PERMEABILIZATION, CELL WALL ULTRASTRUCTURE, AND GERMINATION OF BASIDIOSPORES OF THE ECTOMYCORRHIZAL FUNGUS Pisolithus microcarpus TREATED WITH DIFFERENT COMMERCIAL BRANDS OF BLEACH

Basidiospores of the ectomycorrhizal fungus Pisolithus microcarpus have an impermeable cell wall, a characteristic that is possibly related to the low germination percentages of these propagules, which makes it diffi cult to obtain monokaryons and use these spores in inoculants. The objective of this study was to evaluate the eff ect of diff erent concentrations of commercial bleach on the permeabilization of P. microcarpus basidiospores and to analyze the alterations caused in the cell wall ultrastructure and the viability and germination capacity of these propagules. Fungal basidiospores were collected in eucalyptus plantations and permeabilized using diff erent bleach concentrations and exposure times. The basidiospores were then analyzed by scanning and transmission electron microscopy. The percentage of permeabilized basidiospores varied with the commercial brand, bleach concentration, and exposure time. Basidiospores of diff erent basidiocarps diff ered in susceptibility to permeabilization treatment with bleach. Changes in the ultrastructure of permeabilized basidiospores were observed at bleach concentrations of 15 and 50 % for an exposure time of 40 s, with surface changes and loss of the spicules of the outermost layer of the wall. After permeabilization with 5 % bleach for 40 s, 80 % of the permeabilized spores were viable, resulting in the production of fungal colonies after 15 days of incubation of these propagules in the presence of Corymbia citriodora. However, the germination percentage obtained, 0.001 %, was similar to that of non-permeabilized basidiospores, indicating that other factors, besides cell wall permeability, are determinant for the germination process.


INTRODUCTION
Ectomycorrhizas are symbiotic associations between soil basidiomycete fungi and roots of tree plants, contributing to the promotion of plant growth and mitigation of biotic and abiotic stresses (Smith & Read, 2010;van der Heijden et al., 2015). These associations are frequent in forest species of economic interest, such as eucalyptus (ABRAF, 2013). Among the fungi that form ectomycorrhizas, those of the genus Pisolithus stand out, being associated with roots of several species of gymnosperm and angiosperm plants in several regions of the globe, including Brazil (Martin et al., 2002).
The germination of ectomycorrhizal fungal spores has been studied in several species, and the germination percentages reported in the literature are generally low (Costa, 2002;Pereira, 2004;Pereira et al., 2017). This fact prevents the production of monokaryotic strains intended for studies on the genetics of the ectomycorrhizal association, the application of mutagenesis techniques to spores and the production of spore-based inoculants to be used in seedling production in forest nurseries (Costa, 2002;Pereira et al., 2017). For Pisolithus, the germination percentage of basidiospores ranges from 0.001 to 0.38 % (Kope & Fortin, 1990;Costa, 2002;Pereira, 2004). In part, recalcitrance to germination has been attributed to the impermeability of the cell wall of the spores of this fungus.
Fungal cell wall is composed of glycoproteins and polysaccharides, mainly glucans and chitin (Gow et al., 2017;Ruiz-Herrera & Ortiz-Castellanos, 2019). Chitin is a linear polymer formed by N-acetyl glucosamine molecules, joined by glucosidic bonds. The β-1,4 bond of this polysaccharide form microfi brils in the extracellular space that contribute to the rigidity of the cell wall (Ruiz-Herrera et al., 1996;Gow et al., 2017;Ruiz-Herrera & Ortiz-Castellanos, 2019). Among the proteins present in the cell wall, hydrophobins stand out, which are small and moderately hydrophobic proteins that contain eight conserved cysteine residues (Ruiz-Herrera & Ortiz-Castellanos, 2019), being strongly linked to cell wall polymers (Vries et al., 1993;Ruiz-Herrera & Ortiz-Castellanos, 2019). Glycoproteins are modifi ed proteins of the wall by the binding of oligosaccharides during translocation and cell secretion (Gow et al., 2017;Ruiz-Herrera & Ortiz-Castellanos, 2019).
In studies with the species P. microcarpus, aiming at the description of the process of basidiosporogenesis and meiosis, changes in the fl uorescence emission pattern of the basidiospores throughout development were verifi ed, which was attributed to the deposition of diff erent wall layers in the spores (Campos & Costa, 2010a). It has been suggested that, similarly to P. tinctorius, the basidiospores of P. microcarpus also have four wall layers (Campos & Costa, 2010a). It has been hypothesized that the structure of the cell wall, associated with its hydrophobicity, could be related to the recalcitrance of the basidiospores of this fungal species to germination (Costa, 2002;Campos & Costa, 2010a;Godinho, 2011). Thus, the permeabilization of the cell wall could allow the entry of signals that induce the germination of spores produced by eucalyptus (Costa, 2002).
The permeabilization of the cell wall of fungal spores can be performed by chemical methods, involving the use of lytic enzymes or inorganic substances (Costa, 2002;Godinho, 2011). The treatment of basidiospores with sodium hypochlorite present in commercial formulations of bleach promoted the permeabilization of the cell wall, making it possible to study the diff erent reserve compounds contained in the basidiospores (Godinho, 2011). However, the eff ects of this permeabilization process on the ultrastructure of the wall, as well as the viability and germination capacity of the spores, are not known. Thus, the objective of this study was to evaluate the effi ciency of commercial bleach in the permeabilization of the cell wall of P. microcarpus basidiospores and the eff ects of this treatment on the ultrastructure, viability and germination capacity of these propagules in the presence of C. citriodora.

Microorganism and storage conditions
The study was carried out at the Laboratory of Mycorrhizal Associations of the Department of Microbiology, located at the Institute of Biotechnology Applied to Agriculture (BIOAGRO) of the Federal University of Viçosa (UFV), Viçosa, MG, Brazil. Basidiocarps of P. microcarpus were collected in Corymbia spp. forests in the Forestry Sector of UFV, from August 2012 to June 2013. The basidiocarps were washed externally with distilled water and immersed in 70 % ethanol for 1 min, for superfi cial disinfestation, and air dried in a laminar fl ow cabinet until the solution evaporated. The basidiocarps were sectioned longitudinally with a scalpel, and the spores, collected with sterile spatula, were kept in a refrigerator.

Permeabilization of basidiospores with bleach
Half a gram of basidiospores was suspended in 1 mL of Tween 80 at 0.5 %. The suspension was centrifuged at 10,000 rpm for 8 min and washed three times with distilled water. Finally, the basidiospores were resuspended in distilled water and subjected to the permeabilization treatment with bleach (Costa, 2002). Five brands of bleach, containing 2 to 2.5 % of active chlorine (w/w), were tested as to their capacity of permeabilization of P. microcarpus basidiospores from a single basidiocarp. The bleach brands were identifi ed with numbers from 1 to 5. The concentrations of bleach used were 15 and 50 % for a time of 40 s (Godinho, 2011). 250-μL aliquots of the suspension of basidiospores were transferred to 1.5-mL Eppendorf tubes and treated with 250 μL of bleach solutions, in order to obtain the fi nal concentrations above.
Treatment was interrupted by the addition of 500 μL of sodium thiosulfate at 0.14 mol L -1 . The basidiospores were centrifuged at 10,000 rpm for 2 min, washed twice in distilled water and stained with 250 μL of Sudan Black B dye at 2 % (w/v). To determine the percentage of permeabilized basidiospores, a 10-μL aliquot of each suspension of stained spores was mounted between slide and coverslips and observed under light microscope, counting fi ve fi elds of each slide. Basidiospores stained with Sudan Black B were considered permeabilized. The means were compared by the Tukey test at 5 % probability.

Permeabilization of basidiospores from diff erent basidiocarps
After collecting 10 basidiocarps and cleaning their surfaces with distilled water, the basidiospores were harvested and subjected to permeabilization procedures at concentrations of 15 and 50 % of bleach for 40 s (Godinho, 2011), staining with Sudan Black B at 2 % (v/v) and determination of the percentage of permeabilization, as described in item 2.2. The experiment was set up with three replicates and the obtained data were subjected to analysis of variance. Treatment means of each treatment were compared the Tukey test test at 5 % probability.

Optimization of basidiospore cell wall permeabilization conditions
The optimization of the permeabilization conditions was performed for one of the basidiocarps collected, following the procedures described in item 2.2. The fi nal concentrations of bleach tested were 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 % (v/v). The times of exposure to the permeabilization agent were 5, 10, 20, 30, 40, 50, 60, 70 and 80 s. The experiment was set up in a factorial scheme, with three replicates, and the obtained data were subjected to regression analysis.

Ultra-morphology of basidiospore cell wall determined by scanning and transmission electron microscopy
A sample of basidiospores from a single basidiocarp was permeabilized with bleach at 15 and 50 % for 40 s, fi xed in 10 % formalin (v/v) for 2 h and, subsequently, subjected to drying in an oven at 40 ºC for 24 h. A control, without permeabilization, was also included. The basidiospores were metallized with 150-Aº-thick gold layer and observed in a LEO VP1430 scanning electron microscope. The basidiospores were also subjected to transmission electron microscopy. For this, the examination of cell wall ultrastructure followed the procedure described by Mims & Thurston (1979) and Webster (2007) modifi ed by the processing with 5 pulses of thermal shock for 40 s iin a microwave, 700 W, and 5 min under refrigeration, after each step below: basidiospores permeabilized or not were fi xed in 2.5 % glutaraldehyde solution and in 0.2 M sodium cacodylate, pH 7.2, for 4 h at room temperature, and were subsequently resuspended in 0.1 mol L -1 sodium cacodylate for 5 min (2X). The suspension was centrifuged and resuspended in OsO 4 (1 %) for 2 h at room temperature. After this period, the solution was resuspended in 500 μL of 0.1 mol L -1 sodium cacodylate, centrifuged and subsequently kept in 70 % alcohol for 24 h under refrigeration. The basidiospores were dehydrated dehydrated using an ethanol series of 80, 90, 95 and 100 % for 20 min in each concentration. The dehydrated samples were included in LR White resin and subjected to microtomy. The sections were stained with 1 % uranyl acetate and lead citrate and analyzed under a Zeiss EM109 transmission electron microscope.

Viability and germination of permeabilized basidiospores
Basidiospores from a selected basidiocarp were permeabilized by exposure to bleach at 5, 10, 15, 30 and 50 % for 40 s. For verifying the viability, the basidiospores were stained with the Live/ Dead Fungal Light Yeast Viability Kit, Molecular Probes ® , Life Technologies TM , as instructed by the manufacturer. Cells with intact membrane emit green fl uorescence, while cells with damaged membrane emit red fl uorescence. The samples were analyzed under an Olympux BX 50 fl uorescence microscope with accessories for Y-F epifl uorescence, and the images were captured with an Olympus PM -C35DX digital camera. The experiment was set up with three replicates and the obtained data were subjected to analysis of variance. Means were compared by the Tukey test at 5 % probability.
Newly collected basidiospores were permeabilized as described in item 2.2 by the treatment with bleach at the concentration of 10 % for 40 s. The time of 40 s was the one that made it possible to eliminate contaminant microorganisms and maintain the highest percentage of viable basidiospores, 35 %. 100-μL aliquots of a suspension containing 5 x 10 6 mL -1 permeabilized spores were spread on 25 mL of modifi ed Melin-Norkans medium (Marx, 1969), supplemented with 2 g L -1 of activated carbon, in order to obtain a fi nal density of 5 x 10 5 spores per dish. One seedling of C. citriodora, one week old and free of contamination, was placed in the middle region of the dish containing the spores. C. citriodora seedlings free of contamination were obtained according to the procedures of Pereira (2004). The Petri dishes were sealed with PVC fi lm and incubated in a growth chamber at 25 ºC and with 16 h of light (200 μmol m -2 s -1 ). To determine the germination percentage of basidiospores, the appearance of colonies was evaluated every 2 days by direct observation.

Permeabilization of basidiospores with bleach
The bleach brands tested diff ered ias to their effi ciency at permeabilizing the cell wall of P. microcarpus basidiospores (Figure 1). At bleach concentrations of 15 and 50 % for 40s, brand 1 permeabilized 12 and 78 % of the treated basidiospores, respectively. Given its higher permeabilization effi ciency, the bleach brand 1 was chosen to be used in all other experiments carried out in this study.

Permeabilization of basidiospores from diff erent basidiocarps
The basidiospores from diff erent basidiocarps showed diff erent levels of susceptibility in relation to the treatment of permeabilization with bleach ( Figure  1). In the treatment with 15 % bleach and 40 s, the percentage of permeabilized basidiospores ranged from 4.5 to 26 %, depending on the basidiocarp. In the treatment with 50 % bleach, for the same time of exposure, the values ranged from 70 to 95 %.

Optimization of permeabilization conditions of the cell wall of P. microcarpus basidiospores
The percentage of P. microcarpus basidiospores permeabilized with bleach was proportional to the concentration and time of exposure to the permeabilizing agent (Figure 2). The highest percentage of permeabilization (88 %) was obtained by combining the concentration of 50 % bleach with the time of 80 s (data not shown).

Ultra-morphology of basidiospore cell wall determined by scanning and transmission electron microscopy
Scanning microscopy revealed changes in the spicules contained in the outer layer of the cell wall of the permeabilized basidiospores ( Figure  3). These changes were more pronounced at the highest concentration of bleach used (50 %), with exposure time of 40 s, a condition that led to partial or total loss of the spicules (Figure 3 and 4). The analysis of the ultrastructure of P. microcarpus basidiospores by transmission electron microscopy revealed the following aspects of the cell wall: mature basidiospores have a cell wall consisting of four layers, the outermost, L 1 , is electron-dense and ornated with spicules on its surface, followed by an electron-transparent inner layer, L 2 . The third layer, L 3 , is also electron-dense and the innermost layer, L 4 , is electron-transparent (Figure 4).

Viability and germination of permeabilized basidiospores
The viability of the basidiospores was reduced with the increase in the bleach concentration used in the permeabilization procedure (Figures 5). The use of bleach at concentrations of 5, 10, 15, 30 and 50 % for 40 s resulted in 80, 35, 19 and 2 % of viable basidiospores, respectively. In the treatment of bleach (50 %) for 40 s, no basidiospore remained viable ( Figure 5).
It was possible to obtain fungal colonies from the germination of basidiospores permeabilized with 10 % bleach for 40 s, with 35 % viability. This   treatment was also eff ective for eliminating microbial contaminants that are naturally found in basidiocarps. The fi rst colonies of P. microcarpus from the basidiospores with permeabilized cell wall appeared between the 15th and 18th day of incubation of the basidiospores in the presence of the host plant C. citriodora. The fungal colonies emerged close to the roots. The percentage of germination was low, 0.001 %.

DISCUSSION
In this study, the cell wall of P. microcarpus basidiospores was successfully permeabilized with commercial bleach. The choice of this agent aimed to reduce the costs of permeabilization of spores for the production of commercial inoculants and eliminate contaminant bacteria naturally present in the basidiospores. Previous tests performed with the pure sodium hypochlorite reagent, under the same conditions, resulted in a low percentage of permeabilized basidiospores (data not shown), indicating that other components of the reagent act jointly in the weakening of the cell wall. The diff erences of permeabilization found for each bleach brand may be related to diff erent chemical compositions. The active chlorine content, described in the packaging of the 5 brands tested, was in accordance with the level required by legislation, ranging from 2.0 to 2.5 % (w/w) (INMETRO, 2013). Brand 1 contained sodium hydroxide and sodium carbonate as additional components to sodium hypochlorite and water, while brands 2, 3, 4 and 5 consisted only of sodium hypochlorite and water. Sodium hydroxide and sodium carbonate, as well as sodium hypochlorite, have antimicrobial action (Siqueira Jr & Lopes, 1999;Adner & Zetterlund, 2002;Estrela et al., 2006). The synergistic action of these components associated with the active chlorine of sodium hypochlorite may justify the higher permeabilization effi ciency promoted by brand 1.
The variation in the effi ciency of commercial products in permeabilizing the cell wall of the basidiospores may also be related to the period of collection of basidiocarps and the maturation stage of their basidiospores (Campos & Costa, 2010b), factors that can infl uence the susceptibility of these propagules to the treatment of permeabilization. The maturation stage of basidiospores seems to be decisive, since mature basidiospores are more impermeable (Mims,  1980; Martin et al., 1999;Campos & Costa, 2010b). The tested basidiospores should represent propagules at diff erent stages of development due to the structure of basidiocarps, which does not ensure the collection of material in a single development stage. This fact is highlighted by data from Campos & Costa (2010b), who observed that, even in a single peridiole, the development of basidiospores may be asynchronous.
The results obtained in the present study indicate the need to standardize the permeabilization treatment for each basidiocarp collected.
It was possible to verify a reduction in the hydrophobicity of basidiospores after treatment with bleach, especially at concentration of 50 % for 40 s (data not shown). The presence of hydrophobic compounds in the wall of P. microcarpus basidiospores is known, hindering the production of suspensions of spores and, possibly, the entry of germinationstimulating compounds (Costa, 2002;Pereira, 2004). The presence of hydrophobins in hyphae, spores and ectomycorrhiza of Pisolithus and other fungi has already been reported (Martin et al., 1997;Tagu et al., 2000;Gow et al., 2017).
The percentages of basidiospores with the permeabilized cell wall reported in this study diff ered from those found by Godinho (2011), who reported permeabilization percentages ranging from 51 to 97 % as a function of concentration and time of exposure to sodium hypochlorite. Moreover, in the work of Godinho (2011), mature basidiospores were permeabilized from the concentration equivalent to 2.5 % bleach, with a contact time of 10 s, which was not observed in the present study. The diff erence in permeabilization percentages may result from the morphophysiological variation between diff erent basidiocarps, collected at diff erent sites and times, as demonstrated here.
Alterations of the spicules contained in the last layer of the cell wall of permeabilized basidiospores were observed. These changes were more intense at the highest bleach concentration used, 50 %, with exposure time of 40 s, a condition that led to partial or total loss of spicules. Possibly, these alterations of the cell wall may be associated with losses of viability of the basidiospores observed at this concentration, given the importance of this structure for the performance of functions that are vital to the physiology of the fungus, such as protection against changes in the osmolarity conditions of the medium and in the cellular signaling (Albertsheim & Prouty, 1975;Schoff elmer et al., 1999;Bowman & Free, 2006;Gow et al., 2017).
The cell wall of P. microcarpus was similar to that of P. tinctorius (Mims, 1980), with a cell wall consisting of four layers, the outermost, L 1 , electrondense with spicule-like ornamentations on its surface, followed by an electron-transparent inner layer, L 2 . The third layer, L 3 , is also electron-dense, and the innermost layer, L 4 , is electron-transparent. The combination of the methodologies used by Mims and Thurston (1979) and Webster (2007) with microwave processing allowed the preparation and observation of non-permeabilized basidiospores. In the process of basidiosporogenesis, the outermost layer of cell wall develops fi rst and the other layers progress as the basidiospore matures (Mims, 1980;Campos & Costa, 2010b). As the layers of the cell wall are deposited, the basidiospores of P. tinctorius become increasingly diffi cult to be prepared for transmission electron microscopy analysis (Mims, 1980), due to the reduction of wall permeability. Similarly, mature basidiospores of P. microcarpus are impermeable to stains, making it diffi cult to observe nuclei or reserve materials by light or fl uorescence microscopy (Costa, 2002;Campos & Costa, 2010a;Campos & Costa, 2010b;Godinho, 2011). The data obtained in this study constitute the fi rst report of the structural conformation of the cell wall of P. microcarpus basidiospores.
After permeabilization with 5 % bleach for 40 s, 80 % of the permeabilized spores were viable. These data diff er from those of Godinho (2011), who obtained maximum viability (80 %) with bleach at a concentration equivalent to 2.5 % for 40 s. The results obtained show the need to standardize the permeabilization procedure for each basidiocarp to be used experimentally.
The fi rst colonies of P. microcarpus from the basidiospores with permeabilized cell wall appeared between the 15th and 18th day of incubation of the basidiospores in the presence of the host plant C. citriodora. In previous studies on in vitro germination of non-permeabilized Pisolithus spp. basidiospores, in the presence of diff erent host plants, fungal colonies appeared between 16 and 70 days of incubation (Kope & Fortin 1990;Carvalho et al., 1997;Costa, 2002;Pereira, 2004). Possibly, this variation is due to the diff erent cultivation conditions used. Fungal colonies emerged close to the roots of the host plant, indicating that germination is dependent on the release of stimulating compounds by the root (Fries, 1981;Carvalho et al., 1997;Costa, 2002;Pereira, 2004).
After the treatment of permeabilization with bleach, some basidiospores preserved the capacity to germinate. However, although the emergence of fungal colonies was observed after two weeks, the percentage of germination was low, 0.001 %. This value is similar to those reported by Costa (2002) and Pereira (2004) and indicates that the permeabilization treatment alone was not able to improve the germination percentages of P. microcarpus basidiospores.

CONCLUSIONS
Basidiospores from diff erent basidiocarps have diff erent susceptibilities to the treatment of permeabilization with bleach, highlighting the need to adjust the protocol for each fungal fruiting body. The treatment of basidiospores with bleach causes changes in the ultrastructure of the outermost layer of the cell wall, reducing or eliminating spicules. It was possible to obtain fungal colonies from the germination of permeabilized basidiospores, but the germination percentage was low, indicating that other factors besides the permeability of the spore wall are fundamental for the germination process.

AUTHOR CONTRIBUTIONS
MDC, JES, MRT, and MAMS conceived and planned the experiments. MAMS conducted the experiments and wrote the manuscript with inputs from MDC, JES, and MRT. JMN prepared fi gures and provided inputs to the discussion. All authors provided critical feedback on the manuscript.

ACKNOWLEDGMENTS
The authors express their gratitude to Fundação de Amparo à Pesquisa do Estado de Minas Gerais, FAPEMIG (Project number APQ-00288-08), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Capes, (Finance code 001), and Conselho Nacional de Desenvolvimento Científi co e Tecnológico, CNPq, for providing funds as well as graduate and research scholarships for the conduction of this work.