Pisolithus albus ( Sclerodermataceae ), a first record from Egypt

. Pisolithus albus ( Basidiomycota , Sclerodermataceae ) is reported here for the first time from Egypt where it was found on the Karaman Island in Sohag Governorate. It was found forming an ectomycorrhizal association with Eucalyptus occidentalis and Vachellia nilotica . The fungus was also reported near two potentially new hosts: Paspalum distichum and Dodonaea viscosa ; however, the mycorrhizal connections with these two hosts is not proven. Phylogenetic analyses for the combined ITS and LSU rDNA sequences dataset placed the Egyptian specimen within the Pisolithus albus complex clade. It is described, illustrated, and phylogenetically analyzed in this article. Various hosts and locations of the taxon are reviewed.


Introduction
Pisolithus (Basidiomycota, Sclerodermataceae) is one of the most cosmopolitan mutualistic gasteromycete genera in the world (Marx 1977). It was introduced by Albertini and Schweinitz (1805) to accommodate the type species Pisolithus arenarius. Members of this genus were recorded from various habitats with regard to temperature, nutrient levels and heavy metal concentrations, and has been recorded from tropical, subtropical and temperate regions (Watling et al. 1999;Martín et al. 2013;Rusevska et al. 2015;Jaouani et al. 2015;Crous et al. 2016;Mifsud & Mifsud 2022). Pisolithus species make mycorrhizal associations with different plant species in the families Casuarinaceae, Dipterocarpaceae, Fagaceae, Myrtaceae and Pinaceae (Marx 1977;Malloch & Kuja 1979). Pisolithus is widely used in forestry and horticulture due to growth stimulation properties reported in different tree species, including acacias, eucalypts and pines (Garbaye et al. 1988;Duponnois & Bâ 1999).
Because of the heterogeneity of Pisolithus in terms of sporocarp, spore and mycelial culture morphology, the genus was considered for a long time as monotypic (Coker & Couch 1928;Cunningham 1942;Pilát 1958), and described species within the genus have been widely regarded as conspecific with Pisolithus tinctorius (Chambers & Cairney 1999). In the last two decades, many phylogenetic studies established several species under this genus (Anderson et al. 1998;Martin et al. 1998;Gomes et al. 2000;Díez et al. 2001;Lebel et al. 2018). These studies revealed significant genetic variation within the species, implying that the P. tinctorius group is a complex of several species that cannot be distinguished by morphological studies (Anderson et al. 2001;Martin et al. 2002;Kanchanaprayudh et al. 2003;Moyersoen et al. 2003;Singla et al. 2004). Martin et al. (2002) concluded that there are three distinct lineages of Pisolithus with at least 11 different species. The genus currently comprises 20 species (MycoBank 2022). One of the most common species in this genus is Pisolithus albus, that was collected from a wide range of geographical locations, e.g., Australia, Brazil, China, India, Italy, Morocco, Malta, New Caledonia, New Zealand, Pakistan, Senegal, Spain, Thailand, Tunisia, and the USA. However, this is the first record of this species from Egypt. Most of the collections of Pisolithus albus were in ectomycorrhizal associations with Eucalyptus, Acacia and Kunzea. Hosaka (2009) andJourand et al. (2010) supposed that P. albus may have four genetic ecotypes. These genetic variations and biogeographical patterns may originate from long-distance dispersal, and this may give the species high adaptability to change its mycorrhizal associations (Hosaka 2009).
This work aimed to report the first record of Pisolithus albus from Egypt, based on morphological examination and phylogenetic analyses. Hosts and locations of Pisolithus albus are also reviewed.

Site description
Samples were collected from Karaman Island (26°35′ 26.1″N, 31°42′29.6″E -26°33′48.8″N, 31°42′01.4″E), that is located in the middle of the River Nile at Sohag Governorate, Egypt. It is an uninhabited island, which is characterized by sandy or clay soil with an elevation about 62 m above the sea level, hot desert climate and precipitation less than 2.28 mm per year. The habitats on the island vary from submerged to dry lands. The island vegetation is a mixture of wild plants and urban trees.

Sample collection and morphological description
Basidiomata of Pisolithus albus were collected from Karaman Island several times during the period between December 2018 and November 2020. Metadata including date, geographic coordinates, habitat and associated plant communities were recorded on site. Collections were packed in clean sterilized plastic bags and brought to the laboratory for further examination. Microscopic observations and measurements were taken from tissue sections mounted in 5% KOH and examined using an Olympus SZ61 stereomicroscope. Micrographs were taken using an Olympus BX51 compound microscope (Olympus, Tokyo, Japan) equipped with a Toup Tek XCAM1080PHA (Toup Tek, Zhejiang, China) digital imaging system. Dried basidiocarps were deposited in Sohag University Microbial Culture Collection (SUMCC). Basidiospores from the recorded species did not germinate on potato dextrose agar (PDA; Oxoid, Basingstoke, UK) or MMN agar media (Marx 1969).

DNA extraction, sequencing and phylogenetic analyses
Total genomic DNA was extracted directly from the gleba of fruiting bodies using a microbial DNA extraction kit (MOBIO; Mo Bio Laboratories, Carlsbad, CA, USA) following the manufacturer's protocol. The primer pairs ITS1 and ITS4 (White et al. 1990) and LROR and LR7 (Vilgalys & Hester 1990) were used for PCR amplification and sequencing of internal transcribed spacer (ITS) regions and the partial large subunit nuclear rDNA (LSU), respectively. PCR amplification and DNA sequencing were conducted as described by Abdel-Wahab et al. (2009) by Macrogen Inc., South Korea. Sequences were assembled using Sequencher 4.2.2 (Gene Codes Corporation). Newly generated sequences were deposited in the GenBank (Tab. 1). Sequences of ITS-LSU were aligned with other sequences of Pisolithus albus recorded from different localities and hosts, other species of Pisolithus and the outgroup taxa: Boletinellus merulioides and B. exiguus using Clustal X (Thompson et al. 1997) and optimized manually. The concatenated sequence alignments were obtained from SequenceMatrix v 1.7 (Vaidya et al. 2011). Maximum-parsimony (MP) and maximum-likelihood (ML) phylogenetic analyses were constructed using MEGA X (Kumar et al. 2018).
Bootstrap support (Felsenstein 1985) was obtained by using the Tamura-Nei model. Bayesian inference (BI) analysis was carried out using MrBayes ver. 3.1.2 (Ronquist & Huelsenbeck 2003) with the SYM+G model that was determined by Akaike information criterion (AIC) in MrModeltest 2.2 (Nylander 2004). Four simultaneous Markov chains were run for five million generations and trees were sampled every 100 generations. Trees were visualized using Njplot ver. 2.3 (Perrière & Gouy 1996), and additionally layouts were done in the program of Adobe Illustrator CC (Adobe Systems Inc., CA, USA).

Phylogenetic analysis
The combined ITS and LSU rDNA dataset consisted of 57 taxa, of which 40 belong to the Pisolithus albus complex, 15 representatives of other species of Pisolithus and 2 species from Boletinellaceae used as an outgroup, namely Boletinellus merulioides and B. exiguus. Phylogenetic analyses of individual ITS and LSU rDNA datasets produced similar topology (data not shown). The ITS dataset consisted of 455 characters, which 135 gaps are excluded, 213 constant, 20 variable parsimony-uninformative and 87 parsimony-informative characters. The LSU rDNA dataset consisted of 600 characters, which 49 gaps are excluded, 483 constant, 32 variable parsimony-uninformative characters and 36 parsimony-informative characters.
The Bayesian analysis resulted in 50,000 trees after five million generations. The first 12,500 trees, representing the burn-in phase of the analyses were discarded, while the remaining trees were used for calculating posterior probabilities. The maximum parsimony of combined ITS and LSU rDNA dataset consisted of 1,055 characters that included: 154 gaps (that are excluded), 725 constant, 52 variable parsimony-uninformative and 124 parsimony-informative characters. The most parsimonious tree had a length of 375 steps, a consistency index (CI) of 0.798680, a retention index (RI) of 0.919419 and the composite index is 0.769860. Maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting (SPR) algorithm (Nei & Kumar 2000) with search level 1 in which the initial trees were obtained by the random addition of sequences (10 replicates). Bootstrap analysis was performed with 1000 replicates to assess the statistical support for the tree. Maximum likelihood analysis yielded one tree with -ln likelihood score of 3435.47. ML, MP and Bayesian analyses produced trees with similar topologies. The Bayesian phylogenetic tree is shown in Figure 1. Molecular phylogenetic analyses of the combined ITS and LSU rDNA placed our Pisolithus albus material within the P. albus complex with high statistical support (95/85/99 for MP/ML/BYPP, respectively) (Figure 1). The collection from Egypt is nested in a subclade containing Pisolithus albus collections from Australia, Pakistan, New Caledonia, India, Senegal and Thailand in ectomycorrhizal associations with Acacia, Eucalyptus and unknown hosts. Description. Basidiomata epigeous, club-shaped, with irregular, subglobose, piriform or capitulate shape, 4.5-15 cm wide × 6-18 cm high and with a stout deeply rooting base 3-8 × 4-13 cm in diameter, attached to the substratum by yellowish rhizomorphic strands. Peridium thin, shiny, smooth, membranous, single-layered, leathery when young, dry ruptured at maturity, white to cream and rarely buff in ripe basidiomata, yellowish around the base of fresh specimens, cracked into irregular segments from the top downwards at maturity, exposing the mature spores. Gleba developing within subglobose to ovoid or polygonal peridioles, 1-5 × 1-3 mm (av. = 3.1 × 2 mm, n = 25). Peridioles are larger in the upper parts, while smaller and tightly packed toward the stipe, whitish when young turning into ochre to pulerulent umber at maturity. It is encased by a very thin, yellow-ochre membrane and separated by gelatinous or sticky, carboneous tramal plates. At maturity of the basidiomata, peridioles wall collapse and the gleba turned into powdery mass. Basidia 8-10 × 16-23 µm (av. = 9 × 19.5 µm, n = 10), hyaline,  clavate with a swollen apex, carried 5 or 6 basidiospores. Clamp connection present. Basidiospores 7-11 µm in diameter (av. = 9 µm, n = 45), globose to subglobose, bright yellow when young turned into reddish-brown at maturity, coarsely ornamented with broadly connate structures that form irregular pyramid-like spines. Notes. Pisolithus albus was recorded in several consecutive surveys from Karaman Island in autumn and winter 2018, 2019 and 2020. In this period, temperature ranged from 18 to 32°C; relative humidity was from 22 to 45%.

Discussion
Pisolithus albus is one of the most common gasteromycetes in the world, which is recorded here for the first time from Egypt to extend our knowledge about its distribution in Africa to four countries, namely: Egypt (this study), Morocco (Yakhlef et al. 2009), Senegal (Duponnois & Bâ 1999) and Tunisia (Jaouani et al. 2015). Morphologically, Pisolithus albus specimens from Egypt are slightly different than collections from other localities. Basidiomata in our collection (4.5-15 cm wide × 6-18 cm high) are slightly bigger than that recorded from Tunisia (3-12 cm in diameter) and smaller than that recorded from Italy (3-20 cm in diameter); peridioles in our collection are subglobose to ovoid or polygonal, while they are elliptic-ovoid to lens shape in the Tunisian collection. Basidia were observed clearly in our collection, which carried 5-6 basidiospores, while they were not observed in other collections recorded from Italy, Tunisia, Morocco and Senegal; basidiospores from the Egyptian collection are slightly smaller than those recorded in the Tunisian collection (7-11 µm vs 9-12 μm diameter in Egyptian and Tunisian records, respectively). During this study, P. albus was found associated with Eucalyptus occidentalis and Vachellia nilotica (syn. Acacia nilotica). This fungus was reported previously in ectomycorrhizal association with Acacia and Eucalyptus species from different countries (Díez et al. 2001;Founoune et al. 2002;Martin et al. 2002;Kanchanaprayudh et al. 2003;Singla et al. 2004;Jaouani et al. 2015;Gargano et al. 2018;Lebel et al. 2018;Chouhan & Panwar 2021). During the present study, P. albus basidiomata were reported for the first time in the vicinity of two potential hosts, Paspalum distichum and Dodonaea viscosa; however, the mycorrhizal connections with these two hosts were not investigated. Therefore, detailed studies are required to prove mycorrhizal association of these two hosts with the fungus. Phylogenetic analysis of the combined ITS and LSU sequences placed the Egyptian specimen within P. albus complex clade and formed a distinct lineage with P. arhizus, P. croceorrhizus and P. tympanobaculus, which is similar to the results obtained by Martin et al. (2002) and Lebel et al. (2018). Phylogenetically, the Egyptian specimen showed a high level of genetic similarity with collections of P. albus from Australia (RLB8183) which forms mycorrhizal associations with Acacia sp., Eucalyptus sp., and other collections from Australia (NT03, NT04), Pakistan (Pak IF7) and India (KSRF-0007) where its hosts are unknown.

Conclusion
This study documents the first reports of Pisolithus albus from Egypt. It was found forming ectomycorrhizal associations with Eucalyptus occidentalis and Vachellia nilotica. It was also reported near two potentially new hosts, Paspalum distichum and Dodonaea viscosa. The reported specimens were compared morphologically with other reports of Pisolithus albus from different localities and showed slight differences. Phylogenetic analyses of the combined ITS and LSU sequences placed the Egyptian specimen within P. albus complex clade.