Growth and Tolerance of Pleurotus ostreatus at Different Selenium Forms

Selenium is an important element in physiological and metabolic processes. Due to low Se concentration in most of the soils, strategies as enrichment and biofortification have been used to increase its incorporation in food. The fungus has capacity to absorb, accumulate and transform Se inorganic into organic compounds. However, the concentration and chemical forms of Se used for enrichment can affect the mycelial growth and mushrooms production. Thus, the aim of this study was to analyze the capacity of Pleurotus ostreatus in absorb, accumulate and tolerate growing concentrations of different Se chemical forms. In the disc of agar with mycelium was added 20 mL of PDA medium and Se concentration (0-200 mg L) in the forms of sodium selenite, sodium selenate or selenomethionine (SeMet). The greatest inhibition of mycelial growth and biomass production were observed in highest Se concentration. Regardless of the Se level, SeMet and sodium selenite were more harmful to the P. ostreatus growth than sodium selenate. However, the highest Se accumulation in the mycelium was observed in culture medium with sodium selenite. Thus, Se supplementation in the forms of sodium selenite was more indicated to enrichment of P. ostreatus mushrooms than sodium selenate and SeMet.


Introduction
Selenium (Se) is an essential element to human health.It has antioxidant function and is an immunosystem modulator, and have participation in biosynthesis of ubiquinone, ATP and proteins (Viaro et al., 2001).In the case of chronic diseases such as cancer, cardiovascular, oxidative stress or inflammatory conditions, the Se can act as protective element (Rayman, 2000).However, the Se concentration in food is low (Ferreira et al., 2002).Therefore, enrichment or biofortification have been strategies used to increase the concentration and availability of this element in food (da Silva et al., 2012;Silva et al., 2010;Solovyev et al., 2018).
The Se-enrichment of mushrooms using agriculture residue has been an alternative for accumulation of Se and production of organic Se (da Silva et al., 2012;Falandysz, 2008;Fang et al., 2018;Hu et al., 2018;Nunes et al., 2012;Solovyev al., 2018).Furthermore, mushrooms are much appreciated food, which contain protein, essential aminoacids, fibers, fatty acid and minerals (Manzi et al., 1999).This food is indicated to feeding of people with malnutrition problem (Kane et al., 2017).
The high Se concentration can inhibit the mycelial growth and production of Pleurotus ostreatus mushrooms (Da Silva et al., 2012Silva et al., , 2013)).Therefore, for Se-enrichment of mushrooms it would be interesting to investigate the effect of different Se chemical forms on growth and morphology of mycelium of P. ostreatus and determine the best concentration to be used for enrichment.Furthermore, P. ostreatus is one of the most produced and consumed mushrooms in the world (Azevedo et al., 2012;Furlani & Godoy, 2007) that shows its economic and nutritional importance.
Thus, the aim of this study was to analyze the capacity of Pleurotus ostreatus in absorb, accumulate and tolerate growing concentrations of different chemical forms of Se.

Microorganisms
The isolate of P. ostreatus (PLO 02) of the Department of Microbiology of the Federal University of Viçosa/BIOAGRO, MG, Brazil was used in this study.This isolate was also used in enrichment of mushrooms with selenium, lithium and zinc (da Silva et al., 2012;de Assunção et al., 2012;Vieira et al., 2013).
For inoculum production, this isolate was grown in a Petri dish containing potato dextrose agar (PDA) culture medium, pH 5.8, and incubated at 25±2 ºC for seven days.

Enrichment of P. ostreatus
Two assays were done.In the first test, P. ostreatus was inoculated in PDA media containing 12.5; 25; 50 or 75 mg L -1 of Se, as sodium selenite (Na 2 SeO 3 ), sodium selenate (Na 2 SeO 4 ) or selenomethionine (SeMet).These concentrations were obtaining on previous studies (da Silva et al., 2012Silva et al., , 2013;;Silva et al., 2010).In the second, the fungus was inoculated in PDA with 25, 50, 100, 150 and 200 mg L -1 of Se, as Na 2 SeO 4 .In both tests, the control was PDA without Se.The cultures were incubated for seven days at 25±2 ºC.

Mycelial Growth Velocity and Biomass
The mycelial growth was verified by taken two measurements, perpendicular to each other, at seventh days.The growth rate was calculated by the ratio between colony diameter and time of incubation.This diameter was measured by taken two measurements, perpendicular to each other at seven days.
To evaluate the mycelium dry biomass, all the content of Petri dish was transferred to a flask with 200 mL of distilled water and boiled, in microwave oven, to liquefy agar (Da Silva et al., 2012), followed by filtration and rinsing in distilled water.The retained mycelium was dried at 60 ºC, until constant weight.

Hyphae Diameter and Distance Between Septa
Theses parameters were measured after stained with calcofluor and observed under epifluorescence microscopy (Olympus BX 50).The images were capture by digital camera FUJIX HC-300Z and processed with the software Image Pro Plus.

Selenium Determination in Mycelium
All solutions were prepared from analytical reagent grade chemicals using high-purity deionized water obtained from a Milli-Q water purification system (Millipore, Belford, USA).Selenium analytical solution (Na 2 SeO 3 ) of 1000 mg L -1 was used as standard and its determination by Graphite furnace atomic absorption spectrometry (GF AAS).In the last case, a volume of 10 µL of chemical modifier solution of 5 mg of palladium and 3 mg of magnesium was co-injected with 10 µL of samples or analytical solutions into the graphite furnace.

Statistics
The experiment was a completely randomized design, with three replicates.The assay was for twice.The data were subjected to analysis of variance and mean values were compared by Tukey's test (p < 0.05).

Results and Discussion
Selenium affect the color of colony and the mycelial growth rate (Figures 1 and 2).The change of color from white to orange (Figure 1), mainly in culture medium with sodium selenite, may be due to the precipitation of selenium elemental.In anaerobic conditions, it occurs the reduction of selenite in selenium (Sylvia et al., 1999).Therefore, during P. ostreatus growth in the culture medium may have occurred the anaerobiosis microsites that favor the selenite reduction.Furthermore, the changing in the color of colony is due to metabolites production in Figure 2.
Figure 3 sodium se