Biodiversity, Ecology, and Secondary Metabolites Production of Endophytic Fungi Associated with Amaryllidaceae Crops

Amaryllidaceae family comprises many crops of high market potential for the food and pharmaceutical industries. Nowadays, the utilization of plants as a source of bioactive compounds requires the plant/endophytic microbiome interactions, which affect all aspects of crop’s quantity and quality. This review highlights the taxonomy, ecology, and bioactive chemicals synthesized by endophytic fungi isolated from plants of the Amaryllidaceae family with a focus on the detection of pharmaceutically valuable plant and fungi constituents. The fungal microbiome of Amaryllidaceae is speciesand tissue-dependent, although dominating endophytes are ubiquitous and isolated worldwide from taxonomically different hosts. Root sections showed higher colonization as compared to bulbs and leaves through the adaptation of endophytic fungi to particular morphological and physiological conditions of the plant tissues. Fungal endophytes associated with Amaryllidaceae plants are a natural source of ecofriendly bioagents of unique activities, with special regard to those associated with Amarylloidae subfamily. The latter may be exploited as stimuli of alkaloids production in host tissues or can be used as a source of these compounds through in vitro synthesis. Endophytes also showed antagonistic potential against fungal, bacterial, and viral plant diseases and may find an application as alternatives to synthetic pesticides. Although Amaryllidaceae crops are cultivated worldwide and have great economic importance, the knowledge on their endophytic fungal communities and their biochemical potential has been neglected so far.


Introduction
Amaryllidaceae species have been utilized as vegetables, herbs, spices, and ornamentals in all continents since ancient times. Many of them have shown widespread benefits in cuisine and healing of common diseases like atherosclerosis, diabetes, inflammation, hypertension, and cancer. These protective effects appear to be related to the presence of organosulfur compounds, predominantly allyl derivatives in Allioidae and alkaloids in the Amaryllidoidae subfamily [1]. The economic significance of Amaryllidaceae crops is substantiated by the all-year-round supply and a wide range of cultivars and landraces characterized by plant parts with different shapes and specific taste and flavor.

Biodiversity and Ecology of Endophytes Associated with Crops Belonging to the Amaryllidaceae Family
The endophytic symbiosis could be an implementation of the microorganisms' strategy aimed at reducing the effects of the external changeable environment through the long-term coevolution with plants providing a stable niche in their tissues [47]. The leaf surface is an attractive habitat for endophytic fungi, which are influenced by the possibility of colonization through the epidermal structures, by leaf health and nutritional status, and by competition with the other microorganisms. Several studies have been carried out to characterize the mycobiota of A. cepa rhizosphere and phyllosphere, but much less research has been focused on fungi colonizing internal tissues. Abdel-Gawad et al. [48] isolated and identified, based on macro-and microscopic characters, 24 genera and 38 species of fungi from the rhizoplane of onion, with dominating Aspergillus spp., Cladosporium spp. and Penicillium spp., and 17 genera and 35 species from the phylloplane, with dominating Aspergillus and Penicillium spp. The root and leaf surface of onion hosted a broader spectrum of species than internal tissues, confirming that plants selectively recruit endophytic microorganisms. Moreover, aboveground plant tissues are exposed to rapid fluctuations in temperature, humidity, and solar radiation, so microorganisms colonizing leaves are also affected by abiotic stress, exceeding sometimes their tolerance thresholds. Abdel-Gawad et al. [48] evidenced that the onion's fungal microbiota dependent on temperature, namely the species Humicola grisea (current name Trichocladium griseum), Penicillium mirabile (current name Talaromyces verruculosus), and Rhizoctonia solani, were isolated from leaves at 19 • C, whereas other species, such as Chaetomium brasiliense (current name Ovatospora brasiliensis) and Zopfiella latipes, at 28 • C. Moreover, the mentioned species were not specific for onion but isolated from roots of the other crops in the investigated region, namely Assiut Governorate in Egypt. Only one species, Z. latipes, was isolated from onion leaves for the first time in Egypt [49]. A red spider lily (Lycoris radiata) and golden spider lily (L. aurea) are ornamentals of Asian origin, introduced into many countries all over the world because of decorative flowers, but their bulbs are known as poisonous in traditional medicine systems. Zhou et al. [50] identified, using molecular (polymerase chain reaction-PCR) and morphological characteristics, 27 species of fungal endophytes belonging to 14 genera from L. radiata. Only Fusarium developed hyphae in all organs; Stagonosporopsis and Glomerella (current name Colletotrichum) were isolated from leaf tissues; Phoma from the bulb; Galactomyces, Metacordyceps (current name Metarhizium) and Diaporthe from root tissues. Aspergillus, Colletotrichum, Diaporthe, Fusarium, Penicillium, Phoma, and Phyllosticta were commonly isolated from a wide range of hosts but Cylindrocarpon, Galactomyces, Sarocladium, and Stagonosporopsis were described as endophytes of specific plants. In earlier studies, despite the mentioned species, Trichoderma sp. was isolated from a bulb of L. radiata [51] and Mucor sp. from the bulb of L. aurea [52]. Notably, Metarhizium sp., which was reported as a soil fungus [53], was isolated from L. radiata tissues, so this fungus seems to colonize plants occasionally [50].
The relationships between the endophyte fungi and host plant are very diversified and dynamically change from mutualism, symbiosis, and commensalism to pathogenic during plant ontogeny [54,55]. For example, Colletotrichum, Diaporthe, Fusarium, Phyllosticta, and Phoma, isolated from healthy tissues of L. radiata are commonly recognized as pathogenic, so the antifungal alkaloids can enforce symbiotic lifestyle in plants, maintaining a balance between host and its endophytes/parasites. Regarding endophytes colonization during onion's ontogeny, Mueva et al. [56] stated that the seed inoculation was more effective than seedling inoculation in terms of endophytes recovery in subsequent stages of plant development. Indeed, endophytes inoculated at the seed surface colonized seed radicle and plumule and developed internal mycelia in growing tissues. The fungal colonization and distribution in onion tissues firstly depended on inoculation technique and secondly on the endophyte selection by the host. Independently on the inoculation technique, most of the investigated endophytes, for example Clonostachys rosea, Hypocrea lixii (current name Trichoderma lixii), Trichoderma asperellum, T. atroviride, T. harzianum, and Fusarium spp., were isolated from onion roots, followed by stems and leaves. These differences could be due to tissue morphology and physiology, microbiome interactions, and the influence of external conditions [56,57]. Onions have shallow, weakly branched root systems with sparse root hairs, inefficient in the use of soil nutrient resources. The root endophytic and mycorrhizal fungi play a significant role in supporting onions with mineral salts, that is why this species is among the most symbiosis-dependent crops [7]. Focusing on endophytic fungi colonizing shallot roots, Priyadharsini et al. [58] found that the percent of root length with fungal microsclerotia was significantly and negatively correlated with soil phosphorus level. Similarly, percents of root length with dark septate hyphae and dark septate endophyte total colonization were negatively correlated with soil zinc and copper contents. It can be concluded that colonization of shallot roots by fungal endophytes was reduced in soils rich in mineral salts. Wu et al. [59] hypothesized that the endophytic fungal community may be helpful to symbiotic plants (i.a. Allium mongolicum) for surviving in the extreme environments of Asian deserts. The mycobiota associated with photosynthesizing or storage leaves, for example T. harzianum and T. koningii, could act antagonistically to phytopathogens. On the other side, leaves with disease symptoms, with damaged epidermal cells and the lamellar seta shed releasing nutrients, could be secondarily colonized by opportunistic fungi such as Botrytis cinerea, Penicillium aurantiogriseum, Alternaria alternata, and Cladosporium spp. [20].
Plant storage tissues, including sugar-rich onions' bulbs, can contain specific endophytes, actively reproducing in these tissues without visible damage [60]. One of the main chemoecological roles of Amaryllidaceae alkaloids is the protection of nutrient-rich bulbs against phytopathogens and herbivores. Xiang et al. [61] isolated and sequenced six fungal endophytes from Narcissus pseudonarcissus bulb and only two from leaf tissues. Zhou et al. [19] found that bulbs of L. radiata were exclusively colonized at a higher degree than other tissues, probably because of the perennial life cycle of bulbs and annual cycle of other plant parts [62] and because of the space and carbohydrates provided by bulbs as storage sinks [63].

Biochemistry and Functions of Fungal Endophytes Associated with Allioidae Crops
Abdel-Hafez et al. [20] investigated endophytes colonizing A. cepa leaves, both healthy and infected by purple blotch (Alternaria porri). Fungi were isolated and identified according to their macroscopic and microscopic characteristics. Despite the strains detected from healthy and diseased leaves, belonging to genera Cladosporium, Alternaria, Penicillium, and Stemphylium, five species, namely Absidia corymbifera (current name Lichtheimia corymbifera), B. cinerea, P. aurantiogriseum, P. glabrum, and Syncephalastrum racemosum, were isolated only from infected leaves, while three species (Fusarium oxysporum, Trichoderma harzianum, and T. koningii) were isolated only from healthy ones (Table 1). Trichoderma spp. showed antagonistic potential against A. porri, through competition, lysis, antibiosis, and parasitism [64,65]. The antagonistic effect of Epicoccum nigrum, Penicillium oxalicum, and Stachybotrys chartarum against A. porri was antibiosis caused by effective lytic, as well as antimicrobial secondary metabolites produced by endophytic fungi [20]. Previously, Flori and Roberti [66] noticed the antifungal activity of the endophyte Beauveria bassiana, inoculated to onion roots, against F. oxysporum f. sp. cepae, causing basal rot of onion. The antifungal potential of the endophyte Talaromyces pinophilus (current name Penicillium pinophilum) against B. cinerea was described by Abdel-Rahim and Abo-Elyousr [67]. T. pinophilus was isolated from onion's inflorescences and identified with PCR amplification of the ribosomal internal transcribed spacer (ITS) region. The mycelium of T. pinophilus penetrated intercellularly the hyphae of B. cinerea, involving cell wall degrading enzymes (chitinase, lipase, and protease) in the mycoparasitic process.  A. longicuspis (bulb) Aspergillus. spectabilis Antibacterial against P. aeruginosa and S. aureus Not investigated [74] A. longicuspis (leaf)

Fusarium sambucinum
Antibacterial against E. coli Not investigated [74] Alternaria sp. Antibacterial against E. coli, P. aeruginosa, and S. aureus Not investigated [74] A. terreus Antibacterial against E. coli, P. aeruginosa, and S. aureus, inhibition of α-amylase activity Not investigated [74,75] Aspergillus flavus Antibacterial against P. aeruginosa Not investigated [74] Agriculture 2020, 10, 533 8 of 16 Muvea et al. [56,68] showed the effect of onion inoculation with some strains of endophytic fungi on the proportion of thrips due to reduced feeding and oviposition, caused by antixenotic repellence or higher death rate of thrips. Moreover, the reduced feeding of thrips on endophyte-colonized onions could reduce the transmission of virus diseases, spread by insects.
Among endophytes of garlic that can produce bioactive compounds, Shentu et al. [71] isolated and identified, based on morphological and molecular procedures, Trichoderma brevicompactum with strong antifungal activities. Trichodermin, an antifungal compound of T. brevicompactum inhibited mycelial growth of R. solani, with an EC 50 of 0.25 µg mL −1 , and B. cinerea, with an EC 50 of 2.02 µg mL −1 ( Table 2). A weak inhibition was noted against Colletotrichum lindemuthianum (EC 50 = 25.60 µg mL −1 ). The authors underlined that the relationship between T. brevicompactum and the garlic plant remained unclear. Espinoza et al. [72] investigated the chive's growth parameters and secondary metabolites as affected by inoculation with the endophyte fungus B. bassiana. The fungus applied to the rhizosphere, colonized plant tissues, and finally was isolated from roots and leaves, affecting total alkaloids content but not leaves yield. Koul et al. [73] isolated and morphologically and molecularly identified the fungus Penicillium pinophilum, from bulbs of chive's population native to snow mountain regions of India. P. pinophilum was a source of anticancer anthraquinones, dicatenarin, and skyrin. Both compounds inhibited human pancreatic cancer (MIA PaCa-2) cells with least IC 50 values of 12 µg mL −1 and 27 µg mL −1 respectively, through mitochondrial-mediated apoptotic pathway. Dicatenarin cytotoxic/proapoptotic activity was more pronounced than skyrin due to the presence of an additional phenolic hydroxyl group at C-4, which increased reactive oxygen species generation [73].
Wild and endemic Allium species were also the object of investigation. In the latter respect, Abdulmyanova et al. [74] screened Allium filidens Regel and leaves of A. longicuspis Regel regarding endophytic fungi biodiversity and bioactivity. Among 16 isolates of endophytic fungi obtained from these plants and identified morphologically, the highest biodiversity was determined for bulbs of A. filidens and leaves of A. longicuspis. The Penicillium spp. were the most dominant symbionts of A. filidens, while Aspergillus spp. were commonly isolated from A. longicuspis. Beside cosmopolitan species, the rare endophytes Alternaria tenuissima, Aspergillus spectabilis, and Cladosporium tenussimum were also isolated. The endophytic fungi detected in the same host varied regarding bioactivity. For example, three strains of Penicillium sp. isolated from bulbs of A. filidens were different in cytotoxic, antibacterial, and antiamylase activity, two strains of Alternaria sp. from leaves of A. longicuspis exhibited only antibacterial activity [74,75]. Bulbs of both described Allium species, endemic in Afganistan, have been used in traditional Asian medicine [76].

Biochemistry and Functions of Fungal Endophytes Associated with Amaryllidoideae Crops
Amarylidoideae alkaloids can be involved in chemical crosstalk between host plant and endophytes as communication molecules that are responsible for the shaping of plant-microbe interactions. This phenomenon was more widely investigated for endophytic bacteria, which can promote the synthesis of Amaryllidaceae alkaloids [77,78], but endophytic fungi are also involved in plant-endophyte and endophyte-endophyte interspecies communication. For example, Wang et al. [79] investigated endophytic fungi and bacteria in the bulbs of the Chinese sacred lily (Narcissus tazetta), widely used as an ornamental and medicinal plant in Asia [79]. The authors defined nine hexacyclopeptides produced by fungus and selectively accumulated by an endophytic bacterium Achromobacter xylosoxidans isolated from the same tissue ( Table 2). The production of targeted hexacyclopeptides by F. solani was possible only in planta and decreased in vitro conditions. However, the ecological basis of this chemical cross-talk needs future investigations. Yang et al. [80] isolated and identified, using morphological and molecular methods, 18 strains of endophytic fungi from Narcissus sp. Three species, particularly Rhinocladiella sp., demonstrated significant inhibitory activity against acetylcholinesterase.
Onofri et al. [81] identified, using conventional taxonomic techniques, four strains of Cryptococcus laurentii (current name Papiliotrema laurentii), C1-C3 from root tips, C4 from outer bracts of bulb of daffodil (N. pseudonarcissus). The authors observed that lycorine, an alkaloid of Narcissus bulbs, Agriculture 2020, 10, 533 9 of 16 inhibited the growth of C1-C3 but not C4 strains of fungi. The inhibition was due to destroying the cellular membranes and interfering with the substrate absorption and cell metabolism, namely blocking L-galactonic acid γ-lactone conversion into ascorbate by lycorine. In contrast, C. laurentii, isolated from the lycorine-containing bracts of the bulb, was able to degrade lycorine and to use decomposition products as growth stimulators.
L. radiata is the main source of Amaryllidaceae alkaloids but the low yield and high costs, resulting from its complex procedures and mixed stereoisomers, limit pharmaceutical development of plant-delivered drugs [50]. Lycoris spp. were objects of some investigations regarding endophyte microbiota and assessment of the biological activity of their metabolites. Penicillium sp. isolated from L. aurea was able to produce galanthamine in vitro [82], and the other nonidentified fungus strain L-10 possessed antibacterial and antifungal activity against Staphylococcus aureus and Candida albicans, respectively [52]. This phenomenon confirmed the antagonism between fungal and bacterial symbionts of this plant. Both novel and known compounds, especially alkaloids, could be produced by in vitro grown endophytic fungi isolated from Lycorsis spp. bulbs. Moreover, the inoculation with fungal endophytes enhanced the level of various alkaloids in L. radiata. So, inoculation with particular fungus or consortium of fungi can be used for increasing the content of targeted alkaloids during plant cultivation [50].
Li et al. [83] investigated drimane-type sesquiterpenoids of Aspergillus versicolor. Among the latter compounds, Versicalin A showed moderate cytotoxic activity against HL-60 tumor cells with an IC 50 value of 5.6 µM, while proversilin C and E showed moderate cytotoxicity against human tumor HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cell lines and the normal colonic epithelial cells NCM460 with IC 50 values ranging from 7.3 to 28.4 µM [83,84]. The synthesis of the same chemical compounds by the plant host and endophytic fungus is the phenomenon described for some other species as an example of highly specified coevolution. Moreover, this phenomenon has a great significance in the detection and production of pharmaceutically valuable plant/endophyte derived drugs [85][86][87].

Conclusions
The increasing demand for Amaryllidaceae crops is triggered not only by traditional culinary usage but also by cultural attitudes, social beliefs, modern interest in exotic and ethnic foods, and medicinal applications. However, secondary metabolites responsible for wide applications of crops in every area of human activity are synthesized by both plant and its endopytic microbiota. Indeed, endophytic fungi associated with this group of plants provide host plants with nutrients and water, alleviate biotic and abiotic stresses, increase stress tolerance, and affect metabolome profile. They are a source of metabolites of antifungal and antiparasitic activity and have a promising perspective in the application as effective biocontrol agents, replacing chemical fungicides and pesticides. Moreover, as the chemosynthesis of Amaryllidaceae alkaloids needs complicated and costly procedures, plants remain an exclusive source of these alkaloids for the pharmaceutical industry. Symbiotic endophytic fungi can be used to increase alkaloids yield in plants or as an alternative source of alkaloids and other bioactive compounds in vitro cultures. Taking into account the scant research on endophytic fungi associated with Amaryllidaceae as a prolific source of phytochemicals, the need has raised for screening investigations aimed to identify the endophytic species, as well as the molecular and genetic basis of their relationship with the host plants.

Review Methodology
The present review was based on the literature collected from the leading life science databases, including AGRICOLA, AGRIS, BioOne, CAB Abstracts, PubMed, SciELO, Scopus, and Web of Science. Bibliometric analysis was used for the review, evaluation, and objective representation of the structure within a presented research area, namely Amarylidaceae-fungal endophytes relationship. The most relevant aspects of the evolution, advances, and trends in the reviewed field were presented [88]. References were collected, studied, and selected considering (i) the reports of endophytes isolated from the species of Amaryllidaceae family, (ii) the reports of therapeutic utilization of the host plant or/and an endophyte, (iii) the effects of host plant phylogeny on root microbiome assembly. Plant names were verified according to the Global Biodiversity Information Facility [89] and The Plant List [90]; endophyte taxa were verified according to MycoBank database [91]. Funding: This study was partially supported by the Ministry of Science and Higher Education of the Republic of Poland.

Conflicts of Interest:
The authors declare no conflict of interest.