A new genus of Bambusicolaceae (Pleosporales) on Corylus avellana (Fagales) from Italy

Abstract Background In this study, we introduce Corylicola gen. nov. in the family of Bambusicolaceae (Pleosporales), to accommodate Corylicola italica sp. nov. The new species was isolated from dead branches of Corylus avellana (common hazel) in Italy. The discovery of this new genus with both sexual and asexual characters will contribute to expand the knowledge and taxonomic framework of Bambusicolaceae. New information Corylicola gen. nov. has similar morphological characters compared to other genera of Bambusicolaceae. These are solitary, scattered, globose to subglobose and ostiolate ascomata; anastomosing and branching pseudoparaphyses; cylindrical asci with a well-developed ocular chamber and short furcate pedicel; and single-septate ascospores. The coelomycetous asexual morph of Corylicola has holoblastic, phialidic conidiogenous cells and light brown conidia analogous to other members in the family. Corylicola differs from the other genera of Bambusicolaceae in having yellowish-brown ascospore masses at the top of the ascomatal neck. Detailed morphological illustrations with comprehensive descriptions for the new taxa are provided, as well as a key to the genera of Bambusicolaceae. Maximum Likelihood analysis and Bayesian Inference of a combined SSU, LSU, ITS, RPB2 and TEF1 sequence dataset confirms the placement of this genus as a distinct lineage in Bambusicolaceae.

Species of Bambusicolaceae are characterised by solitary, scattered, immersed, semiimmersed to erumpent and conical or globose to subglobose ascomata; anastomosing, branching interascal filaments; cylindrical to clavate asci with a short furcate or rounded to obtuse pedicel; and slightly broad-fusiform or clavate to ellipsoidal, hyaline or yellowish to brown, single-septate ascospores with gelatinous sheath , Hyde et al. 2013. Coelomycetous asexual characteristics of Bambusicolaceae are pycnothyrial or pycnidial conidiomata, holoblastic or enteroblastic and phialidic or annelidic conidiogenous cells with hyaline or pale to dark brown, cylindrical or oblong to ellipsoidal aseptate to 1-3-septate conidia , Hyde et al. 2013, Jayasiri et al. 2019. Members of the family Bambusicolaceae share morphological characters with families Didymosphaeriaceae, Massarinaceae and Tetraplosphaeriaceae in the order Pleosporales, such as cylindrical to clavate asci and fusiform to ellipsoidal, hyaline to brown, single-septate ascospores (Tanaka et al. 2009, Zhang et al. 2009, Hyde et al. 2013. However, its asexual characteristics are different from these families . Bambusicola was introduced by  and placed in Trematosphaeriaceae, based on the phylogenetic analysis of a large subunit (LSU) ribosomal DNA dataset by maximum parsimony. Bambusicola, with type species B. massarinia , is characterised by small, cone-shaped ascomata; slightly broad and fusiform, hyaline ascospores; and coelomycete asexual morphs with light brown conidia , Yang et al. 2019. A multi-locus phylogenetic analysis conducted by Hyde et al. (2013) resulted in the placement of Bambusicola in Bambusicolaceae. Twelve species are known in this genus according to Index Fungorum (2020). Palmiascoma was introduced by  from palms, based on morpho-molecular analyses. This monotypic genus is characterised by clavate asci with rounded to obtuse pedicels and clavate to ellipsoidal, yellowish-brown to dark brown, echinulate, single-septate ascospores . Leucaenicola is an asexual genus introduced by Jayasiri et al. (2019) from decaying pods of Leucaena species. It is characterised by conidial morphology, size and colour that are similar to those of the micro-conidia of B. thailandica, but are phylogenetically distinct (Jayasiri et al. 2019). Three species are currently described in Leucaenicola (Ariyawansa et al. 2020).
In this study, we introduce Corylicola gen. nov. to accommodate Corylicola italica sp. nov. isolated from Corylus avellana in Italy. We present morphological illustrations of both sexual and asexual morphs, comprehensive descriptions, phylogenetic analyses based on SSU, LSU, ITS, RPB2 and TEF1 sequence data and a key to genera in Bambusicolaceae to confirm the placement of the new genus in Bambusicolaceae.

Specimens collection, examination and isolation
Dead branches with black raised spots on the surface were collected from Corylus avellana trees in Italy (February 2019). Samples were taken to the laboratory in a plastic Ziploc bag and stored inside paper envelopes. Samples were examined and processed following the procedure described by Wijesinghe et al. (2019). Photographs of enlarged host twigs and ascomata were taken using a Motic SMZ 168 compound stereomicroscope. Morphological characters were examined by hand sectioning of fruiting structures on the surface of twigs. The micro-morphological structures inside ascomata were photographed using a Nikon ECLIPSE 80i compound stereomicroscope with a Canon 600D digital camera. The following structures were observed and measured: diameter, height, colour and shape of ascomata and ostiole; peridium width, cell structure and colour; length and width of asci and ascospores (at the longest and widest point, respectively) and width of pseudoparaphyses. Tarosoft (R) Image Frame Work version 0.9.7. programme was used for the measurements of photomicrograph structures. Images used for figures were processed with Adobe Photoshop CS6 Extended version 13.0.1 software (Adobe Systems, San Jose, California).
Single-ascospore isolation was carried out following protocols described by Chomnunti et al. (2014). Single germinated ascospores were aseptically transferred on to potato dextrose agar (PDA) plates, which were incubated at 18°C for 15 to 20 days to obtain pure cultures. Colony characters were observed and measured weekly. After one month, cultures were used to extract DNA. Photographs of enlarged structures in culture were taken using a Motic SMZ 168 compound stereomicroscope. Micro-morphological characters were examined and photographed using a Nikon Eclipse Ni-U microscope with Nikon DS-RI2 microscope camera. All structures (conidiomata, hyphae, conidiomata wall, conidiogenous cells and conidia) were processed for photographs by using water-mounted glass slides. The holotype was deposited at MFLU (Mae Fah Luang University Herbarium, Chiang Rai, Thailand). The ex-type cultures were deposited at MFLUCC (Mae Fah Luang culture collection). Both Facesoffungi and Index Fungorum numbers were obtained , Index Fungorum 2020.

DNA extraction, PCR amplification and sequencing
Genomic DNA was isolated from fruiting bodies and from scraped fresh fungal mycelium grown on PDA media for six weeks at 18°C, using the EZgene Fungal gDNA extraction Kit GD2416 (Biomiga, Shanghai, China), following the manufacturer's instructions. DNA was stored at 4°C for use in regular work and at -20°C for long-term. Sequences were generated for five gene regions, small subunit (SSU), the internal transcribed spacer region (including ITS1, 5.8S, ITS2), large subunit (LSU), RNA polymerase II second largest subunit (RPB2) and translation elongation factor 1-α (TEF1). The following primers were used for PCR amplification: NS1 and NS4 for SSU, ITS5 and ITS4 for ITS, LR0R and LR5 for LSU, fRPB2-5F and fRPB2-7cR for RPB2 and EF1-983F and EF1-2218R for TEF1 (White et al. 1990, Vilgalys and Hester 1990, Hopple 1994, Rehner and Samuels 1994, Rehner 2001.
PCR was carried out in 20 μl reactions, containing 10.0 μl of Bench Top Taq MasterMix PCR mixture (SinoGenoMax, Beijing, China), 1 μl of each forward and reverse primer (10 μM), 1 μl template genomic DNA and 7.0 μl deionised water. PCR protocols were as follows: For ITS and LSU: initial denaturation at 94°C for 2 mins; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 50 s, elongation at 72°C for 90 s; and final extension at 72°C for 10 min. For SSU: initial denaturation at 95°C for 3 mins; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 50 s, elongation at 72°C for 30 s; and final extension at 72°C for 10 min. For RPB2: initial denaturation at 94°C for 2 mins; followed by 35 cycles of denaturation at 95°C for 45 s, annealing at 57°C for 50 s, elongation at 72°C for 90 s; and final extension at 72°C for 10 min. Finally for TEF1: initial denaturation at 94°C for 2 mins; followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 50 s, elongation at 72°C for 1 min; and final extension at 72°C for 10 min. The PCR products were verified by staining with ethidium bromide on 1% agarose electrophoresis gels. Sequencing of PCR amplicons was conducted with the same primers used for PCR. Sequencing of successfully amplified PCR products was outsourced to the SinoGenoMax Sanger sequencing laboratory (Beijing, China). Lasergene SeqMan Pro v.7 software (DNASTAR, Madison, Wisconsin) was used to obtain consensus sequences from generated sequence reads. Resulting sequences were deposited in NCBI GenBank (Table 1) (Zhang et al. 2012. Phylogenetically, however, Corylicola forms a distinct lineage within Bambusicolaceae (Fig. 1 B). Corylicola differs morphologically from Bambusicola and Palmiascoma by the accumulation of ascospores as yellowish-brown masses at the apices of ascomatal necks. Bambusicola, Leucaenicola and Palmiascoma have coelomycetous asexual morphs, which are similar to Corylicola , Jayasiri et al. 2019. Asexually, Corylicola is characterised by holoblastic, phialidic conidiogenous cells similar to Palmiascoma and one-celled, aseptate conidia similar to Palmiascoma and Leucaenicola, whereas Bambusicola has 1-3-septate conidia (Hyde et al. 2013. Based on morphological observations, a key to all four genera of the family is provided.
Culture characteristics: Ascospores germinating on PDA within 24 hours from singlespore isolation. Colonies on PDA reaching 5-10 mm diam. after 14 days at 16°C, circular, crenated edge, flat with dense, whitish-grey in upper and brownish-black in the lower surface of the colony. Sporulated after 12 weeks.

Etymology
Referring to the country where the holotype was collected, Italy

Notes
Corylicola italica sp. nov. shows morphological characters that are similar to other representatives in the family Bambusicolaceae. Based on morphological comparison with the type species of other genera in the family, Corylicola italica is similar to Palmiascoma gregariascomum  in having uni-loculate ascomata, central ostioles with minute papilla, cellular pseudoparaphyses and single-septate, echinulate, brown ascospores . Corylicola italica has cylindrical asci H with short, furcate pedicels similar to Bambusicola massarinia (MFLU 12-0405), while P. gregariascomum has clavate asci with short rounded to obtuse pedicels . The branching and anastomosing pseudoparaphyses above the asci of C. italica are similar to B. massarinia and P. gregariascomum. However, the absence of a mucilaginous sheath around the ascospores in C. italica distinguishes it from both B. massarinia and P. gregariascomum . In addition, B. massarinia has hyaline ascospores, whereas both C. italica and P. gregariascomum have yellowish-brown ascospores .

Discussion
In our multi-locus phylogenetic analysis (Fig. 1), we represent the recognised genera in Bambusicolaceae by letters A, B, C and D. The new genus Corylicola, with a single species C. italica (B), is phylogenetically distinct from other genera in Bambusicolaceae (A, C and D). It is positioned as sister to Bambusicola (A). Nucleotide differences of the extype strain of C. italica (MFLUCC 20-0111) were compared against the type species of other genera in Bambusicolaceae. Number of nucleotide differences (including gaps) by gene region were as follows: for Bamboo is a medicinal plant in which saprobic microfungi are abundant on culms and leaves (Dai et al. 2018, Rathnayaka et al. 2019, Sun et al. 2020. Most species of Bambusicola are reported on dead culms of bamboos , Jayasiri et al. 2019. Recently, Yang et al. (2019) introduced Bambusicola subthailandica and B. sichuanensis from Phyllostachys heteroclada, showing these species could be distributed on a wide range of hosts. Leucaenicola species were isolated from decaying pods of Leucaena sp. (Jayasiri et al. 2019) and leaf spots of Osmanthus fragrans (Ariyawansa et al. 2020). Palmiascoma species were identified from dead palm frond and dead branches of Eucalyptus sp. , Jayasiri et al. 2019). Most species have been isolated from Thailand and a few from China and Taiwan (Yang et al. 2019, Ariyawansa et al. 2020