Designing the Ideotype Mycorrhizal Symbionts for the Production of Healthy Food

The new paradigm in agriculture, sustainable intensification, is focusing back onto beneficial soil microorganisms, for the role played in reducing the input of chemical fertilizers and pesticides and improving plant nutrition and health. Worldwide, more and more attention is deserved to arbuscular mycorrhizal fungi (AMF), which establish symbioses with the roots of most land plants and facilitate plant nutrient uptake, by means of a large network of extraradical hyphae spreading from colonized roots to the surrounding soil and functioning as a supplementary absorbing system. AMF protect plants from biotic and abiotic stresses and are able to modulate the activity of antioxidant enzymes and the biosynthesis of secondary metabolites (phytochemicals), such as polyphenols, anthocyanins, phytoestrogens and carotenoids, that play a fundamental role in promoting human health. An increasing number of studies focused on the use of AMF symbionts for the production of functional food, with enhanced nutritional and nutraceutical value. Yet, while several plant species were investigated, only few AMF were utilized, thus limiting the full exploitation of their wide physiological and genetic diversity. Here, we will focus on AMF effects on the biosynthesis of plant secondary metabolites with health-promoting activity, and on the criteria for a finely tuned, targeted selection of the best performing symbionts, to be utilized as sustainable biotechnological tools for the production of safe and healthy plant foods.


INTRODUCTION
The new paradigm in agriculture, sustainable intensification, is focusing back onto beneficial soil microorganisms, for the role played in reducing the input of chemical fertilizers and pesticides, while improving plant nutrition and health (Philippot et al., 2013). Worldwide, more and more attention is deserved to arbuscular mycorrhizal (AM) fungi (AMF), a key functional group of beneficial soil microbes belonging to the subphylum Glomeromycotina (Spatafora et al., 2016), able to establish a mutualistic symbiosis with the roots of 80% of plant species (Smith and Read, 2008). These encompass the most important crops for human consumption, such as wheat, rice, corn, barley, pulses, oats and millet, grapevine, olive, vegetables like strawberries, potato, tomato, medicinal plants and economically important species, such as sunflower, sugarcane, cotton, tobacco, coffee, tea, cocoa, rubber and cassava. AMF do not show host specificity, but are asexual obligate biotrophs, unable to complete their life cycle without host plants.
AMF life cycle is simple: germinating spores originate a short-lived mycelium able to recognize the roots of host plants and to differentiate appressoria on their surface. Then appressoria produce hyphae growing intercellularly in the roots and arbuscules, a sort of haustoria formed within root cells, where nutrient exchanges between the two symbionts occur. Up to 20% of total plant photosynthates is transferred to AMF, which, as chemoheterotrophs, utilize them as carbon source (Jakobsen et al., 1992;Smith and Read, 2008;Giovannetti et al., 2012); such transfer enables AMF to grow and form new spores. On the other hand, the extensive extraradical mycelium (ERM) explores the surrounding soil beyond the depletion zone around roots, and increases the root absorbing surface (up to 40 times) (Giovannetti et al., 2001). ERM is able to uptake and translocate soil mineral nutrients, such as phosphorus (P), nitrogen (N), sulfur, potassium, calcium, iron, copper, and zinc, thus improving plant growth and biomass production (Lehmann and Rillig, 2015). In addition, AMF provide diverse ecosystem services, enhancing water uptake, and increasing plant tolerance to biotic and abiotic stresses (Gianinazzi et al., 2010), thereby decreasing the need of chemical fertilizers and pesticides inputs (Toussaint et al., 2008;Sikes et al., 2009).
Several studies reported that AMF may modulate the synthesis of secondary metabolites in host plants, leading to a higher activity of antioxidant enzymes and enhancing the levels of diverse phytochemicals with health-promoting activities (Sbrana et al., 2014). Such findings are very important, as worldwide both consumers and producers are increasingly interested in the health-promoting properties of plant-derived foods. Indeed a number of epidemiological studies reported the role played by some plant secondary metabolites, including polyphenols, glucosinolates, flavonoids and carotenoids in the prevention of chronic diseases, arteriosclerosis, heart diseases and cancer (Duthie, 2000;Johnson, 2002;Lund, 2003). For example, theaflavins and thearubigins from black teas showed antiproliferative action (Bhattacharya et al., 2009), grape seed extract exerted preventive effects against human colon carcinoma and lung epithelial cancer (Wang et al., 2007;Lazzè et al., 2009), luteolin, kaempherol, apigenin and myricetin from diverse fruits and vegetables possessed anti-inflammatory and antibacterial activities (Dillard and German, 2000) and essential oils from myrtle displayed antimutagenic and antigenotoxic properties (Mimica-Dukić et al., 2010). In addition, glucosinolates from broccoli, cauliflower and cabbage were able to modulate carcinogens metabolism and detoxification (Dillard and German, 2000;Tang et al., 2010), while allicin and its organosulfur derivatives from garlic showed antitumoral activities in diverse human cancers (Butt et al., 2009;Teiten et al., 2013;Zhang et al., 2015). Although such phytochemicals are expressed mainly depending on plant genotype, their production may be modulated by diverse agronomic and environmental factors, including AMF symbioses. Here, we will focus on AMF ability to modulate the biosynthesis of plant secondary metabolites with health-promoting activity, and on the criteria for a finely tuned, targeted selection of the best performing symbionts, to be utilized as sustainable biotechnological tools for the production of safe and healthy plant foods.

THE PRODUCTION OF PHYTOCHEMICALS BY MYCORRHIZAL PLANTS
A large body of evidence showed that the establishment of AM symbiosis induces changes in plant physiology, modulating the activity of host cell primary and secondary metabolism (Fester and Hause, 2005;Lohse et al., 2005;Schliemann et al., 2008;Wipf et al., 2014;Schweiger and Müller, 2015;Cervantes-Gámez et al., 2016). Many authors investigated the changes induced by AMF in secondary metabolism, in relation to the production of functional compounds in roots, shoots, leaves, fruits and seeds of many different plant species (Sbrana et al., 2014).
Different species of medicinal and aromatic plants were investigated for their phytochemical contents upon mycorrhizal colonization, showing higher shoot levels of antioxidant compounds, such as rosmarinic acid, caffeic acid and essential oils in basil (Copetta et al., 2006(Copetta et al., , 2007Toussaint et al., 2008;Rasouli-Sadaghiani et al., 2010), and anthraquinone derivatives, such as hypericin and pseudohypericin in Hypericum perforatum (Zubek et al., 2012). Also the levels of essential oils showed altered profiles in mycorrhizal Origanum sp. (Karagiannidis et al., 2011), and large increases in the fruits of mycorrhizal Coriandrum sativum, Anethum graveolens, Trachyspermum ammi, in the leaves of Artemisia annua and in the seeds of Foeniculum vulgare (Kapoor et al., 2002a,b;Chaudhary et al., 2008). Moreover, mycorrhizal plants of Stevia rebaudiana showed enhanced levels of the health-promoting compounds steviol glycosides (Tavarini et al., 2018).
Apart from medicinal plants and herbs, works investigating the phytochemical content of mycorrhizal plants cultivated for human consumption encompass a limited number of species, like lettuce, onion, tomato, maize, artichoke, strawberry, pepper and sweet potato ( Table 1). Most of the data available on edible plant products have been obtained by studying single plant varieties, while only few works investigated the differential responses of cultivars/varieties belonging to the same species of food plants. For example, different mycorrhizal strawberry varieties did not show comparable levels of anthocyanins, anthocyanidins and vitamin C in fruits, while only some green and red leaf lettuce varieties contained larger amounts of anthocyanins, carotenoids, chlorophylls, tocopherol, and total phenolics, and showed a higher antioxidant activity, compared with control plants (Table 1). This represents a limitation of the studies performed so far, given the large number of old      Data are reported on a fresh weight (FW) or dry weight (DW) basis; where unspecified, no information was available. The binomial nomenclature of AMF reported in the quoted papers has been maintained. a DPPH, 2,2-Diphenyl-1-picrylhydrazyl assay; b ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay; c ORAC, oxygen radical absorbance capacity assay; d ESR, electron spin resonance spectroscopy; e FRAP, ferric reducing ability of plasma assay.
and new varieties currently grown worldwide, which could be investigated and selected on the basis of their ability to produce beneficial compounds upon mycorrhizal inoculation. Such a selection would be particularly important for some vegetable species considered functional foods, i.e., globe artichoke, for its hepatoprotective, anticarcinogenic, antioxidative and antibacterial activities, and tomato, for its ability to reduce the risks of cancer and cardiovascular diseases (Canene-Adams et al., 2005). Indeed, artichoke and tomato showed higher antioxidant activity and enhanced levels of health-promoting compounds when produced by AMF-inoculated plants ( Table 1).
The mechanistic explanation of the differential biosynthesis of secondary metabolites in mycorrhizal plants involves the activity of diverse enzymes leading to the production of terpenoids, flavonoids and the aminoacids tyrosine and phenylalanine, precursors of polyphenols in the phenylpropanoid pathway (Peipp et al., 1997;Walter et al., 2000;Lambais et al., 2003;Ponce et al., 2004;Herre et al., 2007;Pozo and Azcon-Aguilar, 2007;López-Ráez et al., 2010a). Such molecules, often accumulated in plant resistance reaction to biotic and abiotic stresses, may be modulated by plant hormones such as ABA or jasmonates possibly involved in long distance signaling and in mycorrhizal priming of defense responses (Cameron et al., 2013;Adolfsson et al., 2017). Several gene expression studies showed a differential modulation of genes encoding for key enzymes of biochemical pathways leading to the production of healthpromoting secondary metabolites in food plants ( Table 2) and model plant species Dixon, 1993, 1994;Bonanomi et al., 2001;Liu et al., 2007;Handa et al., 2015). In food plants, the use of the RNA-Seq technology, allowing investigations of whole transcripts, revealed that many genes, belonging to different functional classes, i.e., post-translational regulation, signaling, transport, biotic and abiotic stresses and hormone metabolism, were upregulated upon AMF inoculation and differentially expressed in fruits, leaves and roots, compared with controls ( Table 2). Unfortunately, most of currently available RNA-Seq data assessing mycorrhizal regulated genes derive only from roots of the investigated plants, such as Citrus sinensis, Cucumis sativus, Helianthus annuus, Litchi chinensis, Oryza sativa, and Vitis vinifera ( Table 2). Since the different genes may be differentially expressed in the diverse plant organs, further works should focus on the edible parts of food plants, in order to obtain information on the genes regulating the production of health-promoting compounds, modulated by mycorrhizal symbioses. In addition, the functional significance of fungal symbiont identity in the modulation of phytochemicals production should be deeply investigated, as large variations in gene expression were detected in model plants inoculated with different AMF (Burleigh et al., 2002;Hohnjec et al., 2005;Deguchi et al., 2007;Massoumou et al., 2007).

FUNCTIONAL DIVERSITY OF AMF
A number of studies have been carried out in order to select the optimal host/fungus combinations leading to the best plant performance, in terms of growth and nutrition. Different strategies have been proposed to detect the criteria for the selection of infective and efficient strains to be used for inoculation in diverse host plants and soil conditions. The possibility of implementing AMF inoculation depends first and foremost on the availability of strains able to establish rapidly an extensive colonization in the roots of a host plant and to compete for infection sites with indigenous AMF endophytes. Despite the obligately biotrophic status of AMF, involving labor-intensive experiments, progress has been done in the determination of the parameters that, in many different AMF isolate, affect spore dormancy and germination, pre-symbiotic mycelial growth, appressorium formation and intraradical development . As to the selection of efficient isolates, the great majority of studies assessed their efficiency in terms of host growth responses, nutrient uptake, in particular P and N, and consequently considered the relevant fungal parameters, such as the extent and viability of      The binomial nomenclature of arbuscular mycorrhizal fungi (AMF) reported in the quoted papers has been maintained. Assay methods: RNA sequencing (RNA-Seq), quantitative reverse transcription PCR (RT-qPCR), suppression subtractive hybridization (SSH).
ERM exploring the soil, the rate of absorption, translocation and transfer of mineral nutrients, from hyphae to plant root cells (Giovannetti and Avio, 2002;Ezawa and Saito, 2018).
Only few works studied the differential efficiency of the diverse AMF isolates in relation to the production of health-promoting phytochemicals. The first studies reported higher contents of essential oils in coriander shoots and fruits when inoculated with Rhizoglomus fasciculatum compared with Glomus macrocarpum, that modified also the essential oil profiles, with increased concentrations of linalool and geraniol, respectively (Kapoor et al., 2002b). By contrast, the same AMF species produced completely opposite results with dill and carum plants, where G. macrocarpum was more efficient in enhancing essential oil concentrations, compared with R. fasciculatum (Kapoor et al., 2002a). Experiments with other AMF species confirmed the occurrence of differential activity, as shown by data on the stimulation of root thymol derivative production, which was higher upon inoculation with Rhizoglomus clarum in Inula ensifolia roots (Zubek et al., 2010), and on the enhancement of shoot hypericin and pseudohypericin contents in Hypericum perforatum by Rhizoglomus intraradices (Zubek et al., 2012). Single species inocula showed different results also in artichoke, basil, lettuce, pepper and tomato ( Table 1). When AMF species other than Glomeraceae were used as inoculum, large variations were found in the concentration of basil leaves essential oils: Gigaspora rosea largely increased the concentration of camphor and alfa-terpineol, while Gigaspora margarita highly decreased eucalyptol, linalool, eugenol content, and the total content of essential oils (Copetta et al., 2006). As in agricultural ecosystems many different AMF co-occur in individual plants, it is important to assess plant performance in response to inoculation with multiple AMF species and/or isolates, either originating from natural communities or laboratory assemblages. Only few authors adopted such an approach, revealing the enhancement of some beneficial compounds by AMF mixtures, compared with single-isolate inoculation ( Table 1). For example, total phenolic content (TPC) and antioxidant activity increased in leaf extracts and in flower heads of artichoke plants inoculated with a mixed inoculum, compared with single species inocula, both in microcosm and in the field, suggesting a synergistic effect and/or a functional complementarity between the AMF species (Ceccarelli et al., 2010). In another study the antioxidant activity expressed as the ferric reducing ability of plasma assay (FRAP) was enhanced in onion by a mixed commercial inoculum containing Claroideoglomus etunicatum, Rhizoglomus microaggregatum, R. intraradices, Claroideoglomus claroideum, F. mosseae, Funneliformis geosporus compared with a single species (R. intraradices) inoculum (Albrechtova et al., 2012). The use of mixed inocula vs. single species-inoculum showed in tomato contrasting results, depending on the active molecule involved in the modulation: the mixed AMF species F. mosseae BEG12 and R. irregulare BB-E increased β-carotene and total carotenoids levels with no effects on lycopene content, while an opposite result was obtained using the two species individually (Hart et al., 2015).
One possible mechanism by which AMF stimulate phytochemical production could be through enhanced nutrient uptake, especially P. Basil plants inoculated with Funneliformis caledonius and F. mosseae showed higher levels of rosmarinic acid (RA) in the shoots, compared with control plants of the same P status, suggesting that the increased RA concentrations were not exclusively the results of a better P nutrition (Toussaint et al., 2008). However, the effects of an improved N nutrition remain to be investigated, as the higher N assimilation in mycorrhizal plants could contribute to the synthesis of the aminoacids tyrosine and phenylalanine and to a higher production of phenylalanine ammonia-lyase, an enzyme involved in the production of RA (Petersen and Simmonds, 2003).
The parameters to be taken into account in order to select the best performing AMF isolates, species or communities, should refer not only to the content of the health-promoting secondary metabolites, but also to the levels of transcripts encoding the enzymes of the relevant pathways. Only two studies, related to the trascriptome of tomato (López-Ráez et al., 2010b) and grapevine (Balestrini et al., 2017) roots, used different AMF inocula. In tomato, R. intraradices was able to positively regulate a larger number of genes related to health bioactive molecules, compared with F. mosseae, whereas no differences in the modulation of genes by the two inoculants were observed in grapevine ( Table 2). The ability of different AMF species and isolates to regulate genes related to beneficial phytochemicals should be further investigated by wide transcriptome studies, which could also provide insights into P and N nutrition-dependent effects. In particular, in the years to come metabolomic and transcriptomic analyses should be performed in experiments carried out under commercial production conditions-the normal way to grow plants used for human consumption-in order to avoid false positive responses.

CONCLUSION AND FUTURE PERSPECTIVES
The role played by AMF in the production of health-promoting phytochemicals by host plants has been widely investigated, using multimodal approaches. Although most of the studies showed increases in the levels of several phytochemicals in inoculated plants, some drawbacks hinder the implementation of these beneficial fungi for the production of high quality foods.
One of the main weakness concerning the data obtained so far entails the use of a low number of AMF species (about 24), generally the same ones utilized all over the world, that has limited the full exploitation of their wide physiological and genetic diversity. As the AMF species described so far are about 300, future studies should be performed utilizing the highest possible range of diverse AMF, not only at the species level, but also at the level of isolates and lineages within isolates (Chen et al., 2018;Savary et al., 2018), in order to select the best performing symbionts. Indeed, most of the physiological and functional characteristics of microorganisms, from bacteria to fungi, are properties of each individual strain. Unfortunately, the studies discussed so far often reported just the name of the AMF species utilized, and not always described the isolates, either with their number/name or with the name of the collection where they originated from.
Moreover, some of the studies analyzing the efficiency of laboratory-selected or commercial AMF strains when inoculated in the fields, found that their establishment and persistence were limited, due to the competition with well-adapted indigenous communities (Pellegrino et al., 2012;Loján et al., 2017). Such findings suggest that the selection of native AMF isolates could lead to more successful results. In order to scale up mesocosm experiments, as the success of AMF field inoculation is affected by many factors (Berruti et al., 2016), a more feasible and broadspectrum approach is represented by nursery inoculation of fruit trees or vegetable plant species before field transplant, which may allow early AMF establishment in roots and field persistence (Ceccarelli et al., 2010;Alaux et al., 2018).
Considering that in nature many different species and strains coexist in the same field, it is crucial to increase studies on the different AMF mixtures for detecting possible synergistic effects and functional complementarities among them, leading to a further selection of the best AMF combinations. Moreover, additional works should be performed on the effects of the combined inoculation of AMF with biostimulants and other beneficial microorganisms, such as plant growth promoting bacteria and fungi (i.e., Trichoderma spp.), whose metabolic activities could affect the outcome of the mixed inoculum (Lingua et al., 2013;Colla et al., 2015;Rouphael et al., 2015).
So far, RNA-Seq technology, together with a mechanistic approach, has been utilized for investigating the differential gene expression, mostly in root tissues. Further transcriptomic studies should explore the differential expression of genes involved in the biosynthesis of health-promoting plant compounds in edible tissues of mycorrhizal plants. Such a powerful technology should be applied in order to unravel the mechanisms encompassing the enhancement of health-promoting phytochemicals biosynthesis, as affected by the widest possible range of AMF. This will increase our knowledge on the specific role played by each strain, in order to utilize sound criteria for a finely tuned, targeted selection of the best performing symbionts, to be used as sustainable biotechnological tools for the production of safe and healthy plant foods.