Introduction

Medicinal plants have been a valuable source of therapeutic agents to treat various ailments and diseases such as diarrhea, fever, colds, and malaria since ancient times (Dambisya and Tindimwebwa 2003; Ghiaee et al. 2014; Mathens and Bellanger 2010; Titanji et al. 2008). Nowadays, they also represent a source for the development of new drugs to cure important diseases such as cancer (Newman and Cragg 2007; Beik et al. 2020). Their therapeutic value often is attributed to the presence and richness of active compounds belonging to the secondary metabolism, such as alkaloids, flavonoids, terpenoids, and phenolics (Hussein and El-Anssary 2018). Today, up to 80% of people in develo** countries are totally dependent on herbal drugs for their primary healthcare (Ekor 2014), and over 25% of prescribed medicines in developed countries have been derived from plants collected in the wild (Hamilton 2004).

Numerous methods, such as isolation from plants and other natural sources, synthetic chemistry, combinatorial chemistry, and molecular modeling, have been used for drug discovery (Ley and Baxendale 2002; Geysen et al. 2003; Lombardino and Lowe 2004). However, natural products, and particularly medicinal plants, remain an important source of new drugs, new drug leads, and new chemical entities (Newman et al. 2000, 2003; Butler 2004) because of their cultural acceptability, high compatibility, and adaptability with the human body compared to synthetic chemicals (Garg et al. 2021). According to the International Union for Conservation of Nature and the World Wildlife Fund (Chen et al. 2016), an estimate of as many as 80,000 flowering plant species are used for medicinal purposes. For several thousands of plants worldwide, the activity or composition in bioactive compounds remains poorly documented, requiring further in-depth analysis to fully exploit their medicinal potential (Ali 2019).

In nature, plants are associated with an overwhelming number of beneficial microorganisms (e.g., endophytic or symbiotic bacteria and fungi) that play a significant role in plant health, development, and productivity, and in the modulation of metabolite synthesis (Berendsen et al. 2012; Panke-Buisse et al. 2015; Mendes et al. 2011; Castrillo et al. 2017; de Vries et al. 2020; Brader et al. 2014; Compant et al. 2021). Among these are the arbuscular mycorrhizal fungi (AMF), a ubiquitous group of soil microorganisms, forming symbiosis with more than 70% of vascular plants (Brundrett and Tedersoo 2018). Arbuscular mycorrhizas are characterized by the formation of finely branched structures called arbuscules within root cortical cells of host plants (Coleman et al. 2004), which are the site of bidirectional transport, i.e., minerals from the fungal cell to the plant cell and carbon compounds in the opposite direction.

The establishment of the AMF symbiosis requires recognition between the two partners. Lipochitooligosaccharides, the so-called Myc factors, are perceived by the plant in response to signaling molecules (i.e., strigolactones) released by the roots (Akiyama and Hayashi 2006). After reciprocal recognition, AMF hyphae form a hyphopodium on the root epidermis and colonize the root cortex. At the same time, fungal hyphae spread into the surrounding soil as an extensive extraradical mycelium, representing 9 to 55% of the total soil microbial biomass (Olsson et al. 1999). This dense extraradical mycelium considerably enhances the access of roots to water and mineral nutrients (e.g., P, N, K, Ca, S, Zn, Cu), often increasing plant biomass (Smith and Read 2008; Bowles et al. 2016) and quality of crops (Baum et al. 2015; Bona et al. 2016; Noceto et al. 2021). Moreover, this extraradical mycelium modifies the soil structure (Chen et al. 2018), which improves soil quality and fertility (Zou et al. 2016; Thirkell et al. 2017). AMF also are well known to improve plant resistance or tolerance to stress conditions, such as drought, salinity, nutrient deprivation, extreme temperatures, heavy metals, pests, and diseases (Ahanger et al. 2014; Salam et al. 2017; Porcel et al. 2012; Cicatelli et al. 2014). In addition to these benefits, they also quantitatively and qualitatively could affect the production of secondary metabolites produced by their hosts (Ahanger et al. 2014; Salam et al. 2017; Porcel et al. 2012; Cicatelli et al. 2014; Kaur and Suseela 2020).

Taber and Trappe (1982) were the first to document the presence of AMF in a medicinal plant (in their study conducted on ginger growing in the Fiji Islands and Hawaii). Since then, most medicinal plants were found capable of associating with mycorrhizal fungi (Chen et al. 2014). Recently, single or combinations of AMF have been inoculated to various medicinal plants to investigate their impact on plant biomass as well as on phytochemical constituents in seeds, fruits, leaves, shoots, and roots (e.g., Rydlová et al. 2016; Kapoor et al. 2004; Selvaraj et al. 2009; Dave et al. 2011; Zubek et al. 2012). The majority of studies revealed that AMF were able to enhance plant biomass as well as to promote the accumulation of several active compounds. For example, Lazzara et al. (2017) reported an increased above- and belowground biomass in Hypericum perforatum associated with a mixture of nine different AMF species. Interestingly, the concentrations of pseudohypericin and hypericin, two anthraquinone derivatives that exhibit important photodynamic, antiviral, antiretroviral, antibacterial, antipsoriatic, antidepressant, and antitumoral biological activities (Zubek et al. 2012; Bombardelli and Morazzoni 1995; Gadzovska et al. 2005; Guedes and Eriksson 2005), were increased by 166.8 and 279.2% in the AMF-colonized plants as compared to non-mycorrhizal controls (Lazzara et al. 2017). However, these results should not obviate other studies in which no effects on biomass were reported. For instance, Nell et al. (2010) found that AMF colonization decreased the biomass of rhizomes and roots of Valeriana officinalis, while significantly increasing the levels of sesquiterpenic acids. Another study by Engel et al. (2016) reported an increased content of rosmarinic acid and lithospermic acid A isomer (two phenolic compounds) in Melissa officinalis, while both compounds were diminished in Majorana hortensis, in the presence of three mixtures of AMF. More recently, Duc et al. (2021) showed that a mixture of different AMF species improved the salt stress tolerance of Eclipta prostrata, inducing major changes in polyphenol profile.

In this publication, we provide a thorough review of the literature on AMF mediation of secondary metabolites production in medicinal plants. We also review the different methods that are used to increase/stabilize the production of secondary metabolites. Indeed, the quantity and quality of secondary metabolites obtained from plants grown in natural habitats are critically influenced by various abiotic and biotic stresses (e.g., drought, extreme temperatures, and pathogen attack). This results in high variability of bioactive substances and influences the metabolic pathways responsible for the accumulation of the related natural compounds (Dayani and Sabzalian 2017; Giurgiu et al. 2017; Ramakrishna and Ravishankar 2011). Therefore, we additionally review the most widely used methods of cultivation (i.e., greenhouse, hydroponics, aeroponics, in vitro and hairy root cultures (HRCs)) of medicinal plants, and we investigate their possible application to AMF to further increase the quantity and quality of secondary metabolites produced.

Effect of AMF on growth and secondary metabolite production of medicinal plants

Since the pioneer work of Wei and Wang (1989, 1991), reporting the positive effect of AMF inoculation of Datura stramonium and Schizonepeta tenuifolia on the production of active compounds, numerous studies have been conducted. The literature focusing on AMF in medicinal plants involves 81 plant species belonging to 28 families (Table 1). These medicinal plants present different characteristics to be studied with AMF: important medicinal herbs to treat certain disease such as Artemisia annua producing artemisin to treat malaria in develo** countries (Domokos et al. 2018); important condiment plants such as Allium sativum in India (Borde et al. 2009); rare plant species difficult to culture such as Arnica montana (Jurkiewicz et al. 2021; Kapoor et al. 2004). The content of artemisinin, an important sesquiterpene lactone compound found in Artemisia annua and well known for its effects on malaria and more recently on cancer (Krishna et al. 2008), was increased in leaves of plants colonized by F. mosseae or a combination of Glomus macrocarpum and R. fasciculatus or Diversispora epigaea and R. irregularis grown in pots or under field conditions (Huang et al. 2011; Chaudhary et al. 2008; Domokos et al. 2018). The forskolin content, a diterpene extensively used to treat heart diseases, glaucoma, asthma, and certain types of cancers (Kavitha et al. 2010), was significantly increased in roots of Coleus forskohlii inoculated with Glomus bagyarajii growing under greenhouse conditions (Sailo and Bagyaraj 2005). Similarly, Singh et al. (2013) reported an increased content of forskolin in tubers of Coleus forskohlii associated with R. fasciculatus growing under organic field conditions.

Researchers also have studied the impact of AMF symbiosis on medicinal plants derived from tissue cultures. An example is the increased content of the essential oil carvacrol, a phenolic monoterpenoid with antimicrobial, antioxidant, and anticancer activities (Sharifi-Rad et al. 2018) in micropropagated Origanum vulgare subsp. hirtum after association with the AMF Septoglomus viscosum (Morone Fortunato and Avato 2008).

Phenolics

Phenolics represent a wide group of compounds, sharing one or more phenol groups (Hussein and El-Anssary 2018), among which are flavonoids, curcuminoids, coumarins, tannins, stilbenes, lignans, phenolic acids, and quinones (Cosme et al. 2020).

Arbuscular mycorrhizal fungi have been shown to increase the content of phenols in medicinal plants (Table 1). For instance, the production of formononetin (an antimicrobial, antioxidant, antilipidemic, antidiabetic, antitumor, and neuroprotective compound) (Vishnuvathan et al. 2016), was increased in Medicago sativa grown in the presence of R. intraradices (Volpin et al. 1994). The production of curcumin (an anti-inflammatory, antioxidant, anticancer, antiseptic, antiplasmodial, astringent, digestive, diuretic compound) was increased by circa 26% in Curcuma longa colonized by AMF species belonging to the genera Glomus/Rhizophagus, Gigaspora, and Acaulospora sp., under greenhouse conditions (Dutta and Neog 2016). The concentration of total tannins, used to treat tonsillitis, pharyngitis, hemorrhoids, and skin eruptions (Britannica 2021), was increased by 40% in the fruits of Libidibia ferrea inoculated with Acaulospora longula under field conditions (Santos et al. 2020). Additionally, the concentrations of cichoric acid in Echinacea purpurea colonized by R. intraradices (Araim et al. 2009) and p-hydroxybenzoic acid and rutin in Viola tricolor colonized by R. irregularis (Zubek et al. 2015), and the total content of flavonoids in Libidibia ferrea colonized by Gigaspora albida and gallic acid in Valeriana jatamansi colonized by a consortium of three different isolates of R. intraradices spp. (Silvia et al. 2014; Jugran et al. 2015) were increased by the AMF symbiosis.

Saponins

Saponins are characterized by a polycyclic aglycone moiety with either a steroid (steroidal saponins) or triterpenoid (triterpenoidal saponins) attached to a carbohydrate unit (a monosaccharide or oligosaccharide chain) (Hussein and El-Anssary 2018). Among these compounds, a few have demonstrated pharmacological properties, such as antitumor, sedative, expectorant, analgesic, and anti-inflammatory (Hussein and El-Anssary 2018). Arbuscular mycorrhizal fungi were reported to enhance the production of saponins in medicinal plants (Table 1). For instance, the content of glycyrrhizic acid, a triterpenoid saponin used to alleviate bronchitis, gastritis, and jaundice (Pastorino et al. 2018), was increased by 0.38–1.07-fold and by 1.34–1.43-fold after 4 and 30 months, respectively, in Glycyrrhiza glabra (liquorice) plants colonized by F. mosseae and D. epigaea alone or in combination, grown in sand under greenhouse conditions (Liu et al. 2021) showed that inoculation with F. mosseae significantly improved biomass and essential oil content (mainly thymol, p-cymene and γ-terpinene) of Thymus vulgaris plants grown in a 2-year field experiment in intercrop** with soybean under water deficit conditions. Similarly, Mirzaie et al. (2020) reported that inoculation with F. mosseae significantly increased geranial and β-pinene (both belong to oxygenated monoterpenes essential oils) yields of Cymbopogon citratus grown in a greenhouse pot experiment under moderate water stress conditions (50% field capacity).

Salt stress stimulates the accumulation of phenolic compounds in plants as a general defense mechanism to stress (Parvaiz and Satyawati 2008). Intriguingly, this abiotic stress is a principal elicitor influencing synthesis of compounds in many herbs (e.g., cinnamic, gallic, and rosmarinic acids in Thymus vulgaris; glycyrrhizin in Glycyrrhiza glabra; quinic, gallic, and protocatechuic acids in Polygonum equisetiforme) (Bistgani et al. 2019; Behdad et al. 2020; Boughalleb et al. 2020). A recent study by Amanifar and Toghranegar (2020) reported that moderate salt stress stimulated higher production of valerenic acid in Valeriana officinalis than a situation without salt stress. Interestingly, this increase was significant when the plants were colonized by F. mosseae. Duc et al. (2021) found that a mixture of six AMF species (C. etunicatum, C. claroideum, F. mosseae, F. geosporum, Rhizoglomus microaggregatum, and R. intraradices) increased the tolerance of Eclipta prostrata under moderate salt stress in a pot experiment under controlled conditions, inducing major changes in its polyphenol profile.

Minerals, such as cadmium (Cd) and zinc (Zn), also were reported to impact secondary metabolite production in medicinal plants colonized by AMF. For instance, Hashem et al. (2016) observed that an AMF mixture comprising C. etunicatum, F. mosseae, and R. intraradices enhanced the chlorophyll and protein content and considerably reduced lipid peroxidation in Cassia italica plants under Cd stress in a pot experiment. Moreover, AMF inoculation caused a further increase in proline and phenol content ensuring improved plant growth under stress conditions.

Arbuscular mycorrhizal fungi symbiosis improved the disease tolerance of medicinal plants through the mediation of secondary metabolites. For instance, Jaiti et al. (2007) reported that a complex of native AMF species increased the tolerance of Phoenix dactylifera (a plant characterized by high nutritional and therapeutic value of its fruits (Al-Alawi et al. 2017)) against bayoud disease (the most damaging vascular disease of date palm caused by Fusarium oxysporum f. sp. albedinis) by increasing the enzymatic activities of peroxidases and polyphenoloxidases, which are associated with an increase of phenolic compounds in the cell wall.

Mechanisms by which AMF symbiosis promotes secondary metabolism in medicinal plants

It is often considered that the increased concentrations of various secondary metabolite groups (e.g., flavonoids, phenolics) in AMF-colonized plants are a result of the elicitation of several defense response pathways as reviewed by Zeng et al. (2013). For instance, terpenoids in the carotenoid pathway, flavonoids, phenolic compounds, and some alkaloids (such as hyoscyamine and scopolamine) in the phenylpropanoid pathway are often increased in AMF-colonized plants (Kaur and Suseela 2020). These pathways play different roles in the plant-AMF symbiosis, such as signaling, stress tolerance, nutrient uptake, and resistance against biotic and abiotic stresses. However, it is still not totally clear how AMF trigger changes in the concentrations of phytochemicals in plant tissues (Toussaint et al. 2007).

Many studies have focused on the mechanisms by which AMF modulate the production of terpenoids, phenolic compounds, and alkaloids in plants. Terpenoids are synthesized from isoprene units in the methyleritrophosphate (MEP) and the mevalonic acid (MVA) pathways (Zhi et al. 2007). Phenolic compounds (e.g., phenols, flavonoids, protanthocyanidins, tannins) are synthesized in the shikimic acid pathway where phenylpropanoids are formed and in the malonic acid pathway (Oksana et al. 2012). Most of the alkaloids are synthesized from various biological precursors (most amino acids) such as tyrosine and tryptophane in the shikimic acid pathway (Facchini 2001) (Fig. 1).

Fig. 1
figure 1

Main pathways of secondary plant metabolism resulting in the production of alkaloids, phenolics, saponins, and terpenes (in gray, green, pink, and brown shaded portions, respectively) mentioned in this review. Examples of upregulated compounds or classes of compounds in medicinal plants associated with AMF are highlighted with green type. This figure is modified from Dos Santos et al. (2021)

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Several common nutritional and non-nutritional factors have been proposed to explain the increased production of secondary metabolites in AMF-colonized plants (Kapoor et al. 2017; Sharma et al. 2017; Dos Santos et al. 2021) (Fig. 2).

Fig. 2
figure 2

Non-nutritional and nutritional factors influencing the production of secondary metabolites (i.e., terpenoids, phenolics, and flavonoids) in AMF-colonized plants. Non-nutritional factors (leftside in orange): AMF colonization results in the activation of plant defense mechanisms with the production of phenolics and flavonoids. Change in phytohormone levels, such as jasmonic acid (JA), gibberellic acid (GA3), and 6-benzylaminopurine (BAP), increases the number and size of glandular trichomes and leads to transcriptional activation of sesquiterpenoid biosynthetic gene expression. AMF induce the production of signaling molecules, such as nitric oxide, salicylic acid (SA), and hydrogen peroxide, which influence the activation of key enzymes such as l-phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS), for the biosynthesis of phenolic compounds. Nutritional factors (rightside in blue): AMF colonization increases plant nutrients and water uptake leading to increased plant growth and leaf biomass. This results in enhanced plant photosynthetic capacity and increased production of photosynthates which are precursors of different secondary metabolites. Increased leaf biomass leads to an increased density of glandular trichomes in which terpenoids are synthesized and stored. This figure is adapted with permission from Springer Nature Customer Service Centre GmbHS: Springer Nature, Phytochemistry Reviews. Insight into the mechanisms of enhanced production of valuable terpenoids by arbuscular mycorrhiza (Kapoor et al. 2017). We thank Evangelia Tsiokanou (National and Kapodistrian University of Athens, Greece) for graciously providing the picture of the plant used in this figure

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Regarding nutritional factors, the increase was first attributed to the enhanced uptake of nutrients by AMF-colonized plants (Lima et al. 2015; Oliveira et al. 2015; Riter et al. 2014). For example, the role of phosphorus in the synthesis of terpenoids precursors via the MVA (acetyl-CoA, ATP, and NADPH) as well as the MEP (glyceraldehyde phosphate and pyruvate) pathways is widely recognized (Kapoor et al. 2017). Phosphorus enhances terpenoid biosynthesis by increasing the concentration of pyrophosphate compounds, such as isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) (Kapoor et al. 2002, 2004; Zubek et al. 2010), which contain high-energy phosphate bonds. However, Khaosaad et al. (2006) found that the concentration of essential oils significantly increased in two Origanum sp. genotypes colonized by F. mosseae, while the levels of essential oils in plants treated with P did not change. This suggests that the increased concentration of essential oils in AMF-colonized Origanum sp. plants may directly depend on the association with the fungus. In another study by Zubek et al. (2012), AMF colonization improved hypericin and pseudohypericin concentrations in Hypericum perforatum, probably because of an improved plant P and/or N nutrition in presence of the fungi. The increased growth through improved nutrients and water uptake of AMF-colonized plants also explains the enhanced production of these compounds in plants. It is well known that the AMF symbiosis increases shoot biomass, shoot length, and number of nodes in Ocimum basilicum (Gupta et al. 2002; Khaosaad et al. 2008; Rasouli-Sadaghiani et al. 2010; Copetta et al. 2006). Elevated leaf biomass results in increased photosynthetic capacity (Dave et al. 2011; Zubek et al. 2010), thus increasing the production of total photosynthates (e.g., ATP, carbon substrate, glyceraldehyde-3-phosphate, pyruvate, phosphoenolpyruvate, or erythrose-4-phosphate) required for terpenoids, phenolics, and alkaloid biosynthesis (Cao et al. 2008; Hofmeyer et al. 2010; Niinemets et al. 2002).

Regarding non-nutritional factors, alterations in the levels of phytohormones in AMF-colonized plants may reflect their enhanced production (Mandal et al. 2013, 2015a; Zubek et al. 2012). Indeed, it has been shown that the AMF symbiosis changes the concentrations of phytohormones, such as jasmonic acid (JA), gibberellic acid (GA3), and cytokinins (Allen et al. 1980, 1982; Hause et al. 2002; Shaul-Keinan et al. 2002) in plants. Moreover, it has been reported that phytohormones play a role in the secondary metabolism of plants (An et al. 2011; Maes and Goossens 2010; Maes et al. 2008). For instance, JA has been reported to coordinate transcriptional activation of sesquiterpenoid biosynthetic gene expression in Artemisia annua (Maes et al. 2011). Furthermore, the phytohormonal alterations of GA3, BAP (6-benzylaminopurine), and JA have been reported to promote the formation of glandular trichomes (Maes et al. 2011) which is positively correlated with an enhanced concentration of terpenoids in plant leaves. Glandular trichomes are the epidermal secretory structures in which terpenoids are synthesized and stored in plants (Covello et al. 2007). The enhanced concentration of terpenoids (essential oils) and increased glandular trichome density has been observed in a number of plants (e.g., Mentha x piperita, Phaseolus lunatus, and Lavendula angustifolia) (Ringer et al. 2005; Bartram et al. 2006; Behnam et al. 2006). Thus, an increase in trichome density upon mycorrhization often has been linked with an enhanced concentration of terpenoids (Copetta et al. 2006; Kapoor et al. 2007; Morone-Fortunato and Avato 2008). The modification of these secondary metabolite concentrations in AMF-plants also may be due to signaling mechanisms between host plants and the fungi (Larose et al. 2002; Rojas-Andrade et al. 2003; ** profusely in the HC. At that time, one or several receiver micropropagated plants (Alkanna tinctoria) are placed in the HC with their roots in contact with the extraradical mycelium. The plants are planted inside the HC under a lid. Briefly, the base of a cylinder (150 mm high, 100 mm diameter) matches a hole made in the lid of the 145-mm Petri dish. The cylinder top is glued to a 100-mm Petri dish lid. The culture dishes containing the A. tinctoria plants are sealed and covered, up to the base of the cylinder, by black plastic bags. The systems are incubated in a growth chamber to allow plant and AMF growth (detailed procedures of this system can be found in Lalaymia and Declerck (2020)). For system (c), homogenously chopped agar containing AMF propagules from an AMF in vitro culture is inoculated to the newly growing roots of a micropropagated seedling of Lithospermum erythrorhizon. After a few days, the new hyphae growing from the spores colonize the roots of L. erythrorhizon. In system (e), fine root structures of Ri T-DNA transformed Alkanna tinctoria hairy roots are cut and placed in the RC part of a bi-compartmental Petri dish. Chopped agar containing AMF propagules is spread on the young parts of the hairy roots. After a few days, new hyphae growing from spores colonize the A. tinctoria hairy root, producing new spores and extensive mycelium after several months. All these three techniques should be conducted under a laminar flow hood with sterilized laboratory materials

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