Description of Two Fungal Endophytes Isolated from Fragaria chiloensis subsp. chiloensis f. patagonica : Coniochaeta fragariicola sp. nov. and a New Record of Coniochaeta hansenii

: Prospection of the endosphere of the native plant Fragaria chiloensis subsp. chiloensis f. patagonica from the foothills of the Chilean Andes led to the isolation of two strains of the genus Coniochaeta . We addressed the taxonomic placement of these strains based on DNA sequencing data using the ITS and LSU genetic markers, morphological features, and biochemical traits. One of these strains was identiﬁed as Coniochaeta hansenii , for which the anamorph and teleomorph states were described. The second strain did not seem to match any of the currently described species of this genus; therefore, we propose the name Coniochaeta fragariicola sp. nov.


Introduction
The genus Coniochaeta (Sacc.) Cooke [1] (Coniochaetaceae, Coniochaetales, Ascomycota) is characterized by strongly melanized, cleistothecial or perithecial, usually setose ascomata with cylindrical, unitunicate asci, and one-celled ascospores with a germ slit. Anamorphs are predominantly phialidic and often produce intercalary conidiogenous cells with lateral necks, giving rise to one-celled conidia in slimy masses, with some members developing microcyclic conidiation [2,3]. According to Index Fungorum (www.indexfungorum.org, (accessed on 12 December 2022)), the genus includes 117 species, many of which have only been described based on their morphological characteristics, with no information concerning sequenced DNA regions for phylogenetic analysis. This group of fungi occurs in both terrestrial and aquatic environments and has been reported on bark, dung, lichens, soil, wood, polluted water, submerged plant material, and other substrates [4][5][6].
Previous studies have revealed that members of Coniochaeta may have either beneficial or harmful effects on plants. Many of its members occur as endophytes of phylogenetically diverse hosts, and some species produce antimicrobial secondary metabolites that aid in the protection against plant pathogenic species of Colletotrichum, Verticillium, and other genera [7,8]. On the other hand, certain Coniochaeta species have the ability to infect woody plants, such as apricot and peach [9], while others are lignicolous, humicolous, and coprohilus [10]. However, they are generally regarded as low-virulent, opportunistic pathogens that proliferate on wounded, senescent, or infected plant tissues [4]. Genome mining and transcriptome analyses have discovered that some members of Coniochaeta have exceptional lignocellulolytic machinery to deconstruct lignin [11]. Several Coniochaeta species, mostly known as anamorphs, have been implicated in superficial and deep-seated, life-threatening forms of phaeohyphomycosis in humans and dogs [12][13][14]. Other species are known for their teleomorphs, such as coprohilous Coniochaeta with poly-spored asci [15].
The taxonomic history of Coniochaeta is complex, and early phylogenetic studies found that, under its traditional circumscription, this genus was polyphyletic [16]. Nevertheless, in a molecular study conducted by García et al. [2], a redefinition of the genus was proposed, which included the synonymization of three morphologically similar genera, i.e., Coniochaetidium, Ephemeroascus, and Poroconiochaeta with Coniochaeta. Further taxonomic improvements were made with the transfer of several species originally described in Rosellinia and in the asexually typified genus Lecythophora to Coniochaeta, based on molecular and phenotypic data [12,17]. Despite all these advancements towards a more stable taxonomy of this genus, no DNA sequence data is currently available for many of its members and many species most likely remain undiscovered, especially in poorly studied regions.
During taxonomic studies on the microfungi associated with the native Chilean wild strawberry Fragaria chiloensis (Rosaceae, Rosales), two strains of the genus Coniochaeta were isolated. One of these strains did not seem to match any of the currently described species of this genus. In this paper, we addressed the taxonomic placement of these strains based on the ITS and LSU genetic markers, morphological features, and biochemical traits.

Sampling and Fungal Isolation
In 2020, 12 plants of Fragaria chiloensis subsp. chiloensis f. patagonica were collected from a forested area located in Pinto, Ñuble region, Chile (36.818367 S, 71.622217 W; altitude: 697 m.a.s.l.). All 12 plants were considered as one sample and were placed inside a sterile plastic bag and transported to the laboratory in a dark container. The sample was dissected into three sections: (i) roots, (ii) crown, and (iii) the phyllosphere containing the petioles and leaves. Each section was individually surface sterilized by submerging the tissues for 1 min in 70% ethanol, 3 min in 1.5% NaClO, and 1 min in 96% ethanol; finally, they were rinsed three times in sterile distilled water [18,19]. Nine tissues 5 mm in diameter were cut with a sterile scalpel and distributed on 24-well sterile microplates containing noble agar (NA). The plates were incubated at 25 • C for 2 wk and checked under a microscope every day for the emergence of endophytic fungi from the tissues. Upon emergence, fungal tips were removed using a sterile needle and inoculated on NA plates and incubated for 7 d at 25 • C. Two yeast-like strains were recovered and deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM) of the Institute of Agricultural Research, Chile, and in the Culture Collection of Fungi and Yeasts (CBS) of the Westerdijk Fungal Biodiversity Institute, The Netherlands, under the codes RGM 3301 and RGM 3311, and CBS 149284 and CBS 149292, respectively.

DNA Sequence Analyses
Genomic DNA isolation was carried out with the Wizard Genomic DNA Purification Kit (Promega), following the manufacturer's instructions with the following modifications: 30-60 mg of fungal biomass from a 7 to 14 d old culture, grown in 10 mL of potato dextrose broth, was used as the initial material. The fungal biomass was placed in sterile 1.5 mL microcentrifuge tubes and, after adding a lysis solution, it was ground with a micropestle to disrupt the mycelial cell walls. Samples were then incubated in a water bath at 65 • C for 30 min, and 10 µL of 3 M sodium acetate was added for DNA precipitation. DNA concentration and purity were spectrophotometrically measured using a Take3 microplate reader (Epoch, Biotek Inc., Winooski, VT, USA) and the Gen5 software version 2.09 (Biotek Inc., Winooski, VT, USA).

Macroscopic and Microscopic Characterization
The macromorphological characteristics of the two isolates were described in three different media: potato dextrose agar (PDA, Difco TM ), oatmeal agar (OA), and malt-extract agar (MEA, Difco TM ). Five millimeter-sized discs were excised with a cork borer from the periphery of an actively growing fungal colony of each strain, previously grown on PDA at 20 • C for 7 d, and placed in the center of Petri dishes. The plates were incubated at 25 • C in the dark for 21 d.
The micromorphological characteristics of the strain RGM 3311 were described in OA, rabbit dung agar (RDA), potato carrot agar (PCA), and Leonian's agar. This strain was incubated at 25 • C in the dark for a period of 60 to 75 d. In the case of RGM 3301, the micromorphological characteristics were evaluated in the following media: MEA, V8 agar, OA, corn flour agar, malt agar, Sabouraud (Difco TM )-glucose agar, synthetic poor nutrient agar (SNA) with sterile filter paper, well-water agar (20%), modified Leonian's agar, PCA, RDA, and PDA after incubation at 20 and 25 • C in the dark at 20 • C for 12h/12h light and dark intervals, for up to 60 d. The rabbit dung agar was prepared as follows: 20 g of rabbit dung was sterilized at 121 • C for two 15 min sessions; the dung was then ground and resuspended in 1 L of distilled water and supplemented with 20 g of agar; and RDA was sterilized at 121 • C for 15 min. Other media were prepared according to Crous et al. [60].
Microscopic structures were observed using a Nikon Eclipse 80i microscope and the software NIS-Elements version 2.2 (Nikon Instruments Inc., Tokyo, Japan). Samples were placed in 60% lactic acid and stained with 5% cotton blue. Micromorphometric measurements were reported with a 95% confidence interval.

Phenotypic Characterization
The isolates were examined for phenotypic properties using GEN III microplates (Biolog Inc., Hayward, CA, USA) to assess their ability to oxidize a broad range of carbon sources and resist inhibitory agents [61,62]. Strains RGM 3301 and RGM 3311 were inoculated in PDA and incubated at 25 • C for 10 d for biomass production. The GEN III microplates were inoculated with a conidial suspension of RGM 3301 in the viscous inoculating fluid A (IF-A) at a cell density of 0.025 (600 nm), while RGM 3311 was inoculated in IF-A with a ground mycelium at a cell density of 0.070 (600 nm). The inoculated plates were incubated at 25 • C for 11 d in the dark. Data from microplates were analyzed in an Epoch microplate spectrophotometer (Biotek Inc., Winooski, VT, USA) at 550 nm.

Data and Image Processing
Microscopic measurements and data from phenotypic characterization were processed in Google Spreadsheet. Images were edited using GIMP version 2.10.32 and Inkscape version 1.1.

Taxonomic Position of the Isolates
Both endophyte strains isolated from Fragaria chiloensis subsp. chiloensis f. patagonica were affiliated with the genus Coniochaeta, based on a maximum likelihood (ML) analysis of the concatenated datasets of ITS and LSU ( Figure 1).
A BLAST search using the individual ITS and LSU nucleotide sequences against a local database of Coniochaeta spp. revealed identical sequences between RGM 3311 and C. hansenii CBS 885.68 (ITS identities: 183/183 (100%), no gaps, LSU identities: 456/456 (100%), no gaps), while differences in these nucleotide sequence alignments were found when comparing with the second-best hits, Coniochaeta montana ARIZ-AZ0093 (ITS identities: 346/374 (90.1%), 4 gaps) and Coniochaeta discospora CBS 168.58 T (LSU identities: 864/879 (98.3%), no gaps). Moreover, the strain RGM 3311 formed a single clade with Coniochaeta hansenii CBS 885.68 with a high branch support (92.8/100, SH-aLRT/UFBoot) and a similar branch length, suggesting that both strains represent one and the same species. Taxonomy 2023, 3, FOR PEER REVIEW 9 species. Therefore, we propose the name Coniochaeta fragariicola sp. nov. for the strain RGM 3301 T .  Coniochaeta cruciata FMR 7409 and Coniochaeta gigantospora ILLS 60816 T , the closest neighbors to RGM 3311 in the ML tree, showed branch support values below the reliability threshold. In addition, similarities between the ITS and LSU nucleotide sequences of strain RGM 3311 with the respective genetic marker of C. cruciata FMR 7409 (no ITS sequence was available; LSU: 438/458 (96%), 2 gaps) and C. gigantospora ILLS 60816 T (ITS identities: 495/548 (86.68%), 10 gaps; no LSU sequence was available) lead us to conclude that RGM 3311 is closely related to C. hansenii CBS 885.68 and distant to its closest neighbors. Therefore, it is proposed that strain RGM 3311 corresponds to C. hansenii.
On the other hand, the strain RGM 3301 was placed in the ML phylogenetic tree in a separate clade from RGM 3311. The strain RGM 3301 was grouped in a single clade with Coniochaeta ambigua PAD S00027 T , displaying a node support value that suggests they are related to one another (75/99, SH-aLRT/UFBoot). The nucleotide sequence alignment of the ITS1 and ITS2 of C. ambigua PAD S00027 T compared to the respective sequences of Coniochaeta sp. RGM 3301 indicated low similarity values (ITS1 identities: 65/76 (86%), 3 gaps; ITS2 identities: 153/162 (94%), 6 gaps), suggesting that these two species are not closely associated.

Macromorphological Characterization of the Isolates
Coniochaeta fragariicola sp. nov. RGM 3301 T and C. hansenii RGM 3311 colonies displayed yeast-like growth with entire margins, a circular shape, and sparse aerial mycelium in the three media tested: PDA, OA, and MEA ( Figure 2 and Table 2). Coniochaeta fragariicola sp. nov. RGM 3301 T showed different morphologies depending on the media tested. For instance, in PDA, the colony was rugose, with a pale-yellow pigmentation that changed to a brown-orange in the center after 15 d and yellow towards the margin after 21 d (Figure 2a). In OA and MEA, the colony had a glabrous appearance with a predominate pale orange color in the center and yellow-white towards the margin (Figure 2b,c). Moreover, in the MEA, the margin was more filamentous than in the other media (Figure 2c).
Coniochaeta hansenii RGM 3311 showed similar morphological characteristics in PDA ( Figure 2d) and MEA (Figure 2f). For instance, colonies were shiny, with a moist appearance, radial grooves, and an undulating margin; they were orange in the center and yellow-gray towards the margin. In OA, the colony was drier, with an entire margin (Figure 2e). From the bottom, colonies showed similar characteristics to those observed in the respective media tested.

2c).
Coniochaeta hansenii RGM 3311 showed similar morphological characteristics in (Figure 2d) and MEA (Figure 2f). For instance, colonies were shiny, with a moist ap ance, radial grooves, and an undulating margin; they were orange in the center and low-gray towards the margin. In OA, the colony was drier, with an entire margin (F 2e). From the bottom, colonies showed similar characteristics to those observed in th spective media tested.   Orange-brown in the center and faded-yellow towards the margin, flat colony with entire margin; from the bottom, the same characteristics. A growth diameter of 50 mm. Grows between 15 and 35 • C, optimal between 25 and 30 • C.
Pale orange-pink colony in the center and grayish-white towards the margin, flat with entire margin; from the bottom, the same characteristics. A growth diameter of 86 mm.
Bright pale orange-pink in the center and yellowish-gray towards the margin, flat colony with entire margin; from the bottom, the same color. A growth diameter of 45 mm.

Coniochaeta hansenii RGM 3311
Orange with a slightly irregular margin, radial grooves from the center to the edge of the colony; from the bottom, the same coloration but paler. A growth diameter of 37 mm. Grows between 10 and 25 • C, optimal 25 • C.
Pale orange, flat with entire margin; from the bottom, the same characteristic. A growth diameter of 37 mm.
Pale orange, flat colony with entire margin, underside of the same coloration; from the bottom the same characteristics. A growth diameter of 23 mm. 1 Growth conditions: 25 • C for 21 d.

Micromorphological Characterization of the Isolates
Micromorphological characteristics of Coniochaeta fragariicola sp. nov. RGM 3301 T = CBS 149284 T were evaluated in several media. The anamorph of C. fragariicola sp. nov. RGM Taxonomy 2023, 3 194 3301 T was obtained in PDA, OA, MEA, and PCA, whereas, for the teleomorph, only immature perithecia were produced in PCA and no further development was observed even though they were tested in all of the aforementioned media and conditions.
Comparisons of C. fragariicola sp. nov. RGM 3301 T were carried out with reference strains whose anamorph had been previously described, including the conidiogenous cell, conidia, microcyclic conidiation, and in some cases, the presence or absence of chlamydospores. Only the teleomorph of Coniochaeta ambigua PAD S00027 T had been described; therefore, it was not possible to compare it with other strains. The conidiogenous cell of C. endophytica AEA 9094 T [52] was characterized by reduced cylindrical protrusions from the hyphae or as discrete phialides; phialidic conidiogenous cells ampulliform were (5.2-10.3 × 2.3-2.9 µm wide), similar to C. fragariicola sp. nov. RGM 3301 T . However, the conidia of C. endophytica AEA 9094 T differed from those recorded for C. fragariicola sp. nov. RGM 3301 T , the former being shorter and wider in diameter ((2.5-) 3.1-3.4 (-4.4) µm × (1.3) 1.6-1.8 (2.4) µm) and ellipsoidal to fusiform in shape, while the conidia of C. fragariicola sp. nov. RGM 3301 T were allantoid. Coniochaeta endophytica, unlike C. fragariicola sp. nov. RGM 3301 T , did not develop chlamydospores or microcyclic conidiation.
The conidiogenous cells of Coniochaeta nivea were usually originated from cylindrical protuberances of densely packed hyphae and only occasionally from phialides, which were unusual in C. fragariicola sp. nov. RGM 3301 T [26]. Similarly, the conidiogenous cells in Coniochaeta elegans were mostly reduced to cylindrical protrusions from the hyphae, with collarettes, a condition that was not common in C. fragariicola sp. nov. RGM 3301 T , which presented mostly ampulliform conidiogenous cells with curved apices, regardless of whether or not they formed a collarette. Conidiation in C. fragariicola sp. nov. RGM 3301 T occurs through the conidiogenous cell and by microcyclic conidiation, whereas conidiation in Coniochaeta elegans, as well as in C. nivea, occurs only through the conidiogenous cell [26]. Moreover, the conidia in C. elegans and C. nivea were shorter and wider when compared with C. fragariicola sp. nov. RGM 3301 T (3.0-4.5 × 1.1-1.8 µm in C. elegans; 3.7-4.1 × 1.3-1.6 µm in C. nivea).
The teleomorph of C. hansenii RGM 3311 was observed in OA, PDA, and RDA, while the anamorph was scarcely produced in Leonian's agar. In accordance with the molecular analysis, Coniochaeta hansenii RGM 3311 shared similar micromorphometric characteristics with C. hansenii (≡ Sordaria hansenii Oudem. 1882), as described by Oudemans [65]. The perithecia in this description were subglobose, measuring 350 µm in diameter, and featuring a neck with a short-conical shape, similar to those observed in C. hansenii RGM 3311. The setae in the original description were 23 µm long, which were about threefold shorter than those observed in C. hansenii RGM 3311, though both terminated in bristly hairs. A report of C. hansenii in Italy [15] also corroborates the characteristics of the perithecium of C. hansenii RGM 3311, where both were brown to dark brown to black, pyriform, setose above the perithecium; however, it was bigger in the report from Italy (480-680 µm × 350-430 µm) [15].

Conclusions
This work contributes to expanding our current knowledge regarding the geographic extent and host source of species of the genus Coniochatea. Here, we report the description of two endophytic Coniochaeta strains, isolated from Fragaria chiloensis subsp. chiloensis f. patagonica collected from the foothills of the Chilean Andes.
Phylogenetic studies and macro-and micro-morphological analyses confirmed that strain RGM 3311 corresponded to C. hansenii, which to the best of our knowledge corresponds to the first report of this strain in Chile. Additionally, we described the previously unknown anamorph of this species. The second isolate corresponded to a novel species of Coniochaeta, named C. fragariicola sp. nov. RGM 3301 T = CBS 149284 T that was closely related to C. ambigua PAD S00027 T , C. cephalothecoides L821, C. endophytica AEA 9094 T , and C. simbalensis NFCCI 4236 T . The conidial width of C. fragariicola sp. nov. RGM 3301 T was a distinctive feature of this novel species.
It is worth mentioning that some reference species of the genus Coniochaeta have only been described in morphological terms, and the genetic barcoding scheme of other species of this genus is incomplete, making it difficult to assign names to new isolates. We suggest a revision of the genus Coniochaeta, including the sequencing of genetic markers from reference specimens with their incomplete genetic barcoding as well as the sequencing of other molecular markers. We also suggest including phenotypic analyses to account for another layer of information when analyzing the taxonomic position of new isolates. Further research is needed to understand the interaction between these endophyte species and the Chilean wild strawberry plant.