AMF enhance secondary metabolite production in ashwagandha, licorice, and marigold in a fungi-host specific manner

Highlights

• Inoculation of AMF improved secondary metabolite production in the host plants.

• Ashwagandha and marigold showed maximum secondary metabolites production with Rhizophagus irregularis inoculation.

• Licorice showed maximum secondary metabolites production with Claroideoglomus etunicatum inoculation.

Abstract

Strategies to enhance the production of secondary metabolites, derived from medicinal and agriculturally important plants have been the subject of exploration to enable effective utilization of these biorepositories. Through symbiosis, arbuscular mycorrhizal fungi (AMF), modify plant primary and secondary metabolite biosynthesis. The relationship thus offers the opportunity to exploit combinations of host and fungus that maximize secondary metabolite production. We investigated different AMF host combinations for the enhancement of root-derived secondary metabolites from three plant species - ashwagandha (Withania somnifera (L.) Dunal), licorice (Glycyrrhiza glabra L.), and marigold (Tagetes erecta L.). Each host species was inoculated singly with each of five species of AMF, Glomus hoi, Claroideoglomus etunicatum, Claroideoglomus claroideum, Rhizophagus irregularis, and Acaulospora delicata and secondary metabolite production was assessed. Increased concentrations of the following secondary metabolites were found in roots after AMF establishment: for withaferin-A in ashwagandha (concentrations ranged from 11.5 to 43.5% above than in control non-mycorrhized roots depending on the host and AMF combination); in licorice, glycyrrhizic acid (1.51–3.92% above control) and glabridin (2.85–6.41% above control) and in marigold, alpha-terthienyl (1.51–7.18% above control). Specifically, among the AMF inoculations, the highest levels of secondary metabolite were found in ashwagandha and marigold inoculated with R. irregularis and for licorice following inoculation with C. etunicatum revealing the impact of different AMF species on different plant species. This underpinning knowledge of AMF symbioses with the plant host will augment the development of methods that will provide enhanced concentrations of secondary metabolites of commercial value.

Introduction

Arbuscular mycorrhizal fungi associations play crucial roles in plant nutrient uptake and hence strongly influence productivity, through an enhanced tolerance to abiotic and biotic stress. (Bitterlich et al., 2018; Kumar et al., 2017; Smith and Read, 2008). Furthermore, following AMF inoculation more than 50 medicinal and aromatic plant species have been investigated for potential health-promoting compounds (Golubkina et al., 2020; Zeng et al., 2013). Enhancement in the yield of secondary metabolites (Toussaint et al., 2007; Zubek et al., 2015) following AMF inoculation results through stimulation of secondary metabolism (Schliemann et al., 2008) via up-regulation in the expression of genes that drive key metabolic pathways (Kaur and Suseela, 2020). The increased biosynthesis via these pathways of prominent classes of secondary metabolites such as phenolics (Dos Santos et al., 2017), alkaloids (Andrade et al., 2013), and terpenes (De Souza Ferrari et al., 2020) show the significant influence that AMF has on resource allocation to and by the host.

Arbuscular mycorrhizal fungi partnership with host involves several symbiotic events that are modulated by both the partners eventually giving rise to AMF-host compatibility (Feddermann et al., 2010). The functional aspect of both partners may thus also result in the extent of the strength or weakness of the symbiosis (Campos et al., 2018). Recent reviews (Kaur and Suseela, 2020; Zeng et al., 2013) on the influence of AMF on secondary metabolite production have highlighted the diversity of AMF-host interactions. Several mechanistic studies have investigated the differential expression of plant genes upon AMF inoculation (Battini et al., 2016), and have revealed of expression that may be used to identify plant specificity for increased metabolite production (Rivero et al., 2015). This affinity of certain AMF taxa for specific plant species or cultivars (Avio et al., 2018) is thus necessary to be extensively explored for the selection of effective AMF- host combinations for improved secondary metabolite production. In the present study, we have examined the interaction between five AMF species of broad host range with three plant hosts, Ashwagandha (Withania somnifera (L.) Dunal); Solanaceae), Licorice (Glycyrrhiza glabra; Fabaceae) and Marigold (Tagetes erecta; Asteraceae).

Ashwagandha and licorice are two well-known medicinal plant species (Pastorino et al., 2018; Rayees and Malik, 2017), but the effect of AMF associations on secondary metabolite production has not been comprehensively analysed. For example, studies on ashwagandha and associated AMF have been primarily concerned with the effectiveness of AMF in the promotion of vegetative growth and to the facilitation of increased soil nutrient absorption (Hosamani et al., 2011). However, the influence of AMF on secondary metabolite production in ashwagandha has not been examined, especially for the pharmacologically important steroidal lactones, the withanolides, and withaferin-A in particular (Chirumamilla et al., 2017) which has potential as a therapeutic agent for the treatment of cancer (Dutta et al., 2019). Similarly, licorice is well-known for its extensive use in herbal medicines. One of the most important compounds produced by this species is glycyrrhizic acid (glycyrrhizin), a water-soluble triterpenoid glycoside found in the roots and rhizomes and reported to possess antiviral effects (Sun et al., 2019). Recently, the role of glycyrrhizic acid as a multifunctional drug carrier was also demonstrated (Selyutina and Polyakov, 2019). Further, glabridin, also isolated from the root of licorice has exhibited tyrosinase inhibition and therefore is used widely in the cosmetics industry (Chen et al., 2016). Symbiosis with AMF in licorice promotes growth (Öztürk et al., 2017), increases metabolite production (Orujei et al., 2013), and shortens the acclimatization period during transplantation (Yadav et al., 2013), but its secondary metabolite production, especially of glycyrrhizic acid and glabridin, has not been systematically explored. In the current study, we also investigated marigold, for which AMF associations are known, for example, to alleviate drought stress (Asrar and Elhindi, 2011) and contribute to phytoremediation of soils (Castillo et al., 2011) but importantly, marigold also produces a range of agriculturally important organosulphur compounds, thiophenes (specifically alpha-terthienyl) which have nematicidal properties (Hooks et al., 2010; Marotti et al., 2010). The influence that AMF symbiosis has on alpha-terthienyl production in marigold roots has not been explored.

The objective of the current study was to analyse secondary metabolite production in roots of three host plant species following inoculation individually, of five different AMF species and further, selection of effective AMF-host combinations, which would contribute towards enhanced secondary metabolite production. The plant species investigated in this study produced several metabolites of medicinal and/or agricultural importance and thus an ability to increase their production to enable economies of scale is desirable.