Cryopreservation at −75 °C of Agaricus subrufescens on wheat grains with sucrose

Abstract

Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at −20 °C and at −75 °C for one and two years. Zero, 10% or 20% sucrose was added to potato dextrose agar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrose solutions at 15%, 30% or 45%. After one or two years at −75 °C or at −20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at −20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at −75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at −75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potato dextrose agar is used and unaffected when wheat grain is used after two-year cryopreservation at −75 °C. The fungus genome does not show alteration after two-year cryopreservation at −75 °C.

Semisolid culture medium improves mycelial recovery of Agaricus subrufescens cryopreserved in cereal grains

“…The solid culture medium, conventionally utilized in the recovery of cryopreserved A. subrufescens mycelial viability [13,[16][17][18][19]25], was not effective to recover the mycelial viability (< 75%) of all treatments after 5-year cryopreservation of the fungus (Fig. 1).…”

“…No reports were found for studies evaluating the effect of agar concentration in the culture medium to recover the mycelial viability after cryopreservation. The most utilized culture media to recover basidiomycetes contain from 8 to 20 g L −1 agar such as PDA [13,19,29], MEA [16][17][18], wort agar [14,20], and MGL1 (barley flour, yeast extract, sucrose, and agar) [30]. This makes the comparison between studies difficult, besides the number of different variables involved.…”

“…Most basidiomycete cryopreservation studies evaluate the cryopreservation effect of temperature [12][13][14], substrates for fungal growth before cryopreservation [15][16][17], class and concentration of cryoprotectants [18,19], and maintenance period [18,[20][21][22]. However, few studies assess the effect of cereal grains for cryopreservation and the consistency of recovery culture medium on the mycelial viability after cryopreservation.…”

The Latest Trends in Cryopreservation of Fungi

“…Typically, sterile wheat grains were inoculated on agar discs containing mycelium and kept at 25°C until most of the grains in the tube were colonized with mycelium. Mycelium-grown grains were then used for the freezing process in cryotubes or closed polypropylene straws [18][19][20]35]. Some papers describe cryopreservation without the use of a carrier, where agar discs with grown mycelium were frozen directly in the medium in ampoules or cryotubes [17,18,36].…”

“…A total of 63 genera of fungi (29 genera of basidiomycetes and 34 genera of non-basidiomycete fungi) have been studied in the aforementioned publications from 2017 to 2023 (Table 2). In recent years, the focus has been mainly on ectomycorrhizal species of basidiomycetes [12][13][14]17], which are often more cryosensitive than other basidiomycetes and and thus harder to store at low temperatures; then on to medicinal and edible fungi [18][19][20]. The most often studied and cryopreserved basidiomycete genus was Agaricus, which is widely cultivated around the world, especially in the US, Brazil and in European countries [16] followed by another frequently studied genera such as Amanita, Hebeloma, Kobayasia, Lactarius, Lentinula, Pleurotus, Rhizopogon, Sarcodon, Suillus and Tricholoma (see Table 2).…”