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

This study aimed to evaluate the effects of the solid and semisolid culture medium on the mycelial viability of A. subrufescens after 5-year cryopreservation at - 70 °C. Mycelia were grown in three types of whole or ground grains, with or without 5% glycerol addition in the substrate and/or in a cryotube. After 5 years of cryopreservation at - 70 °C, every treatment was thawed and recovered in malt extract culture medium with 15 (solid culture medium) or 5 g L-1 (semisolid culture medium) of agar. The semisolid recovery culture medium increased the mycelial viability recovery capacity of A. subrufescens cryopreserved for 5 years in grains with glycerol only in the cryotube, and specifically with medium-hard wheat grain without glycerol addition at all. Agar-based substrates such as malt extract agar, agar-ground grain, or the one with glycerol addition to the substrate were not effective to keep the mycelial viability, regardless of the recovery culture medium consistency. Hard and medium-hard endosperm wheat grains or hard endosperm rye grains with addition of glycerol as cryoprotectant only to the cryotube were effective to cryopreserve the fungus for 5 years without cryoprotectant addition in the substrate.

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.

Comparative proteomics analysis of the rice roots colonized by Herbaspirillum seropedicae strain SmR1 reveals induction of the methionine recycling in the plant host.

Although the use of plant growth-promoting bacteria in agriculture is a reality, the molecular basis of plant-bacterial interaction is still poorly understood. We used a proteomic approach to study the mechanisms of interaction of Herbaspirillum seropedicae SmR1 with rice. Root proteins of rice seedlings inoculated or noninoculated with H. seropedicae were separated by 2-D electrophoresis. Differentially expressed proteins were identified by MALDI-TOF/TOF and MASCOT program. Among the identified proteins of H. seropedicae, the dinitrogenase reductase NifH and glutamine synthetase GlnA, which participate in nitrogen fixation and ammonium assimilation, respectively, were the most abundant. The rice proteins up-regulated included the S-adenosylmethionine synthetase, methylthioribose kinase, and acireductone dioxygenase 1, all of which are involved in the methionine recycling. S-Adenosylmethionine synthetase catalyzes the synthesis of S-adenosylmethionine, an intermediate used in transmethylation reactions and in ethylene, polyamine, and phytosiderophore biosynthesis. RT-qPCR analysis also confirmed that the methionine recycling and phytosiderophore biosynthesis genes were up-regulated, while ACC oxidase mRNA level was down-regulated in rice roots colonized by bacteria. In agreement with these results, ethylene production was reduced approximately three-fold in rice roots colonized by H. seropedicae. The results suggest that H. seropedicae stimulates methionine recycling and phytosiderophore synthesis and diminishes ethylene synthesis in rice roots.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.