Diversity of Fusarium Species Isolated from Symptomatic Plants Belonging to a Wide Range of Agri-Food and Ornamental Crops in Lebanon

Lebanon is a small Mediterranean country with different pedoclimatic conditions that allow the growth of both temperate and tropical plants. Currently, few studies are available on the occurrence and diversity of Fusarium species on Lebanese crops. A wide population of Fusarium strains was isolated from different symptomatic plants in the last 10 years. In the present investigation, a set of 134 representative strains were molecularly identified by sequencing the translation elongation factor, used in Fusarium as a barcoding gene. Great variability was observed, since the strains were grouped into nine different Fusarium Species Complexes (SCs). Fusarium oxysporum SC and Fusarium solani SC were the most frequent (53% and 24%, respectively). Members of important mycotoxigenic SCs were also detected: F. fujikuroi SC (7%), F. sambucinum SC (5%), F. incarnatum-equiseti SC (3%), and F. tricinctum SC (4%). Two strains belonging to F. lateritium SC, a single strain belonging to F. burgessii SC, and a single strain belonging to F. redolens SC were also detected. This paper reports, for the first time, the occurrence of several Fusarium species on Lebanese host plants. The clear picture of the Fusarium species distribution provided in this study can pose a basis for both a better understanding of the potential phytopathological and toxicological risks and planning future Fusarium management strategies in Lebanon.


Introduction
Lebanon is one of the smallest countries in the Mediterranean basin. Although it has a limited agricultural area of about 650,000 ha [1], the variety of agricultural land and climatic conditions enables farmers to grow a plethora of plants, including both temperate crops (e.g., grape, olive tree, and solanaceous plants) and tropical crops (e.g., banana and avocado). The territory is quite varied, ranging from the flat coastal area to the mountain chain in the center of the country, where large areas are occupied by cedar and fir woods. Generally, tobacco and figs are grown in the southern part of Lebanon, citrus fruits and banana along the coast, olives and apples in different parts of the Mount Lebanon governorate, and, in the north, fruits, vegetables, and cereals in the fertile Bekaa Valley. Lebanon exports part of its production to other Middle Eastern countries and Arab Gulf states [1]. death syndrome, bean root rot, and multiple vegetable diseases [28,29]. In addition, some members of the FSSC can be involved in human and animal infections.
Since the Fusarium species distribution is widely influenced by environmental conditions, and several species showing different mycotoxin profiles can be largely isolated together from symptomatic plants, a correct identification of Fusarium species can allow for a clear picture of the potential phytopathological and toxicological risks. From this perspective, the aim of this study was to identify a wide set of Fusarium strains isolated from different agriculturally important crops in Lebanon to obtain, for the first time, a large amount of information on the Fusarium species diversity in this country.

Sampling
During a 10-year period, from 2012 to 2021, and within the service of the plant disease diagnostic clinic provided by the Lebanese Agricultural Research Institute (LARI), symptomatic plant tissues were brought by private companies, public institutions, and farmers from all over Lebanon (Figure 1). Fusarium colonies, isolated from different host plants, including agri-food plants, ornamental plants, and forest trees, were selected to obtain a collection of 134 Fusarium strains ( Table 1).

members of the FSSC can be involved in human and animal infecti
Since the Fusarium species distribution is widely influenced by tions, and several species showing different mycotoxin profiles can gether from symptomatic plants, a correct identification of Fusarium a clear picture of the potential phytopathological and toxicologica spective, the aim of this study was to identify a wide set of Fusarium different agriculturally important crops in Lebanon to obtain, for amount of information on the Fusarium species diversity in this cou

Sampling
During a 10-year period, from 2012 to 2021, and within the serv diagnostic clinic provided by the Lebanese Agricultural Research In tomatic plant tissues were brought by private companies, public ins from all over Lebanon (Figure 1). Fusarium colonies, isolated from including agri-food plants, ornamental plants, and forest trees, we collection of 134 Fusarium strains (Table 1).

Morphological Identification of Fusarium Strains
For fungal isolation, after surface disinfection with 70% ethanol or 2% sodium hypochlorite solution and washing twice with sterile distilled water, small portions (about 5 mm × 5 mm) taken from the margin of the infected areas were transferred to potato dextrose agar plates (PDA, Merck, Darmstadt, Germany) and incubated for 5-7 days at 25 • C until colony development.
To obtain pure Fusarium strains from the developing fungal colonies, a mass of conidia was resuspended in sterile distilled water and scattered at low density on 90 mm water agar (WA) Petri dishes. After 18-24 h of incubation at 25 • C in the darkness, germinated conidia were singularly transferred to 60 mm PDA plates using a dissecting microscope.
After 5-7 days of incubation at 25 • C under a 12 h photoperiod, Fusarium strains cultured on PDA and Spezieller Nahrstoffarmer agar (SNA) media were morphologically identified according to Nelson et al. and Leslie and Summerell [30,31].

Molecular Identification of the Fusarium Species by Translation Elongation Factor 1-α Sequencing 2.3.1. DNA Extraction
For each monosporic strain, 5 mycelium plugs were collected from the margin of actively growing colonies on PDA and transferred to a sterilized cellophane disk overlaid on PDA 90 mm plates. After incubation at 25 • C for 2-3 days, the mycelia were collected into 2 mL tubes, frozen, and lyophilized. The DNA extraction was carried out on 15 mg of powdered lyophilized mycelium by using a Wizard Magnetic DNA Purification System for Food kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. The genomic DNA quantity and integrity were checked on 0.8% agarose gel in 1X Tris-Acetate-EDTA (TAE) using a standard 1 kb DNA Ladder (ThermoFisher Scientific, Carlsbad, CA, USA) and a Nanodrop spectrophotometer (ThermoFisher Scientific).

Translation Elongation Factor 1-α (TEF) Amplification and Sequencing Analysis
For each Fusarium strain, the informative fragment of the Translation elongation factor 1-α gene was amplified and sequenced using the primer pair EF-1 and EF-2 [32]. The PCR products, stained with GelRed ® (Biotium Inc., Fremont, CA, USA), were checked after electrophoretic separation on 1.5% agarose gel in 1× TAE buffer under UV light by comparison with a 100 bp DNA Ladder (ThermoFisher Scientific). Before sequencing, the PCR products were purified with an enzymatic EXO/FastAP mixture (ExonucleaseI and FastAP thermosensitive alkaline phosphatase, ThermoFisher Scientific). Sequence reactions were performed for both strands using a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's recommendations. The labeled products were purified by filtration through Sephadex G-50 (5%) (Sigma-Aldrich, Saint Louis, MO, USA) and analyzed using an ABI PRISM 3730 Genetic Analyzer (ThermoFisher Scientific). The FASTA sequences obtained were analyzed and assembled using BioNumerics v. 5.1 software (Applied Maths, Kortrijk, Belgium).
For molecular identification at the species level, the obtained sequences were compared with the sequences available in the NCBI Database through the BLASTN program. To further solve the phylogenetic relationships between and within the Fusarium species detected, the sequences obtained in this study joined to 23 sequences of Fusarium reference strains and to the sequence of Ilyonectria crassa CBS 158.31 strain, used as an outgroup, were aligned by using the MUSCLE algorithm [33]. Phylogenetic relationships were analyzed using the maximum likelihood method with MEGA software version 7 [34]. Bootstrap analyses [35] were conducted to determine the confidence of internal nodes using a heuristic search with 1000 replicates, removing gaps.

Results
In Table 1, 134 Fusarium strains, isolated from 38 different host plants, are listed. In particular, the strains were isolated from different plant parts, such as the roots, crown area, stem, and fruits, showing symptoms of fungal diseases, such as wilting, vascular browning, yellowing, necrotic or chlorotic leaf spots, crown rot, and also decline or death. For each Fusarium strain, the geographical and host plant origin, organ of isolation, and symptoms observed are detailed. The majority of the strains were isolated from plants grown in the South and Bekaa regions (45 and 42 strains, respectively), followed by Mount Lebanon (25 strains) and Akkar (17 strains) regions. Four Fusarium strains were also collected in the North, and a single strain was obtained from an ornamental plant grown in Beirut city (Table 1).
Host plants affected by Fusarium species were grouped considering their botanical and commodity classifications, as shown in Figure 2. Most of the Fusarium strains were isolated from solanaceous plants (34 out of 134), banana (19 out of 134) and lettuce (17 out of 134), together representing half of the Fusarium strains isolated from symptomatic plants. In detail, 20 strains were isolated from potato, 8 strains from tomato, 4 strains from eggplant, and a single Fusarium strain from tobacco and hot pepper, respectively (Table 1). To a lesser extent, Fusarium strains were isolated from forest tree (11 strains), cucurbits (9 strains), citrus trees (8 strains), legume (6 strains), strawberry (5 strains), ornamental plants (5 strains), and onion and olive tree (4 strains, respectively). As reported in Table 1, 12 Fusarium strains were also isolated from other host plants, including apple (3 strains), avocado, parsley, and Paspalum grass (2 strains from each), and basil, cauliflower, and kiwi (a single strain from each). The distributions of Fusarium species in the different geographical regions are reported in Table 2. The single strain isolated in Beirut, from the ornamental Jacaranda tree, was identified as a member of the FSSC. In the North Lebanon region, four Fusarium strains were isolated from tomato, pea, and strawberry, and identified as FOSC. A large number of Fusarium strains were isolated from plants grown in the Akkar, Bekaa, Mount Lebanon, and South Lebanon regions. In all of these regions, the FOSC and FSSC were the most dominant SCs, with values ranging between 43.8 (Akkar) and 59.5% (Bekaa), and between 12.5 (Akkar) and 31% (Bekaa) ( Table 2). In addition to the FOSC and FSSC strains, in the Southern region, great variability among Fusarium species isolated was observed, with high frequency of the FFSC (13.3%), followed by the FTSC and FLSC (4.4%), and, to a lesser extent, by FIESC, FSAMSC, and FBSC (2.2%). Fusarium redolens strains were isolated only from plants grown in the Mount Lebanon region, with a value of 4%. In this region, strains belonging to the FTSC (8%), FIESC (8%), and FSAMSC (4%) were also isolated. In the Bekaa region, strains belonging to the FSAMSC and FFSC, with values of 4.8%, were isolated. In the Akkar region, 6.3% of the strains belonged to the FIESC, and the same value of 12.5% of strains was recorded for each of the following SCs: FSAMSC, FFSC, and FTSC (   ; banana, lettuce, citrus trees (citrus 2, lemon 4, grapefruit 1, and mandarin 1), legumes (bean 2, pea 3, and faba bean 1), cucurbits (melon 4, watermelon 3, and cucumber 2), strawberry, forest trees (cedar 5, abies 1, hornbeam 1, pine 3, and walnut tree 1), ornamental plants (ivy 1, poinsettia 2, Jacaranda 1, and roses 1), olive tree, and onion. To a lesser extent, Fusarium strains were isolated from other host plants (apple 3, avocado 3, kiwi 1, cauliflower 1, basil 1, parsley 1, and paspalum grass 1).
The phylogenetic analysis of the TEF sequences of 134 Lebanese Fusarium strains, 23 Fusarium reference sequences, and 1 sequence of Ilyonectria crassa used as an outgroup taxon resulted in the tree shown in Figure 3. The phylogenetic tree was resolved in 11 well-separated clades, supported by high bootstrap values (over 98%). A large set of 71 strains clustered together and with the reference strain NRRL 52787, belonging to the F. oxysporum species complex (FOSC). Ten strains were identified as belonging to the F. fu- ; banana, lettuce, citrus trees (citrus 2, lemon 4, grapefruit 1, and mandarin 1), legumes (bean 2, pea 3, and faba bean 1), cucurbits (melon 4, watermelon 3, and cucumber 2), strawberry, forest trees (cedar 5, abies 1, hornbeam 1, pine 3, and walnut tree 1), ornamental plants (ivy 1, poinsettia 2, Jacaranda 1, and roses 1), olive tree, and onion. To a lesser extent, Fusarium strains were isolated from other host plants (apple 3, avocado 3, kiwi 1, cauliflower 1, basil 1, parsley 1, and paspalum grass 1).  The phylogenetic analysis of the TEF sequences of 134 Lebanese Fusarium strains, 23 Fusarium reference sequences, and 1 sequence of Ilyonectria crassa used as an outgroup taxon resulted in the tree shown in Figure 3. The phylogenetic tree was resolved in 11 well-separated clades, supported by high bootstrap values (over 98%). A large set of 71 strains clustered together and with the reference strain NRRL 52787, belonging to the F. oxysporum species complex (FOSC). Ten strains were identified as belonging to the F. fujikuroi species complex (FFSC); in particular, two strains were identified as F. steriliphosum, one strain as F. musae, two as F. sacchari, and five strains showed high similarity to the F. proliferatum reference strain. Another 20 strains clustered in six well-supported clades, corresponding to the F. burgessii species complex (FBSC), F. redolens species complex (FRSC), F. tricinctum species complex (FTSC), F. incarnatum-equiseti species complex (FIESC), F. sambucinum species complex (FSAMSC), and F. lateritium species complex (FLSC). The FBSC and FRSC were both represented by only one strain and the corresponding species reference strain (F. algeriense and F. redolens, respectively). In detail, six strains belonged to the FTSC, among which two strains showed homology with the F. acuminatum NRRL 36147 reference strain. Among four FIESC strains, one strain showed high similarity to the F. citri reference strain, one strain to F. pernambucanum, and one strain to F. clavum. The FSAMSC clade included one strain identified as F. culmorum, two strains clustered with the F. sambucinum reference strain, and three strains sharing high homology with the reference strain F. tumidum NRRL 38939. One strain showed similarity to the reference strains NRRL 66923 and NRRL 46662, belonging to the FSAMSC, but this well-supported clade (100 bootstrap value) did not group with the other species of the FSAMSC described previously. Two strains were identified as belonging to the FLSC clade. Another large clade grouping 32 Lebanese strains was the Fusarium solani species complex (FSSC). Most of these strains showed similarity to the F. suttonianum reference strain NRRL 32858; two strains were identified as F. euwallaceae, and three strains clustered with F. solani NRRL 45880.
The occurrence of the different Fusarium species complexes identified through the phylogenetic analysis of TEF sequences in Lebanese plants is shown in Figure 4. The FOSC was the most widespread species complex in Lebanon, representing 53% of the overall 134 collected strains, followed by the FSSC, with a percentage of 24%. Other species complexes occurring in different plants at 7, 5, and 4% are the FFSC, FSAMSC, and FTSC, respectively. To a lesser extent, strains belonging to the FIESC (3%) and FLSC (2%) were identified. Finally, one strain of F. redolens from parsley crown and one strain of F. algeriense from potato crown and roots were isolated.
with the reference strain F. tumidum NRRL 38939. One strain showed similarity to the reference strains NRRL 66923 and NRRL 46662, belonging to the FSAMSC, but this wellsupported clade (100 bootstrap value) did not group with the other species of the FSAMSC described previously. Two strains were identified as belonging to the FLSC clade. Another large clade grouping 32 Lebanese strains was the Fusarium solani species complex (FSSC). Most of these strains showed similarity to the F. suttonianum reference strain NRRL 32858; two strains were identified as F. euwallaceae, and three strains clustered with F. solani NRRL 45880.

Discussion
A wide population of Fusarium strains infecting plants all over Lebanon in the last 10 years has been isolated and identified to achieve, for the first time, extended information regarding Fusarium diversity occurring on Lebanese crops. Furthermore, since coniferous forests are a primary constituent of the Lebanese landscape, the cedar of Lebanon is included in this study, being the national emblem of the country. Indeed, monitoring activities to assess Fusarium's occurrence in a wide range of plants in Lebanon were lacking, as

Discussion
A wide population of Fusarium strains infecting plants all over Lebanon in the last 10 years has been isolated and identified to achieve, for the first time, extended information regarding Fusarium diversity occurring on Lebanese crops. Furthermore, since coniferous forests are a primary constituent of the Lebanese landscape, the cedar of Lebanon is included in this study, being the national emblem of the country. Indeed, monitoring activities to assess Fusarium's occurrence in a wide range of plants in Lebanon were lacking, as well as updated information regarding the risk related to Fusarium mycotoxins. Plants showing mostly symptoms of decline and withering, were collected from different areas of the country and belonged to a wide range of categories, ranging from agri-food to ornamental plants, including herbaceous plants and forest trees. The most represented commodities in the Fusarium host collection were potato, banana, and lettuce. In particular, potato is one of the primary crops grown in Lebanon for local consumption, processing, and export, representing more than 45% of the total area assigned to vegetable crops [1], and banana is one of the main crops intended for both local consumption and export, mostly in South Lebanon. Therefore, it is highly worrisome that such important crops for Lebanese agriculture showed such high Fusarium occurrence.
The molecular identification, based on TEF sequencing, allowed distinguishing several Fusarium species that, according to the contemporary Fusarium species concept [16], have been assigned to given Fusarium species complexes. A total of nine different species complexes occurred among the Fusarium strains herein, demonstrating that a wide biodiversity of Fusarium is detectable in Lebanon.
The most represented species complex (53%) was the FOSC, whose members were isolated from almost all of the plants considered in this study. Members of this complex are well-known causal agents of a wide range of diseases, including Panama disease on banana, and Fusarium wilt and root and crown rot on legumes, lettuce, solanaceous plants, and cucurbits [36]. In our study, the plants from which F. oxysporum strains were recovered are commonly known in the literature as host crops of this ubiquitous species. Furthermore, these strains were isolated from the crowns and roots of plants showing yellowing, decline, wilt, and rot, which are symptoms of diseases caused by F. oxysporum (Table 1). Besides the economic damage caused by their pathogenicity to plants, FOSC members are able to produce toxic metabolites, such as beauvericin (BEA), a phytotoxic compound contributing to pathogenicity in tomato; fusaric acid (FA), known for its ability to induce wilt symptoms in several plants; and enniatins (ENNs). In addition, each of these metabolites has been proven to be toxic to human and animal cell lines or animals fed with contaminated feed [23]. These results encourage the consideration of prevention measures to be adopted by studying these pathogens and control methods aimed to prevent heavy deterioration and yield losses in Lebanese fields.
The second-most-occurring species complex in Lebanon in the present study was the FSSC (24%). Similar to FOSC members, members of this complex are known to include species causing root and crown rot. Consistently, Lebanese FSSC strains were isolated from several different host plants showing browning, necrosis, decline, and rot. Although the FSSC does not contribute to mycotoxin contamination, this complex includes more than 80 species sharing very high genetic variability and is considered among the most relevant pathogenic group of species in the genus.
Both the FOSC and FSSC are present worldwide, but F. oxysporum has been previously reported in Lebanon only associated with Panama disease in banana [12] and in chickpea [13]. In the present investigation, nine strains belonging to FFSC were isolated from banana, potato, lettuce, strawberry, olive tree, and the cedar of Lebanon. Among the four strains isolated from banana tree, the only one from fruits was identified as F. musae, confirming the strong association of this species with banana fruits, whereas the other strains isolated from banana stems were F. sacchari, F. steriliphosum, and F. proliferatum. Only one FFSC strain from potato tubers was identified as F. proliferatum, similar to the other strains from strawberry, olive tree, and cedar. Although the number of occurring strains is limited, the presence of F. proliferatum is of great concern for its ability to produce different mycotoxins, mainly FBs, which have been associated with a wide number of animal diseases and are classified by IARC as potentially carcinogenic [37].
Two other species complexes identified in Lebanese strains of great importance for the presence of toxigenic species are the FSAMSC and FIESC. However, except for one F. culmorum strain from eggplant, which produces DON, the species identified within the SAMSC were mainly F. sambucinum and F. tumidum, both producing trichothecene type A mycotoxins. Conversely, FIESC species, detected in citrus trees, melon fruits, and ornamental plants are known to produce multiple mycotoxins, including type A and B trichothecenes, zearalenone, BEA, and ENNs.
On the other hand, 4% of the studied Fusarium strains belonged to the FTSC, characterized by widespread species, but producing only BEA and ENNs. These strains were recovered only from citrus trees and from apple fruits. Similarly, F. avenaceum was reported as a postharvest pathogen of apples in Italy [38] and the Netherlands [39].
Furthermore, sporadic strains belonging to the FLSC (two strains from lemon), FRSC (a single strain from parsley), and FBSC (a single F. algeriense strain from potato root and crown) were identified. These Fusarium species can produce mycotoxins, such as BEA, ENNs, FA, and MON. Fusarium algeriense, is a recent species identified only on durum wheat in Algeria, as far as we are aware [40]. On the contrary, F. redolens, causing wilt decline and death in the parsley plants investigated in our study, has been previously reported in Lebanon on chickpea [14].

Conclusions
Lebanon produces several important agricultural commodities, due to the climatic conditions favorable to agriculture, that are even exported to several other countries. Since investigations into the occurrence of Fusarium in Lebanon are limited, an extended investigation of Fusarium occurrence was conducted, focused on a widespread monitoring of several Lebanese plants of agri-food and ornamental interest. The Fusarium genus was proven to be widely spread, with very high biodiversity, including several species that could represent a risk to consumers due to their potential mycotoxin production. The collected strains were distributed in nine different species complexes, with the FOSC and FSSC being the most present. Other important species complexes of great concern, such as the FFSC, FSAMSC, FIESC, and FTSC, were also identified. To a lesser extent, a few strains belonging to the FLSC, FBSC, and FRSC were detected. Most of the species detected in the present investigation are reported for the first time in Lebanon, since few investigations focused on the Fusarium genus in this country. The wide distribution and diversity of Fusarium around the country suggest a lack of effective disease management, correct identification, and good agricultural practices to prevent any new occurrence and dispersal of pathogens between fields. In addition, this investigation shows that a policy to evaluate the regulation of Fusarium mycotoxins in Lebanon is needed and must be undertaken.

Data Availability Statement:
The data presented in this study are available within the article.