Arbuscular mycorrhizal fungi enhance monoterpene production in red clover (Trifolium pratense L.): a potential tool for pest control

Abstract The possibility of modifying terpene production in plants is a defensive strategy that has been studied in conjunction with their biosynthetic pathways. A biotic factor such as Arbuscular Mycorrhizal Fungi (AMF) could modify terpene production in Trifolium pratense L. In this work, the enzymatic production of monoterpenes in Superqueli INIA cultivar with two AMF was evaluated via HeadSpace-Gas Chromatography (HS-GC). A significant increase of (S)-limonene was found in plants inoculated with Claroideoglomus claroideum as well as with the AMF mix (genera Scutellospora, Acaulospora and Glomus). Moreover, significant increases in other monoterpenes such as (-)-β-pinene, myrcene, linalool, were observed. Results showed higher monoterpene production capacities in the Superqueli-INIA cultivar, suggesting the participation of monoterpene synthases (MTS). The significant rise of (S)-limonene in red clover plants inoculated with AMF suggests this strategy could be implemented in an agronomical manage for controlling the H. obscurus, the primary pest. Graphical Abstract


Introduction
Trifolium pratense L., commonly named as red clover, is an important leguminous forage used for livestock. It is classified as perennial plant, but its survival does not exceed three years because it is strongly affected by the red clover root borer, Hylastinus obscurus Marsham (Ortega et al. 2003). Adult and larval states of H. obscurus feed on the main root of this plant, affecting plant development and growth. Hitherto, biological, and chemical strategies for controlling this pest are limited. Interestingly, studies have shown that secondary metabolites can mediate the chemical communication between plants and insects (Chen et al. 2011). For instance, (S)-limonene, a monoterpene released from T. pratense has a natural repellent effect on H. obscurus (Parra et al. 2013). The biosynthesis of these compounds can be affected by biotic factors, such as the arbuscular mycorrhizal fungi (AMF). In fact, the AMF is known to induce morphological, physiological, and biochemical changes, including secondary metabolites (Kobae 2019). The relationship between H. obscurus and (S)-limonene present in red clover, stimulates the following question: Will it be possible to increase the (S)-limonene production in AMF-inoculated red clover plants? We hypothesize that symbiosis associated with AMF C. claroideum (AMF 1 ) and mix (genera Scutellospora, Acaulospora, Glomus) (AMF 2 ) positively affect the biosynthetic pathway of monoterpene production in red clover (Superqueli cultivar). The evaluation of the enzymatic activities of secondary metabolites associate to defensive semiochemicals in red clover have been explored previously by our research group (Muñoz et al. 2021). However, there are no studies of enzymatic activities associating the effect of AMF on terpenoids production. Thus, the aim of this work was to establish the effect of AMF on monoterpene production determining the respective enzymatic activities in red clover.

Identification and quantification of monoterpenes by enzymatic activity
Enzymatic activities, expressed as mg monoterpene/mg total protein per hour (mg mg À1 h À1 ), were performed to evaluate the effect of AMF on monoterpenes production. A total of 10 monoterpenes were identified: geraniol, linalool, b-pinene, dihydromyrcenol, myrcene, a and b-phellandrene, (S)-limonene, (À)-isopiperitenone and perillyl alcohol. All the monoterpenes identified are generated by the action of monoterpene synthases (MTSs), a group of key enzymes transforming geranyl pyrophosphate (GPP) to different monoterpenes (Chen et al. 2011). The MTSs (or cyclases) are involved in the conversion of the acyclic isoprenoid diphosphate precursor to cyclic hydrocarbon (Gao et al. 2012). This reaction depends on Mn þ2 or Mg þ2 and it is initiated by the ionization of GPP to generate an allylic carbenium ion intermediate (terpinyl cation) (Lesburg et al. 1998).
The AMF-plant symbiotic relationships play a key role in more than 80% of terrestrial plants resulting in an enhancing of their mineral nutrition (Smith and Read 2008;Kobae 2019). The effect of AMF in increasing terpenoids production has been well stablished (Zeng et al. 2013;Welling et al. 2015;Kapoor et al. 2017). However, most of the studies have been focalized in increasing the terpenoids components of essential oil from medicinal plants. Kapoor et al. (2017) reported the analysis of essential oils from 27 plants inoculated with AMF, where 89% of these species showed an increase in the essential content obtained from fruits, seeds, leaves, roots, shoots, and rhizome. We report huge increment of individuals monoterpenes, such as b-pinene (1,042%), myrcene (4,655%), limonene (471%) among others (Tables S1 and S2). Perusal literature reveals the AMFs Glomus macrocarpum and G. fasciculatum increase the content of b-pinene, myrcene, limonene, a-phellandrene, linalool and geraniol in 29, 221, 76.9, 123, 9.1 and 408.7% respectively (Kapoor et al. 2002a(Kapoor et al. , 2002bHabeeb et al. 2020). Different AMFs species can change the metabolome of the host plant (Zeng et al. 2013;Kaur and Suseela 2020). The relative percentage of terpenes present in dill, carum and coriander essential oil (Kapoor et al. 2002a(Kapoor et al. , 2002b was dependent of the Glomus species inoculated, and moreover in some cases negative effects were observed related to the controls. This effect has been related to genetic composition of specific AMF genotypes (Welling et al. 2015). These results suggest that the AMF 1 and AMF 2 genotypes are ecologically compatible, reducing the variation in their symbiosis with red clover and the subsequent biosynthesis of monoterpenes.
Finally, the literature indicates that the increase in the content of secondary metabolites emerge as a defense response to fungal colonization (Zeng et al. 2013), as is the case of herbivory by insects (Shrivastava et al. 2015). Our results show that the increase in limonene, myrcene and b-pinene concentration by AMF colonization could provide protection to the red clover against insect attack. Yildirim et al. (2013) reported that myrcene presents an insecticidal effect on the curculionid Sitophilus zeamais, finding 33% mortality, whereas the treatment with b-pinene showed 53% mortality after 96 hours of exposure. We believe that a potential toxic effect of myrcene or b-pinene would represent an opportunity for controlling H. obscurus in addition to the repellent (S)-limonene (Parra et al. 2013).

Conclusions
Our results show that the SQ cultivar, when inoculated with AMF, significantly increases the total production of the identified monoterpenes, with emphasis on the production of (S)-limonene and other monoterpenes, such as myrcene and b-pinene. This suggests that the respective enzymatic complexes associated with MTS are improved in the same extension by AMF 1 and AMF 2 symbioses. Future studies will be carried out to propose or identify the action mechanism of monoterpene synthase in T. pratense and to establish the roles of myrcene and b-pinene in the behaviors of H. obscurus, as well as to assess the toxicity and environmental impact of these metabolites.

Funding
Financial support was provided by FONDECYT N 1181697.