration, which is consistent with the central hypothesis proposed in this study. The net photosynthesis rate (Pn) demonstrated a concentration-dependent response (P < 0.01). The Pn increased by 46.3% and 22.1% in the CL and CM groups, respectively, in comparison with the CK group (5.469 ± 0.527 μmol·m⁻²·s⁻¹), but it decreased by 31.9% in the CH group (Figure 1F). A similar trend was observed for stomatal conductance (Gs) (P < 0.01). In comparison with the CK group, Gs in the CL and CM groups increased by 30.0% and 12.0%, respectively (0.056 ± 0.003 mol·m⁻²·s⁻¹), whereas this factor decreased by 31.9% in the CH group (Figure 1G). The transpiration rate (Tr) also significantly increased in the CL and CM groups by 50.0% and 42.0%, respectively, in comparison with the CK group (1.329 ± 0.083 mmol·m⁻²·s⁻¹; P < 0.01; Figure 1H). In contrast, the intercellular CO₂ concentration (Ci) significantly decreased in all P. oxalicum-treated groups in comparison with the CK group. Specifically, Ci decreased by 25.6% and 23.5% in the CL and CM groups, respectively, and by 10.6% in the CH group in comparison with the CK group (1.329 ± 0.083 µl·L⁻¹; Figure 1I; P < 0.05). P. oxalicum dose-dependently modulated photosynthesis; namely, it enhanced carbon fixation at low-moderate doses but triggered stress at high doses.

Effects on the Chlorophyll Fluorescence Kinetic Parameters

Fluorescence parameter measurements provided further validation for the photosynthetic adjustments, indicating enhanced photochemical efficiency at beneficial fungal concentrations. The CL group exhibited reduced initial fluorescence (Fo), suggesting decreased nonphotochemical quenching, whereas the CM group exhibited increased maximum fluorescence (Fm), indicating augmentation of tis light absorption capacity (Figure 1J-K). Both the CL and CM groups exhibited elevated Fv/Fo and Fv/Fm ratios, which are key indicators of PSII activity and maximum photochemical efficiency, and the CM group presented the most pronounced increase (Figure 1L-M). These findings, which were consistent with the pigment and gas exchange data, demonstrated that moderate fungal concentrations improved energy conversion efficiency. P. oxalicum also primed photosynthesis to allocate resources to growth/secondary metabolism.

Antioxidant Enzyme System and Related Biochemical Parameters

Antioxidant enzyme activities and oxidative stress markers were analyzed to investigate stress responses associated with secondary metabolism. CAT and POD activities increased significantly in all the treatment groups, in which context the CM group exhibited the highest CAT (30.61 ± 1.65 U/mL) and POD (146.49 ± 14.59 U/mg prot) activities, suggesting enhanced hydrogen peroxide scavenging capacity (P < 0.01, Figure 2A–C). SOD activity exhibited a U-shaped response, decreasing in the CL group but increasing in the CM and CH groups, suggesting a concentration-dependent regulation of superoxide radical detoxification (Figure 2B). GR exhibited a similar pattern to those of CAT and POD, such that the highest activity was detected in the CL group (929.18 ± 66.45 U/mg prot), indicating the increased efficiency of the ascorbate–glutathione cycle (Figure 2D). APX activity was significantly greater in the CL group than in the CK group (P < 0.01), whereas such activity was significantly lower in both the CM and CH groups (P < 0.01; Figure 2E). NR activity was significantly lower in all the treatment groups than in the CK group (P < 0.05), and the lowest level of activity was detected in the CL group (Figure 2F). MDH activity was significantly reduced in the CL group compared with the CK group (P < 0.01; Figure 2G). ACP activity was significantly reduced in the CH group compared with the CK group (P < 0.01; Figure 2H). Notably, the level of MDA, a marker of lipid peroxidation, decreased significantly in all the treatment groups, in which context the CL group exhibited the lowest level (P < 0.01; Figure 2I), indicating reduced oxidative damage. These enzymatic responses suggested that P. oxalicum activated the antioxidant defense system at low to medium concentrations, thus establishing a favorable redox environment for secondary metabolite biosynthesis.

Figure 2.

Impacts on Antioxidant Enzyme Activities. CK: Distilled Water; CL: P. oxalicum (10³ CFU/mL); CM: P. oxalicum (10⁴ CFU/mL); CH: P. oxalicum (10⁵ CFU/mL). Group Comparisons Were Performed on the Basis of a One-Way ANOVA with Duncan's HSD Test for Multiple Comparisons. Data: Mean ± SD (n = 6). *P < .05, **P < .01.

Effects on the Contents of Soluble Sugars, Starch, and Soluble Protein

The increase in photosynthesis resulted in altered carbon partitioning, as evidenced by the accumulation of soluble sugars and starch.^38,39 The soluble sugar content in the CL group (3188.25 ± 115.75 μg/mg protein) was more than five times greater than that observed in the CK group, in which context the CM and CH groups also exhibited elevated sugar contents (Figure 3A), suggesting the increased production of photoassimilates. Starch, a major carbohydrate, increased in both the CL and CH groups, which indicated different allocation strategies: a low concentration promoted rapid sugar utilization, whereas a high concentration favored storage (Figure 3B). The soluble protein content was highest in the CM group (52.72 ± 1.37 mg prot/L), potentially reflecting increased enzyme biosynthesis and supporting improved metabolic activity (Figure 3C). Fungal-enhanced photosynthesis increased carbohydrate/protein levels, supplying precursors for secondary metabolites.

Figure 3.

Impacts on Carbohydrate Metabolism and Secondary Metabolites: (A) Soluble Sugars, (B) Starch, (C) Soluble Protein, (D) Total Phenols, (E) Flavonoids. CK: Distilled Water; CL: P. oxalicum (10³CFU/mL); CM: P. oxalicum (10⁴CFU/mL); CH: P. oxalicum (10⁵CFU/mL). Group Comparisons Were Performed on the Basis of a One-way ANOVA with Duncan's HSD Test for Multiple Comparisons. Data: Mean ± SD (n = 6). *P < .05, **P < .01.

Total Phenol and Flavonoid Contents

The total phenolic content was significantly affected by fungal treatment (Figure 3D). The highest value was recorded in the CL group (12.90 ± 1.13 mg/g), where this value was significantly higher than that observed in the CK group (8.44 ± 0.48 mg/g; P < 0.01). Similarly, the total flavonoid content differed significantly among the treatment groups (Figure 3E). The CL group exhibited the highest flavonoid content (9.14 ± 0.54 mg/g), which was more than twice the amount of that observed in the CK group (4.18 ± 0.24 mg/g; P < 0.01). Compared with the control group, the CM group also presented a significant increase in flavonoid content. Collectively, these results indicated that P. oxalicum, particularly at low concentrations, strongly promoted the accumulation of phenolic and flavonoid compounds in E. senticosus leaves, thereby increasing the plant's antioxidant potential and medicinal value.

Discussion

Dose-Dependent Modulation of Growth

The results of this study demonstrated that P. oxalicum had a concentration-dependent effect on the growth of E. senticosus leaves. At low (CL) and medium (CM) concentrations, P. oxalicum increased the fresh and dry leaf biomass, potentially through growth-promoting signals, although phytohormone levels were not quantified. These findings are consistent with previous reports demonstrating that endophytic fungi stimulate shoot elongation and biomass accumulation in various medicinal plants.^40,41 Notably, the CM group exhibited the highest leaf drying rate and protein content, suggesting optimal carbon and nitrogen utilization. In contrast, high-dose (CH) treatment resulted in a reduction in leaf area and overall growth, suggesting that P. oxalicum becomes phytotoxic at a specific threshold. Excessive fungal metabolites may interfere with host metabolism or overstimulate defense signaling pathways, thereby suppressing growth,^42,43 although this hypothesis requires further validation. These highlight the need to optimize microbial biostimulant doses, suggest that careful dose titration is required in field applications. Compared to root-associated strains,^44,45 P. oxalicum applied directly to the leaves exhibits strong regulatory potential without the need for root colonization, thereby offering a practical approach for foliar crop treatment. Overall, Low to moderate concentrations appear to support productivity, while excessive doses may trigger stress responses that limit growth, potentially by altering resource allocation.

Modulation of Photosynthetic Performance

Photosynthetic performance responded sensitively to varying P. oxalicum concentrations. The CL group exhibited the greatest increases in chlorophyll a, chlorophyll b, and carotenoids, alongside significant increases in Pn, Gs, and Tr, suggesting improved light harvesting and CO₂ assimilation. ⁴⁶ These changes were accompanied by elevated Fv/Fm and Fv/Fo ratios, indicating enhanced PSII efficiency. Notably, CM had the highest biomass, but reduced pigment contents compared with CL. In the CH group, photosynthesis was impaired, as evidenced by the reduced chlorophyll content, a decrease of ∼32% in Pn, and increased Fo, thus indicating photoinhibition.^47–49 Notably, Ci also decreased despite a reduction in Gs, revealing nonstomatal limitations, likely due to Rubisco downregulation or disruption of the Calvin cycle. ⁵⁰ These results are consistent with the findings reported by studies on Trichoderma-treated cucumber plants. ⁵¹ Elevated SOD and CAT activities in the CH group further support stress-induced repression of photosynthesis. ⁵² These results indicate that fungal applications in moderate concentrations optimize photosynthetic capacity and growth efficiency, whereas high concentrations activate oxidative signaling cascades that inhibit carbon fixation. Recognizing this biphasic photosynthetic response is critical to the tasks of determining optimal spore dosages and mitigating phototoxic effects during large-scale medicinal plant cultivation.

Antioxidant Regulation: Enzymatic and Non-Enzymatic Defense

P. oxalicum modulated the antioxidant defense system of E. senticosus in a dose-dependent manner. In the CL group, APX activity increased, suggesting a potential role in H₂O₂ detoxification, possibly involving the ascorbate–glutathione cycle. ⁵³ SOD activity was lowest in the CL group. However, MDA levels were also minimal, indicating efficient ROS scavenging via alternative antioxidant mechanisms.^37,54 The total phenolic and flavonoid contents peaked in the CL group, which correlated with increased nonenzymatic antioxidant capacity in the context of mitigating oxidative damage ⁵⁵ These findings demonstrated that low-dose P. oxalicum application promoted the biosynthesis of low-molecular-weight antioxidants as a primary defense strategy. In contrast, the CM and CH treatments primarily activated enzymatic defenses, as evidenced by elevated SOD and CAT activities, reflecting increased superoxide and hydrogen peroxide stress. ⁵⁶ However, APX activity decreased in the CH group, potentially due to feedback inhibition or substrate depletion, thus indicating impaired redox cycling under stress conditions. ⁵⁵ While feedback inhibition or substrate limitation is hypothesized, further biochemical evidence is needed. These results highlight the dose-dependent reconfiguration of antioxidant pathways in response to fungal colonization. Low concentrations were associated with phenolic-mediated ROS scavenging, whereas higher concentrations were linked to enhanced enzymatic defenses. ⁵⁷

Reconfiguration of Primary and Secondary Metabolism

The observed metabolic reprogramming reflects complex trade-offs between plant growth and defense mechanisms. Low and medium doses of P. oxalicum increased the levels of soluble sugars, starch, and proteins, thus indicating stimulation of photosynthetic activity, ⁵⁸ carbon fixation^59,60 and nitrogen assimilation.^61,62 Notably, the CM group exhibited maximal accumulation of proteins and starch, potentially supporting cell proliferation and metabolic enzyme biosynthesis. ⁶³ The CL group exhibited the highest levels of total phenolics and flavonoids, which coincided with increased starch and soluble sugar accumulation. This stimulatory effect is consistent with previous findings involving endophytic fungi in other medicinal species, such as Dendrobium officinale and Houttuynia cordata.^64,65 Conversely, the CM group prioritized biomass and protein synthesis over secondary metabolite production, suggesting a metabolic shift toward primary metabolism. ⁶⁶ At high concentrations, CH-treated plants exhibited decreased phenolic and flavonoid contents, potentially due to carbon competition between the host and fungus or the oxidative inhibition of biosynthetic enzymes.^67,68 These findings suggest a dose-dependent metabolic transition, namely, from defense activation at low doses (CL), to growth prioritization at moderate doses (CM), and ultimately to stress-induced resource limitation at high doses (CH). Understanding these metabolic trade-offs is essential for efforts to optimize the application of P. oxalicum to increase both biomass yield and bioactive compound accumulation in medicinal plant production systems.

Application Potential and Comparisons with Other Endophytes

Compared with well-characterized endophytes such as Trichoderma spp. and Serendipita indica, P. oxalicum exhibited strong biostimulant activity in E. senticosus, particularly through foliar application. ⁴³ The ability of P. oxalicum to promote plant growth, photosynthetic efficiency, antioxidant activity, and secondary metabolism makes it a promising candidate for sustainable biofertilizer development. Unlike root-associated fungi such as T. harzianum, ⁵⁹ P. oxalicum is effective through foliar application, thereby offering practical advantages in terms of delivery and broader crop applicability. However, field-level application requires careful consideration. In natural soil environments, microbial competition may reduce colonization efficiency, whereas abiotic stressors could alter fungal behavior or exacerbate phytotoxicity at high application doses. Moreover, the formulation stability, spore viability, and regulatory compliance of P. oxalicum preparations must be addressed prior to large-scale implementation. Multiomics approaches, including transcriptomics and metabolomics, could elucidate the underlying molecular mechanisms and guide the selection of effective strains or the design of microbial consortia. When applied appropriately, P. oxalicum may complement or even outperform conventional endophytes, thus contributing to the increased productivity and quality of medicinal plants. However, its practical deployment requires rigorous ecological validation and controlled formulation strategies to ensure consistent efficacy across diverse agricultural systems.

Integrative Summary

Collectively, our findings support a triphasic response model in E. senticosus leaves following foliar application of Penicillium oxalicum, as illustrated in Figure 4. At low concentrations (CL), P. oxalicum increased the photosynthetic pigment accumulation, activated nonenzymatic antioxidant defenses, and promoted phenolic biosynthesis. Overall, the physiological performance and phytochemical quality observed in this context exhibited considerable improvement. At medium concentrations (CM), P. oxalicum promoted maximal biomass accumulation and protein synthesis via efficient carbon–nitrogen allocation and enhanced enzymatic antioxidant activity, despite reduced pigment levels. At high concentrations (CH), excessive fungal colonization triggered photoinhibition, suppressed APX activity, and decreased phenolic content, which indicated the presence of oxidative stress and metabolic burden. These dose-dependent outcomes highlight the dual role played by P. oxalicum as both a promoter of plant growth and a potential phytotoxin when it is applied in excess. Figure 4 presents a conceptual framework that integrates these outcomes, thus illustrating the tasks of dose-specific coordination of photosynthesis, antioxidant regulation, and metabolic reprogramming. These mechanistic insights provide a foundation for efforts to optimize the use of endophytic fungi in medicinal plant biotechnology and supportguide the development of precision bioformulations for sustainable agriculture.

Figure 4.

Concentration-Dependent Regulatory Mechanism of P. oxalicum.

This schematic diagram illustrates the physiological responses triggered by the foliar application of P. oxalicum spore suspensions at various concentrations (CL: 10³ CFU/mL, CM: 10⁴ CFU/mL, CH: 10⁵ CFU/mL) and their subsequent colonization of the leaf surface of E. senticosus. Six major regulatory effects are presented: (1) growth response; (2) photosynthesis; (3) chlorophyll fluorescence; (4) carbohydrate and protein metabolism; (5) antioxidant enzyme activity; and (6) accumulation of secondary metabolites. The integrated outcomes induced by different concentrations are summarized on the right: low concentrations promote growth, photosynthetic activity, and antioxidant defense; medium concentrations significantly increase biomass accumulation, enzymatic activity, and protein levels; and high concentrations exhibit phytotoxicity, which is characterized by pigment degradation and oxidative stress.

Study Limitations

This study has several limitations. First, the experiment was conducted under controlled greenhouse conditions, which differ from complex field environments (eg, in terms of temperature fluctuations, microbial competition, and soil heterogeneity). Thus, the practical efficacy of P. oxalicum requires further field validation. Second, only three concentrations were tested over a short period (30 days); long-term cumulative effects and precise definitions of concentration thresholds require further investigation. Additionally, this study focused solely on leaf physiological responses, while its impacts on E. senticosus roots and overall plant development remain unexplored. Finally, the synergistic effects between P. oxalicum and other endophytes have not been explored, which may limit our ability to obtain a comprehensive understanding of microbial interaction networks. These limitations highlight directions for future research, such as studies on multiple environments, multiple concentrations, long-term field trials, and molecular mechanism analysis on the basis of multiomics techniques.

Conclusions

This study demonstrated that P. oxalicum dose-dependently affected the physiological performance of E. senticosus leaves. At low and medium concentrations, P. oxalicum increased growth, photosynthetic efficiency, antioxidant activity, and secondary metabolite accumulation, whereas high concentrations induced photoinhibition and oxidative stress. Dose optimization is critical for endophyte-based biostimulants. P. oxalicum is a promising avenue for efforts to improve the quality and bioactive compound content of medicinal plants through foliar application. Further field trials and molecular investigations are needed to validate its efficacy and elucidate the underlying mechanisms, thus laying a foundation for the integration of P. oxalicum into sustainable phytopharmaceutical production systems. Consequently, comprehensive assessments of application protocols and environmental interactions are essential to facilitate the development of standardized, efficacious, and environmentally sustainable biostimulant formulations incorporating P. oxalicum.