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Bioactive Products from Endophytic Fungi of Sages ( Salvia spp.)

: In the aim of implementing new technologies, sustainable solutions and disruptive innovation to sustain biodiversity and reduce environmental pollution, there is a growing interest by researchers all over the world in bioprospecting endophytic microbial communities as an alternative source of bioactive compounds to be used for industrial applications. Medicinal plants represent a considerable source of endophytic fungi of outstanding importance, which highlights the opportunity of identifying and screening endophytes associated with this unique group of plants, widespread in diverse locations and biotopes, in view of assessing their biotechnological potential. As the ﬁrst contribution of a series of papers dedicated to the Lamiaceae, this article reviews the occurrence and properties of endophytic fungi associated with sages ( Salvia spp.).


Introduction
Endophytic fungi are defined as fungi inhabiting tissues and organs of healthy plants during certain stages of their life cycle without causing apparent symptoms. The concept of endophytism, introduced by De Bary in 1866 and almost completely neglected for over a century, has recently become of common usage concomitantly to advances in knowledge on occurrence and functions of this component of biodiversity. Increasing attention by the scientific community is boosted by the opportunity to exploit the unique aptitudes and properties of these microbial associates of plants [1].
As a consequence of the long-term association of endophytes with medicinal plants, based on mutually beneficial relationships, the former may also participate in metabolic paths and boost their own natural biosynthetic activity, or may gain some genetic information to synthesize biologically active compounds closely related to those directly produced by the host plant [2,3]. Endophytic fungi derived from medicinal plants are becoming more and more popular, due to specific modes of action and the ability to provide multiple benefits, which make them relevant for both agricultural and pharmaceutical applications [3]. This review is devoted to an analysis of the biochemical potential of endophytic fungi reported from species of sage (Salvia spp.), examining the advances in this particular field made by the scientific community in recent years.

Ecology and Occurrence
As introduced above, endophytic fungi are polyphyletic groups of microorganisms, which asymptomatically colonize healthy tissues of different parts of living plants such as stems, leaves or roots. Their diversity is huge, and it has been estimated that every plant hosts several endophytic species, among which at least one shows host specificity [13,14]. Through the evolutionary processes, endophytic fungi have developed different symbiotic relationships with their host plants [3]. Furthermore, many species are reported to exhibit multiple ecological roles as both endophytes and pathogens. However, it is not clear whether the same genotypes can play both these roles with equal success. Understanding the mechanisms responsible for the conversion between so different outcomes of the ecological interaction represents one among many frontiers in endophyte biology [15,16]. One of the mechanisms developed by plants during the long-term co-evolution with microbial associates is the ability to produce antibiotic compounds. Simultaneously, many endophytes have developed an important transformative capacity and/or tolerance to these products which in a large part determines the colonization range of their hosts [17]. In turn, endophytes can influence growth and development of host plants, and enhance their resistance to biotic and abiotic stresses by releasing bioactive metabolites [18], to such an extent that in natural habitats some plant species require to be supported by endophytic fungi for stress tolerance and survival [19].
The diversity of endophytic fungi associated with medicinal plants is largely affected by ecological or environmental factors. Particularly, temperature, humidity and soil nutritional conditions influence the quality and quantity of secondary metabolites synthesized by the hosts, which in turn affect the population structure of endophytic fungi. The species composition of endophyte communities also differs in organ and tissue specificities, as a result of their adaptation to different physiological conditions in hosts [3,20].
The analysis of the recent literature shows that species of sage (Salvia spp.) host diverse communities of fungal endophytes. As many as 64 different taxa belonging to 38 genera, with a clear prevalence of Ascomycetes, have been reported so far (Table 1). Most observations concern the species Salvia miltiorrhiza and S. abrotanoides, respectively, with 28 and 24 records. There is an evident correspondence between the Salvia species and the geographical area. In fact, all isolations concerning S. miltiorrhiza come from several provinces of China, while the available findings from S. aegyptiaca come from Egypt, and those referring to S. abrotanoides derive from an Iranian study and from the activity currently in progress at our laboratories. Despite the fact that isolations have been carried out from any plant organ (Figures 1 and 2), no indications concerning a specific association with roots or the aerial parts can be inferred. The access to biomolecular methods as a taxonomic tool has generally enabled to perform reliable identification at the species level, with the exception of a Chinese study concerning seeds of S. miltiorrhiza, where sorting was basically limited to the class level despite the wide variation observed [21]. The repeated findings in several species/locations mostly refer to strains provisionally identified at the genus level, particularly Alternaria, which seems to be of quite common occurrence on sages regardless to the plant part used for isolations. At the species level, there are only two cases with more than just one record-that is, Chaetomium globosum and Didymella (=Phoma) glomerata, both from S. miltiorrhiza at two different locations in China. However, the recovery of these species from both roots and leaves may represent a possible indication of a more regular association with this plant, which should be taken into consideration in further studies.     Table 1.

Biochemical Properties
A large part of literature on the occurrence of endophytic fungi of Salvia spp. deals with their ability to produce bioactive compounds (Figure 3), focusing on structure elucidation and possible applications. Some studies have been limited to a partial characterization of culture filtrates or their   Table 1.

Biochemical Properties
A large part of literature on the occurrence of endophytic fungi of Salvia spp. deals with their ability to produce bioactive compounds (Figure 3), focusing on structure elucidation and possible applications. Some studies have been limited to a partial characterization of culture filtrates or their  Table 1.
Strains of Alternaria and Chaetomium also yielded products, such as alternariols, altenuisol, chaetoviridin and chaetoglobosins, whose antibiotic, antifungal, antiproliferative and radical scavenging properties have been previously described, and reviewed in recent papers [47,48]. More known products previously reported from other fungi are the tetramic acid derivative equisetin and the isocoumarin diaporthin, originally described as phytotoxins [49,50], and griseofulvin, a compound which has found application in dermatology and displayed interesting antitumor properties [51]. Other secondary metabolites which are used as pharmaceuticals are paracetamol, nipecotic acid, mandelic acid and azelaic acid. The latter has displayed antibiotic properties and antiproliferative effect on malignant melanocytes, and is commonly used in dermatology as an antiacne [52]. Moreover, in plants it is reported to be involved in the defense response against disease agents [53]. Other products to be mentioned are the plant hormone indole-3-acetic acid (IAA) and a couple of analogue auxins, which are considered key intermediates in the mutualistic relationship between endophytes and their host plants [2,16].
However, probably the most interesting products of endophytic fungi of Salvia species are a series of compounds previously identified as plant metabolites, which are treated in further detail in the next chapter.

Biotechnological Implications
Long-lasting evolutionary processes taking place together with their host plants have allowed endophytic fungi to work out various strategies enabling them to keep an equilibrium between virulence and plant defense in order to share common habitat. Endophytic fungi not only are able to influence plants' metabolism and physiology by producing unique and specific secondary metabolites, they were also found to produce bioactive natural products originally known exclusively from their host plants, and have elaborated strategies for detoxification by exploiting their biotransformation abilities. These properties make endophytes a perfect target for various biotechnological approaches and further commercial exploitation.

Endophytic Fungi as In Vitro Production Platforms for Plant Secondary Metabolites
The ever-increasing demand for bioactive natural compounds cannot be met at the desired levels by just relying on their extraction from plants, considering that in most instances they are produced at a specific developmental stage or under specific environmental condition, stress, or nutrient availability [54]. Medicinal plants from the Salvia genus are often shrubs, thus they may need several years to attain a suitable growth phase for bioactive product accumulation and extraction. Moreover, harvesting medicinally important plants from the wild makes them critically endangered and affects the environmental biodiversity [39]. As for crop plants, although cultivated in a large scale, they often produce the desired metabolites in a low yield, making the production unprofitable. Considering the limitations associated with productivity and vulnerability of plants, fungal endophytes may serve as a renewable and inexhaustible source of bioactive compounds. Many endophytes have experienced long-term symbiotic relationships with their host plants, and through long-term coexistence and direct contact, they have exchanged genetic material [17]. Horizontal gene transfer (HGT), an important evolutionary mechanism observed in prokaryotes, is also thought to be the phenomenon responsible for transmission of genetic material across phylogenetically distant species [55]. As an increasing number of reports indicate a physical clustering of genes for specialized metabolic pathways in plant genomes [56], the HGT phenomenon is believed to be responsible for rapid transfer of whole gene clusters from host plants, conferring "novel traits" to the associated fungi. As a consequence, many endophytic fungi have developed the ability to produce bioactive substances originally known from their hosts, thus raising the prospect of using such organisms as alternative and sustainable sources. HGT has been proposed to explain the production of tanshinone I, tanshinone IIA and their precursor ferruginol by Trichoderma atroviride D16, an endophytic fungus in S. miltiorrhiza [39].
Daidzein and glycitein are naturally occurring compounds found in soybeans and other legumes which are produced in plants through the phenylpropanoid pathway and structurally belonging to a class of compounds known as isoflavones. Daidzein is a phytoestrogen with possible pharmaceutical application as menopausal relief, osteoporosis, blood cholesterol lowering, and it is thought to reduce the risk of some hormone-related cancers and heart disease [57], while glycitein has a weaker estrogenic activity [58]. They both were found to be produced by endophytic fungi of S. abrotanoides, that is Penicillum canescens for daidzein and Talaromyces sp. for glycitein [22]. The latter is also able to synthesize trigonelline, an alkaloid originally extracted from Trigonella foenum-graecum, known for its antidiabetic properties [59] as well as solanidine, a potato alkaloid. Stachydrine, another alkaloid known from Medicago sativa, was found to be synthetized by a strain of Fusarium dlaminii inhabiting S. abrotanoides [22].
Danshen, dried roots and rhizomes of S. miltiorrhiza, is a well-known traditional Chinese herbal medicine [60]. It contains two kinds of bioactive compounds: tanshinones and hydrophilic phenolic acids, the latter being represented by rosmarinic acid, salvianolic acids B-C, and others. Salvianolic acids are mainly responsible for the favorable activities on cardiovascular and cerebrovascular diseases of danshen [61]. Salvianolic acid C was found in both mycelium and fermentation broth of strain D14 of D. glomerata in very low yields [35]. This indicates the opportunity to optimize fermentation conditions for achieving its efficient production, or alternatively to enhance its production via regulating the key enzymes involved in the biosynthetic pathway.
Caffeic acid was found in the metabolome profiles of isolates of Talaromyces and Paraphoma endophytic in S. abrotanoides [22]. Besides rosmarinic acid and salvianolic acid B, it is regarded as the major phenolic acid in S. miltiorrhiza [62]. A series of caffeic acid derivatives, obtained from Salvia officinalis [63,64], showed pronounced leishmanicidal activity, as well as immunomodulatory effects on macrophage functions [65]. Moreover, antibacterial, antifungal and modulatory effects of caffeic acid have been shown in recent studies [66].
Tanshinones are a group of abietane-type norditerpenoid quinones, originally found in danshen [62]. More than 40 structurally diverse tanshinones have been isolated and identified [67], among which cryptotanshinone, tanshinone IIA, and tanshinone I are the main active ingredients [68]. Although many biotechnological improvements have been implemented to increase tanshinone production from plants, at present no mature hairy root, suspension cell line, or culture system of S. miltiorrhiza have been developed. Thus, the extraction from roots and rhizomes of S. miltiorrhiza still represents the main source of tanshinones [62]. Salvia yangii has also been found to produce a range of tanshinones [69][70][71][72], as well as S. abrotanoides, although the compound assortment was found to be considerably different according to the preliminary data obtained by our working group.
As introduced above, tanshinone IIA is currently in short supply because of overcollection of the wild plants and environmental change [28], so that endophytic fungal strains represent an alternative source. In this respect, tanshinone I and tanshinone IIA production has been confirmed by T. atroviride D16 from S. miltiorrhiza [39]. Moreover, strain TR21 of Aspergillus foeniculicola was shown to produce low amount of tanshinone IIA [84]. Production of this compound by TR21 was increased in the NU152 mutant, obtained by traditional mutagenesis using ultraviolet radiation and sodium nitrate treatment [28], and in strain F-3.4 through genome shuffling [85], providing a yield of tanshinone IIA which is over 11 times higher than the original strain TR21. This study showed that the genetic basis of high-yield strains can be achieved through genome shuffling, which can shorten the breeding cycle and improve the mutagenesis efficiency in obtaining strains with good traits, to be used for industrial production.
Cryptotanshinone, another nor-abietanoid diterpenoid, which is a main bioactive compound of S. abrotanoides known for leishmanicidal, antiplasmodial and cytotoxic activity [86] has been found to be produced also in roots of S. yangii [69]. Very recently, this compound has been reported as a secondary metabolite of endophytic strains of P. canescens, Penicillium murcianum, Paraphoma radicina, and Coniolariella hispanica, independently of the host plant. Moreover, the effect of exogenous gibberellin (GA3) on S. abrotanoides and endophytic fungi was shown to have a positive effect on increasing the cryptotanshinone production in the plant as well as in endophytic fungi cultivated under axenic conditions [22]. Exogenous gibberellin treatment was also previously observed to promote the production of cryptotanshinone, tanshinone I and tanshinone II in S. miltiorrhiza [87].

Endophytic Fungi as Biotic Elicitors
Indiscriminate collection and cutting down of medicinal plants from the wild for extraction of medicinal products have almost led to the extinction of certain plant species, making them either vulnerable or critically endangered. The biotechnological approaches involving plant cell, organ and hairy root cultures appeared to fulfill the ever-increasing demand up to a certain level [54]. Endophytes could possibly be used as alternative or more efficient elicitors, compared to other biotic and abiotic elicitation methods.
A tanshinone IIA-producing endophytic strain of A. foeniculicola (U104) was demonstrated to elicit production of this compound in sterile seedlings of S. miltiorrhiza through upregulation of several enzymes involved in its biosynthesis [94]. Likewise, mycelium extract and its polysaccharide fraction (PF) produced by T. atroviride D16 promoted root growth and stimulated the biosynthesis of tanshinones in hairy roots. Moreover, the transcriptional activity of genes involved in the tanshinone biosynthetic pathway increased significantly after treatment with PF, which could be effectively utilized for large-scale production of tanshinones in the S. miltiorrhiza hairy root culture system [95]. Later on, PF was found to more deeply regulate the metabolic profiling of roots of this plant [96]. The main component of PF resulted to be an heteropolysaccharide (PSF-W-1), whose structure has been elucidated [97]. Moreover, an enhancing role by jasmonic acid on production of tanshinone I by this fungal strain was demonstrated [26], along with Ca 2+ triggering, peroxide reaction and protein phosphorylation, leading to an increase in leucine-rich repeat (LRR) protein synthesis [98].
Another endophytic strain from S. miltiorrhiza (Phoma herbarum D603) was found to stimulate growth and root development by producing IAA and siderophores and improving nutrition through phosphorus solubilization; moreover, it promoted the synthesis and accumulation of tanshinones by regulating the expression level of key genes in the synthetic pathway [37].
Eliciting effects on the synthesis of salvianolic acids and tanshinones, particularly dihydrotanshinone I and cryptotanshinone, have been also reported by a strain of Chaetomium globosum and its mycelial extract [29]. The effect of the mycelial extract was much stronger than that of the live fungus on tanshinones synthesis, which significantly increased the transcriptional activity of key genes in tanshinone biosynthetic pathway. Thus, C. globosum D38 was proposed to be supplemented as a biotic fertilizer in S. miltiorrhiza seedling culture, as it not only significantly promoted growth of the host plant, but also notably enhanced the accumulation of tanshinones and salvianolic acids.
Alternaria sp. A13 has been shown to simultaneously enhance the dry root biomass and secondary metabolite accumulation of S. miltiorrhiza, thus demonstrating its application potential as a bio-fertilizer in the cultivation of this plant [26]. Compared to uninoculated seedlings, S. miltiorrhiza seedlings colonized by Alternaria sp. A13 showed significant increment in the contents of total phenolic acids and lithospermic acids A and B. Examination of the related enzyme activities showed that the elicitation effect of A13 on lithospermic acid B accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. A similar effect was demonstrated for a strain of Paecilomyces sp. which increased content of salvianolic acid B in S. miltiorrhiza and promoted plant growth [36].

Biotransformation/Detoxication Abilities of Endophytic Fungi
To be able to colonize host tissues, endophytes developed a strong tolerance toward host's defensive metabolites. The detoxification of plant bioactive compounds is an important transformation ability of many endophytes which, to a certain extent, decides the colonization range of their hosts [17]. Biotransformation abilities of endophytes help in detoxification of antifungal metabolites produced by the host plant, and may intervene in the production of some novel bioactive compounds [54,99,100].
Trichoderma hamatum, an endophytic fungus inhabiting roots of Salvia officinalis alongside other microorganisms, was found to be able to degrade caffeine [40]. Aromatic plants such as sage have been used as intercrops in coffee plantations. Salvia officinalis was proved to absorb caffeine from the incubation media and store it mainly in roots. The cited study demonstrated that the degradation of caffeine was initiated by the ability of the microorganisms to perform demethylations, whereas xanthine degradation may be attributed to either the plant or the microorganisms. The existence of a beneficial biochemical interaction in caffeine degradation between endophytic T. hamatum and sage root was proposed. Using sage with its endophyte T. hamatum as an intercrop may become an ecologically friendly strategy to reduce caffeine accumulation in soil.

Conclusions
Endophytic fungi are prospective producers of both known and novel bioactive compounds. However, to ensure feasibility of industrial application, yield and productivity enhancement strategies at several levels are required [101]. A combination of genetic, metabolic and bioprocess engineering may be used to sustain and enhance production of high value secondary metabolites from selected strains, whose biosynthetic abilities can be improved through physical and chemical mutagenesis, or various methods for genetic transformation. Improved strains can be in turn subjected to various bioprocess optimization strategies for further enhancement in yield and productivity of selected compounds.
This review of the available literature specifically concerning endophytic fungi of sages highlighted that research in the field is quickly progressing, with the aim of both refining biotechnological applications concerning tanshinone production and prospecting novel strains for further applications. The spread of reliable methods for detection and characterization of both the endophytic strains and their bioactive secondary metabolites is expected to further improve the translational perspectives.