Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

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.

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.

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.

Growth inhibition and morphological alterations of Trichophyton rubrum induced by essential oil from Cymbopogon winterianus Jowitt ex Bor

Trichophyton rubrum is one of the most common fungi causer of dermatophytosis, mycosis that affect humans and animals around the world. Researches aiming new products with antifungal activity become necessary to overcome difficulties on treatment of these infections. Accordingly, this study aimed to investigate the antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte T. rubrum. The antifungal screening was performed by solid medium diffusion method with 16 T. rubrum strains, minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined using the microdilution method. The effects on mycelial dry weight and morphology were also observed. Screening showed essential oil in natura inhibited all the tested strains, with inhibition zones between 24-28 mm diameter. MIC50 and MIC90 values of the essential oil were 312 µg/mL for nearly all the essayed strains (93.75 percent) while the MFC50 and MFC90 values were about eight times higher than MIC for all tested strains. All tested essential oil concentrations managed to inhibit strongly the mycelium development. Main morphological changes on the fungal strains observed under light microscopy, which were provided by the essential oil include loss of conidiation, alterations concerning form and pigmentation of hyphae. In the oil presence, colonies showed folds, cream color and slightly darker than the control, pigment production was absent on the reverse and with evident folds. It is concluded that C. winterianus essential oil showed activity against T. rubrum. Therefore, it could be known as potential antifungal compound especially for protection against dermatophytosis.

Vegetative compatibility and heterokaryon formation between different isolates of Colletotrichum lindemuthianum by using the nit mutant system

Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability.

Kinetic analysis of batch ethanol acetylation in isothermal non-stationary multiphase systems by lyophilized mycelium of Aspergillus oryzae

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.

New hosts of Myrothecium spp. in Brazil and a preliminary in vitro assay of fungicides

Myrothecium roridum and M. verrucaria are two plant pathogenic species causing foliar spots in a large number of cultivated plants. This paper aims to study the causal agents of foliar spots in vegetable crops (sweet pepper, tomato and cucumber), ornamental plants (Spathiphyllum wallisii, Solidago canadensis, Anthurium andreanum, Dieffenbachia amoena) and a solanaceous weed plant (Nicandra physaloides). Most of the isolates were identified as M. roridum; only the isolate 'Myr-02' from S. canadensis was identified as M. verrucaria. All the isolates were pathogenic to their original plant hosts and also to some other plants. Some fungicides were tested in vitro against an isolate of M. roridum and the mycelial growth recorded after seven days. Fungicides with quartenary ammonium, tebuconazole and copper were highly effective in inhibiting the mycelial growth of M. roridum. This paper confirms the first record of M. roridum causing leaf spots in sweet pepper, tomato, Spathiphyllum, Anthurium, Dieffenbachia and N. physaloides in Brazil. We also report M. roridum as causal agent of cucumber fruit rot and M. verrucaria as a pathogen of tango plants.

Comparison of methodologies for conidia production by Alternaria alternata from citrus / Comparação de metodologias para produção de conídios por Alternaria alternata do citros

Conidia production is a problem in the study of Alternaria alternata from citrus. Thus, this study aimed to compare existing methodologies for conidial production of A. alternata isolated from Ponkan tangerine (2 isolates), Cravo lemon (1 isolate), Pêra orange (2 isolates) and Murcott tangor (1 isolate). The methodologies used were conidia production with 12 and 24 hours under white fluorescent light, evaluation with 24 and 48 hours after applying fungal mycelium stress technique, cold stress followed by injury of mycelium and evaluation with 24 hours, using healthy vegetable tissue and the use of black fluorescent near ultraviolet (NUV) lamp. Satisfactory result was obtained with A. alternata isolate from Murcott tangor, with the production of 2.8 x 10(5) conidia mL-1, when fungal mycelium was stressed (Petri dish with 66.66 percent of fungi growth) and subsequently 24 h of growth. The use of white light (24 h) and black fluorescent NUV lamp also induced expressive conidia production by one isolate of Ponkan tangerine, which produced 17.2 x 10(5) and 10.1 x 10(5) conidia mL-1 and another of Murcott tangor, which produced 13.9 x 10(5) and 10.1 x 10(5) conidia mL-1, respectively. The remaining methodologies analyzed in this study were not able to induce conidia production in satisfactory quantity. The use of both mycelium stress technique and white light (24 h) and black fluorescent NUV lamp allowed the production of enough quantities of conidia to be used in vitro (detection of fungitoxic substances)and in vivo (pathogenicity test)assays, respectively.

A produção de conídios consiste em problema no estudo de Alternaria alternata do citros. Assim, este estudo objetivou comparar metodologias existentes para a produção de conídios de A. alternata por dois isolados de tangerina Ponkan, um de limão Cravo, dois de laranja Pêra e um de tangor Murcott. As metodologias empregadas foram a produção de conídios com 12 e 24 horas sob luz branca, avaliação com 24 e 48 horas após estressamento do micélio do fungo, choque térmico com imediato estressamento do micélio e avaliação com 24 horas, produção de conídios pelo emprego de tecido vegetal sadio e o emprego de luz negra ultravioleta. Produção satisfatória de conídios foi obtida com o isolado de A. alternata de tangor Murcott, a qual foi de 2,8 x 10(5) conídios mL-1, mediante emprego da técnica de estressamento da colônia e cultivo do fungo por 24 horas. Os empregos de luz branca (24 h) e negra ultravioleta promoveram expressiva produção de conídios por um isolado de tangerina Ponkan, a qual foi de 17,2 x 10(5) e 10,1 x 10(5) conidios mL-1 e por outro de tangor Murcott, a qual foi de 13,9 x 10(5) e 10,1 x 10(5) conídios mL-1, respectivamente. As outras metodologias analisadas neste estudo não foram capazes de induzir a produção de conídios em quantidade satisfatória. Com o emprego das técnicas de estressamento do micélio e a utilização de luz branca (24 h) e negra ultravioleta, tornou-se possível obter quantidades de conídios suficientes para serem utilizadas em testes in vitro (detecção de substâncias fungitóxicas)e in vivo (testes de patogenicidade), respectivamente.

Supplementation of sugarcane bagasse with rice bran and sugarcane molasses for shiitake (Lentinula edodes) spawn production

The purpose of this study was to assess the myceliation rate, mycelial vigor and "estimated biomass" of Lentinula edodes (Berk.) Pegler, grown on a sugarcane bagasse substrate enriched with rice bran and sugarcane molasses for spawn production. The proportions of rice bran used were 0, 10, 15, 20, 25, 30 and 40 percent (dry weight/dry weight of bagasse) and the sugarcane molasses concentrations tested were 0, 10, 20, 30, 40, 50 and 60 g/kg (dry weight/dry weight of bagasse plus rice bran). The myceliation rate was decreased by the addition of the higher quantities of rice bran. The 25 and 30 percent rice bran proportions induced the highest stimulation of mycelial vigor. The addition of sugarcane molasses did not change myceliation rate or mycelial vigor. The "estimated biomass" values were similar when intermediate rice bran proportions were used and for all sugarcane molasses concentrations. Based on response surface obtained for the "estimated biomass" data, higher values were obtained with substrates containing 20 to 25 percent rice bran combined with 10 to 30 g sugarcane molasses, although the latter supplement was not considered to stimulate L. edodes growth.