A Survey of Endophytic Fungi Associated with High-Risk Plants Imported for Ornamental Purposes

: An extensive literature search was performed to review current knowledge about endophytic fungi isolated from plants included in the European Food Safety Authority (EFSA) dossier. The selected genera of plants were Acacia , Albizia , Bauhinia , Berberis , Caesalpinia , Cassia , Cornus , Hamamelis , Jasminus , Ligustrum , Lonicera , Nerium , and Robinia. A total of 120 fungal genera have been found in plant tissues originating from several countries. Bauhinia and Cornus showed the highest diversity of endophytes, whereas Hamamelis , Jasminus , Lonicera , and Robinia exhibited the lowest. The most frequently detected fungi were Aspergillus , Colletotrichum , Fusarium , Penicillium , Phyllosticta , and Alternaria. Plants and plant products represent an inoculum source of several mutualistic or pathogenic fungi, including quarantine pathogens. Thus, the movement of living organisms across continents during international trade represents a serious threat to ecosystems and biosecurity measures should be taken at a global level.


Introduction
Endophytic fungi are ubiquitous to plants, and are mainly members of Ascomycota or their mitosporic stage, but they also include some taxa of Basidiomycota, Zygomycota, and Oomycota. Endophytes are organisms living within the tissues of plants [1] establishing stable relationships with their host, ranging from non-pathogenic to beneficial [2,3]. The endophytic fungi communities represent an enormous reserve of biodiversity and constitute a rich source of bioactive compounds used in agriculture [4,5]. For these reasons, they have attracted the attention of the scientific community worldwide. By definition, all or at least a significant part of the endophytic fungi life cycle occurs within the plant tissues without causing symptoms to their host [6][7][8]. A wide range of fungi, including pathogens and saprophytes, may be endophytes. Several pathogens live asymptomatically within plant tissues during their latency or quiescent stage, while some saprobes can also be facultative parasites [1,8,9]. Fungal endophytes are influenced by abiotic and biotic factors, occupying different habitats and locations during their life cycle phases. Even if host plants do not show any symptoms, they may represent a source of inoculum for other species [10][11][12][13]. Furthermore, changes in environmental conditions or species hosts may modify the fungal behavior, thus producing Table 1 summarizes the abundance of endophytic fungi reported in association with High-Risk Plants for ornamental purposes. Herein, the number of endophytic species found in association with the examined plant genera has been taxonomically grouped by fungal genus. There are important differences in terms of fungi recovered per specific plant genus (SP) as well as in the frequency of a specific fungal genus (SF). These discrepancies could be explained by the different availability of literature data on these specific plants. Table 1. Endophytic fungi isolated from Acacia (AC), Albizia (AL)., Bauhinia (BA), Berberis (BE), Caesalpinia (CP), Cassia (CS), Cornus (CO), Hamamelis (HA), Jasminus (JA), Ligustrum (LI), Lonicera (LO), Nerium (NE), Robinia (RO). Columns report the number of isolated fungal species. The total number of records calculated per fungal genus is indicated as Tot. SF. The total number of records per plant genera is indicated as Tot. SP. Fungal genera are sorted by alphabetic order.

Acacia
The Acacia, commonly known as wattle, belongs to the family Mimosaceae. The genus comprises more than 1350 species found throughout the world: almost 1000 are native of Australia, up to 140 species occur in Africa, 89 from Asia, and about 185 species are found in North and South America. Some Australian wattles are naturalized beyond their native range and have become invasive in many parts of Europe, South Africa, and Florida, especially in conservation areas [32]. Aboriginal communities use some Acacia species as sources of food and medicine. Australian acacias are widely used as wood products, ornamental plants, commercial cut flowers, and perfume crops [33].

Albizia
The genus Albizia (Mimosaceae) comprises almost 150 species, mostly trees and shrubs native to tropical and subtropical regions of Asia and Africa. They are common components of timber plantations, cropping, and livestock production systems [45]. Albizia synthesizes numerous bioactive compounds with pharmacological properties such as saponins, alkaloids, flavonoids, and phenolics [45]. The species A. lebbeck has been extensively introduced in seasonally dry tropical regions of Africa, Asia, the Caribbean, and South America, mainly as an ornamental plant, and has become naturalized in many areas [46]. Table 3 reports endophytes isolated from Albizia genera. These fungi belong to 14 different genera, most of them found in leaves and twigs of A. lebbeck originating from Iraq, India, Indonesia, and Egypt. Isolated fungi included different species of Aspergillus (9 isolates), which are dominant in comparison to other genera, followed by Fusarium (4 isolates), Penicillium (3 isolates), and Paecilomyces (2 isolates). One isolate for each of the following genera Neocosmospora, Bipolaris, Colletotrichum, Diaporthe, Lasiodiplodia, Rosellinia, Acremonium, Trichoderma, Verticillium, Curvularia, and Nigrospora has been detected.

Bauhinia
The genus Bauhinia, commonly known as the orchid tree, belongs to the family Fabaceae. It comprises more than 500 species of shrubs, and small trees mostly native to tropical countries (Africa, Asia, and South America). Many species are widely used as ornamental plants, forage, human food, and in folk medicine [51,52].

Berberis
The genus Berberis (Berberidaceae) comprises almost 500 species of deciduous or evergreen shrubs, which occur in the temperate and subtropical regions of Europe, Asia, Africa, and America [67]. This genus has remarkable pharmacological properties [68]. Berberine and Berbamine are the main compounds produced by these plants, together with alkaloids, tannins, phenolic compounds, sterols, and triterpenes [69].

Caesalpinia
The genus Caesalpinia (Fabaceae) includes approximately 200 species, mainly arboreal and shrubby species, distributed in seasonally dry tropical forests, as well as in tropical and warm temperate savannas, tropical wet forests, and tropical coastal habitats [75]. Several classes of compounds, mainly flavonoids, diterpenes, and steroids, have been isolated from Caesalpinia species, which have shown various medicinal properties [75]. The most common species cultivated as ornamental plants are C. pulcherrima and C. echinata.

Cornus
The genus Cornus (Cornaceae) consists of over 50 species of woody plants, many of which are cultivated as ornamental and medicinal trees [93]. The most widespread ornamental plants of the genus are C. florida and C. stolonifera, called the flowering dogwood, native to northern and central America [93]. The species C. officinalis is widely distributed in China, Korea, and Japan, and used for its several pharmacological activities. Among bioactive compounds, iridoids, tannins, and flavonoids are the major components [94].

Hamamelis
Hamamelis (Hamamelidaceae), commonly known as witch hazel, comprises six species of ornamental shrubs. This genus is distributed across North America and eastern Asia. Bark extracts contain proanthocyanidins and polyphenolic fractions, with medicinal properties [107,108].
Fungal endophytes belonging to genera Colletotrichum, Nigrospora, Pezicula, and Phyllosticta have been isolated from Hamamelis plant tissues in the USA, China, Netherlands, Canada, and Japan (Table 9).

Jasminum
The genus Jasminum (Oleaceae) includes more than 200 species distributed in China, Africa, Asia, Australia, South Pacific Islands, and Europe. Jasmines are widely cultivated for ornamental, medical, and cosmetical uses. The species J. sambac, commonly known as Arabian Jasmine, is cultivated throughout India and tropical regions. This genus has been reported for several uses due to the following pharmaceutical activities: antimicrobial [115], antioxidant [116], antidiabetic [117], antiviral [118], and antitumor [119]. Seven species of endophytic fungi of the genus Colletotrichum have been reported from J. sambac in India and Vietnam (Table 10).

Ligustrum
Ligustrum (Family Oleaceae) is a genus of about 50 species of shrubs and trees from warm areas of Europe to Asia [122]. Several species of the genus have been cultivated in many areas of the world as urban ornamental hedge and street trees. In particular, the most widespread species L. lucidum compete with and inhibit the regeneration of native flora, becoming invasive in many areas with a subtropical and temperate climate, such as North America, South America, Europe, Asia, Africa, and Oceania [123]. Due to its active constituents such as glycosides, flavonoids, phenylpropanoids, phenylethanoid, and terpenoids, Ligustrum spp. have been widely used as a health remedy in European, Chinese, and Japanese communities [124,125].

Robinia
Robinia is a genus of flowering plants of the family Fabaceae. R. pseudoacacia, called black locust, grows naturally on a wide range of sites. It is considered to be one of the 40 most invasive woody species all over the world [147] and it is included in the invasive alien species list of the EU [148,149]. It is used for many purposes, such as ornamental plant, for shelterbelts, land reclamation, fuelwood, and pulp production [147]. Six species of endophytic fungi were isolated from R. pseudoacacia in Germany, Slovakia, Hungary, and China (Table 14).

An Overview of Fungal Diversity and Frequency
Investigations on the mycobiota of plants frequently reported new taxa or new species distribution, and several fungi are still undiscovered or undetected. Numerous higher plants have developed a variety of resistance mechanisms to prevent fungal infections. However, the presence of weakly pathogenic fungi in healthy plant tissues highlights the evolutionary continuum between latent pathogens and symptomless endophytes [15]. Generally, all plants have symbiotic interactions with fungal endophytes which can influence host performance in terms of disease resistance [154][155][156], stress tolerance [157], and biomass accumulation [158]. Fungal endophytes may also change according to plant tissues colonized [159], phenological growth stages, host genotypes [160], and geographical distribution areas [161].
In this review, a total of 428 endophytic species belonging to 122 fungal genera have been found in association with 13 plant genera ( Table 1). The greatest level of fungal diversity was reported in in association with Bauhinia with 43 fungal genera and 94 fungal species, and Cornus with 44 fungal genera and 78 fungal species. The degree of fungal recovery from Acacia (29 genera, 51 species), Albizia (14 genera, 27 species), Berberis (17 genera, 29 species), Caesalpinia (19 genera, 42 species), Cassia (15 genera, 19 species), Ligustrum (20 genera, 29 species), and Nerium (21 genera, 37 species) was nearly half in comparison to the abundance noted in the genera Bauhinia and Cornus. Nonetheless, the lowest diversity showed for Hamamelis (4 species/genera), Jasminus (7 species, 1 genera), Lonicera (3 species/genera), and Robinia (6 species/genera) was also due to the lack of published research about fungal endophytes in these plant genera.
It is worth noting the relative homogeneity in distribution of fungi such as Colletotrichum, Fusarium, and Alternaria among these plant genera. In fact, Colletotrichum was undetected only in Lonicera and Robinia, Fusarium in Caesalpinia, and Hamamelis, Jasminus, and Alternaria in Cassia and Lonicera. Although scarcely abundant, the fungal genus Phyllosticta was almost reported for all selected plants except for Albizia, Jasminus, Robinia, and Hamamelis. Other endophytic fungi were detected more occasionally. Future surveys may reveal the presence of additional fungal species also from less investigated plants, such as Robinia, Jasminum, and Lonicera.
The presence of pathogenic or saprotrophic fungi has already been discussed by several authors [165,166]. Table 1 shows that several of the listed fungi were apparently restricted to a single plant genus or at least exhibit some preference for a particular one. Some common and ubiquitous pathogens have been recovered in more than one plant host. This is the case of F. oxysporum (8 host plant species belonging to 7 different genera), A. alternata, A. niger, C. gloeosporioides (7 host plant species), N. oryzae (4 host plant species), B. dothidea, C. globosum, C. acutatum (3 host plant species), A. ochraceus, A. pullulans, and C. truncatum (3 host plant species).

The Most Common Plant Pathogens
The most frequent endophytes detected from the investigated plants are cosmopolitan and ubiquitous pathogens that may cause severe yield losses. In detail, F. oxysporum is responsible for the wilt of vascular tissues on numerous crops that may result in plant death, even if several strains have proved to be non-pathogenic [167]. It has been isolated from 8 different plant species belonging to 7 genera, namely A. hindsii, A. julibrissin, B. malabarica, B. phoenicea, B. aristata, C. officinalis, L. lucidum, and N. oleander. The fungus A. alternata may infect over 380 host plant species causing leaf spots, rots, and blights. It includes opportunistic forms in developing field crops as well as saprophytic strains that may cause harvest and post-harvest spoilage of harvested products. One of the major concerns represented by its infection is related to the production of mycotoxins that may be introduced in the food chain [168]. In this review, A. alternata has been found in association with 3 genera, in 7 plant species (B. malabarica, B. racemosa, B. poiretii, B. aristata, Cornus sp., L. lucidum, and C. pulcherrima). The saprophytic pathogen A. niger is responsible for the spoilage of a wide range of fruit, vegetable, and food products. It is also the causal agent of the black rot of onion bulbs, the kernel rot of maize, and the black mold rot of cherry [169,170]. It has been found within plant tissues of A. arabica, A. lebbeck, B. fortificata, B. malabarica, B. racemosa, C. pulcherrima, and N. oleander (7 plant species or 4 genera). Furthermore, three different species of Colletotrichum have been isolated from reviewed plants. C. gloeosporioides has been isolated from 7 plant species (3 genera), namely A. hindsii, B. racemosa, B. aristata, C. echinata, C. officinalis, C. stolonifera, and L. lucidum, whereas C. acutatum has been found in Cornus spp., Hamamelis sp., and H. virginiana (3 species; 2 genera). Both Colletotrichum species may cause severe fruit rot mainly occurring in pre-and post-harvest [171]. Moreover, C. truncatum, the causal agent of anthracnose disease affecting several leguminous crops [171], has been collected from 2 plant genera, namely A. hindsii and J. sambac. Furthermore, C. lunata, was isolated from the tissues of 4 plant species (2 genera), including B. malabarica, B. racemosa, B. phoenicea, and C. sappan, is the causal agent of seed and seedling blight in several crops, such as rice, millet, sugarcane, and rice, and of maize leaf spot [172]. Besides, B. dothidea reported in association with A. karroo, Cornus sp., and C. officinalis may cause cankers, dieback, fruit rot, and blue stain in woody plants, including Acacia, Eucalyptus, Vitis, and Pistachio [12]. Concerning the species F. lateritium, it has been extensively investigated as the causal agent of chlorotic leaf distortion on sweet potato (Ipomoea batatas) in the USA [173]. This fungus has been isolated from three different plant species and genera (B. aristata, C. controversa, and L. lucidum). Moreover, the common soil-borne fungus G. candidum, found in association with B. vahlii, C. sappan, and L. lucidum, is the causal agent of sour-rot of tomatoes and citrus fruits, and it is also one of the most economically important post-harvest diseases of citrus [174]. Also, C. cladosporioides, detected in B. racemosa, C. echinata, and C. stolonifera, is the causal agent of blossom blight in strawberries [175]. Other pathogenic fungi associated with these selected plants are less widespread and some of them are subjected to containment measures in some countries. This is the case of N. parvum, N. oryzae, L. theobromae, and D. destructiva. In particular, N. parvum, isolated as an endophyte in three Acacia species (A. heterophylla, A. karroo, and A. koa), is one of the most aggressive causal agent of Botryosphaeria dieback on the grapevine and it is known as an aggressive polyphagous pathogen attacking more than 100 plant hosts [176]. Also, N. oryzae, reported from H. mollis, B. phoenicea, B. racemosa, and B. fortificata, may reduce plant growth and seed quality of rice plants as well as Brassica spp., maize, and cotton [177]. Moreover, L. theobromae, found in association with six different plant species (A. karroo, A. koa, B. racemosa, C. echinata, L. lucidum, and N. oleander), is the causal agent of dieback, root rot, and blights for a wide range of plant hosts, mainly located in tropical and subtropical regions [178]. Finally, D. desctructiva, recovered from three different species of Cornus, is the causal agent of the dogwood anthracnose, a devastating disease that was firstly documented in the USA and then introduced into Europe [179].
Generally, closely related organisms, including pathogenic fungi as well as those non-pathogenic, may share similar ecological niches and may potentially interact among themselves. Their co-occurrence could be due to phylogenetic evolution or some unclear biological benefits gained [180,181]. The effects of this interaction may lead to a definition of spaces for development and survival. Nevertheless, it is widely known that non-indigenous species represent one of the greatest threats to native biodiversity [11,[23][24][25]. In fact, a fungal invasion into a new ecosystem may change the native endophytic community structure, leading to the extinction of host-specialized fungi [182]. This antagonistic phenomenon is regulated by the production of antifungal compounds, mycoparasitism, or competition for space and resources [180], as well as a synergy of these interactions [181]. Biological invasions may set in motion a long-lasting cascade of effects on the plant host and associated species in unpredictable ways. Generally, the ecological importance of native species prior to the invasion may not be quantified because of the lack of information on fungal communities, especially for non-pathogenic fungal species. As a consequence of global trade and climatic or environmental changes, studies about the impact of new organisms on the ecosystem represent innovative challenges worldwide. In view of these considerations, even if fungal pathogens found in association with investigated plants are widely distributed in the EU [182][183][184][185][186][187][188][189][190], the risk posed by the introduction of potentially noxious species may be very high. Thus, our results suggest the importance of monitoring imported material to avoid the introduction of such alien species.

Emerging and Potential Threats Due to Commercial Trade
Several species reported in this review are Quarantine Pests (QP) or Regulated as Non-Quarantine Pests (RNQP), as defined by containment measures within the importing country [191]. Among the fungal pathogens found in Cornus species, Elsinoe fawcettii is listed as a QP in the EU, Tunisia, and Israel. This fungus is the causal agent of Citrus scab and it is one of the most important pathogens in many areas of citrus production [192]. E. fawcettii is common in South America and its presence has been detected in other areas such as Central and South Africa, India and South-Eastern Asia, and Australia [192].
Organisms that move across continents may or may not become dangerous depending on several factors, and unexpected consequences may occur [193,194]. The current knowledge about the fungal community associated with ornamental plants and their interaction with the environment is fragmentary. Fungi species generally well known as pathogens, are not necessarily pathogenic when isolated as endophytes [6][7][8]. Genetic mutation can occur in virulent pathogens, transforming the original pathogen into a nonpathogenic strain [9]. Likewise, even though some endophytes are mutualistic, this does not imply that they will not have negative impacts if introduced in a new ecosystem [6,9]. Alien pathogens can often encounter more susceptible host plants and different microbial and abiotic environments without their own 'natural enemies'. The so-called 'risk pathway' defined by international protocols tend to assume that the pathogen will attack a plant host taxonomically similar to that of the susceptibile plant species in its native countries. However, an invasive pathogen may spread to new target hosts, when introduced in a new ecosystem, and novel pathogen combinations can occur [11]. The disease outcomes of these combinations may be extremely complex and the invasive pathogen populations can reach explosive distribution levels that are usually difficult to eradicate once established [23][24][25]. Beyond the damage which may occur on the host plant species and local microbial communities, biological invasions may affect entire ecosystems and the connected ecosystem processes and services, such as soil fertility, fire control, hydrology, and recreation and tourism amenities [23][24][25]. In response to expanding global trade, several EU regulations [27][28][29] and international protocols [195,196] are aimed at regulating over-dissemination and accidental introduction of plant diseases. However, despite existing laws and efforts to prevent the introduction of potential pathogens at ports of entry, many of them will evade detection and establish alien populations [197,198]. Many pathogenic fungi may be undetected, transported in the form of inocula as endophytes, propagules, mycelium, or spores of vegetative material. In addition, large import volumes often permit the inspection of only a small proportion of the introduced plants. According to the precautionary principle, all imported plant species should be considered as a potential threat (vectors of fungi), therefore the presence and establishment may not depend on the number of arrivals. As a consequence, even a reduced amount of infected plants, which can easily escape phytosanitary inspections, may cause the introduction and the spread of diseases with devastating outcomes [199]. The development of tools, such as new molecular diagnosistics [200] and volatile compounds detection devices [201], that allow the rapid and on-site identification of potentially invasive species and the screening of large volumes of plants, clearly appears to be essential [202]. Despite increasing trade, targeted investment in biosecurity may be effective to reduce pathogen introduction and limit the establishment of alien microorganisms. Thus, we highlight the importance of surveillance due to the potential risk of accidental introductions in the absence of effective biosecurity measures.

Conclusions
Globalization has led to intensified movement of people, plants, and plant products, and an increase in the unintentional introduction of non-native fungal species into new ecosystems. Many plant pathogens are biological opportunistic invaders causing several billion dollars in losses to crops, pastures, and forests annually, worldwide. Consideration needs to be given to building resilience in the new environments, from the perspective of pathogen introductions. In particular, the monitoring of plants and plant products, plus early identification-detection of pathogen risks are key steps towards ensuring successful regulation to exclude potential disorders caused by pathogens. This review demonstrated the broad fungal diversity recovered from a small group of ornamental plants that have been relatively unexplored as fungal hosts. Even if the reviewed plant genera are not recognized as sources of significant forest diseases, that have had an ecosystemic impact on a continental scale in the past, we highlight the risk represented by plants as inoculum sources of potentially harmful organisms. Overall, many other species not listed by the EU have represented or may cause important impact in many ecosystemic, environmental, and ecological issues. Our literature search revealed that fungal species may also be introduced through a few hundred plants and invade new ecosystems. In this context, it is important to underline that the amount of imported plant material may not be related to a specific risk, but needs to be considered and evaluated to estimate the negative impacts on agriculture, forestry, and public health, associated with non-indigenous species in European ecosystems. For example, little is known about the effects of invasive species on ecosystem services, although some historic pest invasions (e.g., chestnut blight from North America to Europe) have destroyed host tree species in their locations. The true challenge lies in preventing further damage to natural and managed ecosystems. For this reason, preventative policies need to take into account the means through which pathogens gain access to the EU. The accidental introduction of potentially harmful pathogens also links to other issues of major policy concern (i.e., biotechnology, human health, climate change, etc.) that should be addressed through improved international cooperation and a holistic approach. We should expect that some strategies should be continued or further established to prevent or monitor future introductions, especially at airports, seaports, and other ports of entry, to reduce risks to an acceptable level and preserve natural and agricultural ecosystems.

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