An Italian Research Culture Collection of Wood Decay Fungi

: One of the main aims of the University of Pavia mycology laboratory was to collect wood decay fungal (WDF) strains in order to deepen taxonomic studies, species distribution, o ﬃ cinal properties or to investigate potential applications such as biocomposite material production based on fungi. The Italian Alps, Apennines and wood plains were investigated to collect Basidiomycota basidiomata from living or dead trees. The purpose of this study was to investigate the wood decay strains of the Mediterranean area, selecting sampling sites in North and Central Italy, including forests near the Ligurian and Adriatic seas, or near the Lombardy lakes. The isolation of mycelia in pure culture was performed according to the current methodology and the identity of the strains was conﬁrmed by molecular analyses. The strains are maintained in the Research Culture Collection MicUNIPV of Pavia University (Italy). Among the 500 WDF strains in the collection, the most interesting isolates from the Mediterranean area are: Dichomitus squalens (basidioma collected from Pinus pinea ), Hericium erinaceus (medicinal mushroom), Inocutis tamaricis (white-rot agent on Tamarix trees), Perenniporia meridionalis (wood degrader through Mn peroxidase) and P. ochroleuca . In addition, strains of species related to the Mediterranean climate (e.g., Fomitiporia mediterranea and Cellulariella warnieri ) were obtained from sites with a continental-temperate climate.


Introduction
Wood decay fungi provide an extraordinary model both for pure and applied research, as well as a food or medicinal mushroom resource.
From an ecological point of view, wood decay fungi have a fundamental role, since they are important degraders of lignocelluloses. Heterogeneity in degradation strategies consists in different enzymatic pools, conditions for secretion and catalysis, alternative non-enzymatic pathways and strategy-switch depending on environmental conditions [1,2]. Consistently, wood decay fungi shift from necrotrophism to pure saprotrophism, sometimes at an intraspecific level.
Systematic revisions based on multi-locus or genomic approach have revealed an extremely complex scenario concerning both biochemical features and morphology. Similar degradation strategies and similar morphologies are widespread, even among phylogenetically distant taxa, whereas the

Sampling Sites and Field Work
Basidiomata were mostly (but not exclusively) collected in North and Central Italy. Sampling stratification was selectively applied, i.e., specific areas have been more frequently and strictly examined than others and sampling effort was not equal among different species [32]. The different environments examined are resumed hence: (a) highly-fragmented marginal woodlands and shrublands placed into an agricultural landscape, particularly referring to vegetation surrounding the hydrographic network, including major lakes; (b) mountain continuous woodlands and shrublands, both managed and unmanaged; (c) woody cultures (e.g., poplar plantations and vineyards), tree rows and hedges in agricultural landscape; (d) urban and suburban environments (tree rows, parks, private and public gardens).
Environments a, c, and d are mostly related to basal altitudinal belt and upper hill altitudinal belt in Po Plain, Apennines and Prealps (lower mountain thermal belt), as well as Adriatic, Tirrenian and Ligurian coasts; Environment b is mostly related to the lower and upper montane belt in the North and Central Apennines.
The basidiomata were completely or partially harvested by knife, gently brushed to eliminate debris and stored in paper bags until laboratory operations. The collecting sites were geolocalized, and the host species and general features were detected.

Experimental Procedures
Basidiomata identification was carried out by macro and micro-morphological analysis [6,33,34]; stereo and light microscopy were performed by Zeiss Axioplan and Zeiss Stemi 2000-C.
According to Stalpers [35] and Gams et al. [36], as well as Stamets [37], isolation of mycelia in pure culture was obtained in sterile conditions by inoculating small portions of the basidioma context into Petri dishes containing MEA medium and antibiotic (malt extract 2% + agar 1.5% + cloramphenicol 50 ppm). The incubation was carried out at 24 • C in the dark and each strain growth was checked constantly for a month. Based on the above, all the mentioned strains are to be regarded as dikaryotic.
Besides the morphological checks, molecular identifications of isolates were carried out on mycelia cultured in liquid medium (malt extract 2%). DNA was extracted from lyophilized mycelia by Nucleospin Plant II kit (Macherey-Nagel). Amplification by Polymerase Chain Reaction (PCR) used the primer pair ITS1 (19bp) and ITS4 (20bp)-that is, Internal Transcribed Spacer of ribosomal DNA; this region has been widely used for different fungal taxa [38,39]. PCR protocol exploited Dream Taq Mastermix (Promega) and was performed in a thermocycler, as reported in Table 1. The qualitative checking of DNA (5 µL/sample) was performed both after extraction and amplification by DNA run (30 min, 100 V) on electrophoretic gel (1% agarose). SYBR Safe-DNA Gel Stain (Invitrogen) was used as an intercalant; GeneRuler 1kb (Thermo Scientific, Waltham -USA) was used as a ladder; BlueJuice (Invitrogen) was used as a gel loading buffer. The imaging was performed by Gel Doc (Biorad, Berkeley, CA, USA).
ExoSAP-IT (Applied Biosystems, Foster City, CA, USA) was used for the purification of amplification products. According to the suggested protocol, the sample/ExoSAP ratio was 5:2 µL; the reaction was carried out in a thermocycler in two steps-15 min at 37 • C and 15 min at 80 • C.
The sequencing was ordered to Macrogen (The Netherlands). Sequence analysis was performed by Sequencher 5.0 Demo. The sequences were finally matched with the ones available in the molecular identification facility of Mycobank [40].
Strains in pure culture were stored by different methods: (a) on malt extract agar (MEA) in a aPetri plate at 3 • C; (b) in a glass tube corked with cotton at room temperature; (c) colonized paper discs in demineralized water at 4 • C; (d) at −80 • C in glycerol (selected strains only).
Periodic checking and refreshment of cultures was performed to avoid contamination and devitalization.
The strains are maintained in the Fungal Research Culture Collection (MicUNIPV) of Department of Earth and Environmental Sciences of University of Pavia (Italy); each strain is included in a private database with all the information regarding sampling sites, data of collection and ecological notes.

Results and Discussion
MicUNIPV includes species related to plant pathology, soil, extreme environments, fresh and marine water, monuments and cultural heritage. As previously mentioned, different working groups within the Laboratory of Mycology (DSTA-University of Pavia) are engaged in the management, preservation and improvement of each MicUNIPV section. The section regarding wood decay species has up to now achieved 500 strains belonging to 110 different species [41][42][43]. The broad focus on wood decay led us to include in this section species related to different applications such as nutraceuticals, forest pathology, wood degradation and biocomposite materials.
The distribution of most species exceeds the Mediterranean area; nevertheless, several of them also display wide spatial gaps among stations and clear heterogeneity in host preference depending on the geographic location of the population.
Here, we present the species that have a distribution strongly related to the Mediterranean region and/or Southern Europe and/or warm climates, according to Ryvarden and Melo [6] and Bernicchia [33,34,44]. The species related to the Mediterranean diversity are reported in Table 2 and the most peculiar are discussed below.      Table 2 represent about one in five of the comprehensive collection of wood decay species in MicUNIPV. The temperate region and Mediterranean region in Italy are reciprocally intersected and several phytoclimates are represented based on both thermal-pluviometrical parameters and floristic-vegetational ones. According to our field observations, this has the consequence that several species can be found in different phytoclimates and on different hosts, whereas a minor fraction is strictly related to one or few hosts.
Daedaleopsis confragosa and D. tricolor are easily distinguished by morphology; nevertheless, ITS sequences are important to discriminate the species strain. D. tricolor seems more common in Central and Southern Europe; in Italy, it has been reported in seven out of 20 regions [45]. Our strain (MicUNIPV D.tric.1) comes from the lower Apennines in Pavia Province; other field observations suggest that Prunus avium is the favourite host of D. tricolor in North and Central Italy. The strain has not been characterized yet, although pharmacological effects have been reported [46].
Daedalea quercina has been reported in 11/20 Italian regions [45]; as expected, all MicUNIPV strains were isolated from Quercus spp. Nevertheless, strain MicUNIPV D.q.1 efficiently colonized poplar wood chips and confirmed that this species is a typical brown rot agent [47].
Despite being apparently cosmopolitan, Desarmillaria tabescens is strictly related to Quercus in warm climates, where it behaves as a secondary pathogen [48]. D. tabescens has been reported in 16/20 Italian Regions [45]. Accordingly, our strain was isolated from roots of Q. robur in RNIS Bosco Siro Negri (Pavia, Italy), which is a significant, unmanaged residue of typical forest of the western Po Plain.
Fomitopsis iberica is a rare species, reported in three Italian regions [45]. All the strains in MicUNIPV were isolated close to Varese lakes, either on broadleaves or conifers.
Ganoderma is represented in MicUNIPV by seven species: G. adspersum, G. applanatum, G. carnosum, G. pfeifferi, G. lucidum, G. resinaceum and G. valesiacum. This genus has been intensely studied due to its wide range of secondary metabolites, including several bioactive compounds [49]. According to Ryvarden and Melo [6], Ganoderma is one of the most difficult genera to identify at species level. As reported in Table 2, we obtained strains of G. adspersum, confirming that is a southern species in Europe [50]. G. carnosum is usually located in the G. lucidum complex due to its morphological similarity, despite it showing clear differences in host relationship. Our strain was isolated from its type-locality in Italy, i.e., a forest of Abies alba, that is likely to be its preferred host in South Europe [33]. Molecular identification by ITS region met difficulties in discriminating G. carnosum from G. tsugae and G. oregonensis; this topic presents questions about the real interspecific diversity within this conifer-related species in Ganoderma. Strains in pure culture will thus help us to investigate both the molecular and morphological nested diversity in this complex. An analogous problem concerns G. pfeifferi, as it partially shares its trophic niche with G. lucidum and G. resinaceum.
L. sulphureus is particularly related to Quercus according to our observations in North Italy, although Castanea sativa is also represented. Interestingly, C. sativa is also one of the favourite hosts of Phellinus torulosus, according to field observations, despite strain MicUNIPV Ph.tor.1 being isolated from P. avium.
Genus Perenniporia is represented in MicUNIPV by P. fraxinea, P. meridionalis and P. ochroleuca. According to our field observation, P. fraxinea is more common and widespread than expected, particularly in urban areas. We have focused our attention in indentifying strains, which, to date, number 27. Some of the isolated strains have been used for population studies and tests on heavy metal bioaccumulation [16,51].
Pleurotus eryngii is a typically Mediterranean species as well as its herbaceous hosts in Apiaceae. Consistently, the MicUNIPV Pl.e.1 and Pl.e.2 strains were isolated in properly Mediterranean areas (Sardinia and Sicily).
Punctularia strigosozonata is a rare, poorly studied species, typically related to the Mediterranean area; its resupinate morphology increases the difficulty in achieving pure isolates.
Spongipellis pachyodon has a mainly central-southern distribution in Europe; according to Onofri et al. [45], in Italy it is known in five out of 20 Regions, not including either Lombardy or Liguria. This species is reported as uncommon but locally abundant; regarding this, our field observations suggest that the population in the Pavia-Piacenza Apennines is particularly related to P. avium.
Further species listed in Table 2 are reported below in more detail owing to their taxonomic controversy or potential applications.

Background
As detailed in Table 2, the basidiomata of some species were collected in the Mediterranean area and others that are known to prefer warm environment, even if they were collected in continental or temperate zones. An example is Cellulariella warnieri, a poorly investigated species related to warm climates, according to Bernicchia and Gorjón [44]; despite not strictly being related to the Mediterranean region, Ryvarden and Melo [6] reported it as a southern and rare species.
The notable scarcity of data about this species has probably contributed to its uncertain systematic and taxonomic status. Currently it is reported as: Lenzites warnieri Durieu and Mont. by Mycobank [40], C. warnieri by Index Fungorum [52] and Trametes warnieri (Durieu and Mont.) Zmitr., Wasser and Ezhov by Ryvarden and Melo [6]. The latter indication is suggested also by Justo and Hibbett [53] based on a five marker-based phylogenetic classification of Trametes. Significantly, only 108 records for this species have been reported by the GBIF (Global Biodiversity Information Facility) [54] and only 10 sequences are available in GenBank, almost half of them being critical as they are reported from South East Asia [55]. Further analyses on a more representative number of strains are thus needed to clarify the position of C. warnieri.
Strain MicUNIPV L.w.1 was tested for the evaluation of lignocellulolytic activity and resulted in a very low production of Mn peroxydase and lack of lignin peroxydase, whereas cellulase and hemicellulase had the highest presentation among the species under examination [47]. This was also confirmed when testing the effect of its colonization on Medicago sativa for pre-treatment, as cellulose and hemicellulose were preferentially removed [56].

MicUNIPV WDF Strains Results
According to our field observations, localities are distributed as small local clusters which are very scattered in turn. Thus, the strains MicUNIPV L.w.1, L.w.4 and L.w.5 were collected from Po plain areas (Pavia and RNIS Bosco Siro Negri), whereas strains the MicUNIPV L.w.2, L.w.3 and L.w.6 were collected from hill area (Oltrepo Pavese).
The six strains of MicUNIPV were collected from Quercus, Ulmus, Populus and Robinia; interestingly, our field observations pointed out some preference for Ulmus, which was not previously reported as a host in Italy. As expected, all the strains were isolated from individuals behaving as saprotrophs. It should be noted that, since C. warnieri develops basidiomata in late autumn but it releases spores in spring, the basidioma itself remains vital even at low temperatures and under the snow.

Background
Dichomitus squalens is a model species for studies about the selectivity of white rot and its enzymatic basis [57][58][59][60]. Despite being reported all throughout the boreal emisphere, it appears scattered and is commonly found in the northern parts of Europe, North America and Asia [61]; the GBIF [54] places the wide majority of records in the Fennoscandian region. The host relationship is apparently controversial and surprising: Ryvarden and Melo [6] assumed Pinus as the only European host species, whereas Bernicchia and Gorjón [44] recorded Picea abies for the Italian sample and Niemelä [62] assigned most samples to Pinus and a smaller fraction to Picea abies in Białowieża Forest (Poland / Belarus). Nevertheless, it should be noted that American samples have been reported on six different genera in Pinaceae. Furthermore, young basidiomata of D. squalens are easily misidentified due to the close morphological resemblance with Neoantrodia serialis (Fr.) Audet and related species. Consistently, a remarkable intraspecific variability in growth and enzyme profiles was revealed by testing different monokarya strains [61]. This is also consistent with the numerous mating types deriving from tetrapolarity [61].

MicUNIPV WDF Strains Results
Strains MicUNIPV D.sq.1 and MicUNIPV D.sq.2 were recovered from Pinus pinea and Cedrus sp. respectively near the Adriatic Sea and Varese Lake. The Italian strains have not yet been investigated for their enzymatic properties, so they may provide an additional tool to explore the diversity in degradation potential of this selective decayer.

Background
Hericium erinaceus (Bull.) Pers. is one of the most famous cultivated medicinal species in the world; a wide range of peculiar compounds, both related to primary (e.g., β-glucans) and secondary metabolism (e.g., erinacines and hericenones) have been up to now characterized and screened for bioactivity [7,[63][64][65][66]. GBIF [54] places the wide majority of H. erinaceus sites in Europe, North America and North Eastern Asia. According to the phylogenetic study by Cesaroni et al. [67], a subclade containing European and American ITS sequences is well distinguished from the Asian clade. Despite relying on ITS region only, these data suggest the possibility to differentiate H. erinaceus strains also by the phylogeographic structure. Notwithstanding the scarcity of available data for Asian samples, H. erinaceus apparently has a quite broad trophic niche including several host species in Fagaceae, and Aceraceae to lesser extent, and particularly showing a preference for Quercus all throughout its distribution area [37]. Consistently, European samples have mainly been recovered from Quercus and Fagus, the former likely being the exclusive host in Italy and the only known host in North Africa [34,68]. Strain MicUNIPV H.e.2 was analyzed for the production of erinacine A and hericenes (presumably A, B, C, D). Thus, a complete quali-quantitative comparison of these selected metabolites was provided throughout different growth stages but within the same strain, which is a powerful tool for the standardization of bioactive products [69].
Strain MicUNIPV H.e.1 was selected to test the effect of oral supplementation on mice memory. The results indicate an improvement in recognition memory and induction of hyppocampal and cerebellar neurogenesis during aging. This strain has therefore contributed to pointing out which areas are directly involved in the neuroactivity of H. erinaceus compounds, highlighting which type of memory is increased [70].

MicUNIPV WDF Strains Results
Accordingly, all four strains in the MicUNIPV collection were recovered in the municipality of Siena from Q. ilex, that is, a featuring species in the flora of Mediterranean area often forming homogeneous woodlands. It is noteworthy that the Mediterranean basin hosts a great variety of Quercus species, whose phylogenetic and systematic relationships are still controversial, with particular concern to the Q. ilex group [71][72][73].

Background
The relationship of Inocutis tamaricis with Tamarix is apparently so strict to be regarded as a discriminant character in identification [6,33,44]. Although the genus Tamarix consists of 72 accepted species in Europe, Asia and Africa [74], I. tamaricis is restricted to the Mediterranean basin and Macaronesia. Here, it grows on different Tamarix species according to their availability but shows a preference for T. gallica [75,76]. Consistently, strains MicUNIPV I.t.1 and I.t.2 were both isolated from T. gallica. As a whole, the intra-familiar phylogeny of Hymenochaetaceae is still to be clarified; multiple revisions have tried to point out nested diversity within polyphyletic taxa, such as Inonotus [77,78]. Thus, the genus Inocutis is nowadays accepted to be distinct from Inonotus itself, as formerly suggested by Fiasson and Niemelä [79]. Interestingly, the type-species for Inocutis is I. rheades (Pers.) Fiasson and Niemelä, which is morphologically very similar to I. tamaricis and is mostly distinguished by host and distribution [6]. Thus, I. tamaricis may be regarded as the Mediterranean counterpart of I. rheades. As a whole, only 12 sequences have been up to now deposited in GenBank [55] as belonging to this species, some of which are lacking data to assess their effective reliability. Further sequences from the Mediterranean area, equipped with information about host and geographic origin, are needed to support studies about intrageneric diversity in Inocutis in the light of biogeographic patterns.

MicUNIPV WDF Strains Results
Strains MicUNIPV I.t.1 and I.t.2 were both isolated from T. gallica, forming in both cases ornamental rows along the sea coast.

Background
As mentioned for Inonotus, the genus Phellinus is increasingly revealing its hidden diversity; recognized as being polyphyletic, several species have been distributed into other genera, such as Fomitiporia Murrill. Fomitiporia mediterranea is a peculiar example due to its morphology, being actually indistinguishable from P. punctatus. According to Fischer [80], these two species also show differences in growth rate at selected temperatures and mating behaviour. The same study provides strong evidence for dichotomy in host selection by F. mediterranea depending on biogeography, i.e., this species grows on several tree species in Italy [81,82], whereas north of the Alps it apparently grows on Vitis vinifera exclusively [83,84].
It should be considered that misidentification with P. punctatus has probably led to the underestimation of F. mediterranea in the Mediterranean area [85]. Analogously, Polemis et al. [86] suggested that the relationship with P. pseudopunctatus A. David, Dequatre and Fiasson should be reconsidered as well, enclosing the latter in F. mediterranea clade. It may be observed that the P. pseudopunctatus is apparently more related to the South Mediterranean region and climates [87,88]. Further analyses on strains from different geographic origins and hosts are thus needed to clarify both phylogenetic relationships and biogeographic patterns. As a whole, this species complex is characterized by intense necrotrophic white rot; F. mediterranea in particular is regarded as one of the main agents responsible for wood rot in V. vinifera, Corylus avellana and Olea europaea [82,83,85].

MicUNIPV WDF Strains Results
All of the 17 Italian strains up to now attained by the Laboratory of Mycology DSTA-University of Pavia were assigned to F. mediterranea instead of P. punctatus and recovered from different substrates in North Italy. Even within one province (Pavia), F. mediterranea was located on five hosts, namely Q. robur, Hedera helix (State Natural Strict Reserve Bosco Siro Negri), Salix alba (University of Pavia courtyard), R. pseudocacacia and V. vinifera (Oltrepo Pavese hills).
The identified strains thus provide a tool to deepen pathology dynamics and different susceptibility depending on host species and cultivar.

Background
Genus Perenniporia Murrill sensu lato is large, cosmopolitan and supposed to be polyphyletic, and thus is in need of further phylogenetic analysis based on sequences from different species [6]. In turn, an example of intrageneric complexity is provided by P. meridionalis, within P. medulla-panis (Jacq.) Donk group. Actually, the complete revision by Decock and Stalpers [89] arose the doubt that several records, as well as specimens in herbaria, are to be referred to P. meridionalis instead of P. medulla-panis (or closely related species), particularly when coming from the Mediterranean area. Up to now, only a partial and fragmentary investigation into the intra-generic diversity in Perenniporia has been carried out from a molecular and phylogenetic point of view [90]. It is noteworthy that no sequences at all are available in GenBank by the name of P. meridionalis, whereas 40 sequences are referred to P. medulla-panis [55]. Strains from culture collections are thus needed as basic material for this purpose. According to the indications by both Bernicchia and Gorjón [44], as well as Ryvarden and Melo [6], P. meridionalis is particularly related to Quercus (more than P. medulla-panis), the holotype having been isolated from Q. ilex in Sardinia [62,89].

MicUNIPV WDF Strains Results
Strains MicUNIPV P.m.1 and P.m.2 strains were respectively isolated from Q. robur (North Italy, near a lake) and Q. ilex (Central Italy). Besides considerations on biodiversity, P. meridionalis has a great applicative potential. MicUNIPV P.m.1 showed remarkable selectivity as a white rot agent and versatility when inoculated onto unusual substrates such as grass. The selective removal of lignin by this species contemporarily relies on high activity for Mn peroxidases and very low for one cellulase; the final delignification in the substrate is clear both in thermogravimetric analysis and FTIR spectroscopy [12,47].

Background
Perenniporia ochroleuca is another example of the unsolved intra-generic diversity within Perenniporia. This species is suspected to hide a complex, and transfer to Truncospora Pilát ex Pilát has thus been suggested [91]. According to the same authors, the Iberian/Macaronesian clade gives T. atlantica Spirin and Vlasák, whereas the status of Australian samples is more uncertain, which would mainly belong to T. ochroleuca. Nevertheless, the new taxonomy has not yet been fully accepted, neither by Mycobank [40] nor by Index Fungorum [52]. These hypotheses therefore need to be supported by entering further sequences into the phylogenetic analyses from an exhaustive geographic range. P. ochroleuca was reported by Bernicchia and Gorjón [44] and Ryvarden and Melo [6] as tropical and growing on several hosts, whereas in Europe it is particularly related to the Mediterranean area. Nevertheless, Bernicchia and Gorjón [44] report a range of typically Mediterranean hosts, whereas Ryvarden and Melo [6] also include host plants whose distribution exceeds the Mediterranean area to include samples from the coasts of South England and Wales. Further phylogenetic analyses focused on the Mediterranean region versus the adjacent Atlantic ones are needed to test the monophyly of the proposed T. atlantica.

MicUNIPV WDF Strains Results
Both strains MicUNIPV P.och.1 and P.och.2 were isolated from Q. ilex in Central Italy and the Ligurian west coast, respectively.

Conclusions
At the moment, MicUNIPV, the fungal research culture collection of University of Pavia (Italy), maintains 500 strains from wood decay species. Examples particularly correlated to the Mediterranean area were discussed and their roles in accomplished research were mentioned in this study.
Culture collections of wood decay fungi are an important tool both for systematic and applied studies. Strains in pure culture are more easily and reliably identified and analyzed for metabolic activities and competitivity. The environmental features of the strain origin place have often been underestimated; nevertheless, the diversity of wood decay fungi strongly depends on biogeography and is related to host distribution. This also highlights the need for an investigation including a wider concept of the Mediterranean region than one strictly limited by climate or phytoclimate classification, i.e., even continental regions surrounding the Mediterranean area contribute to the explanation of Mediterranean diversity.
The Laboratory of Mycology DSTA-University of Pavia (Italy) has up to now successfully collaborated with both researchers from other universities and amateurs in order to increase the diversity richness and geographic origin range of strains, as well as to enter these strains in original pure and applied research such as MATER and CE4WE (grants from Cariplo Foundation and Regione Lombardia).