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.

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

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.(AU)

Micorrização e atividade microbiana de um solo cultivado com coentro e camomila / Mycorrhization and microbial activity from a soil cultivated with coriander and chamomile / Micorrización y actividad microbiana de suelo cultivado con manzanilla y cilantro

Fungos micorrízicos arbusculares (FMAs), entre outros micro-organismos do solo, desempenham papel fundamental no crescimento de plantas. O objetivo deste trabalho foi avaliar a densidade de esporos e a colonização radicular por FMAs em solos cultivados com coentro ( Coriandrum sativum L.) e camomila (Matricaria chamomilla L.), bem como a atividade microbiana do solo medida por meio da respiração basal do solo (RBS) e matéria orgânica do solo (MOS). Foram coletadas quatro amostras de solo e raízes no horto medicinal da Universidade Paranaense, Umuarama-PR e de uma área adjacente de mata nativa. A densidade de esporos de FMAs no solo cultivado com camomila foi três vezes maior (44,2 esporos g–1 solo) do que no solo cultivado com coentro (16,8 esporos g–1 solo) e de mata (9,9 esporos g–1 solo). A colonização radicular por FMAs não diferiu significativamente. O solo da mata apresentou maior atividade microbiana (2,0 mg CO 2 kg–1 solo h–1) comparado com os solos cultivados com camomila e coentro (1,56 e 1,13 mg CO 2 kg–1 solo h-1, respectivamente). O mesmo comportamento foi observado com a MOS. Conclui-se que o solo cultivado com camomila apresentou maior densidade de esporos de FMAs, comparado com os solos da mata e coentro. A maior atividade microbiana no solo da mata é possivelmente em virtude do maior fornecimento de matéria orgânica e ciclagem do carbono.(AU)

The arbuscular mycorrhizal fungi (AMFs), among other soil microbial microrganisms, has a fundamental role in plant growth. The objective of this study is to evaluate the spore density and root colonization by AMFs and microbial activity measuring basal soil respiration (BSR) and soil organic matter (SOM) from soil and root collected from coriander (Coriandrum sativum L.) and chamomile (Matricaria chamomilla L.). Four soil and root samples were collected in a medicinal botanical garden at the Paranaense University, Umuarama PR, and soil samples from a nearby forest was used as control. The spore density of AMF in the soil differed significantly among treatments. In the soil under chamomile, the amount of spores found was over three-fold the amount of spores found in relation to forest and soil cultivated with coriander, but soil under coriander and forest did not differ significantly. The assessment of root colonization by AMF was not significantly different among treatments. In soil from the forest, a higher soil microbial activity (2.0 mg CO 2 kg–1 solo h–1) was found when compared to soil cultivated with chamomile and coriander (1.56 and 1.13 mg CO 2 kg–1 soil h–1, respectively). The same behavior was observed with SOM. It can be concluded that the soil cultivated with chamomile showed higher spore density of AMF compared to coriander and forest. A higher forest soil microbial activity is possibly due to a major input of SOM ecarbon cycling.(AU)

Hongos micorrízicos arbusculares (HMA) y otros micro-organismos del suelo, desempeñan papel fundamental en el crecimiento de las plantas. El objetivo de este estudio fue evaluar la densidad de esporas y la colonización de las raíces por HMAs en suelos cultivados con cilantro ( Coriandrum sativum L.) y manzanilla (Matricaria chamomilla L.), así como la actividad microbiana del suelo medido por medio de la respiración basal del suelo (RBS) y materia orgánica del suelo (MOS). Se colectó cuatro muestras de suelo y raíces en el jardín de plantas medicinales de la Universidad Paranaense, Umuarama - PR, y de una zona adyacente de mata nativa. La densidad de esporas de HMAs en el suelo cultivado con manzanilla fue tres veces mayor (44,2 esporas g-1 de suelo) que en el suelo cultivado con cilantro (16,8 esporas g-1 de suelo) y de mata (9,9 esporas g-1 suelo). La colonización radicular por HMAs no difirió significativamente. El suelo de la mata presentó mayor actividad microbiana (2,0 mg CO 2 kg-1 suelo h-1) en comparación con los suelos cultivados con manzanilla y cilantro (1,56 y 1,13 mg CO2 kg-1 suelo h-1, respectivamente). El mismo comportamiento se observó con el MOS. Se concluye que el suelo cultivado con manzanilla presenta mayor densidad de esporas de AMFs, en comparación con los suelos de mata y cilantro. La mayor actividad microbiana en el suelo de mata es posiblemente en virtud de mayor oferta de materia orgánica y el ciclo del carbono.(AU)

Viabilidade de agaricus blazei em diferentes condições de preservação / Viabilidad de agaricus blazei en diferentes condiciones de preservación / Viability of agaricus blazei under different preservation conditions

A técnica de transferências periódicas de fragmentos de micélio para novo meio de cultura é a mais utilizada para a preservação de Agaricus blazei. Entretanto, esta técnica apresenta maior risco de contaminação, degeneração genética e perda de caracteristicas biológicas. O desenvolvimento de técnicas de preservação que permitam a manutenção da viabilidade da espécie por mais tempo e a um menor custo é de interesse biotecnológico. Desse modo, o objetivo deste trabalho foi avaliar a viabilidade de A. blazei crescido em dois meios de cultivo e preservado à +4 ºC ou -20 ºC em diferentes recipientes de contenção. O fungo foi crescido em meio de ágar-extrato de malte ou ágar-grão de trigo moído e preservado à +4 ºC ou -20 ºC em diferentes recipientes de contenção, simples ou duplos, com adição de soluções aquosas de glicerol, sacarose, glicose, água ultrapura ou sem adição de crioprotetor. Após 1 ou 12 meses o micélio preservado foi transferido para ágar-extrato de malte para avaliação da viabilidade micelial. Os crioprotetores glicerol, sacarose e glicose, associados com o meio de cultura ágar-extrato de malte ou ágar-grão de trigo moído, em recipiente de contenção simples ou duplo são efetivos para preservação à +4 ºC por períodos curtos, um mês, mas não são efetivos para períodos longos, 12 meses. Os crioprotetores, meios de cultivo e recipientes de contenção simples ou duplos não são efetivos para criopreservação do fungo à -20 ºC. Os recipientes simples são tão eficientes quanto os recipientes duplos para evitar contaminações e preservar o fungo.(AU)

Continuous mycelial subculturing is frequently used for the preservation of Agaricus blazei. However, this technique has a higher risk of contamination, genetic degeneration and loss of biological characteristics. The development of preservation techniques that allow maintaining the viability of this species longer and at lower costs is of biotechnological interest. Thus, the objective of this study was to evaluate the viability of A. blazei grown in two culture media and preserved at +4 ºC or -20 ºC in different containment vessels. The fungus was grown on malt extract agar or grounded wheat grain agar culture medium and preserved at +4 ºC or -20 ºC in different containment vessels, single or double ones, with the addition of aqueous solutions of glycerol, saccharose, glucose, ultrapure water or without addition of cryoprotectant. After 1 or 12 months, the preserved mycelium was transferred to malt extract agar for assessment of mycelial viability. Glycerol, saccharose and glucose associated with malt extract agar or grounded wheat grain agar culture medium, in single or double containment vessels, are effective for preservation at +4 ºC for a short period, one month, but they are not effective for a longer period, 12 months. Cryoprotectants, culture media and single or double containment vessels are not effective for fungus cryopreservation at -20 ºC. Simple containment vessels are as efficient as double ones to prevent contamination and to preserve the fungus.(AU)

La técnica de transferencias periódicas de fragmentos de micelio para nuevo medio de cultura es la más utilizada para la preservación de Agaricus blazei. Sin embargo, esta técnica presenta mayor riesgo de contaminación, degeneraciones genéticas y pérdidas de características biológicas. El desarrollo de técnicas de preservación que permitan la manutención y viabilidad de la especie por más tiempo y con un costo más bajo es de interés biotecnológico. De esta manera, el objetivo de este estudio fue evaluar la viabilidad de A. blazei sembrado en dos medios de cultivo y preservados en +4 ºC o -20 ºC en diferentes recipientes de contención. El hongo se cultivó en medio de extracto de agar de malta o agar de grano de trigo molido y preservado en +4 ºC o -20 ºC en diferentes recipientes de contención simple o doble, con adición de soluciones acuosas de glicerol, sacarosa, glucosa, agua ultra pura o sin adición de crioprotector. Después de 1 o 12 meses, el micelio preservado fue transferido para extracto de agar de malta para evaluación de la viabilidad del micelio. Los crioprotectores glicerol, sacarosa y glucosa, asociados con el medio de cultura extracto de agar de malta o de agar de grano de trigo molido, en recipiente de contención simple o doble son eficaces para la preservación a +4 ºC por períodos cortos, un mes, pero no son eficaces por períodos largos, como 12 meses. Los crioprotectores medios de cultivo y recipientes de contención simples o dobles no son eficaces para la criopreservación del hongo a -20 ºC. Recipientes simples son tan eficaces como los dobles para evitar contaminaciones y preservar el hongo.(AU)