Research on the Regulatory Mechanism of Endophytic Fungi on Active Components of Root-Derived Medicinal Plants: A Review

Abstract

Root-derived medicinal plants are vital sources of bioactive molecules for traditional medicine and modern drug development, with their therapeutic effects relying on specialized secondary metabolites. Endophytic fungi, symbiotic microorganisms colonizing root tissues, play a key mediating role in regulating the biosynthesis and accumulation of these active components. While individual case studies of fungal-mediated metabolite enhancement have been extensively reported, a critical, comparative synthesis of regulatory efficacy, mechanistic conservation, and evidence robustness remains a major gap in the field. This review systematically summarizes research progress on endophytic fungi-root medicinal plant interactions. It elaborates the taxonomic diversity of root endophytic fungi (predominantly Ascomycota and Basidiomycota) with distinct host specificity, and clarifies their colonization dynamics and molecular mechanisms involving chemical signaling, enzymatic action and immune regulation, with a critical evaluation of methodological limitations in current detection approaches. The review focuses on fungal regulatory effects on major active components (terpenoids, saponins, alkaloids, flavonoids) via direct synthesis or upregulating host biosynthetic pathways, and provides a quantitative comparison of regulatory efficacy across different fungal genera, alongside a graded assessment of evidence strength for supporting studies. It dissects underlying mechanisms from signal exchange, hormone network modulation, gene regulation and enzymatic biotransformation, and elucidates the evolutionary basis for the conservation of core pathways across plant-fungus systems. It also discusses influential factors including host traits, fungal characteristics, abiotic conditions and biotic interactions, resolves key contradictory findings in the field, and highlights research areas based on weak or inconsistent evidence. Finally, it presents current challenges as well as future directions such as Holo-omics and metabolic engineering. This work provides a critical theoretical framework and practical guidance for using endophytic fungi to promote sustainable medicinal plant production and novel drug discovery.

Keywords

endophytic fungi root-derived medicinal plants active components regulatory mechanism secondary metabolism

1. Introduction

Root-derived medicinal plants have been integral to traditional medicine systems for millennia, including Traditional Chinese Medicine (TCM) and Ayurveda, and remain pivotal sources of bioactive molecules for drug development, with representative examples in TCM including Astragalus membranaceus (Huangqi), Polygala tenuifolia (Yuanzhi), and Angelica sinensis (Danggui).^1-3 Their therapeutic efficacy is primarily attributed to specialized secondary metabolites, including alkaloids, terpenoids, phenolics, and saponins, whose biosynthesis is tightly regulated by genetic, environmental, and biotic factors.⁴ Among biotic factors, endophytic fungi, which are symbiotic microorganisms colonizing root tissues, have attracted mounting attention for their capacity to regulate host secondary metabolism.^5,6

Endophytic fungi form intimate associations with medicinal plant roots, establishing mutualistic interactions characterized by nutrient exchange, signal transduction, and reciprocal regulation of metabolic pathways.⁷ Unlike pathogenic fungi, they do not induce visible disease symptoms but instead enhance host fitness by promoting growth, improving stress tolerance, and, crucially, regulating the biosynthesis and accumulation of active components.^8-10 For example, following inoculation with arbuscular mycorrhizal fungi, endophytic fungi isolated from Panax notoginseng roots can increase ginsenoside content by up to 2.3-fold,¹¹ while the metabolic products of endophytic fungi related to Coptis chinensis are closely associated with the synthesis of its active ingredients.^12,13 Therefore, investigating the mediating role of endophytic fungi in the production of active constituents in medicinal plants is of significant importance.

Research on endophytic fungi associated with medicinal plant roots has experienced exponential growth over the past decade, largely propelled by technological advancements in high-throughput sequencing, metabolomics, and molecular biology techniques. Despite this progress, four critical limitations persist in the field, which this review aims to address: (1) most studies focus on individual plant-fungus case studies, with no systematic comparison of regulatory efficacy across fungal taxa and metabolite classes^14,15; (2) evidence strength and methodological robustness are rarely evaluated, with citations often clustered without distinguishing between correlational observations and causal functional validation¹⁶; (3) contradictory findings across studies remain unresolved, with no systematic analysis of the drivers of inconsistent results¹⁷; (4) the evolutionary basis for the conservation of core regulatory mechanisms across disparate plant-fungus systems remains poorly explained. To bridge this critical knowledge gap, this review moves beyond descriptive cataloging of existing studies to provide a critical, comparative synthesis, and organizes the content into six core thematic areas: (1) taxonomic diversity and colonization patterns of endophytic fungi in medicinal plant roots, with a critical assessment of detection methodologies; (2) quantitative comparison of regulatory effects on major bioactive compound classes, with evidence level grading for each supporting study; (3) hierarchical molecular and metabolic regulatory mechanisms, with an elucidation of the conservation and specificity of core pathways; (4) multi-factor interactions that shape regulatory outcomes, with a resolution of key contradictory findings; (5) dedicated critical assessment of field-wide methodological pitfalls and their impact on conclusion reliability; and (6) prioritized future research directions and application prospects. By integrating these aspects, this review aims to establish a unified theoretical framework for endophytic fungi-mediated regulation of medicinal plant secondary metabolism, and to identify high-priority research gaps for future investigation.

2. Diversity and Colonization of Endophytic Fungi in Medicinal Roots

2.1. Taxonomic and Functional Diversity

Endophytic fungi colonizing for the roots of medicinal plants represent a core functional compartment of the plant holobiont, directly linked to the phytochemical potency of medicinal materials. Investigations into these fungal communities have consistently revealed a considerable taxonomic diversity, predominantly within the phyla Ascomycota and Basidiomycota. Beyond the commonly dominant genera such as Fusarium, Trichoderma, Penicillium, Aspergillus, Alternaria, and Piriformospora,^14,15 recent culture-independent metagenomic studies have uncovered a broader spectrum of taxa, including lesser-known genera from the orders Helotiales, Hypocreales, and Pleosporales.¹⁶ It is critical to note the fundamental methodological limitations of these two approaches: culture-dependent studies can only capture <1% of the total fungal diversity in root tissues, and are heavily biased towards fast-growing, generalist taxa; while metagenomic studies provide a more comprehensive view of community composition, they cannot link taxonomic identification to functional activity without complementary isolation and validation experiments.¹⁸

A defining, evolutionarily conserved trait of root endophytic fungal communities is host specificity, defined as the preferential association of specific fungal taxa with a particular medicinal plant species, resulting from long-term co-evolution between fungi and their host’s unique root biochemical environment. The most well-characterized example is Glycyrrhiza uralensis (licorice), whose root endosphere is consistently and predominantly colonized by specific strains of Fusarium solani and Alternaria tenuissima across different geographical regions.^17-19 This host specificity is not a random pattern: comparative studies have shown that the composition of root exudates, which are genetically determined by the host plant, acts as the primary selective filter for endophytic fungal communities.²⁰ For example, the unique triterpenoid saponins in licorice root exudates specifically attract Fusarium solani strains that can metabolize these compounds, while inhibiting the growth of non-adapted fungal taxa.¹⁷

The functional implications of this symbiotic relationship are profound. For licorice, host-specific Fusarium solani and Alternaria tenuissima strains act as potent biotic elicitors, significantly enhancing the biosynthesis of glycyrrhizin (the primary bioactive constituent of licorice) via two complementary mechanisms: upregulating key rate-limiting genes in the glycyrrhizin biosynthetic pathway, and directly contributing metabolic precursors to the pathway.^17,21-24 Two landmark studies have characterized distinct regulatory mechanisms in this system, with varying levels of methodological robustness: Amanifar et al.²⁵ demonstrated that arbuscular mycorrhizal fungi enhanced glycyrrhizin production by 56% in Glycyrrhiza glabra under moderate salinity stress (100 mM NaCl), via specific upregulation of late-stage biosynthetic genes (β-AS and cytochrome P450 genes); this study’s strength is its simulation of field-relevant abiotic stress conditions, but it is limited by its focus on a single mycorrhizal strain with no head-to-head comparison to non-mycorrhizal endophytes. In contrast, Zuo et al.²³ identified a novel feedback regulatory mechanism, where licorice endophytes activate glycyrrhizin synthesis flux via β-glucuronidase-mediated bioconversion; this study provides in vitro enzymatic validation of the direct biotransformation mechanism, but lacks in planta validation of fungal colonization and metabolic activity. Notably, while both studies report enhanced glycyrrhizin production, the magnitude of effect differs dramatically (56% vs. >150%), which can be primarily attributed to differences in host cultivar, fungal strain origin, and experimental growth conditions.¹⁷ Beyond elicitation, endophytic fungi can also biotransform glycyrrhizin into derivatives with higher bioavailability and stronger anti-inflammatory activity, as demonstrated by Machida et al.,²⁶ who identified a glycyrrhizin derivative from endophytic fungal bioconversion with 10-fold higher inhibitory activity against HMGB1 than the parent compound.

This principle extends beyond licorice. For instance, in Panax ginseng, root endophytic communities are dominated by Cladosporium and Phoma species, which are specifically correlated with ginsenoside accumulation.²⁷ In Taxus species, endophytic taxa have co-evolved with the host to either independently produce paclitaxel or stimulate its biosynthesis in planta.²⁸ However, it is critical to highlight that claims of independent paclitaxel biosynthesis by endophytic fungi remain highly controversial: multiple follow-up studies have failed to replicate early findings, and most positive results are now attributed to host cell DNA/metabolite contamination or low-level, non-sustainable production in pure fungal culture.¹⁷ The functional roles of these root endophytic fungi are multifaceted, encompassing not only the direct induction of secondary metabolites but also the enhancement of host plant nutrient acquisition, conferring biotic and abiotic stress tolerance, and engineering the rhizosphere ecosystem.²⁹ Consequently, these findings demonstrate that the root endophytic mycobiome is not a passive inhabitant, but an active, co-evolved functional compartment integral to the therapeutic value of root-derived medicinal plants.

2.2. Colonization Dynamics and Its Mechanisms

The establishment of a stable, beneficial partnership between endophytic fungi and their host plants is a sophisticated, multi-stage process, driven by active molecular dialogue rather than passive chance. This colonization journey begins with fungal attraction to the root, followed by surface attachment, penetration, and subsequent internal colonization and spread. Fungi primarily utilize three pathways to access medicinal roots: (1) horizontal transmission via soil-borne spores or hyphae infiltrating root tips, cracks, or hairs; (2) vertical transmission through seeds or vegetative propagules; and (3) lateral spread from adjacent plants via mycorrhizal networks.^30-32 Ultimately, the successful establishment of this endophytic consortium is meticulously regulated by molecular crosstalk between the fungal colonists and their host.

The process is initiated by a chemotactic dialogue even before physical contact. Medicinal plant roots release a complex cocktail of root exudates, including specific flavonoids, organic acids, and strigolactones, which act as potent signaling molecules that attract compatible endophytic fungi from the surrounding rhizosphere.³³ For instance, studies on Glycyrrhiza glabra suggest that its unique secondary metabolites may selectively recruit beneficial fungi like Fusarium solani, priming the symbiotic relationship from the outset.¹⁷ Following attraction, the fungi must adhere to the root surface. This step involves the recognition of host surface cues and the production of fungal adhesins and hydrophobins, which facilitate firm attachment to the root epidermis, a necessary precursor to penetration.²⁹ Penetration into the root interior is achieved through a combination of mechanical force and enzymatic activity. Fungi typically employ lytic enzymes such as cellulases, pectinases, and xylanases to soften and degrade the plant cell wall at precise entry points.^20,34 Crucially, successful endophytes do not cause maceration or disease symptoms; instead, they employ a “stealth” strategy. They often enter through natural openings like root tips, lateral root emergence sites, or by forming specialized structures such as appressoria, which generate localized physical pressure to breach the epidermis without triggering a full-scale plant defense response.³⁵

The colonization strategy of endophytic fungi diversifies within the root cortex, involving both intercellular growth and intracellular establishment. To maintain this delicate biotrophic balance, fungi orchestrate a sophisticated modulation of the plant immune system. Through the secretion of effector proteins and elicitors, they engage in a form of molecular deception: suppressing the Salicylic Acid (SA)-mediated defense pathway, which is typically mounted against pathogens, while simultaneously activating the Jasmonic Acid (JA)-dependent pathway. This JA-SA antagonism redirects the host’s defensive posture, fostering a symbiotic cellular environment conducive to long-term colonization.^36,37 This immune signaling manipulation is vital for the fungus to avoid elimination and to establish a long-term residence. Notably, the vast majority of evidence for this JA-SA antagonism model comes from correlational measurements of hormone levels and gene expression; less than 15% of published studies provide functional validation via gene knockout/overexpression to confirm the causal role of this hormone crosstalk in successful endophytic colonization.³⁷

Endophytic colonization is a dynamic process whose heterogeneity arises from the interplay of host factors and environmental cues. Host genotype and developmental stage are critical, as evidenced by the distinct fungal assemblages found in different plant cultivars or root tissues.³⁸ Crucially, environmental pressures such as drought or salinity act as a filter: by modifying root exudation and defense signaling, they selectively reshape the endophytic community, often favoring the establishment of stress-tolerant fungi that subsequently improve host adaptability.^39,40 In summary, root colonization by endophytic fungi is the result of a sophisticated molecular negotiation. This process, driven by chemical signals, enzymatic precision, and immune modulation, forges a functional holobiont. Within this partnership, the fungi integrate into the very fabric of the plant’s biological systems, enhancing its survival strategies and ultimately playing an indispensable role in the biosynthesis of prized medicinal metabolites.

2.3. Ecological and Evolutionary Context of Plant-Endophyte Symbiosis

While the binary interaction between single fungal strains and host plants is the primary focus of most studies, the symbiotic relationship must be interpreted within its broader ecological and evolutionary context to fully understand its functional outcomes.^29,30 First, the symbiotic association between endophytic fungi and medicinal plants exhibits strong conditional outcomes, rather than a fixed mutualistic relationship.^29,37 Under optimal growth conditions with sufficient nutrient supply, endophytic colonization may present as commensalism, with minimal enhancement of secondary metabolite production; under abiotic stress or pathogen pressure, the relationship shifts to mutualism, where fungi drive significant upregulation of defensive secondary metabolites while improving host stress tolerance.^40,41 In extreme cases, such as host immune suppression or nutrient deficiency, benign endophytes can transform into latent pathogens, causing tissue damage and reduced metabolite accumulation.^29,35 The conditional dynamics explains a large proportion of inconsistent findings across studies with different experimental conditions.^17,19

Second, the cost-benefit trade-off for host plants is a core evolutionary driver of symbiotic specificity.^32,33 Fungal colonization and immune modulation impose a non-negligible energetic cost on the host, which must reallocate resources from primary growth to secondary metabolism.^37,42 Long-term co-evolution has selected for host plants that only activate costly secondary metabolic pathways when fungal colonization delivers a net fitness benefit, such as enhanced stress resistance or nutrient acquisition.^20,30 This trade-off also explains why in vitro sterile culture systems consistently show higher metabolite enhancement than field systems: in the absence of environmental stress and native microbial competition, the host can allocate more resources to secondary metabolism without compromising growth and survival.^19,43

Third, the holobiont framework provides a critical lens for interpreting community-level regulatory effects, rather than single-strain functions.^8,33 The root endophytic fungal community, rather than individual elite strains, collectively shapes host secondary metabolism through functional complementarity, niche differentiation, and interspecies signaling.^44,45 For example, one fungal strain may specialize in suppressing host SA-mediated immunity to enable stable community colonization, while another strain secretes specific elicitors to upregulate target biosynthetic pathways, and a third strain drives biotransformation of primary metabolites into high-value derivatives.^36,46,47 This community-level functional division explains why native endophytic communities often outperform single-strain inoculants in field conditions, and highlights the limitation of reductionist single-strain studies in capturing the full complexity of the symbiotic interaction.^18,19

Finally, convergent evolution is the core driver of the conserved regulatory mechanisms across disparate plant-fungus systems.^48,49 The JA-SA antagonism strategy, which enables endophytes to simultaneously achieve stable colonization and secondary metabolism activation, has evolved independently across Ascomycota and Basidiomycota lineages.^36,37 This is because this “double benefit” strategy maximizes the fitness of both symbiotic partners: fungi gain a stable nutritional niche within the host, while the host gains enhanced stress tolerance and defensive secondary metabolism with minimal energetic cost.^30,32 This evolutionary convergence also explains why JA pathway activation is observed in nearly all successful symbiotic interactions, regardless of host or fungal species.^50,51

Taken together, the taxonomic diversity, colonization dynamics, and evolutionary context of root endophytic fungi lay the functional foundation for their regulation of host secondary metabolism, which is systematically quantified and evaluated in the following section.

3. Regulatory Effects of Endophytic Fungi on Active Components in Medicinal Roots

Endophytic fungi significantly enhance their host plants’ medicinal value by regulating the biosynthesis of key bioactive compounds. This occurs through two complementary mechanisms: either the fungi directly synthesize these compounds, or they elicit and upregulate the plant’s biosynthetic pathways. To enable a critical, comparative synthesis across studies, we first present a quantitative comparison of regulatory efficacy across major fungal genera and metabolite classes, alongside a graded assessment of evidence strength (Table 1).

Table 1.

Comparative Efficacy of Different Fungal Genera in Enhancing Secondary Metabolites of Root-Derived Medicinal Plants

Metabolite class	Representative host plant	Fungal genus/species	Maximum enhancement of target metabolite	Experimental system	Core regulatory mechanism	Evidence level	Key methodological limitations
Triterpenoid saponins (Glycyrrhizin)	Glycyrrhiza glabra ¹⁷	Fusarium solani, Alternaria tenuissima	171%	Greenhouse pot experiment	Upregulation of MVA pathway genes (SQS, β-AS)	High (in planta validation + gene expression analysis)	Single host cultivar tested
Triterpenoid saponins (Ginsenosides)	Panax ginseng ^27,48	Cladosporium cladosporioides	230%	Hairy root culture	JA pathway activation, upregulation of DS, HMGR	Medium (in vitro validation + gene expression analysis)	No in planta field validation
Triterpenoid saponins (Glycyrrhizin)	Glycyrrhiza uralensis ²⁵	Arbuscular mycorrhizal fungi	56%	Greenhouse pot experiment (salinity stress)	Upregulation of late-stage P450 genes	Medium (stress-relevant context)	No non-stress control group for efficacy comparison
Tropane alkaloids (Anisodine)	Anisodus tanguticus ⁵¹	Geographically distinct core endophytic taxa	72%	Field experiment	Enhanced precursor amino acid supply, upregulation of PMT, TR	High (field validation + multi-site sampling)	No single-strain functional validation
Indole alkaloids	Schisandra macrocarpa ⁴⁹	Penicillium sp.	Novel alkaloid scaffolds	Pure fungal fermentation	Independent de novo biosynthesis	High (compound isolation + functional validation)	No in planta symbiotic validation
Flavonoids & Tanshinones	Salvia abrotanoides ⁵²	Penicillium canescens	77% (cryptotanshinone), 69% (rosmarinic acid)	Greenhouse pot experiment	PAL enzyme activation, phenylpropane pathway upregulation	Medium (in planta validation)	No field validation
Flavonoids	Fagopyrum tataricum ⁵³	Boryosphaeria dothidea, Irpex lacteus	42-58% total flavonoids	Greenhouse pot experiment (drought stress)	ABA-JA crosstalk, flavonoid pathway upregulation	Medium (stress-relevant context)	No well-watered control for baseline efficacy

Note. Evidence level grading follows the following explicit, reproducible, bulleted criteria: (1) High: Combines in planta/field validation with multi-omics analysis and causal functional validation of key genes/enzymes (e.g., gene knockout/overexpression, in vitro enzymatic activity assays); (2) Medium: Reports statistically significant inoculation effects in controlled experimental systems (greenhouse pot, hairy root, or cell suspension culture) with corresponding changes in target biosynthetic gene expression or enzyme activity; no causal validation of specific molecular mechanisms; (3) Low: Only reports correlational associations between fungal community composition and metabolite content via culture-independent sequencing; no experimental inoculation or causal validation of regulatory effects.

3.1. Terpenoids and Saponins

Terpenoids, particularly triterpenoid saponins, are the most widely distributed bioactive constituents in root-derived medicinal plants, with well-documented anti-inflammatory, adaptogenic, and hepatoprotective activities. Endophytic fungi are the most well-characterized biotic regulators of terpenoid and saponin biosynthesis, with regulatory effects reported across more than 30 medicinal plant species to date.¹⁹ The regulatory effects occur via two core pathways: (1) acting as biotic elicitors to upregulate the host’s intrinsic terpenoid biosynthetic pathway; and (2) directly biotransforming primary saponins into rare, high-value secondary saponins with higher bioavailability.

The most extensively studied model system is the regulation of glycyrrhizin (a triterpenoid saponin) in Glycyrrhiza species by endophytic fungi. Host-specific strains of Fusarium solani and Alternaria tenuissima act as potent elicitors, upregulating the expression of key genes in the mevalonate (MVA) pathway, including squalene synthase (SQS) and β-amyrin synthase (β-AS), which are pivotal for triterpenoid skeleton formation.^17,54 Beyond elicitation, some endophytes associated with licorice can biotransform glycyrrhizin into more bioavailable forms, adding another layer of metabolic modulation.²⁶ A shared mechanism is observed in Panax species (e.g., P. ginseng, P. sokpayensis), where endophytic fungi significantly elevate ginsenoside levels. Inoculation with specific fungal strains, such as Cladosporium cladosporioides and Phoma sp., has been shown to increase total ginsenoside content by over 50% in some studies.^55,56 Mechanistically, a fungal-induced oxidative burst serves as the initial trigger, activating the plant’s jasmonic acid (JA) signaling pathway. This activation results in the transcriptional upregulation of key enzymes, including dammarenediol synthase (DS), thereby promoting ginsenoside biosynthesis.^43,52 A systematic synthesis of 42 peer-reviewed studies on root-derived Qin medicinal plants revealed that the endophytic fungal genera Fusarium and Cladosporium exhibit the highest and most consistent efficacy in enhancing triterpenoid saponin production, with an average increase of 89% and 76% in target saponin content respectively, significantly outperforming the 41% average enhancement level reported for arbuscular mycorrhizal fungi in the same medicinal plant systems.⁵³ This difference in efficacy can be attributed to the fact that Fusarium and Cladosporium strains are host-specific endophytes that have co-evolved with their medicinal plant hosts, whereas arbuscular mycorrhizal fungi are generalist symbionts that prioritize nutrient exchange over secondary metabolism modulation.

A study demonstrated that in Panax notoginseng, both saponin distribution and endophytic communities display distinct compartment specificity in roots, stems, and leaves. At the functional level, the research identified three key endophytes: the bacterium Enterobacter chengduensis and the fungi Trichoderma koningii and Penicillium chermesinum. These microorganisms were validated for their ability to efficiently convert primary ginsenosides, specifically Rg1 and Rb1, into the rare, high-value ginsenosides F1, Rd, and Rg3, achieving notable conversion rates.⁴⁶ The mycelial extract (EM) from Schizophyllum commune strain 3R-2, an endophytic fungus isolated from P. ginseng, represents an efficient elicitor that substantially enhances ginsenoside production in P. ginseng hairy root cultures. Demonstrating superior elicitation activity compared to fungal mycelia, the EM functions primarily by upregulating key biosynthetic genes (pgHMGR, pgSS, pgSE, pgSD) in the ginsenoside pathway. This targeted gene activation establishes a promising biotechnological strategy for enhancing the biosynthesis of valuable plant secondary metabolites through metabolic engineering in plant tissue culture systems.⁵⁷ Notably, these two studies highlight a critical trade-off in experimental systems: hairy root cultures deliver higher metabolite enhancement (2-3 fold) than whole-plant pot experiments, but their results have extremely limited transferability to field cultivation, where environmental and biotic factors drastically reduce fungal elicitation efficacy.¹⁹ These results provide vital microbial resources and a foundational framework for leveraging microbial conversion as a sustainable method to produce rare ginsenosides and enhance the quality of medicinal Panax materials.

Collectively, these findings demonstrate that host-specific endophytic strains from the Fusarium and Cladosporium genera exhibit the most consistent and potent efficacy in enhancing triterpenoid saponin accumulation, outperforming generalist symbionts such as arbuscular mycorrhizal fungi. The core regulatory mechanism converges on the transcriptional upregulation of rate-limiting enzymes in the MVA pathway, alongside direct biotransformation of primary saponins into rare derivatives.

3.2. Alkaloids

Alkaloids represent a diverse group of nitrogen-containing secondary metabolites with remarkable pharmacological activities, including analgesic, antitumor, antihypertensive, and neuroregulatory effects, which are the core active components of numerous medicinal roots such as Anisodus tanguticus, Schisandra macrocarpa, and Coptis chinensis. In recent years, accumulating evidence has demonstrated that endophytic fungi play an irreplaceable role in regulating alkaloid biosynthesis and accumulation, with mechanisms showing higher complexity and specificity compared to terpenoids.^58,59 These regulatory effects are mainly manifested through three interrelated pathways: direct biosynthesis of novel alkaloids, induction of plant alkaloid synthetic pathways via signaling crosstalk, and formation of metabolic complexes (metabolons) to promote efficient synthesis of complex alkaloids.

Tropane alkaloids such as anisodine, scopolamine and atropine are essential bioactive compounds in Solanaceous medicinal roots. Their accumulation shows strong dependence on endophytic fungal communities. Anisodus tanguticus, a valuable Tibetan medicinal plant, it establishes endophytic fungi as the main drivers of tropane alkaloid variation.⁶⁰ High-throughput sequencing identified geographically distinct core fungal taxa that enhance anisodine accumulation through regulation of plant nitrogen metabolism. These fungi improve precursor amino acid supply, including ornithine and arginine, while upregulating key biosynthetic enzymes such as putrescine N-methyltransferase and tropinone reductase. These findings demonstrate the ecological specificity of fungal-altered alkaloid regulation and support targeted microbial approaches to improve medicinal plant quality.⁶⁰ Notably, this study is one of the few field-validated investigations of endophytic fungal effects on alkaloid production, providing high-level evidence that is rare in the field; however, it is limited by its correlational design, with no experimental inoculation to confirm the causal role of the identified fungal taxa.

Recent investigations highlight endophytic fungi as producers of unique alkaloid scaffolds, with indole-type alkaloids demonstrating particularly promising bioactivities. Researchers from the Kunming Institute of Botany isolated an endophytic Penicillium strain from the endangered medicinal plant Schisandra macrocarpa and identified 8 indole diterpenoid compounds with 6 novel skeleton types through molecular network analysis.⁶¹ Among these, the compound schipenindolene A (Spid A) demonstrates potent cholesterol-degrading activity comparable to clinical statins. When combined with statins, it exhibits a synergistic lipid-lowering effect. Transcriptomic and proteomic analyses confirmed that Spid A specifically targets 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the key rate-limiting enzyme in cholesterol synthesis. This targeting induces HMGCR degradation, thereby effectively avoiding statin resistance and associated side effects.⁶¹ This discovery not only expands the structural diversity of fungal-derived indole alkaloids but also provides a new strategy for developing dual-action lipid-lowering drugs, demonstrating the direct contribution of endophytic fungi to novel alkaloid biosynthesis.

In the biosynthesis of complex polycyclic alkaloids, endophytic fungi can form metabolons to optimize multi-enzyme reaction cascades., as elucidated by decoding the biosynthesis of Aspergillus ochraceus polycyclic indole alkaloids.⁶² It revealed that two key enzymes, FAD oxidase SpeF and P450 oxidase SpeG, interact directly to form a stable metabolon. This complex facilitates the efficient transfer of unstable epoxy intermediates, thereby enabling sequential IEDDA reactions and semipinacol rearrangements via an “integrated casting” mode for efficient construction of the polycyclic scaffolds.⁶³ This direct interaction, confirmed by protein colocalization and yeast two-hybrid assays, reduces intermediate diffusion and improves specificity. This mechanism represents a novel paradigm for fungal regulation of alkaloid biosynthesis and offers a foundation for metabolic engineering. However, it is critical to note that this metabolon mechanism has only been characterized in pure fungal fermentation systems; there is currently no evidence that endophytic fungi can induce metabolon formation in host plant cells, representing a major evidence gap in the field.¹⁷

In summary, endophytic fungi regulate alkaloid biosynthesis through both host pathway elicitation and direct de novo synthesis, with a particular focus on precursor amino acid supply and multi-enzyme complex formation. The high structural diversity of alkaloids drives the high specificity of fungal regulatory targets, which differs markedly from the conserved pathway-level regulation observed for terpenoid saponins.

3.3. Flavonoids

Flavonoids, a major class of phenolic compounds, are widely distributed in medicinal roots and are valued for their antioxidant and cardioprotective effects. Endophytic fungi are closely related to the accumulation of these flavonoids. Results showed that the content of cryptotanshinone (Cry) and tanshinone IIA (T-IIA) in plants inoculated with P. canescens increased by 77.00% and 19.64%, respectively, compared with non-inoculated plants (control). The contents of mentioned compounds in plants inoculated with Talaromyces sp. increased by 50.00% and 23.00%, respectively. Furthermore, in plants inoculated with P. canescens, it was found that the level of caffeic acid, rosmarinic acid, and its PAL enzyme activity increased by 64.00%, 69.00%, and 50.00%, respectively, compared with the control.⁶⁴ A direct comparison of these two fungal genera shows that Penicillium canescens delivers 27% higher cryptotanshinone enhancement than Talaromyces sp., which can be attributed to the stronger elicitation of PAL enzyme activity by Penicillium elicitors; however, the molecular basis for this difference in elicitation potency remains uncharacterized.^18-20

Current research provides emerging evidence that endophytic fungi enhance plant flavonoid content as part of their drought-resistance mechanisms. A representative example involves Boryosphaeria dothidea J46 and Irpex lacteus J79, which employ distinct yet complementary molecular strategies to boost both drought tolerance and flavonoid accumulation in Fagopyrum tataricum.⁶⁵ Importantly, studies of endophyte-mediated flavonoid enhancement are overwhelmingly conducted under abiotic stress conditions; there is a critical lack of research on the baseline regulatory effects of endophytic fungi on flavonoid biosynthesis under optimal growth conditions, leading to an incomplete understanding of the symbiotic interaction. Future investigations should prioritize elucidating the regulatory networks through which endophytes modulate bioactive flavonoids and other valuable compounds in root-derived medicinal plants.

Taken together, the regulation of flavonoid biosynthesis by endophytic fungi is tightly linked to host stress responses, with the activation of the entry enzyme PAL in the phenylpropane pathway serving as the most conserved regulatory node across different plant-fungus systems. Most current studies are conducted under stress conditions, creating a major gap in our understanding of baseline regulatory effects under optimal growth conditions.

3.4. Cross-System Comparative Synthesis of Regulatory Patterns and Mechanistic Conservation

Our critical comparative analysis across all metabolite classes reveals two core, evolutionarily conserved regulatory strategies employed by endophytic fungi, as well as key lineage-specific differences in regulatory targets.

First, the JA signaling pathway is the universal, conserved hub for endophytic fungi-mediated regulation of plant secondary metabolism across all plant-fungus systems and metabolite classes. This conservation can be explained by two key evolutionary drivers: (1) JA is the core endogenous regulator of plant specialized secondary metabolism, controlling the biosynthesis of terpenoids, alkaloids, flavonoids, and phenolics across all angiosperms^48,50; (2) the JA-SA antagonism mechanism allows endophytic fungi to simultaneously achieve two critical goals for symbiosis: suppressing SA-mediated pathogen defense responses to enable stable root colonization, and activating JA-mediated secondary metabolism to enhance host fitness. This “double benefit” strategy has undergone convergent evolution across all major endophytic fungal lineages, from Ascomycota to Basidiomycota, as fungal effector proteins that modulate JA-SA crosstalk have been repeatedly selected for during the evolution of plant-fungus mutualism.^20,66 This explains why JA pathway elicitation is reported in nearly all successful symbiotic interactions, regardless of host plant or fungal species.

Second, fungal regulatory targets exhibit clear specificity for different metabolite classes: (1) For terpenoid saponins, fungi predominantly target the rate-limiting enzymes of the core carbon skeleton biosynthetic pathway (MVA/MEP pathway), such as HMGR, SQS, and DS; (2) For alkaloids, fungi primarily regulate the supply of precursor amino acids (ornithine, arginine, tryptophan) and the first committed enzyme of the alkaloid biosynthetic pathway; (3) For flavonoids and phenolics, fungi mainly activate the entry enzyme of the phenylpropane pathway (PAL) and key branch-point enzymes of the flavonoid pathway.

This specificity is determined by the biosynthetic architecture of each metabolite class: terpenoid and flavonoid pathways are controlled by a small number of rate-limiting entry enzymes, while alkaloid biosynthesis is primarily limited by precursor availability. Endophytic fungi have evolved to target the most “cost-effective” regulatory node for each metabolite class, maximizing the enhancement of target metabolites with minimal energetic cost to the host.¹⁹

Third, the experimental system directly determines the magnitude of regulatory effects: in vitro hairy root/cell suspension cultures consistently show higher metabolite enhancement (100–300% increase) than greenhouse pot experiments (40–80% increase) and field experiments (20–50% increase). This gradient is explained by the absence of environmental variability, biotic interference, and resource trade-offs in in vitro systems. This finding highlights a critical gap in the field: most studies report high efficacy in controlled in vitro systems, but less than 5% have validated these effects under field conditions, which is the ultimate bottleneck for agricultural application.⁴³

3.5. Analysis of Contradictory Findings in the Field

Our systematic synthesis identifies three core drivers of contradictory findings across studies, which have been largely overlooked in previous descriptive reviews.^18,29First, fungal strain specificity is the primary cause of inconsistent results: even within the same fungal species, different strains can exhibit dramatically different regulatory efficacy, ranging from significant enhancement to no effect, or even inhibition of target metabolite biosynthesis.^46,60For example, different Fusarium solani strains isolated from licorice roots have shown glycyrrhizin enhancement effects ranging from 28% to 171% in independent studies,^17,23,25 which is attributed to differences in elicitor secretion capacity, host adaptation, and functional gene content between strains.^17,24This extreme strain specificity means that findings from one strain cannot be generalized to other strains within the same genus, a common oversight in many meta-analyses.^19,53

Second, experimental conditions and host genotype drive large variations in regulatory outcomes.^19,38,40The same fungal strain can produce opposite effects in different host cultivars of the same medicinal plant species, due to genetic polymorphisms in pattern recognition receptors (PRRs) and key biosynthetic enzyme genes.^36,51Similarly, growth conditions such as nutrient availability, temperature, and abiotic stress can completely alter the symbiotic outcome^40,65,67:a strain that significantly enhances metabolite production under moderate drought stress may have no effect under well-watered conditions, due to the host’s shifted resource allocation strategy.^64,65

Third, methodological differences in detection and validation create apparent contradictions.^18,29Studies using culture-independent metagenomic sequencing often report correlational associations between fungal taxa and metabolite content,^16,18 while inoculation-based experimental studies provide causal evidence for regulatory effects.^9,17These two approaches can produce seemingly contradictory results: a fungal taxon that is positively correlated with metabolite content in community studies may have no direct regulatory effect in inoculation experiments, as its correlation may be driven by indirect interactions with other community members.^18,44,68Similarly, differences in metabolite detection methods and sampling time points can lead to significant variations in reported enhancement levels.^11,52

4. Mechanisms Underlying Endophytic Fungi-Mediated Regulation of Active Components

Endophytic fungi-mediated enhancement of bioactive compound biosynthesis in medicinal plant roots is a highly coordinated, hierarchical process rather than a random, isolated event. Here, we synthesize current mechanistic findings into a unified four-tiered regulatory framework, which sequentially comprises molecular signal perception and defense priming, hormone signaling network modulation, transcriptional reprogramming of biosynthetic pathways, and enzymatic biotransformation with metabolic flux redirection. This framework fully explains both the conservation of core regulatory pathways and the metabolite targeting specificity across diverse plant-fungus symbiotic systems.

4.1. Tier 1: Molecular Signal Perception and Elicitor-Induced Defense Priming

The symbiotic relationship is initiated and maintained by a well-documented but incompletely resolved molecular dialogue, rather than a fully characterized “sophisticated molecular interaction”. Endophytic fungi secrete a diverse array of Microbe-Associated Molecular Patterns (MAMPs), such as chitin oligosaccharides, glycoproteins, and fungal cell wall components, which are recognized by specific plant pattern recognition receptors (PRRs) on the root surface.^51,69 This recognition triggers a mild, non-destructive “defense priming” immune response, the critical first step for subsequent metabolic regulation. Unlike pathogenic fungi that induce full hypersensitive responses, compatible endophytes have evolved to modulate this immune response, avoiding host cell death while activating downstream signaling cascades that converge on secondary metabolism.^49,50

Beyond MAMPs, fungi release specific elicitors including small peptides, effector proteins, and low-molecular-weight metabolites, which act as highly specific signals for metabolic regulation.^36,49,70 For instance, the mycelial extract from Schizophyllum commune alone can upregulate ginsenoside biosynthetic genes in P. ginseng hairy roots without physical fungal colonization, confirming the sufficiency of fungal elicitors to trigger metabolic reprogramming.⁵⁷ Notably, current research has critical limitations: less than 10% of studies have identified specific fungal elicitors driving metabolic regulation, and very few have functionally validated the corresponding host PRRs. Most existing evidence remains correlational, with no causal validation of elicitor-PRR interactions, representing the most fundamental knowledge gap in symbiotic mechanism research. This initial signal exchange sets the stage for a reallocation of the plant’s resources from primary growth to defense-associated secondary metabolism.

4.2. Tier 2: Modulation of Plant Hormone Signaling Networks

The interplay between plant hormone pathways acts as a central hub, translating fungal signals into metabolic reprogramming. The core, conserved mechanism across all symbiotic systems is the targeted modulation of jasmonate (JA) and salicylate (SA) signaling: endophytes suppress SA-mediated defenses against biotrophic pathogens to enable stable root colonization, while simultaneously activating the JA pathway, the master regulator of plant secondary metabolism.^36,37

JA-mediated elicitation is universally observed across all metabolite classes, as JA signaling activation upregulates the biosynthesis of terpenoids, alkaloids and flavonoids in nearly all medicinal plants studied to date.^50,52 As noted previously, this conservation is driven by convergent evolution: JA-SA antagonism enables endophytes to achieve both successful colonization and enhanced host secondary metabolism, a win-win strategy for mutualistic symbiosis. Beyond JA-SA crosstalk, endophytes also modulate ethylene (ET) and abscisic acid (ABA) pathways to fine-tune metabolic regulation under abiotic stress. For example, under drought stress, endophyte-induced ABA biosynthesis further amplifies JA-mediated flavonoid production via ABA-JA crosstalk, simultaneously enhancing host drought tolerance and medicinal value.^48,65

4.3. Tier 3: Transcriptional and Post-transcriptional Regulation of Biosynthetic Genes

The fungal-initiated signal transduction cascade culminates in targeted transcriptional reprogramming of the host genome, rather than fully characterized “precise transcriptional control”, driving systematic upregulation of genes encoding rate-limiting enzymes in bioactive compound biosynthesis, which serves as the direct, proximal mechanism for enhanced metabolite production. Two consistent regulatory patterns are observed across all plant-fungus systems. First, endophytic fungi coordinately upregulate all key genes in the target biosynthetic pathway, from precursor supply to final product formation, rather than targeting a single gene. For example, Fusarium solani inoculation in licorice upregulates all core genes in the glycyrrhizin biosynthetic pathway (SQS1, SQS2, CYP88D6, CYP72A154), while downregulating genes in competing pathways (LUS, CAS) to redirect metabolic flux toward glycyrrhizin production.¹⁷ Second, endophytes exhibit high specificity in targeting rate-limiting and branch-point genes that have the greatest impact on metabolite yield. In the glycyrrhizin pathway, for instance, late-stage cytochrome P450 genes (CYP88D6, CYP72A154) that control triterpenoid skeleton modification are the most strongly upregulated by endophytic fungi.²⁴

At the transcriptional regulation level, functionally validated host transcription factors targeted by endophytic fungi include MYB, bHLH, and WRKY family members, which are the core transcriptional regulators of plant secondary metabolism.^4,62For example, endophytic fungal elicitation has been shown to activate MYB transcription factors that directly bind to the promoter regions of SQS and β-AS genes in the glycyrrhizin biosynthetic pathway, driving their coordinated upregulation.^17,24Similarly, JA-responsive MYC2 transcription factors, the master regulators of JA signaling, are consistently activated by endophytic fungi across different plant–fungus systems, serving as the core link between hormone signaling and transcriptional reprogramming of biosynthetic pathways.^36,48,50

Beyond transcriptional regulation, fungi can enhance metabolic efficiency through the formation of metabolons, transient multi-enzyme complexes that channel unstable intermediates directly between active sites. This mechanism has been well characterized in fungal alkaloid biosynthesis, where direct protein-protein interactions between FAD oxidase and P450 oxidase form a metabolic channel that reduces intermediate diffusion and improves reaction efficiency.^62,63 However, it is critical to emphasize that this metabolon model has not yet been validated in plant-fungus symbiotic systems, with no direct evidence of protein-protein interactions between plant biosynthetic enzymes induced by endophytic fungal colonization. It remains a plausible but unproven hypothesis for the high efficiency of fungal-mediated metabolic enhancement, representing a high-priority research direction.¹⁷

Notably, the vast majority of studies only characterize transcriptional regulation, with almost no investigation of post-transcriptional, epigenetic, or non-coding RNA-mediated regulation by endophytic fungi. This represents a major frontier for future research: emerging evidence in other plant-microbe systems shows that endophytic fungi can modulate host DNA methylation, histone modification, and small interfering RNA (siRNA) trafficking to regulate gene expression, but these mechanisms remain completely uncharacterized in medicinal plant-endophyte symbiotic systems.^4,29 Cross-kingdom RNA trafficking, in particular, may serve as a key mechanism for fungal manipulation of host gene expression, but no functional studies have been conducted to date.^36,62

4.4. Tier 4: Enzymatic Biotransformation and Metabolic Flux Redirection

The final tier of regulation involves direct enzymatic modification of plant metabolites by endophytic fungi, via two complementary pathways: biotransformation of primary metabolites into rare, high-value derivatives with higher bioavailability, and feedback activation of the host biosynthetic pathway through metabolite conversion. For example, endophytic fungi can convert primary ginsenosides into rare ginsenosides with enhanced anticancer activity,⁴⁶ and transform glycyrrhizin into derivatives with stronger anti-inflammatory effects.²⁶ This direct biotransformation mechanism holds significant biotechnological potential, enabling in vitro production of rare medicinal compounds without whole-plant cultivation.

5. Factors Influencing the Regulation of Active Components Mediated by Fungi

The regulatory effects of endophytic fungi on bioactive metabolite biosynthesis in medicinal plant roots are not uniform but are dynamically modulated by complex interactions between host plants, fungal symbionts, and their surrounding environments. Understanding these influential factors is crucial for optimizing the application of endophytic fungi in medicinal plant cultivation and secondary metabolite production.

5.1. Host-Related Factors

The host genotype serves as a primary driver of the differential outcomes in endophyte-mediated metabolite regulation observed among intraspecific variants of medicinal plants. These variations largely stem from genetic polymorphisms in two key components: the genes encoding plant pattern recognition receptors (PRRs), which influence fungal signal perception, and those encoding key enzymes within secondary metabolic pathways, which directly shape the efficiency of metabolic reprogramming.^66,71,72 These findings highlight the necessity of matching fungal strains with appropriate host cultivars for optimal metabolite enhancement.

The regulatory efficacy of endophytic fungi varies significantly across the host’s life cycle, mirroring changes in root structure, physiology, and resource allocation. During the seedling stage, rapid root elongation and low secondary metabolite biosynthesis limit fungal colonization and regulatory effects. In contrast, the mature stage (3–5 years for most medicinal roots) is characterized by enhanced root lignification, increased root exudation of signaling molecules, and elevated metabolic flux toward secondary pathways, creating a favorable niche for endophytic fungi.^38,73

Root exudates serve as chemical mediators of host-fungus communication, shaping endophytic community structure and functional interactions. Specific compounds in exudates, such as flavonoids, strigolactones, and organic acids, act as chemoattractants for compatible fungi and trigger the secretion of fungal elicitors.^33,67,68,74 Crucially, the composition of these root exudates is modulated by both host genotype and environmental conditions, thereby forming a dynamic feedback loop that fine-tunes fungal-mediated metabolite production in the host plant.⁷⁵

5.2. Fungal-Related Factors

Even within the same fungal genus, different strains display distinct abilities to regulate host secondary metabolism, attributed to variations in elicitor production, nutrient utilization, and immune modulation strategies.⁶⁰ Similarly, Penicillium chermesinum strain P1 from Panax notoginseng efficiently converted ginsenoside Rb1 to Rd (conversion rate 35%), whereas the closely related Penicillium citrinum strain P2 lacked this biotransformation capability.⁴⁶ Further demonstrating strain-specific functionality, the endophytic fungus Fusarium sp. DN689 has been established as a sustainable microbial platform for dendrobine production, providing the first genomic evidence of its biosynthetic capacity. Genomic mining of this strain uncovered 13 terpenoid biosynthetic gene clusters, and functional analysis identified a key sesquiterpene cyclase while revealing a positive correlation between the expression of four cyclase/oxidase genes and alkaloid accumulation.⁷⁶ Together, these findings establish a foundational genomic framework for metabolic engineering and alternative production of this pharmaceutically valuable compound. This extreme strain-specificity means that findings from one fungal strain cannot be generalized to other strains within the same genus or species, a critical point that is often overlooked in the literature.⁴³

5.3. Abiotic Environmental Factors

Abiotic conditions modulate the host-endophyte interaction by influencing fungal colonization, host physiology, and metabolic pathway activation, with key factors including stressors, soil properties, and nutrient availability. Abiotic stress often acts as a “double-edged sword”: moderate stress can enhance fungal-mediated metabolite regulation by priming host defense responses, while severe stress inhibits both fungal growth and host metabolism.⁴⁰ This synergy is mediated by ABA-JA cross-talk, where stress-induced ABA upregulates JA biosynthesis, amplifying fungal elicitation of flavonoid pathways.⁴¹ Similarly, moderate salinity (100 mM NaCl) enhanced glycyrrhizin production by 56% in Glycyrrhiza uralensis inoculated with arbuscular mycorrhizal fungi, by upregulating bAS and P450 genes.²⁵ However, high salinity (>200 mM) or extreme temperatures (°C or >35°C) reduced fungal colonization and metabolic activity, diminishing regulatory effects.³⁹

Nitrogen (N), phosphorus (P), and other nutrients modulate the allocation of host resources between primary and secondary metabolism, indirectly affecting fungal-mediated regulation. Low N availability enhances secondary metabolite production by redirecting metabolic flux away from protein synthesis, and endophytic fungi further amplify this effect by improving N uptake and assimilation.^42,77 For Anisodus tanguticus, low N conditions combined with endophytic fungal inoculation increased anisodine content by 72%, as fungi enhanced ornithine and arginine supply for tropane alkaloid biosynthesis.⁷⁸ Similarly, P deficiency induces the expression of fungal phosphatases, which solubilize soil P and promote host secondary metabolism.^79,80 However, excessive N suppresses fungal-mediated metabolite regulation by favoring primary growth over secondary metabolism.⁴².

5.4. Biotic Interactions

Rhizosphere bacteria often synergize with endophytic fungi to enhance metabolite production, by promoting fungal colonization, modifying host signaling, or supplementing metabolic precursors.⁴⁴ In Citri Grandis Exocarpium, long-term organic fertilization enriched the functional attributes of endophytic bacteria and fungi. This shift in the endophytic community broadened the metabolic profile of the fruit peels and led to a marked increase in the fruit’s content of key medicinal compounds, namely flavonoids and polysaccharides.⁴⁵ A study reveals that bacterial-fungal mutualism hinges on the host fungus’s specific adaptation to a conserved bacterial signal. Bacteria employ a common set of symbiosis factors, but whether the interaction becomes mutualistic or antagonistic is determined by the fungal ROS response. Mutualistic fungi suppress reactive oxygen species to enable bacterial persistence, whereas antagonistic fungi elevate ROS for defense. Thus, compatibility is defined by this differential fungal adjustment.⁴⁷

6. Challenges and Future Research

Despite substantial advances in deciphering endophytic fungi-mediated regulation of secondary metabolism in root-derived medicinal plants, several critical unresolved challenges remain, which severely limit the translation of fundamental research into practical applications. First, the pronounced host-fungal specificity of beneficial symbioses restricts broad applicability: the metabolite-enhancing effects of elite fungal strains are rarely transferable across heterologous host species, or even between different cultivars of the same medicinal plant.^60,76 Second, the molecular mechanisms underpinning stable symbiosis remain incompletely elucidated, with major knowledge gaps persisting in epigenetic regulation and non-coding RNA-mediated post-transcriptional control of host metabolic reprogramming during fungal colonization.^4,29 Third, the translation of laboratory findings to field cultivation is significantly hampered by environmental heterogeneity and native rhizosphere biotic interference, both of which drastically reduce the stability and reproducibility of endophyte-mediated metabolite enhancement in real agricultural settings.^40,47

To address these bottlenecks, future research should prioritize four key directions: first, deploying holo-omics approaches to systematically disentangle the multi-partite interactions among host plants, endophytes, pathogens, and the abiotic environment.⁸ This integrated approach is uniquely positioned to answer two critical unresolved questions regarding the emergent properties of the plant-fungus holobiont that cannot be addressed by single-strain or single-omic studies: (1) how the entire root endophytic fungal community, rather than individual strains, collectively modulates host secondary metabolism through functional complementarity and interspecies signaling; and (2) the molecular basis underlying the conditional shift of symbiotic outcomes from commensalism to mutualism and ultimately to latent pathogenicity across different environmental contexts.

Second, the rational design of synthetic microbial consortia that integrate complementary endophytic fungi and rhizobacteria to achieve robust, tailored functional outcomes.^44,45

Third, applying metabolic engineering and synthetic biology tools to optimize high-efficiency fungal elicitors, or to establish heterologous biosynthesis platforms for rare high-value medicinal metabolites.^61,76

Fourth, developing standardized field inoculation protocols that fully account for host developmental stage and environmental adaptability to ensure consistent performance in large-scale cultivation.^38,73

To explicitly resolve the translational gap between in vitro and field results, we propose that standardized, multi-location field trials across at least 3 geographically distinct sites with consistent experimental design should be established as a minimum requirement for validating “High” evidence-level claims regarding endophyte-mediated metabolite enhancement. These integrated research efforts will help translate foundational insights into sustainable cultivation practices and novel drug discovery.

Footnotes

ORCID iD

Wangsuo Liu

Author Contributions

YL: Investigation, Data curation, Methodology, Visualization, Writing-review & editing. WL: Funding acquisition, Project administration, Supervision, Writing-original draft, Writing-review & editing.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the High-Level Talent Introduction Research Start-up Funding of Hetao College of China [HYRC202401], Project for the Inner Mongolia Natural Science Foundation [2025LHMS03017], National Natural Science Foundation of China [32360327], and the Project for Ecological Environment Innovation Team Building of Hetao College of China [HTKCT-A202408].

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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