Cereal grains and glycerol in Agaricus blazei cryopreservation / Grãos de cereais e glicerol na criopreservação de Agaricus blazei

Agaricus blazei is an important commercial basidiomycete studied for its biological activity. However, little has been studied about the preservation techniques of this basidiomycete, and cryopreservation is one alternative. The aim of this study was to evaluate the physical and chemical effects of different cultivation media and glycerol on A. blazei cryopreservation at -20 ºC and at -70 ºC. A solid cultivation medium consisting of agar with ground whole cereal grains (hard endosperm wheat, medium hard endosperm wheat or hard endosperm rye) or malt extract agar, or whole cereal grains (hard endosperm wheat, medium hard endosperm wheat or hard endosperm rye) without agar was used. Cultivation media had zero or 5% glycerol addition. Cultivation medium grains or disks containing mycelia were transferred to cryotubes with glycerol at 5% with or without water and then were cryopreserved at -20 ºC or at -70 ºC. After one-year or two-year cryopreservation, fungus viability was evaluated. The results showed that the physical structure of whole grains of hard and medium hard wheat is effective (p≤0.01) for two-year cryopreservation at -70 ºC as well as the use of glycerol in the cultivation medium or as a cryoprotectant (p≤0.01) at -70 ºC. Cryopreservation at -20 ºC was ineffective (p≤0.01) to preserve fungus viability.

Agaricus blazei é um basidiomiceto de importância comercial amplamente estudado quanto à sua atividade biológica. Entretanto, pouco foi estudado sobre as técnicas de preservação deste basidiomiceto, sendo a criopreservação uma alternativa. O objetivo deste trabalho foi avaliar os efeitos físicos e químicos de diferentes meios de cultivo e do glicerol na criopreservação de A. blazei à -20 ºC e à -70 ºC. Foi usado meio de cultivo sólido composto por ágar com farinha de grãos inteiros de cereais (trigo textura dura ou semidura ou centeio textura dura) ou ágar extrato de malte, ou grãos de cereais inteiros (trigo textura dura ou semidura ou centeio textura dura) sem ágar. Os meios de cultivo tiveram adição de glicerol (zero ou 5%). Grãos ou discos de meio de cultivo contendo o micélio foram transferidos para criotubos com glicerol a 5%, ou com ou sem água, sendo, em seguida, criopreservados (-20 ºC ou -70 ºC). Após um ou dois anos de criopreservação foi avaliada a viabilidade do fungo. Os resultados mostraram que a estrutura física de grãos inteiros de trigo semiduro ou duro é efetiva (p≤0,01), quando em -70 ºC para criopreservação por dois anos, assim como o uso de glicerol tanto no meio de cultivo ou como solução crioprotetora (p≤0,01). A criopreservação a -20 ºC mostrou-se ineficaz (p≤0,01) na manutenção da viabilidade do fungo.

Cryopreservation of Agaricus blazei in liquid nitrogen using DMSO as cryoprotectant / Criopreservação de Agaricus blazei em nitrogênio líquido usando DMSO como crioprotetor

The preservation of Agaricus blazei is generally done using successive subcultivations that are laborious and are subject to contaminations or genetic degenerations, resulting in loss of biotechnological interest characteristics. An alternative process would be cryopreservation, but there are no reports of methodologies for this basidiomycete in liquid nitrogen. Thus, the objective of this study was to evaluate mycelial viability of A. blazei strains after cryopreservation in liquid nitrogen in order to establish the initial parameters of species preservation. Five strains grown on malt extract agar (MEA) were used. Disks of MEA containing A. blazei mycelium were transferred for screwcap cryovials containing the cryoprotectant, 10% dimethyl sulfoxide. Then, they were cooled at 8 ºC for 30 min and kept at -196 ºC with liquid nitrogen. After 1.5 year of cryopreservation, the cryovials were thawed in water bath at 30 ºC for 15 min. The disks with mycelia were transferred to MEA culture media without cryoprotectant and kept at 28 ºC for 30 days. A. blazei strains respond differently to the cryopreservation method at -196 ºC by varying mycelial viability recovery. Cryopreservation with liquid nitrogen, using dimethyl sulfoxide as cryoprotectant, is not the most appropriate one for A. blazei preservation.

A preservação de Agaricus blazei ocorre usualmente por subculturas sucessivas que são laboriosas e estão sujeitas a contaminações ou degenerações genéticas, com eventual perda das características de interesse biotecnológico. Uma alternativa a este processo é a criopreservação, no entanto não existem relatos de metodologias para este basidiomiceto com uso de nitrogênio líquido. Desta forma, o objetivo deste trabalho foi avaliar a criopreservação em nitrogênio líquido de linhagens de A. blazei, visando estabelecer os parâmetros iniciais de criopreservação para a espécie. Foram utilizadas cinco linhagens do fungo crescidas em meio de ágar-extrato de malte (MEA). Os discos de MEA contendo o micélio crescido foram transferidos em temperatura ambiente (25 ºC) para criotubos com rosca contendo o agente crioprotetor dimetilsulfóxido a 10%. Em seguida foram resfriados a 8 ºC por 30 min e mantidos a -196 ºC com nitrogênio líquido. Após 1,5 anos de criopreservação os criotubos foram descongelados por imersão em água a 30 ºC por 15 min. Os discos com micélio foram transferidos para meio de cultura MEA, sem o agente crioprotetor, e mantidos por 30 dias a 28 ºC. As linhagens de A. blazei respondem de forma diferente ao processo de criopreservação a -196 ºC com variação na recuperação da viabilidade do micélio. A criopreservação em nitrogênio líquido de A. blazei, com dimetilsulfóxido como crioprotetor, não é a forma mais adequada para a preservação desta espécie.

Production flush of Agaricus blazei on Brazilian casing layers

This study aimed to verify the biological efficiency and production flushes of Agaricus blazei strains on different casing layers during 90 cultivation days. Four casing layers were used: mixture of subsoil and charcoal (VCS), lime schist (LSC), São Paulo peat (SPP) and Santa Catarina peat (SCP); and two genetically distant A. blazei strains. The fungus was grown in composted substratum and, after total colonization, a pasteurized casing layer was added over the substratum, and fructification was induced. Mushrooms were picked up daily when the basidiocarp veil was stretched, but before the lamella were exposed. The biological efficiency (BE) was determined by the fresh basidiocarp mass divided by the substratum dry mass, expressed in percentage. The production flushes were also determined over time production. The BE and production flushes during 90 days were affected by the strains as well as by the casing layers. The ABL26 and LSC produced the best BE of 60.4 percent. Although VCS is the most used casing layer in Brazil, it is inferior to other casing layers, for all strains, throughout cultivation time. The strain, not the casing layer, is responsible for eventual variations of the average mushroom mass. In average, circa 50 percent of the mushroom production occurs around the first month, 30 percent in the second month, and 20 percent in third month. The casing layer water management depends on the casing layer type and the strain. Production flush responds better to water reposition, mainly with ABL26, and better porosity to LSC and SCP casing layers.