Additions to Italian Pleosporinae, including Italica heraclei sp. nov.

Abstract Background In the last few years, many microfungi—including plant-associated species—have been reported from various habitats and substrates in Italy. In this study of pleosporalean fungi, we researched terrestrial habitats in the Provinces of Arezzo (Tuscany region), Forlì-Cesena and Ravenna (Emilia-Romagna region) in Italy. New information Our research on Italian pleosporalean fungi resulted in the discovery of a new species, Italica heraclei (Phaeosphaeriaceae). In addition, we present a new host record for Pseudoophiobolus mathieui (Phaeosphaeriaceae) and the second Italian record of Phomatodes nebulosa (Didymellaceae). Species boundaries were defined, based on morphological study and multi-locus phylogenetic reconstructions using Maximum Likelihood and Bayesian Inference analyses. Our findings expand the knowledge on host and distribution ranges of pleosporalean fungi in Italy.


Introduction
A number of prominent scholars contributing to the foundation of fungal classification were of Italian origin. Among the most important mycologists of the 19 century are Giuseppe De Notaris and Pier Andrea Saccardo, who were the earliest mycologists to validate microscopic characteristics as important features in fungal taxonomy (Onofri et al. 1999). Currently, fungal taxonomy benefits from a combination of morphology and DNA-based molecular analyses to resolve species limits (e.g. Alors et al. 2016, Skrede et al. 2017, Haelewaters and De Kesel 2020. In the last few years, a high number of microfungal taxa have been recorded in different Italian habitats (Jensen et al. 2010, Rodolfi et al. 2016,Wanasinghe et al. 2018a, Liu et al. 2019, Marin-Felix et al. 2019, Hyde et al. 2020b). Currently, a database (https://italianmicrofungi.org/) for plantassociated Italian microfungi is being developed with past, present and upcoming studies being added.
The order Pleosporales is amongst the most family-rich ones in Dothideomycetes (Mugambi and Huhndorf 2009, Brahmanage et al. 2020, with 91 families and 566 genera . Luttrell (1955) suggested that Pleosporales should contain dothideomycetous species with perithecioid ascomata and pseudoparaphyses amongst the asci. After investigations by Luttrell (1973) and Barr (1983), the Pleosporales order was established by Barr (1987), based on Pleosporaceae with the type species Pleospora herbarum. The order includes taxa characterised by perithecioid ascomata with perithecia that have a papillate apex and ostiole, with or without periphyses, cellular pseudoparaphyses, fissitunicate asci and ascospores with variable pigmentation, septation and shape and usually with bipolar asymmetry (Barr 1987, Hyde et al. 2013. In this study, we investigated three fungal taxa in pleosporalean families, two taxa belonging to Phaeosphaeriaceae and one to Didymellaceae. th Phaeosphaeriaceae was introduced by Barr (1979). The family was typified by Phaeosphaeria and P. oryzae is the type species . Members of this family can be saprotrophic, endophytic, pathogenic on economically-important plants and crops and hyper-parasitic on living plants and humans (Kirk et al. 2010, Bakhshi et al. 2019, Phookamsak et al. 2014, Senanayake et al. 2018, Roels et al. 2020. Phaeosphaeriaceous species associated with monocotyledons have been often described as having small to medium-sized ascomata and septate, ellipsoidal to fusiform or filiform ascospores (Zhang et al. 2012, Hyde et al. 2013. Some species of Phaeosphaeriaceae have been recorded from dicotyledonous plants (Ariyawansa et al. 2015a, Hyde et al. 2020b, Brahmanage et al. 2020. Didymellaceae is another family in Pleosporales introduced by De Gruyter et al. (2009) to accommodate Ascochyta, Didymella (type), Phoma and Phoma-like species (Chen et al. 2015. It is a species-rich family containing numerous plant pathogenic, saprotrophic and endophytic species associated with a wide range of hosts (Aveskamp et al. 2008, Aveskamp et al. 2010, Wanasinghe et al. 2018a. Species of Didymellaceae are cosmopolitan and have been reported from inorganic materials, water, air, soil and different environments, such as deep-sea sediments, deserts and karst caves (Wanasinghe et al. 2018a).
Currently, a total of 83 and 35 genera are accounted for Phaeosphaeriaceae and Didymellaceae, respectively . New additions of phaeosphaeriaceous and didymellaceous species have been recorded from Italian localities in the last few years, from multiple hosts, substrates and geographical locations (Ariyawansa et al. 2015b, Marin-Felix et al. 2019, Farr and Rossman 2020. Here, we present the characterisation of three fungal strains isolated from dead attached stems of different dicotyledon hosts collected in Italy.

Sample collection, morphological studies and specimen deposition
Strains were isolated from dead stems of different host plants belonging to Apiaceae, Asteraceae and Urticaceae (dicotyledons) collected in the Provinces of Arezzo, Forlì-Cesena and Ravenna (Italy) from September to December 2018. Samples were preserved in sterile Ziploc bags in the laboratory at 18°C. Macromorphological characters of the samples were observed using a Motic SMZ 168 compound stereomicroscope and micromorphology was examined from hand-sectioned structures using a Nikon ECLIPSE 80i compound stereomicroscope, equipped with a Canon 600D digital camera. The measurements of photomicrographs were obtained using Tarosoft (R) Image Frame Work version 0.9.7. Images were edited with Adobe Photoshop CS6 Extended version 13.0.1 software (Adobe Systems, San Jose, California).
Single-spore isolation was carried out as described by Chomnunti et al. (2014). Germinated spores were aseptically transferred into fresh potato dextrose agar medium (PDA, Merck KGaA of Darmstadt, Germany). Culture plates were incubated at 18°C for six weeks and inspected every week. Herbarium specimens are preserved at Mae Fah Luang University Herbarium (MFLU) in Chiang Rai, Thailand. All living cultures are deposited at Mae Fah Luang Culture Collection (MFLUCC). Facesoffungi and Index Fungorum numbers for new taxa were obtained (Jayasiri et al. 2015, Index Fungorum 2020. The administrative boundaries of Italy and geocodes for collecting sites related to our newly-isolated species were mapped by using QGIS version 3.14 (QGIS Geographic Information System, Open Source Geospatial Foundation Project. http://qgis.org/). Geocodes of collecting locations were confirmed with GoogleEarthPro version 7.3.3 (the data providers were: Image Landsat/Copernicus, Data SIO, NOAA, US. Navy, NGA, GEBCO, US Dept. of State Geographer, https://www.google.com/earth/). The data files (.cvs and .shp) for administrative boundaries were downloaded from DIVA-GIS for mapping and geographic data analysis (Hijmans et al. 2001, https://www.diva-gis.org/).

DNA extraction, PCR amplification, sequencing and molecular analyses
The methodology for DNA extraction, PCR, gel electrophoresis and sequencing was followed, as detailed in . The genomic DNA was extracted from fresh mycelium using EZgeneTM Fungal gDNA extraction Kit GD2416 (Biomiga, Shanghai, China), following the guidelines of the manufacturer. DNA sequences were obtained for the internal transcribed spacer region (ITS1, 5.8S, ITS2), the small subunit (SSU) and large subunit (LSU) of the nuclear ribosomal RNA gene, translation elongation factor 1-α (TEF) and β-Tubulin (TUB2). PCR thermal cycle programmes for each locus region are presented in Table 1. Purification and sequencing were outsourced to the SinoGenoMax Sanger sequencing laboratory (Beijing, China). Newly-generated sequences were submitted to NCBI GenBank (https://www.ncbi.nlm.nih.gov/genbank/).
From a comparison of ITS and LSU sequences between P. mathieui (type) and MFLUCC 20-0150 strain, both were identical. However, seven nucleotide differences (1.13%) were found between the TEF sequences of two strains. Following the integrative taxonomic approach with both morphological data and molecular    higher altitude (296 m a.s.l.), compared to the previous Italian record on the same host, but from the Province of Forlì-Cesena (34 m a.s.l.) (Hyde et al. 2020a). Considering the results of our integrative taxonomic approach, we report this strain as a new record of P. nebulosa, the first for the Province of Arezzo and the second for Italy, widening its geographic distribution in the country.

Discussion
The pleosporalean fungal collections in this study originated from terrestrial habitats in the Provinces of Arezzo (Tuscany region), Forlì-Cesena and Ravenna (Emilia-Romagna region) in Italy (Fig. 6). The fungal isolates were associated with hosts in Apiaceae, Asteraceae and Urticaceae, which are economically and ecologically valuable plants (Simpson 2010, Bennett 2011. The expansion of ecological and mycogeographical knowledge, other than the taxonomic knowledge, are prerequisites to understand fungal biology, diversity and conservation. Our new species (Italica heraclei) and the new record ( Pseudoophiobolus mathieui) of Phaeosphaeriaceae, reported in this study, led to an expansion of knowledge about the family Phaeosphaeriaceae. Pseudoophiobolus mathieui strain was found on a new host, Artemisia sp. (Asterales, Asteraceae), enlarging the host distribution of this species in Italy. The record of Phomatodes nebulosa (Didymellaceae) for the Province of Forlì-Cesena represents the second record for Italy, widening the geographical range for this species.
At times, members of these fungal families are able to have pathogenic relationships with different host plants in different environments . Therefore, the accurate reporting of host-fungal records with their geographical locations is highly recommended to gain a better understanding of emerging plant pathogens (Dugan et al. 2009). In this study, we highlighted the expansion of the taxonomic framework and host- fungal relationships of those studied taxa in different Italian geographic regions. Additionally, we combined morphological data and multi-locus phylogenetic analyses to verify their identities and assess their taxonomic placement amongst other pleosporalean taxa.