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.

Abstract Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.

ABSTRACT Gallesia integrifolia (Phytolaccaceae) is native to Brazil and has a strong alliaceous odor. The objective of this study was to identify the chemical composition of G. integrifolia fruit essential oil and evaluate fungicidal activity against the main food-borne diseases and food spoilage fungi. The essential oil was extracted by hydrodistillation and identified by GC–MS. From 35 identified compounds, 68% belonged to the organosulfur class. The major compounds were dimethyl trisulfide (15.49%), 2,8-dithianonane (52.63%) and lenthionine (14.69%). The utilized fungi were Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceus, Aspergillus versicolor, Penicillium funiculosum, Penicillium ochrochloron, Penicillium verrucosum var. cyclopium, and Trichoderma viride. Minimal fungicidal concentration for the essential oil varied from 0.02 to 0.18 mg/mL and bifonazole and ketoconazole controls ranged from 0.20 to 3.50 mg/mL. The lower concentration of the essential oil was able to control P. ochrochloron, A. fumigatus, A. versicolor, A. ochraceus and T. viride. This study shows a high fungicidal activity of G. integrifolia fruit essential oil and can support future applications by reducing the use of synthetic fungicides.

Anthracnose, caused by Colletotrichum sublineolum Henn. ex Sacc. & Trotter, is one of the most important sorghum [Sorghum bicolor (L.) Moench] diseases in Brazil. This fungus exhibits conidial dimorphism, producing either falcate or oval conidia on solid and liquid media, respectively. We compared patterns of the initial infection events by these two types of conidia on sorghum leaves using light microscopy and scanning electron microscopy. The infection events during the first 24 h were similar for both oval and falcate conidia. Globose and melanized apressoria were formed at 24 h after inoculation (hai) regardless of the conidia type. Dense mycelium and oval conidia developed from germinated falcate conidia at 32 hai. Hyphal mass displaying acervuli filled with falcate conidia and surrounded by setae, developed from germinated oval conidia at 48 hai. Oval conidia were as capable as falcate conidia of infecting sorghum leaves. The inherent ability to grow faster and the easeness with which oval conidia can be produced in vitro as compared to falcate, make the former a preferred choice for studies on the C. sublineolum-sorghum interaction. It would be instructive to further investigate the potential role of the oval conidia in epidemics.

O uso da biobalística na transformação genética de plantas requer a otimização de diversos parâmetros, entre eles, o desenvolvimento de protocolos para regeneração de plantas a partir de células transformadas. Este trabalho teve como objetivo avaliar a eficiência de regeneração de plantas de milho a partir de calos embriogênicos e de explantes sementes divididas, submetidos à transformação por biobalística, utilizando o gene bar como marcador de seleção. Foram submetidos 2000 agrupamentos de calos embriogênicos de milho e 2000 explantes sementes divididas à transformação por biobalística. A seleção dos eventos de transformação foi realizada em meios de cultivo suplementados com glifosinato de amônio. As plântulas selecionadas foram aclimatadas e transplantadas para vasos em casa de vegetação. Com a utilização de embriogênese somática, a eficiência de regeneração foi de 0,2%, enquanto que, com a utilização de sementes divididas, a eficiência de regeneração foi de 1,3%. Embora ambos os protocolos possibilitem a regeneração de plantas de milho submetidas à transformação por biobalística, há a necessidade de aumentar sua eficiência.

The use of biolistic on genetic transformation of plants requires an optimization of several parameters, including the protocol development for plant regeneration from transformed cells. The objective of this research was to evaluate the regeneration efficiency of maize plants from embryogenic callus and explants from split seeds submitted to transformation by biolistic using the gene bar as a selectable marker. It was submitted 2000 clusters of embryogenic callus and 2000 divided maize seeds to transformation by biolistic. The selection of transformation events was in culture medium supplemented with ammonium glifosinate. Selected seedlings were acclimatized and transplanted to pots in a greenhouse. Using somatic embryogenesis, the regeneration efficiency was 0.2%, while using split seeds explants the regeneration efficiency was 1.3%. Although both protocols allow the maize plant regeneration after transformation by biolistic, there is a need to increase its efficiency.

The essential oil of Lippia alba is reported as an antifungal against human pathogenic microorganisms but few articles report its use as an alternative to synthetic fungicides on green mould control. The objective of this study was to determine chemical characteristics of L. alba essential oil and its antifungal activity against green molds as an alternative to synthetic fungicides. Essential oil was extracted by Clevenger hydrodistillation, characterized by GC-MS analysis, and the structure of the main compounds confirmed by ¹H and 13C-NMR spectroscopy. Microdilution assays evaluated the essential oil minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Commercial fungicides Ketoconazole and Bifonazole were used as control. Essential oil yield is of 0.15% and the major components are neral (33.32%) and geranial (50.94%). The L. alba essential oil has MIC of 0.300-1.250 mg/mL and MFC of 0.600-1.250 mg/mL. Ketoconazole and Bifonazole show MIC ranging from 0.025-0.500 to 0.100-0.200 mg/mL, and MFC ranging from 0.250-0.100 to 0.200-0.250 mg/mL, respectively. L. alba essential oil is classified as citral type and the results indicate that it is a potential alternative to synthetic fungicides.

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%. 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% of the mushroom production occurs around the first month, 30% in the second month, and 20% 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.

Basidiomicetos são produtores de lacases utilizadas na hidrólise de subprodutos lignocelulósicos em processos fermentativos e pode ser utilizado na produção de biocombustíveis ou na alimentação de ruminantes. Avaliou-se o efeito da adição de fontes e concentrações de nitrogênio não-protéico na produção de lacase e no crescimento micelial de Pleurotus ostreatus, Lentinula edodes e Agaricus blazei. Os fungos foram cultivados em cascas de soja com adição de uréia (U), sulfato de amônio (AS) ou AS:U (1:1) de forma a manter a relação carbono/ nitrogênio (C/N) de 5, 15, 20 ou 30. O crescimento micelial longitudinal médio foi medido, e a atividade da lacase foi determinada pela oxidação do ácido 2,2'-azino-bis-3-etilbenzotiazolina-6-sulfônico. O crescimento micelial foi diretamente proporcional à relação C/N, enquanto a atividade de lacase foi inversamente proporcional. Os maiores valores de atividade foram obtidos para a relação C/N de 5. As melhores fontes de N para a produção de lacase por P. ostreatus, L. edodes e A. blazei foram, respectivamente, AS, AS:U e U. Relações C/N menores que 30 induziram a síntese de lacase e inibiram o crescimento micelial, proporcionando condições ideais para a pré-hidrólise de resíduos vegetais.

Basidiomycetes are laccase producers used for hydrolysis of lignocellulosic byproducts in fermentative processes and could be used on biofuel production or ruminant feeding. The objective of this study was to evaluate the effect of concentrations of non-protein nitrogen sources on laccase production and mycelial growth of Pleurotus ostreatus, Lentinula edodes and Agaricus blazei. The fungi were grown on soybean hulls to which urea (U), ammonium sulfate (AS) or mixture of AS:U (1:1) were added to achieve carbon/nitrogen (C/N) ratios of 5, 15, 20 or 30. The average longitudinal mycelial growth was measured and laccase activity was determined by the oxidation of 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid. Higher C/N ratios increased mycelial growth and decreased laccase production. The highest activities were obtained with a C/N ratio of 5. P. ostreatus, L. edodes and A. blazei produced more laccase when AS, AS:U and U, respectively, were added. In addition, C/N ratios lower than 30 induced laccase syntheses, inhibited mycelial growth and were a better condition for pre-hydrolysis of plant residues.

Different maturation phases of basidiocarp could affect the bioactivity and concentration of some active substances. A. brasiliensis Wasser et al.(A. blazei Murrill) has shown antitumor activity that could be related to the antioxidant activity. However there is no information of the best basidiocarp maturation phase for extracting antioxidant substances in order to determine the moment of harvesting in mushroom cultivation. The objective of this work was to evaluate the antioxidant activity of A. brasiliensis strains on different basidiocarp maturation phases. The best condition for extraction of A. brasiliensis antioxidants is with methanol as solvent at 60 ºC for 60 min. Strains with closed basidiocarp have higher antioxidant activity than with opened basidiocarp. Antioxidant activity varies in each strain. It was concluded that A. brasiliensis is a natural source of antioxidant compounds. Also there is higher antioxidant activity in closed than opened caps and consequently higher functional activity. It reinforces the synergic action among different A. brasiliensis compounds as a functional food and the importance of further investigation for isolation and characterization of antioxidant substances of A. brasiliensis. It also determines the best harvest period in order to obtain the highest antioxidant activity from basidiocarp.

A escolha da camada de cobertura é uma das mais importantes etapas do cultivo de Agaricus brasiliensis. Apesar dessa importância, poucos estudos relatam o uso de diferentes tratamentos térmicos para o controle da microbiota em camadas de cobertura alternativas. Assim, o objetivo deste trabalho foi avaliar o efeito da pasteurização e da autoclavagem do material alternativo calxisto para utilização como camada de cobertura no cultivo de A. brasiliensis. O fungo foi inicialmente crescido em grãos de trigo e transferido para meio de cultivo previamente compostado. Após a completa colonização, a camada de cobertura (calxisto) pasteurizada ou autoclavada foi adicionada. Avaliaram-se a eficiência biológica, o número e a biomassa de cogumelos produzidos e o fluxo de produção. Concluiu-se que a camada de cobertura com calxisto autoclavado reduzem o tempo de produção, a eficiência biológica e o número e a biomassa de cogumelos cultivados. Entretanto, a camada de cobertura com o calxisto pasteurizado é a mais eficiente para o cultivo de A. brasiliensis.

Casing layer choice is one of the most important phases on Agaricus brasiliensis cultivation. Besides the importance of it few studies report the use of different heat treatments to control the microbiota in alternative casing layers. Thus, the objective of this work was to evaluate the effect of pasteurized or autoclaved lime schist as an alternative casing layer on A. brasiliensis cultivation. The fungus was previously grown on wheat grains and transferred to a substratum previously composted. After substratum mycelium colonization a pasteurized or autoclaved lime schist casing layer was added on. It was evaluated the biological efficiency, the number and mass of produced mushroom and the production flush along cultivation. It was concluded that autoclaved lime schist casing layer decreases period of production, biological efficiency, number and mass of cultivated mushrooms. However pasteurized lime schist casing layer is the most efficient on A. brasiliensis cultivation.

As técnicas de cultivo do Agaricus brasiliensis (método de compostagem) e formulação de substrato são as mesmas utilizadas para o Agaricus bisporus. A maioria das relações carbono/nitrogênio (C/N) relatadas para A . brasiliensis são similares às usadas para A. bisporus no método de compostagem. Há poucos estudos sobre a variação da relação C/N para A. brasilienses para o método axênico. O objetivo deste trabalho foi verificar o efeito de diferentes relações C/N no crescimento micelial de Agaricus brasiliensis utilizando subprodutos regionais como substrato pelo método axênico. As relações C/N no substrato (misturas de fibra de soja e de mandioca) estudadas variaram de 11 a 248 com consequentes concentrações de nitrogênio de 4,25 a 0,20%, respectivamente. Concluiu-se que os substratos somente com fibra de soja propiciam maior crescimento micelial que qualquer mistura com fibra de mandioca; o crescimento micelial é maior no substrato com relação C/N de 11 (N = 4,25%), intermediário na faixa de relação C/N entre 15 e 50 (N = 3,31 e 0,98%) e menor na relação C/N de 100 ou superior (N < 0,50%).

Cultivation techniques for Agaricus brasiliensis (composting method) and substratum formulations are the same ones used for Agaricus bisporus. Most of the carbon-to-nitrogen (C:N) ratios reported for A. brasiliensis are similar to those used for A. bisporus on the composting method and there are few studies about the variation of C:N ratios for A. brasilienses on the axenic method. The objective of this study was to verify the mycelial growth of A . brasiliensis on different C:N ratios using regional by-products as substrate formulation on the axenic method. Studied C:N ratios of substrate (mixture of soybean and cassava fibers) ranged from 11:1 to 248:1, with nitrogen content ranging from 4.25 to 0.20%, respectively. It was concluded that substrate with only soybean fiber generates higher mycelial growth than any formulation with cassava fiber; the highest mycelial growth on substrate is with C:N ratio of 11:1 (N = 4.25%); the intermediate growth is with C:N ratio range from 15:1 to 50:1 (N from 3.31 to 0.98%); and the lowest growth is with C:N ratio of 100:1 or higher (N < 0.50%).

The fungus Agaricus brasiliensis is a Basidiomycete studied because of its immunomodulation and/or antitumor substances. The objective of this study was to verify the Agaricus brasiliensis antineoplasic activity in vivo on different basidiocarp maturation phases on Sarcoma 180 cells implanted in mice. Sarcoma cells were implanted in mice and after seven days mice were divided in three groups. The first group was treated with saline solution, the second group was treated with closed basidiocarp extract solution and the third group was treated with opened basidiocarp extract solution. After 30 days of being daily orally treated with these three solutions all animals suffered euthanasia, and the splenic index, tumor mass and volume were determined. No significant differences of the tumor growth inhibition in function of the different basidiocarp maturation phases for the Agaricus brasiliensis strain were observed. The in vivo basidiocarp antineoplasic average activity was 89.22%.

O objetivo deste trabalho foi determinar a influência de variáveis de processo na produção das enzimas poligalacturonase (PG) e polimetilgalacturonase (PMG) por cultivo semi-sólido utilizando bioreator de coluna. Um planejamento fatorial fracionário foi utilizado para determinar o efeito das variáveis "microrganismo" (Aspergillus oryzae e Aspergillus niger), "substrato" (farelo de trigo e farelo desengordurado de arroz), "aeração" (40 e 60 ml h-1 g-1), "pectina" (5 e 10 g g-1) e "nitrogênio" (uréia e sulfato de amônia) para a produção de PG. Microrganismo, aeração e pectina foram significantes (p<0,05) para a produção de PG. Utilizando-se um planejamento composto central com quadruplicata no ponto central concluiu-se que Aspergillus niger apresenta maior produção de PG, o substrato e a fonte de nitrogênio não afetam (p<0,05) a produção de PG, a aeração afeta positivamente a produção de PG e negativamente a de PMG, e a concentração inicial de pectina afeta positivamente a atividade de ambas enzimas. Os pontos ótimos de aeração e de concentração inicial de pectina para a produção de PG são 66,13 ml h-1 g-1 e 12,8 g g-1, respectivamente, e para a produção de PMG são 40 ml h-1 g-1 e 15,0 g g-1, respectivamente.

This study aimed to determine the influence of process variables and production, of polygalacturonase (PG) and polymetylgalacturonase (PMG) by solid substrate cultivation using a fixed bed column bioreactor. A fractional factorial design (FFD) was used to study the effect of the following variables: microorganism (Aspergillus oryzae and Aspergillus niger), substratum (wheat bran and defatted rice bran), aeration (40 and 60 ml h-1g-1), pectin (5 and 10 g g-1) and nitrogen (urea and ammonium sulfate). Microorganism, aeration and initial pectin were identified in FFD as significant variables (p<0.05) on PG production. A central composed design to optimize PG and PMG productions indicated that Aspergillus niger presents higher PG production; substratum and nitrogen do not affect PG production; the aeration rate affects positively the production of PG and negatively the production of PMG and the initial pectin concentration affects positively both PG and PMG production. The optimal point of aeration and initial pectin concentration for PG production are 66.13 ml h-1 g-1 and 12.8 g g-1, respectively and for PMG production are 40 ml h-1 g-1 and 15.0 g g-1, respectively.

Determinou-se a divergência genética entre cinco isolados do fungo Agaricus blazei pela técnica de RAPD. Dos cinco isolados três não apresentaram qualquer divergência, sendo caracterizados como isolados de uma mesma origem, embora obtidos em locais diferentes do país. Outros dois isolados mostraram-se diferentes do primeiro grupo e divergentes entre si.

The genetic divergence of five isolates of Agaricus blazei was determined based on RAPD data. Results indicate that there is little genetic variability among the commercialized strains and that RAPD is a feasible and low cost technique that can be used to characterize this fungus.