Taxonomic Advances from Fungal Flora Associated with Ferns and Fern-like Hosts in Northern Thailand

Ferns are one of the most significant plant groupings that comprise a substantial proportion of the plant flora due to the fact of their great diversity, especially in tropical areas. The biodiversity of fungi associated with ferns and fern-like hosts has also received little attention in studies. Plant samples were collected from diseased and dead plants of ten fern or fern-like species from Chiang Rai in northern Thailand. Forty-one isolates were selected from the obtained isolates for molecular and morphological analysis, with a focus on pathogenic fungal genera and consideration of the diversity in host and geographical location. Twenty-six species belonging to seven genera (Colletotrichum, Curvularia, Diaporthe, Fusarium, Lasiodiplodia, Neopestalotiopsis, and Pestalotiopsis) in six families were identified. Thirty new hosts, eight new geographical hosts, and one new species, Colletotrichum polypodialium, are described. Nepestalotiopsis phangngaensis, N. pandancola, Diaporthe tectonendophytica, D. chiangraiensis, and D. delonicis were isolated for the first time from leaf spots. Additionally, new reservoirs and geographical locations for species previously isolated from leaf spots or whose pathogenicity was established were found. However, more studies are necessary to prove the pathogenicity of the fungi isolated from the leaf spots and to identify the fungi associated with other species of ferns.


Introduction
Ferns are considered an important component of plant diversity in Thailand, which consists of approximately 5-7% of the total flora in this country, and 670 taxa have been estimated [1]. Ferns also comprise a large proportion of the world's plant flora (9000-12,000 species) [2,3]. They are used as ethnomedicine, pests controllers, food, and ornamental plants [4]. Additionally, they play a crucial role in ecology and interact with a variety of organisms, including insects and saprobic, endophytic, pathogenic, and mycorrhizal fungi [5].
Endophytes related to ferns have not been extensively studied. An investigation of the endophytes of seven fern species in Costa Rica revealed that more than 95% of the fungi belonging to Ascomycota, with Dothideomycetes, Eurotiomycetes, and Sordariomycetes dominating [6], with a prevalence of Xylariales [6]. Pathogenic Ascomycota and Basidiomycota have been isolated from ferns worldwide [7]. Inocyclus angularis was reported to cause tar spots on Pleopeltis astrolepis in Brazil [8] and Milesina dryopteridis to cause rust on Rumohra adiantiformis and Pteris fauriei in Japan [9]. Pestalotiopsis maculans causes leaf spots on Lygodium venustum in Argentina [10] and Colletotrichum gloeosporioides on Lygodium microphyllum and L. japonicum in the USA [11]. Additionally, C. acutatum causes anthracnosis in leather ferns in the USA [12] and Fusarium thapsinum brown rot in Azolla microphylla in India [13].
In contrast to endophytes and pathogens, saprobes have been frequently identified in ferns [5], accounting for 21 ascomycetes on the fronds and rachises of various ferns in Mexico [14]. More recently, new species have been introduced from this plant group:   Pseudosporidesmiaceae, Pseudotruncatellaceae, Sporocadaceae, Vialaeaceae, and Xyladictyochaetaceae. The taxonomic treatment of this order is based on Hyde et al. [27].
This family included 23 genera, including endophytes, saprobes, plant pathogens, and parasites of animals and humans. Members of Sporocadaceae are characterized by acervuli, septate, hyaline, and pale to dark brown conidia. Additionally, they are distinguished by the sequence data of ITS, LSU, and rpb2 [27].
Culture characteristics: The colonies reached 72-80 mm after seven days of growth on PDA at 28 °C; cottony, irregular shape, dull surface, undulated edge, fluffy margin, medium density, and without pigmentation in the medium and conidial mass. Upper view is white, reverse pale luteous in the center, and primrose in other areas.
Material Comparison of the DNA sequences of N. guajavicola strains (ex-type FMBCC 11.4 and MFLUCC 22 -0134) revealed 0.59% nucleotide differences in tef1 (1 gap) and 0.24% in tub2 (one nucleotide) genes, while the sequences of ITS were identical. This species was first reported from a guava tree in Pakistan [29] and, here, it was isolated from Nephrolepis sp.
In Thailand. Herein, we provided a new host and a geographical record for N. guajavicola.
Culture characteristics: The colonies reached 65-67 mm after seven days of growth on PDA at 28 °C; cottony, entire edge, fluffy margin, medium density, and without pigmentation in the medium and fruiting body. Upper view white and the reverse primrose.
Culture characteristics: Colonies reached 72-80 mm after seven days of growth on PDA at 28 • C; cottony irregular shape, dull surface, undulate edge, medium density, and without pigmentation in the medium and conidial mass. Upper view white and the reverse primrose.
Culture characteristics: Colonies reached 32-36 mm after seven days of growth on PDA at 28 • C; felted, regular and entire edge, dull surface and well-defined margin, medium density and without pigmentation in medium and conidial mass. Upper view white, and the reverse greenish black, buff, and primrose circles from center to margin.
Notes: Isolated from a blight on leaves'margin of Nephrolepis cordifolia. The isolate obtained in this study (MFLUCC 22-0145) clustered with P. dracontomelon in the same clade by 99% ML bootstrap support and 1.0 BYPP ( Figure 13). Comparison of the DNA sequences of P. dracontomelon strains (ex-type strain MFLUCC 10-149 with MFLUCC 22-0145) showed 1.02% nucleotide differences in ITS (five nucleotides) and 0.23% in tef1 (one gap). Sequence data for tub2 were unavailable for the type strain (MFLUCC 10-149). This species was introduced from Dracontomelon dao in Thailand [33]. Here, we provide a new host record for P. dracontomelon. clade by 99% ML bootstrap support and 1.0 BYPP ( Figure 13). Comparison of the DNA sequences of P. dracontomelon strains (ex-type strain MFLUCC 10-149 with MFLUCC 22 -0145) showed 1.02% nucleotide differences in ITS (five nucleotides) and 0.23% in tef1 (one gap). Sequence data for tub2 were unavailable for the type strain (MFLUCC . This species was introduced from Dracontomelon dao in Thailand [33]. Here, we provide a new host record for P. dracontomelon .
Culture characteristics: Colonies reached 73-82 mm after seven days of growth on PDA at 28 • C; felted, circular shape, dull surface, entire edge, fluffy margin, medium dense density, and without pigmentation in medium and conidial mass. Upper view white and the reverse primrose.
Notes: Th strain MFLUCC 22-0150 was isolated from necrotic brown leaf spots on Cyclosorus sp. The isolate obtained in this study (MFLUCC 22-0150) clustered with P. hydei in the same cluster by 100% ML bootstrap support and 1.0 BYPP ( Figure 13). Comparison of the DNA sequence data of P. hydei strains (ex-type strain MFLUCC 20-0135 and MFLUCC 22-0150) revealed 0.62% nucleotide differences in ITS (three nucleotides), while the sequences of tef1 and tub2 were identical. This species was first reported from Litsea petiolata in Thailand [30] and, here, it was isolated from Cyclosorus sp., providing a new host record for this species. Diaporthales Nannf., Nova Acta Regiae Societatis Scientiarum Upsaliensis. 8 (2): 53 (1932). Diaporthales comprises 30 families and 181 genera and is characterized by solitary or aggregated perithecia, sometimes with long papilla, unitunicate asci, with a conspicuous refractive ring. The asexual morph is mostly coelomycetes and rarely hyphomycetes [27]. Species of this order can be plant and animal pathogens and are found in the soil and as saprobes or endophytes [34].
Diaporthe Nitschke, Pyrenomycetes Germanici. 2: 240 (1870). Diaporthe comprises 13 species complexes and nine singleton species based on phylogenetic analyses of ITS, tef1, tub2, cal, and his3 [35]. Diaporthe species can live on various hosts as saprobes, endophytes, or pathogens [35]. The present study identified four species (D. chiangraiensis, D. delonicis, D. heveae, and D. tectonendophytica) from the D. arecae and D. sojae complexes on ferns, based on the morphology and phylogeny of the ITS, tef1, tub2, cal, and his3 sequence data. aggregated perithecia, sometimes with long papilla, unitunicate asci, with a conspicuou refractive ring. The asexual morph is mostly coelomycetes and rarely hyphomycete [27]. Species of this order can be plant and animal pathogens and are found in the soi and as saprobes or endophytes [34].
Culture characteristics: Colonies filled a 90 mm Petri dish after seven days of growth on PDA at 28 • C; fluffy, circular shape, dull surface, undulate edge and fluffy margin, medium sparse density, and without pigmentation in the medium and fruiting body. Upper view with circles of white and olivaceous buff and the reverse primrose with greenish olivaceous areas.
Notes: Isolated from dark-brown spots on Pteris grandifolia leaves. The isolate obtained in this study (MFLUCC 22-0133) clustered with D. delonicis by 100% ML bootstrap support and 0.98 BYPP ( Figure 16). The sequence data of cal and tef1 are not available for the type strain of D. delonicis. Comparing sequences of the ex-type strain and our strain (MFLUCC 22-0133) revealed 1.32% nucleotide differences in ITS (six nucleotides) and 0.66% in tub2 (three nucleotides). This species was first reported from dried seed pods of Delonix regia in Thailand [36]. It was isolated from Pteris grandifolia, providing a new host record for D. delonicis.
Culture characteristics: Colonies filled a 90 mm Petri dish after seven days of on PDA at 28 °C; fluffy, circular shape, dull surface, undulate edge and fluffy m medium sparse density, and without pigmentation in the medium and fruiting Upper view with circles of white and olivaceous buff and the reverse primro greenish olivaceous areas.
Material available for the type strain of D. delonicis. Comparing sequences of the ex-typ and our strain (MFLUCC 22 -0133) revealed 1.32% nucleotide differences in I nucleotides) and 0.66% in tub2 (three nucleotides). This species was first reporte dried seed pods of Delonix regia in Thailand [36]. It was isolated from Pteris gra providing a new host record for D. delonicis.
Culture characteristics: Colonies reached 33-43 mm after seven days of growth on PDA at 28 • C; felted, dull surface, circular shape, entire edge, fluffy margin, puckered aspect, medium density without pigmentation in the medium and fruiting body. Upper view white with olivaceous buff areas and the reverse primrose and buff areas.
Notes: Isolated from a blight on the leaf margin of Nephrolepis cordifolia. The isolate obtained in this study (MFLUCC 22-0146) was clustered with D. heveae by 100% ML bootstrap support and 1.0 BYPP ( Figure 16). The sequence of his3 is not available for the strain MFLUCC 22-0146 in this study, while the reference sequences of D. heveae had this gene region. Comparison of the sequence data of the reference strain for D. heveae with our strain (MFLUCC 22-0146) showed 2.87% nucleotide differences in cal (7 nucleotides and four gaps), 1.10% in ITS (5 nucleotides), 3.22% in tef1 (10 nucleotides), and 3.36% in tub2 (15 nucleotides). This species was first reported from Hevea brasiliensis as a die-back agent of the seedlings of the mentioned host in Thailand, Brazil, China, Indonesia, Malaysia, and Sri Lanka [37]. Here, it is first reported in Nephrolepis cordifolia. Malaysia, and Sri Lanka [37]. Here, it is first reported in Nephrolepis cordifolia.   Diaporthe chiangraiensis (Senan & Hyde) Norph., 2022. Facesoffungi number: FoF 13407. Assocciated with Asplenium nidus leaf spot. Sexual morph: not observed. Asexual morph: conidiomata, pycnidium, immersed and semi-immersed, a few, aggregated and black. Sporulation, not observed (for the conidial morphology, see the description of Chiangraiomyces bauhiniae [38]).
Culture characteristics: Colonies reached 75-80 mm after seven days of growth on PDA at 28 • C; felted, dull surface, circular shape, undulate edge, fluffy margin, and medium sparse density without pigmentation in the medium and fruiting body. Upper view with circles of white and primrose and the reverse with circles of straw and olivaceous buff.
Notes: Isolated from a small blight on leaves of Asplenium nidus. The isolate obtained in this study (MFLUCC 22-0136) grouped with D. chiangraiensis with 100% ML bootstrap support and 1.0 BYPP ( Figure 19). Sequence data of cal and tub2 were unavailable for the ex-type strain of D. chiangraiensis. Comparing sequences of D. chiangraiensis strains (MFLUCC 22-0136 and ex-type strain MFLUCC 17-1669) revealed DNA sequence differences (including gaps) in 2% ITS (nine nucleotides) and 4.73% in tef1 (nine nucleotides and six gaps) genomic regions. This species was first reported from dead twigs of Bauhinia sp. (Fabaceae) in Thailand [38], and it was synonymized as D. chiangraiensis based on the phylogenetic analysis [35]. Here, we provide a new host record for D. chiangraiensis.
Culture characteristics: Colonies filled a 90 mm Petri dish after seven days growth on PDA at 28 • C; fluffy to felted, dull surface, and fluffy margin, medium sparse density without pigmentation in medium and fruiting body. Upper view white and primrose circles and the reverse view, honey in the center and primrose in other areas with honey spots. Gamma conidia, absent.
Notes: Isolated from a blight on Nephrolepis cordifolia leaves. The isolate obtained in this study (MFLUCC 22-0140) was grouped with D. tectonendophytica by 100% ML bootstrap support and 1.0 BYPP ( Figure 19). Comparison of the DNA sequence data of D. tectonendophytica strains (MFLUCC 22-0140 and ex-type strain MFLUCC 13-0471) showed 1.46% nucleotide differences in cal (seven nucleotides), 0.67% in ITS (three nucleotides), 1.02% in tef1 (two nucleotides and one gap), and 0.98% in tub2 (two nucleotides and two gaps) genomic regions. This species was first introduced from Tectona grandis in Thailand [39]. Here, we provide a new host record for D. tectonendophytica. The taxonomic structure of this order was clarified, and its name was published validly by Réblová et al. [40]. This order comprises five families: Australiascaceae, Glomerellaceae, Malaysiascaceae, Plectosphaerellaceae, and Reticulascaceae [27]. The members of this order are characterized by apostromatal and endostromatal ascomata and hyaline aseptate ascospores, which can be endophytes or plant parasites [40]. This is a monotypic family with Colletotrichum asexual morph and Glomerella sexual morph. Members of this family are plant endophytes, pathogens, and saprobes [27] . The taxonomic structure of this order was clarified, and its name was published validly by Réblová et al. [40]. This order comprises five families: Australiascaceae, Glomerellaceae, Malaysiascaceae, Plectosphaerellaceae, and Reticulascaceae [27]. The members of this order are characterized by apostromatal and endostromatal ascomata and hyaline aseptate ascospores, which can be endophytes or plant parasites [40].
Colletotrichum based on morphological and phylogenetic data [41]. In this study, seven species (Colletotrichum polypodialium, C. fructicola, C. gigasporum, C. orchidearum, C. pandanicola, C. plurivorum, and C. truncatum) belonging to C. gloesporioides, C. gigasporum, C. orchidearum, and C. truncatum complexes reported on ferns and fern-like hosts based on the morphology and combined phylogeny of the ITS, tub2, act, gapdh, and chs-1 sequence data.  Culture characteristics: Colony reached 59-68 mm after seven days of growth on PDA at 28 • C; cottony, circular shape, dull surface, entire edge, well-defined margin, and medium density, without conidial mass and pigmentation. Upper view pale olivaceous gray in the center, smoke gray in the middle, and white margin, and the reverse with circles of dull green, greenish gray, and primrose margin.
Notes: Isolated from a blight on the leaf margin of Nephrolepis cordifolia. The isolate obtained in this study (MFLUCC 22-0147) clustered with C. fructicola by 99% ML bootstrap support and 1.00 BYPP (Figure 21). The DNA sequence data of C. fructicola strains (MFLUCC 22-0147 and ex-type strain ICMP 18581) differed 1.05% in act (one nucleotide and one gap) and 0.41% in tub2 (two nucleotides), while the sequences of chs-1, gapdh, and ITS were identical. This species was first reported from Coffea arabica in Thailand [42]. It was also isolated from Capsicum annuum, Carica papaya, Cymbopogon citratus, Dendrobium sp., Dimocarpus longan, Freycinetia sp., Freycinetia sp., Pandanus sp., and Pennisetum purpureum in Thailand [43]. Here, we provide a new host record for C. fructicola.  (Figure 21). The DNA sequence data of C. fructicola strains (MFLUCC 22 -0147 and ex-type strain ICMP 18581) differed 1.05% in act (one nucleotide and one gap) and 0.41% in tub2 (two nucleotides), while the sequences of chs-1, gapdh, and ITS were identical. This species was first reported from Coffea arabica in Thailand [42]. It was also isolated from Capsicum annuum, Carica papaya, Cymbopogon citratus, Dendrobium sp., Dimocarpus longan, Freycinetia sp., Freycinetia sp., Pandanus sp., and Pennisetum purpureum in Thailand [43]. Here, we provide a new host record for C.
Culture characteristics: Colonies reached 47-52 mm after seven days of growth on PDA at 28 • C; cottony, circular shape, dull surface, entire edge, and well-defined margin with medium density and without pigmentation in media and conidial mass. Upper view white and the reverse dull green in the center and primrose in other parts.
Culture characteristics: Colonies reached 47-52 mm after seven days of PDA at 28 °C; cottony, circular shape, dull surface, entire edge, and well-defin with medium density and without pigmentation in media and conidial ma view white and the reverse dull green in the center and primrose in other part  (Figure 26). The DNA sequence data of C. gigasporum strains 22 -0158 and CBS 133266) differed 2.13% in ITS (seven nucleotides), 1.94% in gap and four nucleotides), 2.83% in gapdh (seven nucleotides), and 1.44% in gap and six nucleotides). The sequence of act for the type strain is not Colletotrichum gigasporum was first introduced from Centella asiatica, S guianensis, and Coffea arabica from Madagascar, Mexico, and Colombia [48].
reported on Acacia auriculiformis, Alocasia sp. and Hibiscus rosa-sinensis in [43,47,49]. Here, we first report it from N. cordifolia.   Notes: The isolate obtained in this study (MFLUCC 22-0152) clustered with C. orchidearum in the same clade by 100% ML bootstrap support and 1.0 BYPP ( Figure 28). The DNA sequence data of C. orchidearum strains (ex-type strain CBS 135131 and T22-0385) showed 1.17% nucleotide differences in chs-1 (three nucleotides), 0.48% in gapdh (one gap), and 1.03% in tub2 (three nucleotides and two gaps), while the sequences of ITS and act were identical. Colletotrichum orchidearum was revised using obtained isolates from Eria javanica, Epipremnum aureum, Dendrobium nobile, and Hymenocallis sp. in Germany, Iran, Netherlands, and Thailand [50]. Here, we provided a new host record for this species.
Notes: The isolate obtained in this study (MFLUCC 22 -0152) clustered with C. orchidearum in the same clade by 100% ML bootstrap support and 1.0 BYPP (Figure 28).
Here, it was isolated from Cyclosorus sp., providing a new host of C. truncatum.   This family, comprising 64 genera, are saprobes, pathogens, and endophytes on human, plant, and insect substrates in aquatic and terrestrial habitats [27]. They also cause important plant diseases and are significant from this point of view [27]. The asexual morph of them is mostly hyphomycetous and, rarely, coelomycetous [27].
Fusarium Link, Magazin der Gesellschaft Naturforschenden Freunde Berlin. 3 (1): 10 (1809). This genus is characterized by thin-or thick-walled macroconidia with various basal or apical cell shapes, production of trichothecene mycotoxin, and Giberrella sexual morph [53]. Species of this genus were identified by morphological characters and phylogenetic data of the tef1, rpb1, and rpb2 gene regions [53]. This study reports four species (Fusarium ipomoeae, F. nirenbergiae, F. pernambucanum, and F. sulawesiense) of the F. incarnatum-equiseti and F. oxysporum complexes obtained from fern and fern-like hosts using morphological data and multilocus phylogeny.
Culture characteristics: Colonies reached 38-39 mm after seven days of growth on PDA at 28 • C; fluffy, dull surface, entire edge, fluffy margin, medium density, and without pigmentation in the medium. Upper view was white and the reverse primrose.
Microconidia not observed. Sporodochium on PDA and SNA not observed.
Culture characteristics: Colonies reached 38-39 mm after seven days of growth on PDA at 28 °C; fluffy, dull surface, entire edge, fluffy margin, medium density, and without pigmentation in the medium. Upper view was white and the reverse primrose.
Culture characteristics: Colonies reached 47-68 mm after seven days of growth on PDA at 28 • C; fluffy, circular shape, dull surface, entire edge, fluffy margin, medium density, and without pigmentation in the medium. Upper view is salmon in the center, white in other areas, and the reverse in salmon and primrose areas.
Notes: Strain MFLUCC 22-0148 was isolated from dry stems of Asparagus setaceous. MFLUCC 22-0148 clustered with F. pernambucanum in the same clade with 100% ML bootstrap supports and 1.0 BYPP (Figure 33). Comparison of the sequences of the ex-type strain (URM 7559) of this species with strain MFLUCC 22-0148 showed 0.64% nucleotide differences in tef1 (four nucleotides), while no differences were found in the rpb1 and rpb2 genes. Fusarium pernambucanum was introduced from Aleurocanthus woglumi and Dactylopius opuntiae in Brazil [55]. Here, we provide a new host and geographical records for F. pernambucanum.
Culture characteristics: Colonies reached 47-68 mm after seven days of growth PDA at 28 °C; fluffy, circular shape, dull surface, entire edge, fluffy margin, mediu density, and without pigmentation in the medium. Upper view is salmon in the cent white in other areas, and the reverse in salmon and primrose areas.
Material      Figure 37). The sequence of the rpb1 gene is not available for the ex-type strain of this species (CBS 840.88). Comparison of the sequences of the ex-type strain with strain MFLUCC 22-0155 revealed 0.58% nucleotide differences in rpb2 (five nucleotides) and 0.16% in tef1 (one nucleotide). The pairwise comparison results between the ex-type and strain MFLUCC 22-0156 were 0.57% nucleotide differences in rpb2 (five nucleotides) and 0.33% in tef1 (two nucleotides). This species was first reported from Dianthus caryophyllus, Passiflora edulis, Bouvardia longiflora, Solanum lycopersicum, Agathosma betulina, amputated human toe, Solanum tuberosum, Chrysanthemum sp., tulip roots, human leg ulcer, Secale cereal, and Musa sp. [57]. Here, we provide a new host and geographical record for F. nirenbergiae.  (Figure 37). The sequence of the rpb1 gene is not available for the ex-type strain of this species (CBS 840.88). Comparison of the sequences of the ex-type strain with strain MFLUCC 22 -0155 revealed 0.58% nucleotide differences in rpb2 (five nucleotides) and 0.16% in tef1 (one nucleotide). The pairwise comparison results between the ex-type and strain MFLUCC 22 -0156 were 0.57% nucleotide differences in rpb2 (five nucleotides) and 0.33% in tef1 (two nucleotides). This species was first reported from Dianthus caryophyllus, Passiflora edulis, Bouvardia longiflora, Solanum lycopersicum, Agathosma betulina, amputated human toe, Solanum tuberosum, Chrysanthemum sp., tulip roots, human leg ulcer, Secale cereal, and Musa sp. [57]. Here, we provide a new host and geographical record for F. nirenbergiae.

Discussion
Ferns and fern-like species comprise a significant portion of the world's flora and are crucial for economic and ecological reasons [2,4,58]. In this study, we collected fungi associated with ferns and fern-like species in Chaing Rai, Thailand, including ornamental (Asplenium nidus, Asparagus setaceus, Nephrolepis cordifolia, Nephrolepis spp., Phymatosorous sp., Pteris ensiformis, and P. grandifolia) and wild (Cyclosorus spp.). We introduce one new species and report 30 new hosts of 25 fungal species.
Colletotrichum fructicola, C. polypodialium, C. gigasporum, C. orchidearum, and C. truncatum species were isolated from the anthracnose symptoms (Figures 21, 22, 24 and 26-31). Considering the isolation of these species from anthracnose spots on the other hosts [42,50,52,59] and the proof of pathogenicity of some species by other studies [60], our results indicate that ferns can be important hosts for Colletotrichum species, contributing to their evolution and fitness. This study obtained multiple associations from anthracnose of three hosts: Nephrolepis cordifolia (C. fructicola, C. pandanicola, and C. gigasporum); Cyclosorus spp. (C. pandanicola, C. plurivorum and C. truncatum, C. Orchidearum, and C. truncatum). Therefore, further studies are necessary to identify the dominant species on these hosts to ease controlling the fungus. Moreover, we demonstrated that C. polypodialium is able to complete its life cycle as a saprophyte or parasite on Phymatosorus sp., characterizing an important characteristic of the biology of this in ferns.
We isolated and identified C. lunata from leaf spots on Pteris grandifolia (Figures 3 and 4) in Thailand. Curvularia lunata was first introduced as a saprobe on decaying leaves of sugarcane [23], while it was also introduced as the leaf spot agent in many hosts, such as corn [61], mulberry [25], and olive [62]. Even though we did not do pathogenicity proofs, based on the mentioned studies, the occurrence of C. lunata in P. grandifolia characterizes a reservoir for this pathogen and should be noted in the quarantine program and for managing the disease. In addition, the phylogenetic analysis of this article suggested synonymizing some species in the C. gigasporum complex and Curvularia genus. It is recommended that C. lunata and C. chiangmiensis be labeled the same species, because they cannot be distinguished by phylogenetic analysis (Figure 3), as also shown by Garcia-Aroca et al. [61]. It also appears that C. gigsporum and C. zhaoqingense ( Figure 26) are essentially one species, which is supported by the nonseparation of the different isolates of these two species in the phylogenetic tree constructed for the C. gigasporum complex.
We isolated D. heveae and D. tectonendophytica associated with leaf spots on N. cordifolia (Figures 16 and 19), and D. delonicis and D. chiangriensis were associated with leaf spots on Pteris grandifolia and Asplenium nidus, respectively (Figures 16, 17 and 19). Among the Diaporthe species that have been reported with different lifestyles, D. delonicis and D. chiangriensis have been reported as saprobe on dried seed pods of Delonix regia and dead twigs of Bauhinia sp., respectively [36,38], while D. tectonendophytica was introduced as the endophyte [39]. However, all obtained Diaporthe species in this study were isolated from leaf spots. Therefore, this indicates a lifestyle switch from endophytes to pathogens or saprophytes [63], representing an increase in their fitness.
Species of Fusarium were isolated from leaf blight on a dry stem of A. setaceous (F. ipomoeae), dead leaf and leaf spot on A. nidus (F. pernambucanum), leaf spot on N. cordifolia (F. nirenbergiae), and dry leaves of A. setaceous (F. sulawesiense) (Figures 33, 34, 37 and 38). Fusarium sulawesiense and F. ipomoeae are pathogenic on sesame [64] and peanut [65], respectively. Additionally, F. pernambucanum and F. nirenbergiae were obtained from disease symptoms of Musa sp., Passiflora edulis [66], and potato [57]. This article completed our knowledge of the host range of these species by isolating them from symptoms on new hosts. Furthermore, F. nirenbergiae and F. pernambucanum were isolated from the dried disease branches of Asparagus setaceous, indicating that it is essential to identify the dominant species on this host before treating the related disease on this host.
This article reports L. thailandica from a leaf spot on A. nidus and a dead leaf of N. cordifolia (Figures 1 and 2). Lasiodiplodia thailandica was first reported from symptomless twigs of Mangifera indica [21]. It was also isolated from canker on branches of Podocar-pus macrophyllus and Albizia chinensis in China [67]. Hence, pathogenicity assays should be carried out to confirm that this species possesses both endophytic and pathogenic phases.
Several species of Neopestalotiopsis (N. guajavicola, N. hydeana, N. musae, N. pandanicola, N. phangngaensis, N. psidii, and N. saprophytica) were also isolated from leaf spots on ferns and fern-like species (Figures 5-12). These species have been reported from several hosts worldwide [30,31]. Neopestalotiopsis musae was isolated from a leaf spot on Musa sp. [31], while N. guajavicola and N. psidii were obtained from symptomatic guava tree branches [29]. Additionally, N. hydeana was shown to cause fruit rot in Annona squamosal and Garcinia mangostana and leaf spots on a fern, Alpinia malaccensis, and Garcinia mangostana [30]. However, N. saprophytica was isolated as the saprobe on Litsea rotundifolia and Magnolia [28] and as the pathogen on Elaeis guineensis and Paphiopedilum micranthum [68,69]. Neopestalotiopsis phangngaensis and N. pandanicola were introduced as the saprobes [32]. A novel lifestyle or extending host range was thus discovered for all Neopestalotiopsis species listed in this study, which were all isolated from leaf spots of various ferns. Three species N. phangngaensis, N. pandanicola, and N. saprophytica, were isolated from Cyclosorus spp., a wild fern in Thailand, and should be regarded as the reservoir of these fungi. We also obtained the closely related species, P. dracontomelon and P. hydei, associated with leaf spots on N. cordifolia and Cyclosorus spp., in the present study (Figures 13-15). These species were also reported from leaf spots on Dracontomelon dao [33] and Litsea petiolata [30]. The new host revealed the new reservoirs for these fungi, which should be considered in quarantine programs and disease management.
Ninety percent (37 out of 41 strains) of the fungi reported in this article were isolated from leaf spots and blights using the tissue culture method. All strains were collected during the dry season from Mueang district in Chiang Rai Province, Thailand. Among the 41 isolates, 26 species were identified, which shows great diversity. Each isolate of Pestalotiopsis, Neopestalotipsis, and Diaporthe isolated from ferns represented single species, showing 100% biodiversity when compared with the other recorded genera. Only approximately 4% of the fungi identified in this paper represent new species. However, Hyde et al. [43] predicted that 96% of the fungi in northern Thailand are new. Thus, limiting the lifestyle (fungi that cause leaf spot), environmental conditions (e.g., dry or wet season), and geographic area also limits identifying the flora of a given area, such as northern Thailand, which has a high fungal diversity [70][71][72][73][74]. In addition, it was documented that the sampling time on fern species affected the diversity, abundance, and composition of fungal flora [75]. Similar to our findings, only one new species was found in the biodiversity survey of associated fungi with ferns in Taiwan, although it has been estimated that between 800 and 8000 new species can be found in the country [7]. Moreover, only one new saprobe was detected in Alsophila costularis in Thailand [17]. In addition, the fungal biodiversity of two collections of fern in Mexico revealed 14 records among 21 taxa, and no new species was found [14]. However, 15% of species novelty (two new species among 13 strains) and 92% diversity (12 species among 13 strains) were recorded by Kirschner and Liu [15] in Taiwan, and 76% of the novel taxa (15 new species and on new genera among 21 taxa) was detected on amazon ferns [76] and five new Pseudocercospora species were discovered on ferns [77]. Our study showed a snapshot of fungi that can cause spot or blight on ferns and the low or high species novelty on ferns. However, wide collections must be performed in pristine tropical environments to reveal the fungal diversity of ferns and fern-like species.
This study revealed new hosts for 25 fungal species and new lifestyles for some of them (D. delonicis, D. tectonendophytica, D. chiangriensis, N. phangngaensis, and N. pandanicola). Identifying the host range of associated fungi with plants is important from several points of view. To manage plant disease using different strategies, including resistant cultivars, quarantine regulations, crop rotations, eradication of reservoirs, disease forecasting, landscape planning, risk modeling, and knowledge of pathogen host range, is necessary [78]. Furthermore, most pathogens have host specificity, meaning they cannot cause damage to nontarget plants. Therefore, they can be employed as a biocontrol agent alongside herbicides or physical techniques to manage exotic plants in agricultural ecosystems [79]. Furthermore, identifying new endophyte hosts is important because some have both pathogenic and no-pathogenic phases, while others only have nonpathogenic phases [63]. As nonpathogenic endophytes are the cause of resistance to heat, salinity, and heavy metals, identifying endophytes whose advantages have been proven in the past leads to the identification of plants that are resistant to salt and heat stress, as well as the identification of plants that are involved in bioremediation [80,81]. In addition, identifying the pathogenic forms of fungi previously reported as saprophytic, provided that their pathogenicity is proven, is necessary to control plant diseases, because it reveals unrecognized gaps in the disease cycle. No correlation between biodiversity and geographic location was found in the hosts where more than one species of the same genus was isolated from leaf spots. Only a small portion of the fungi associated with ferns in northern Thailand was identified in this work. Given the biodiversity of ferns in tropical regions and the paucity of research in this area, it is advised that additional hosts be collected and surveyed to investigate the diversity of fern-associated fungi.

Sampling and Fungal Isolation
Infected leaves and debris of 11 fern and fern-like species were collected from three locations in Chiang Rai Province, Thailand, in December 2021. For the fungal pathogens isolations, they were cut into 4 mm square pieces and sanitized using 1:1 70% alcohol: 1% sodium hypochlorite solution for 1-2 min, followed by a double wash in sterilized distilled water for 1 min [82]. Finally, they were air-dried on sterilized filter paper for 5 min, placed on PDA plates, and incubated in darkness at room temperature. From the debris, cultures were obtained using the imprint or direct culture methods [82]. Pure isolates were obtained using the hyphal tip method.

Culture Characteristics and Morphological Investigations
Fungal structures were obtained from the sporulating cultures on PDA and SNA, and the morphology of the isolates was investigated. The necessary morphological characters, such as asci, ascospores, conidiomata, conidiophores, phialides, conidiogenous cells, conidia, appressoria, and chlamydospores, were examined. All microscopic characters were observed with a microscope (Nikon Y-TV55, Nikon coopration, Tokyo, Japan), and digital images were captured with a Nikon DS-Ri2 camera. All measurements were made using image framework Version 0.9.7. Images used for photoplates were processed with Adobe Photoshop CS6 v. 13.1.2 (Adobe Systems, USA).
The culture morphology was investigated for all isolates, and the colony character and color were recorded after seven days growth on PDA at 28°C. The color was recorded based on Rayner's color chart [83].

DNA Extraction and PCR Amplification
The DNA was extracted using the DNA extraction kit (Omega Bio-Tek). Polymerase chain reaction (PCR) amplifications were performed in a total of 25 µL: 1 µL DNA (50-700 ng/µL), 1 µL of each primer, 12.5 µL Go Taq Green Master Mix 2x (PROMEGA), and 9.5 µL molecular grade water [84] and amplified using the primers and conditions listed in Table 2. Table 2. PCR conditions and used primers in this study.

Pairwise Homoplasy Index Test
To compute the recombination level between the closely related species on the phylogenetic tree, the pairwise homoplasy index (PHI) test [110] was performed by Splits Tree4 v. 4.18.2 [110], using concatenated datasets of the five loci of Colletotrichum species. The relationship between species was observed by the splits tree graph and activation of the splits decomposition and logDet options.