**Ecology, Phylogeny, and Potential Nutritional and Medicinal Value of a Rare White "Maitake" Collected in a Mediterranean Forest**

**Maria Letizia Gargano <sup>1</sup> , Georgios I. Zervakis <sup>2</sup> , Omoanghe S. Isikhuemhen 3, Giuseppe Venturella <sup>4</sup> , Roberta Calvo 4,\*, Anna Giammanco 5, Teresa Fasciana <sup>5</sup> and Valeria Ferraro <sup>4</sup>**


Received: 4 May 2020; Accepted: 4 June 2020; Published: 8 June 2020

**Abstract:** Albino *Grifola frondosa* (Dicks.) Gray "maitake" mushrooms (described as *G. albicans* Imazeki and then placed in synonymy with *G. frondosa*) are particularly rare, and the few pertinent records are not treated in scientific publications. A field investigation carried out in Sicily (Italy) led to the collection of an unusual white *Grifola* specimen at the base of a living tree of *Quercus pubescens* Willd. s.l. The outcome of sequencing the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) indicated that it belongs to *G. frondosa* and provided an insight to the phylogenetic relationships within the genus. The results of nutritional composition analysis showed that the albino basidioma possesses relatively high contents of Ca, Fe, K, and Cu and is rather low in Na when compared with literature data on edible mushrooms. Vitamin (B1, B2, B3, B5, B9, and D2) contents ranged from 0.15 to 3.89 mg per 100 g of mushroom dry weight. The cold-water extract of this specimen was effective at inhibiting the growth of *Staphylococcus epidermidis* ATCC 12228 and *Pseudomonas aeruginosa* ATCC 15442 at the maximum screening concentration of 50% *v*/*v*. In addition, the extract slowed down the ability of *Staphylococcus aureus* ATCC 43300 to form biofilms. According to data hereby reported, the albino *G. frondosa* is a culinary-medicinal mushroom with a promising exploitation potential.

**Keywords:** *Grifola frondosa*; fungal diversity; Mediterranean forest; medicinal mushroom; bioprospecting; ITS rDNA; phylogenetics; basidiomycete; polypore fungus; *Quercus pubescens*

#### **1. Introduction**

The family Grifolaceae Jülich (Polyporales, Basidiomycota) comprises only the genus *Grifola* Gray [1], which consists of nine species according to Index Fungorum. *Grifola frondosa* (Dicks) Gray includes several forms and varieties, such as *G. frondosa* f. *frondosa* (Dicks.) Gray, *G. frondosa* f. *intybacea* (Fr.) Pilát, *G. frondosa* var. *frondosa* (Dicks.) Gray, and *G. frondosa* var. *intybacea* (Fr.) Cetto, while *G. intybacea* (Fr.) Imazeki is considered as a synonym. The culinary-medicinal mushroom

*G. frondosa*, widely known as "maitake", is a white-rot polypore associated primarily with deciduous trees of different genera (*Quercus* L., *Acer* L., *Carpinus* L., *Castanea* Mill., *Fagus* L., *Ulmus* L.) while it appears more rarely on conifers. This species is distributed in temperate regions of North America, Europe, and Asia (Japan and China) [2]. Albino maitake mushrooms (described as *G. albicans* by Imazeki in 1943 [3] and then placed in synonymy with *G. frondosa*) are quite rare and the few available pertinent records do not appear in scientific publications. Only recently, Kawaguchi et al. [4,5] reported on melanin biosynthesis in *G. frondosa* and suggested that the albino mutation is caused by a single base deletion in the coding region of the tyrosinase 2 (*tyr2*) gene. It is noteworthy that white strains of *G. frondosa* are much sought after since they can be used for culinary purposes without the unwanted dark brown pigment resulting from processing the common form of this mushroom [4].

Edible mushrooms are widely acknowledged for their nutritional and medicinal properties [6]; however, their potential market is still far from being fully developed considering that there is a high consumer demand. Consequently, the private sector is eager to generate new relevant products. In the frame of a research project aiming at collecting mushrooms with bioprospecting potential, a field investigation carried out in Sicily (Italy) led to the collection of an unusual *Grifola* basidioma of white color and considerable weight (ca. 7 kg). The objective of the present work was to provide morphological, molecular/phylogenetic, and ecological data about this rare albino *Grifola* specimen. Information on the nutritional value and antimicrobial activity is also reported.

#### **2. Materials and Methods**

#### *2.1. Sample Collection, Habitat Details, and Evaluation of Morphological Characters*

Research carried out in forest ecosystems in Sicily (Italy) led to the collection of a single albino *Grifola* basidioma found at the base of a monumental downy oak tree (*Quercus pubescens* Willd. s.l.), 10 January 2016, Castelbuono, Madonie Regional Park (province of Palermo, Sicily), 800 m a.s.l., 37◦54 48" N, 14◦04 41" E. In Castelbuono, summers are rather brief, hot, and dry, with clear skies and condensation often appearing in the late afternoon, while winters are long, cold, rainy, and windy. Annual temperature ranges from 7 to 29 ◦C (rarely below 4 ◦C or above 32 ◦C) while the average value is 15.8 ◦C. The average annual rainfall is 515 mm. In January, the month when the specimen under study was harvested, the average temperature is 8.9 ◦C, and the respective maximum and minimum values are 11.5 and 6.3 ◦C; the rainfall is 67 mm. The forest is characterized by evergreen and deciduous oak vegetation with a prevalence of *Quercus ilex* L. and *Q. pubescens* Willd. s.l. In the upper part of the mountain, the vegetation is characterized by mixed woods of oaks, *Fagus sylvatica* L. and *Ilex aquifolium* L. The specimen was transferred to the Department of Agricultural, Food, and Forest Sciences (SAAF) of the University of Palermo, and stored at <4 ◦C for up to 24 h prior to morphological examination, which was carried out according to Bernicchia [7]. Observations on macromorphological characters (pileus, cuticle, pores, stipe, context, etc.) were performed on fresh material, while microscopic characters (hyphal system, generative hyphae, basidia, sterigmata, cystidia, cystidioles, and basidiospores) were studied by using 3% potassium hydroxide and ammoniacal Red Congo. In addition, the reaction of the flesh to iodine-potassium iodide, potassium hydroxide, and iron salts was also evaluated.

#### *2.2. Establishment of Pure Cultures and Mushroom Cultivation Trials*

The collected basidioma was dried and then deposited in the Herbarium of the Department of Agricultural, Food, and Forest Sciences (SAAF 450). Prior to this, a piece of pseudo-tissue was removed from the fresh specimen, placed on potato dextrose agar (PDA) in Petri dishes under aseptic conditions, and incubated for 15 days at 25 ± 2 ◦C. The established pure culture was stored in the Mycotheca of the Herbarium SAF (SAF 323), and it was subsequently used for the inoculation of mushroom cultivation substrates composed of either holm oak (*Quercus ilex* L.) or chestnut (*Castanea sativa* Miller) wood residues. The former (holm oak wood shavings) was filled in plastic bottles (volume: 1 L each), while the latter (chestnut wood chips of ca. 2–3 cm and sawdust) was filled in plastic bags (volume: 6 L each) after their moisture content was adjusted to 50–60%; in both cases, non-hydrophilic cotton filters were used to allow air exchange. Sterilization was performed twice at 120 ◦C, 1.1 Atm for 20 min with a 24-h interval between each cycle. After the final cooling, substrates were inoculated using actively growing mycelium and incubation was carried out at 25 ◦C in the dark.

#### *2.3. DNA Extraction, PCR Amplification, and Sequencing*

Total genomic DNA was extracted from the herbarium specimen using the Nucleospin Plant II DNA kit (Macherey and Nagel, Düren, Germany) following the manufacturer's protocol. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) was amplified using the primer combination ITS1/ITS4 [8]. Polymerase chain reactions (PCRs) were performed in 50 μL containing 50 ng DNA template, 0.25 μM of each primer, 0.2 mM of each dNTP, 1 × HiFi Buffer (Takara BIO INC., Shiga, Japan), and 1 U HiFi Taq DNA polymerase (Takara BIO INC., Shiga, Japan). Conditions for PCR amplification were as follows: 94 ◦C for 5 min, followed by 35 cycles of 94 ◦C for 30 s, 50 ◦C for 30 s and 72 ◦C for 1 min, and a final extension at 72 ◦C for 10 min.

PCR products were purified using an Invitrogen PureLink kit (Thermo Fisher Scientific, Seoul, Korea) and were submitted for sequencing to CeMIA S.A. (Larissa, Greece). The resulting chromatograms were proofread, and the sequence generated was deposited in GenBank under the accession number MN944407.

#### *2.4. Phylogenetic Analysis of Sequence Data*

In addition to the biological material examined in this study, 53 additional ITS sequences were included in the phylogenetic analysis; 38 of them representing other *Grifola* Spp., i.e., all available sequences of *G. colensoi* (Berk.) G. Cunn., *G. gargal* Singer, and *G. sordulenta* (Mont.) Singer, and the rest corresponding to two other species used as outgroups, i.e., *Polyporus umbellatus* (Pers.) Fr. (syn. *Grifola umbellata* (Pers.) Pilát) and *Rhodonia placenta* (Fr.) Niemelä, K.H. Larss. & Schigel (syn. *Oligoporus placentus* (Fr.) Gilb. & Ryvarden). Sequence alignment was performed through the aid of the Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/); alignments were inspected and manually adjusted at misaligned sites using MEGA X (Pennsylvania State University, State College, PA, USA) [9].

Phylogenetic relationships were inferred by using the maximum likelihood (ML) method based on the Kimura 2-parameter model [10]. Initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with the superior log likelihood value. Phylogenetic analysis was conducted in MEGA X [9].

#### *2.5. Evaluation of Nutritional Value and Determination of Antimicrobial Activity*

The powder resulting after freeze-drying part of the collected basidioma was also subjected to analysis for the evaluation of the nutritional value (proximate composition, and content in elements and vitamins) according to standard methodologies reported by Palazzolo et al. [11], AOAC [12], Thompson et al. [13], and Loewus [14].

With regard to the antibacterial properties, the study initially included the evaluation of the activity of the lyophilized powder deriving from the basidioma against four Gram-positive and Gram-negative bacteria strains: *Staphylococcus aureus* ATCC 25923, *S. epidermidis* ATCC 12228, *Pseudomonas aeruginosa* ATCC 15442, and *Escherichia coli* ATCC 25922. Tryptic soy broth (Sigma-Aldrich, Darmstadt, Germany) containing glucose (2% *w*/*v*) or Mueller Hinton (Sigma-Aldrich, Darmstadt, Germany) was used as growth media. The lyophilized sample (3 g) was placed in a beaker with 200 mL of demineralized water, and then stored at −20 ◦C.

The minimum inhibitory concentration (MIC) was determined in a Minisart syringe with a 2.5-μm cellulose acetate filter by preparing solutions in vitro at increasing concentrations, incubating them with separate lots of cultivated bacteria, and measuring the results using agar dilutions. In addition, the effect of extract obtained from the basidioma powder was examined in respect to the biofilm produced by strains of *S. aureus* ATCC 43300, *Enterococcus faecalis* ATCC 29212, *P. aeruginosa* ATCC 27853, *E. coli* ATCC 25922, and *Klebsiella pneumoniae* ATCC 700603.

The bacterial strains were inoculated in Trypticase™ Soy Agar (BBL™; Becton, Dickinson and Company, Sparks, MD, USA) media and incubated at 37 ◦C for 24 h. In total, 100 μL of overnight grown culture (0.5 McFarland in tryptose broth, BT) was added in a sterile 96-well flat bottom microtiter plate (Biosigma S.r.l. Dominique Dutscher Group, Brumath, France). The negative control was composed by BT medium only; to demonstrate biofilm production, excess bacterial suspension was removed. Subsequently, the extract from the basidioma powder (25% *v*/*v*) was re-suspended in BT, 100 μL were added in the wells, and the cultures were incubated at 37 ◦C for 24 h. Finally, the biofilm was stained with crystal violet dissolved in ethanol (0.5% *w*/*v*). The optical density was measured at 540 nm (Spectrophotometer Multiskan Go; Thermo Fisher Scientific, Waltham, MA, USA). The experiment was conducted in triplicate [15,16].

#### **3. Results and Discussion**

#### *3.1. Morphological Description of the G. frondosa Specimen and Cultivation Tests*

Basidioma is composed of multiple pilei in the form of a rosette, sharing a branched stem-like structure (Figure 1a). Individual pilei were found to be more or less fan-shaped or deltoid, entirely white and yellowing with age, and finely velvety or bald, with wavy margins, 4 to 10 cm wide and 5 to 10 mm thick. The pore surface was shown to be white, staining yellowish when ripe, 2 to 3 mm deep, and irregular, and varying in shape from round to elongated (Figure 1b); it is slightly decurrent to the stipe. The stipe structure was found to be branched, whitish, tough, and often off-center. The flesh was firm, white, and unchanging when sliced. The odor and taste were found to be mild and pleasant, although less so when decaying or acrid. The chemical reaction of flesh to iodine-potassium iodide and potassium hydroxide was negative. The iron salts reaction on the pileus and flesh was negative. Basidiospores, slightly reduced when compared to the brown maitake (5–7 × 3.2 μm), were found to be broadly ellipsoidal, smooth, 5.0–7.0 × 3.2–4.0 μm, slightly narrower than those of the brown maitake, and inamyloid. Basidia were 25–30 × 6–8 μm; clavate; and 4-sterigmata (Figure 2a). Clamp connections were found to be present on generative hyphae but are rare or absent on skeletal hyphae. Hymenial cystidia and cystidioles were absent. The hyphal system is dimitic, and the spore print white. Sterigmata were found to be up to 6 μm long, and robust, with a base diameter up to 1.6 μm (Table 1). Relevant information referring to the description of common brown form [7,17] is included in Table 1. It should be mentioned that the length of the sterigmata and the diameter of their base were not reported by Ryvarden [17], while Bernicchia [7] stated that sterigmata appear "thin and very divaricate".

**Figure 1.** The albino maitake specimen: (**a**) Basidioma in situ; (**b**) Pore surface under the stereomicroscope (scale bar = 1 mm).

**Figure 2.** (**a**) Microscopic features (basidia and spores) of albino maitake. (**b**) Pure culture isolated from fresh basidioma.

**Table 1.** Main macro- and micromorphological features of brown [7,17] and albino (this study) forms of maitake.


Mycelium growth was very slow in the pure cultures established, and the surface of the Petri dish (diameter 90 mm) was completely overgrown in 30 days. The colony was found to be slightly floccose and flat, with dense white hyphae without any zonation and with a filiform margin (Figure 2b). Regarding the cultivation tests performed either in holm oak or in chestnut-based substrates, colonization was slow and ca. two months elapsed before they were completely overgrown by mycelium. At this time, a few primordia were formed on the substrate surface, which, however, failed to produce fully developed basidiomata.

#### *3.2. Phylogenetic Analysis*

The main objective of the molecular analysis was to identify the biological material under examination (albino maitake). Following BLAST, the sequence (MN944407) from our specimen showed high identity (>99%) to several other sequences deposited as *G. frondosa* in GenBank. The generated ML trees had similar topologies, and only the tree with the highest log likelihood (−7662.14) was selected to be presented (Figure 3).

**Figure 3.** Maximum likelihood tree produced on the basis of ITS (internal transcribed spacer) sequence analysis presenting the phylogenetic position of the albino *G. frondosa* specimen (underlined in red and marked with an asterisk) among members of the genus *Grifola*; GenBank accession numbers are quoted together with information of their geographic origin (when available). Sequences of *Polyporus umbellatus* (syn. *G. umbellata*) and *Rhodonia placenta* were used as outgroups. The percentage of trees in which the associated taxa clustered together is shown next to the branches when ≥70% (ML bootstrap support values derived from a total of 1000 replicates). The tree is drawn to scale, with branch lengths measured as the number of substitutions per site.

To the best of our knowledge, the sequence obtained in this study is the only one available from an albino *Grifola* specimen. The outcome of the phylogenetic analysis revealed that the albino maitake clusters within the *G. frondosa* terminal clade, which receives high bootstrap support (100%), and forms a subgroup (84%) together with other sequences from material collected in Europe; the rest of the *G. frondosa* sequences derive almost exclusively from east Asia (China, Japan, South Korea, Taiwan) and the USA. Although this subgroup could represent a distinct taxonomic entity, the information generated from our work (through the examination of one basidioma and the use of a single marker only) does not allow such inferences, at least before additional specimens are included and analyzed in the frame of a multigene approach. However, it is noteworthy that *G. frondosa* sequences from Asian and eastern North American isolates were separated through the use of a partial beta-tubulin gene and ITS [18]. *G. frondosa* forms a sister group to *G. sordulenta* (94%). The latter is highly supported (99%) and consists of three sequences under this name and one representing *G. gargal* (AB539700). Hence, the distinct taxonomic position of the latter is dubious by examining the ITS marker alone. Of interest was also the position of the sole *G. colensoi* sequence available (MH856542; this species is known so far from Australia and New Zealand only), which appears to be quite distant from the rest of the material placed in the genus *Grifola*. Lastly, sequences deposited either as *G. umbellata* or *P. umbelatus* form a distinct highly supported group (100%), which is well separated from the clade corresponding to *Grifola* Spp. To the best of our knowledge, the sequence generated in the frame of this work is the first one deriving from an albino *G. frondosa* specimen.

#### *3.3. Mushroom Proximate Composition and Content in Elements and Vitamins*

In the frame of this work, it was not possible to analyze (and hence directly compare to our albino specimen) the composition of wild basidiomata of brown maitake since they were not available during the period of the study, this species being very rare in Sicily. On the other hand, only limited literature data are available referring to the chemical composition and vitamin contents in cultivated *G. frondosa* [19,20]; the respective values reported—apart from a few exceptions—are considerably lower than those found in the white maitake. However, such information should be evaluated with care since it is known that the nature of the cultivation substrate could considerably affect the mushroom content [21,22]. Similarly, metal accumulation in wild edible mushrooms was found to be influenced by various factors, including the species, the available concentration of elements in soil substrates, the substrates' properties (e.g., pH, organic matter content), the antagonistic and/or synergistic effects among elements, and the concentration of other elements in mushrooms [23–26].

The results of the proximate composition analysis of the albino maitake showed rather low values of protein, fat, and carbohydrate contents (Table 2), while the calorie content was 35.84 kcal·100 g−1. In addition, element analysis showed that this specimen presents relatively high contents in Ca, Fe, K, and Cu, and is rather low in Na (Table 2). The Ca content is higher than in some baby foods, such as oatmeal and whole milk, as well as ham, cream, spread cheese, white chocolate, and others [27]. The content in Fe is considerably higher than in most other foods; it is noteworthy that dried thyme (i.e., one of the richest foods in Fe) contains about 50 mg less Fe per 100 g when compared to the albino *G. frondosa*. The K content in albino maitake is lower only to that of cremor tartar and much higher than that of dried spirulina algae [27], while the Na content is lower than that of miso soup and baking powder [27]. The Cu content of albino maitake is higher than most foods, including dark chocolate, oysters, liver, and lobster [27].


**Table 2.** Outcome of proximate composition analysis (% for nitrogen, proteins, fats, carbohydrates, and ash), and content in elements and vitamins (mg·100 g<sup>−</sup>1) of the albino maitake specimen. Values are expressed as means ± standard error, *n* = 3.

The content in vitamins was: B1, 0.15 mg 100 g−1; B2, 0.36 mg·100 g−1; B3, 3.89 mg·100 g−1; B5, 0.68 mg 100 g−1; B9, 0.38 mg 100 g−1; and D2, 0.41 mg 100 g−<sup>1</sup> (Table 2). Noteworthy is the value of vitamin B9, or folic acid, since the daily requirement is about 0.2 mg, which doubles for pregnant women [28].

#### *3.4. Antibacterial and Antibiofilm Activity*

The minimal inhibitory concentration (MIC) of the extract deriving from the albino maitake was determined by a micro-method, i.e., by diluting the protein extract in the range from 25% to 0.025% *v*/*v* as previously described [29].

The antibacterial activity test carried out on albino maitake showed that the cold water extract was effective in inhibiting the growth of two bacterial strains, i.e., *S. epidermidis* ATCC 12228 and *P. aeruginosa* ATCC 15442, at the maximum screening concentration of 50% *v*/*v*. *S. epidermidis* is a Gram-positive bacterium typically present on the skin, which could be particularly dangerous in surgical procedures, while *P. aeruginosa*, is a ubiquitous bacterium and opportunistic pathogen in humans. As regards the effect of the water extract from the albino *G. frondosa* on biofilm formation, the results revealed that it decreased the biofilm produced by *S. aureus* ATCC43300 [30], whereas it slightly enhanced biofilm formation by *E. faecalis* ATCC 29212, *E. coli* ATCC 25922, *K. pneumoniae* ATCC 700603, and *P. aeruginosa* ATCC 15442 (Figure 4).

**Figure 4.** Effect of the cold water extract from the albino *Grifola frondosa* basidioma on biofilm production (expressed as optical density, OD) by five bacterial species. BT: biofilm produced in the absence of the fungal extract; BT+GF: biofilm produced in the presence of the fungal extract. The experiment was conducted in triplicate.

#### *3.5. Ecological and Conservation Issues*

The characterization of mushrooms growing in the Mediterranean area is one of the main targets of studies carried out by our research group. The principal objective is to provide reliable/robust scientific data amid the considerable confusion existing in the commercial exploitation of pertinent material in Italy and elsewhere, since accurate identification and/or origin of products placed on the market are often missing or of ambiguous validity. Previous results have highlighted the importance of mushrooms of the genus *Pleurotus* in human nutrition and in the feeding of farm animals [31–35]. Particular attention is also drawn to edible mushroom species that are infrequent/rare or linked to particular habitats (e.g., *Pleurotus nebrodensis* and *P. opuntiae*) [36,37], and for which only few or no studies exist regarding their nutritional and medicinal properties.

Although *G. frondosa* is widely distributed in the northern hemisphere, it is considered very rare in Italy. Particularly in Sicily, the only reports available concern the brown maitake found in the northeastern part of the island and date back to the end of the 19th century [38]; more recent informal records by amateur mycological groups derive from the same wider region (however, no pertinent samples are available for further study). Hence, the occurrence of an albino maitake is of apparent importance in a biogeographical, ecological, and applied context. In addition, the presence of this fungus on a monumental *Q. pubescens* tree of high significance points out the potential risks for host plants in the area because *G. frondosa* demonstrates a parasitic action resulting in root and butt decay [39,40]. At the same time, it is necessary to ensure the necessary conditions for its ex situ conservation due to the exploitation potential it exhibits as food and source of bioactive compounds as it was reported for wild mushrooms in general [41]. We have therefore adopted well-established protocols for its long-term maintenance [42] since our investigation showed that fructification of the albino maitake is not constant over time and the ability of the strain to be preserved under laboratory conditions—without significant degeneration of its properties—is lower than in other mushroom species.

#### **4. Conclusions**

An albino maitake specimen was reported for the first time in Italy. The examined basidioma differs morphologically from the common brown/gray form, with the most significant differences being the color and shape of the pileus, the type of pores, the shape of the stipe, and the period of fructification. Phylogenetic analysis evidenced that the specimen under study groups among other European isolates within a larger terminal clade corresponding to *G. frondosa*. Moreover, the nutritional composition

and the potential of the albino maitake to serve as source of functional compounds is of interest, and further investigation is in progress on the characterization of polysaccharides and at evaluating the antitumor activity against several types of cancer cells. The outcome of tests performed using the mushroom water extract against human pathogenic bacteria demonstrated its efficacy at inhibiting the growth of *S. epidermidis* and *P. aeruginosa* strains, while it also hindered biofilm formation by *S. aureus*, i.e., the causal agent of serious infections in immunocompromised patients. The antibacterial agents present in the albino maitake are promising candidates for dealing with pathogens that show resistance to a large spectrum of antibiotics and are widespread in hospitals.

**Author Contributions:** Conceptualization, G.V. and M.L.G.; methodology, G.V., G.I.Z., M.L.G. and O.S.I.; validation, R.C. and V.F.; formal analysis, G.I.Z., M.L.G. and O.S.I.; investigation, G.V., G.I.Z., M.L.G. and O.S.I.; data curation, A.G. and T.F.; biofilm analysis, R.C. and V.F.; writing—original draft preparation, G.V. and M.L.G.; writing—review and editing, G.V. and G.I.Z.; visualization, G.V.; supervision, G.V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **An Italian Research Culture Collection of Wood Decay Fungi**

#### **Carolina Elena Girometta 1,\* , Annarosa Bernicchia 2, Rebecca Michela Baiguera 1, Francesco Bracco <sup>1</sup> , Simone Buratti 1, Marco Cartabia 1,3, Anna Maria Picco <sup>1</sup> and Elena Savino <sup>1</sup>**


Received: 29 December 2019; Accepted: 30 January 2020; Published: 1 February 2020

**Abstract:** 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, officinal 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.

**Keywords:** wood decay fungi (WDF); culture collection; fungal strain; host; Italy; morphological and molecular identification

#### **1. 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 same taxonomic group may include species displaying different strategies and different morphologies. As a whole, wood decayers appear to be a pivotal model in the study of the evolutionary relationships of both Dikarya and extra-Dikarya taxa [3–5]. Consistently, the incipient molecular-based biogeography of wood decayers seems to display distribution patterns strongly affected by preferred host species (trees or shrubs), which are apparently followed throughout [6].

Wood decay fungi include several edible species as well as species reported to be the source of bioactive compounds, related to either primary (e.g., β-glucans) or secondary metabolism (terpenoids, phenolics, acids, superior alcohols, etc.). Structural diversity, occurrence and distribution among taxa, synthesis stimulation factors and correlation to growth stage, bioactivity pathways, standardization of products and crude extracts are the main current topics under investigation [7–9].

Due to their relatively easy reproduction in culture, several other applications are being developed on wood decay fungi: degradation of organic pollutants and bioremediation [10]; pretreatment of biomasses for production of sugars and bioethanol [11,12]; production of enzymes for industrial purposes, namely Mn peroxidases, laccases, cellulases and hemicellulases [13]; and bioadsorption and bioaccumulation of metal ions either in living or dead biomass [14–16].

Last but not least, necrotrophic wood decay means a loss of harvest in forestry and woody cultures, whereas in public and private green areas it means destabilization and consequently risk for people and objects [17].

Culture collections are an important reference, since availability and exchange of authenticated, quality-guaranteed pure cultures are increasingly needed by researchers at an international level [18,19]. Above all, tests on different species and strains (at intraspecific level) are required since biochemical differences are often not negligible [20–22].

Actually, only a few research centers can afford to structure their culture collection in conformity to international guidelines provided by the World Federation for Culture Collections (WFCC) [23,24]. The strains maintained in many universities or research centers can be considered an important source of experimental material, even without a WFCC certification. Since small uncertified collectors are geographically widespread, their contribution may be significantly representative of local ecosystems and biodiversity [25].

The Mycology Laboratory at the Botanical Garden of Pavia University (Italy) has a long tradition of the isolation, identification and preservation of fungal strains in various areas of mycology. This is supported by the numerous publications from the middle of the last century [26] up to now. Currently, the fungal strains collection is named MicUNIPV and each working group preserves and enriches the collection.

Although the definition of ecotypes is usually hard, a remarkable intraspecific variability is well documented in several fungal species and it may be particularly true for rare species, whose populations are supposed to be more isolated [27]. This highlights the value of Italian territory for fungal biodiversity and the great potentiality for research [28].

Italy has a wide variety of climates and morphologies, both due to its remarkable latitudinal range (about 13◦) and structural-topographic complexity, including the presence of four different seas and two main mountain chains. According to the official maps of MATTM (Ministero dell'Ambiente e della Tutela del Territorio e del Mare), 28 different phytoclimatic classes are recognized, five of which are specifically referred to as Mediterranean [29], also taking into account the biogeographic reference map suggested by Rivas-Martínez [30]. Nevertheless, the pluri-millennial stratification of human impact has made it difficult to distinguish between actual and potential ecosystem features. As a consequence, the classification and mapping of either Italian ecoregions or phytoclimates provide a tool for the comprehension of biodiversity instead of a strict map of biodiversity itself [31].

The present article reports the results obtained by the researchers of the Laboratory of Mycology in DSTA-University of Pavia (Italy) who continuously collect new cultures of wood decay fungi, focusing on fungal biodiversity of species related to the Mediterranean area and climates.

#### **2. Materials and Methods**

#### *2.1. 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:


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.

#### *2.2. 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.


**Table 1.** Thermocycling protocol for PCR.

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:


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.

#### **3. 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 [41–43]. 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.









The species reported in 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. pfei*ff*eri*, *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. pfei*ff*eri,* 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.

#### *3.1. Cellulariella Warnieri (Durieu and Mont.) Zmitr. and V. Malysheva*

#### 3.1.1. 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].

#### 3.1.2. 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.

#### *3.2. Dichomitus squalens (P. Karst.) D.A. Reid*

#### 3.2.1. Background

*Dichomitus squalens* is a model species for studies about the selectivity of white rot and its enzymatic basis [57–60]. 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 Białowie ˙za 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].

#### 3.2.2. 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.

#### *3.3. Hericium Erinaceus (Bull.) Pers.*

#### 3.3.1. 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 [7,63–66]. 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].

#### 3.3.2. 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 [71–73].

#### *3.4. Inocutis Tamaricis (Pat.) Fiasson and Niemelä*

#### 3.4.1. 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.

#### 3.4.2. 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.

#### *3.5. Fomitiporia Mediterranea M. Fisch.*

#### 3.5.1. 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].

#### 3.5.2. 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.

#### *3.6. Perenniporia meridionalis Decock and Stalpers*

#### 3.6.1. 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].

#### 3.6.2. 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].

#### *3.7. Perenniporia ochroleuca (Berk.) Ryvarden*

#### 3.7.1. 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*.

#### 3.7.2. 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.

#### **4. 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).

**Author Contributions:** Conceptualization, E.S. and C.E.G.; Methodology, E.S., A.B., A.M.P., C.E.G., R.M.B. and M.C.; Investigation, E.S., A.B., C.E.G., R.M.B., S.B. and M.C. Resources, E.S., A.M.P. and F.B.; Data Curation, E.S., A.B. and C.E.G.; Writing – Original Draft Preparation, C.E.G., S.B.; Writing – Review and Editing, C.E.G., E.S., A.B., A.M.P.; Supervision, E.S., A.M.P.; Project Administration, E.S.; Funding Acquisition, E.S., F.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This project has been funded by: Fondo Ricerca e Giovani dell'Università degli Studi di Pavia (Savino 2019); Fondazione Cariplo, grant n◦ 2018-1765 entitled "Myco-advanced leather materials (MATER)".

**Acknowledgments:** The State Natural Strict Reserve (RNIS) Bosco Siro Negri – Zerbolò for support and authorization for sampling; the Botanical Garden of Pavia (Orto Botanico di Pavia) for authorization for sampling. The authors are also grateful to C. Perini, University of Siena (Italy), Valentina Cesaroni and Fabio Savino for providing some original specimens.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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### *Article*
