Growth of the Ectomycorrhizal Fungus Pisolithus Microcarpus in different nutritional conditions

The most important plant species employed in reforestation programs depend on ectomycorrhizal fungi for their establishment and growth. The exploitation of this symbiosis to improve forest productivity requires fungal inoculants in a large scale level. To develop such a technology it is necessary to define the optimal composition of the culture medium for each fungus. With these objectives in mind, the effect of the composition of the culture medium on biomass production of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) was studied. The original composition of two culture media, already employed for cultivation of ectomycorrhizal fungi, was submitted to several variations with the C/N ratio as the main variable. A variation of the Pridham-Gottlieb medium was the most efficient for the production of biomass. Therefore, it was submitted to a factorial assay where glucose, peptone and yeast extract components were the factors analyzed. Results showed that the glucose concentration may be increased up to 40 % in order to promote higher biomass production. Peptone had a positive effect on this variable, whereas yeast extract promoted a deleterious effect. These results indicate that it is advisable to eliminate yeast extract from the medium and replace it with peptone prior to use.

Growth of the Ectomycorrhizal Fungus Pisolithus Microcarpus in different nutritional conditions

The most important plant species employed in reforestation programs depend on ectomycorrhizal fungi for their establishment and growth. The exploitation of this symbiosis to improve forest productivity requires fungal inoculants in a large scale level. To develop such a technology it is necessary to define the optimal composition of the culture medium for each fungus. With these objectives in mind, the effect of the composition of the culture medium on biomass production of the ectomycorrhizal fungus Pisolithus microcarpus (isolate UFSC-Pt116) was studied. The original composition of two culture media, already employed for cultivation of ectomycorrhizal fungi, was submitted to several variations with the C/N ratio as the main variable. A variation of the Pridham-Gottlieb medium was the most efficient for the production of biomass. Therefore, it was submitted to a factorial assay where glucose, peptone and yeast extract components were the factors analyzed. Results showed that the glucose concentration may be increased up to 40 % in order to promote higher biomass production. Peptone had a positive effect on this variable, whereas yeast extract promoted a deleterious effect. These results indicate that it is advisable to eliminate yeast extract from the medium and replace it with peptone prior to use.

Production of endoglucanase by the native strains of Streptomyces isolates in submerged fermentation

Cellulase is a complex enzyme system, commercially produced by filamentous fungi under solid-state and submerged cultivation. It has wide applicability in textile, food and beverage industry for effective saccharification process. In this study, cellulolytic enzyme activity, particularly endoglucanase of 26 Streptomyces strains isolated from garden soil was examined, including two isolates selected on the basis of potential cellulolytic activity on Bennett's agar medium. To enhance the endoglucanase formation in broth culture, different conditions including carbon and nitrogen sources, and growth conditions were tested. The maximum endoglucanase activity (11.25-11.90 U/mL) was achieved within 72-88 h in fermentation medium containing Tween-80, followed by phosphate sources. Both cellulolytic Streptomyces isolates gave almost equal quantity of enzyme in all trials. However the effect of medium ingredients on endoglucanase induction diverged with strains in some extent.

A celulase é um sistema enzimático complexo, produzido comercialmente a partir de fungos filamentosos através de cultivo em estádio sólido e submerso. Tem uma grande aplicação na indústria têxtil e de alimentos e bebidas no processo de sacarificação. Nesse estudo, examinou-se a atividade celulolítica, especialmente de englucanase, de 26 cepas de Streptomyces isoladas de solo, incluindo duas cepas selecionadas por sua atividade celulolítica no ágar Bennett. Para estimular a produção de englucanase em meio de cultura, diferentes condições de cultivo, incluindo fonte de carbono e nitrogênio e condições de crescimento, foram avaliadas. A atividade máxima de glucanase (11,25 a 11,90 U/mL) foi obtida em 72-88h em meio de cultura contendo Tween-80, seguido por fontes de fosfato. Ambas as cepas celulolíticas de Streptomyces produziram quase a mesma quantidade de enzima em todos os experimentos. Entretanto, o efeito dos ingredientes do meio na indução da glucanase divergiu de acordo com a cepa.

Viability and infectivity of an ectomycorrhizal inoculum produced in an airlift bioreactor and immobilized in calcium alginate

The viability and infectivity of an ectomycorrhizal inoculum (isolate UFSC-Rh90, Rhizopogon nigrescens), produced by submerged cultivation in an airlift bioreactor and immobilized in beads of calcium alginate gel, was studied. Inoculum remained 100% viable after 18 months in a 0.85% NaCl solution at 8 ± 1ºC. Mycelium grew from the beads after 48 h when they were placed on a solid culture medium at 25 ± 1ºC. Viability of pellets of non-immobilized mycelium stored under the same conditions decreased gradually after the third month of storage, reaching 0% by the 12th month. These pellets presented a gradual darkening, which was more intense in those located near the surface of the NaCl solution. In culture medium, these dark pellets showed no viability. Gel immobilization helps to maintain mycelium viability during storage and offers a physical protection when the inoculum is applied to the planting substrate. After eight months refrigeration, the immobilized inoculum was still able to infect Pinus taeda seedlings, colonizing an average of 37% of the root tips when inoculated in the plant growth substrate under greenhouse conditions. This inoculum presents a commercial potential to be produced and applied in forest nurseries.

Estudou-se a viabilidade e a infectividade de inoculante fúngico ectomicorrízico (isolado UFSC-Rh90, Rhizopogon nigrescens), produzido através de cultivo submerso em biorreator airlift e encapsulado em gel de alginato de cálcio. O inoculante permaneceu viável após 18 meses em solução de NaCl (0,85%) a 8 ± 1ºC. O micélio emergiu dessas cápsulas após 48 h de incubação a 25 ± 1ºC em meio de cultura sólido. A viabilidade dos pellets de micélio não imobilizado, armazenados sob as mesmas condições, reduziu-se gradualmente após três meses de armazenamento e atingiu 0% aos 12 meses. Esses pellets apresentaram um escurecimento gradual que foi mais intenso naqueles localizados próximos à superfície da solução de NaCl. Em meio de cultura, os pellets escurecidos mostraram-se inviáveis. A imobilização em gel mantém a viabilidade do micélio durante o armazenamento, além de oferecer uma barreira física quando aplicado ao substrato de plantio. Após oito meses de armazenamento sob refrigeração, o inoculante imobilizado colonizou uma média de 37% das raízes curtas de mudas de Pinustaeda, quando aplicado ao substrato de plantio sob condições de casa-de-vegetação. Esse inoculante apresenta potencial para produção comercial e aplicação nos viveiros florestais.

Metodologia para produção de Metarhizium anisopliae (METSCH.) sorokin em cultivo submerso: esporulação da biomassa, efeito da concentração de açúcar e custo do inoculant

A method of cultivation and a culture medium were developed aiming at the mass production of fungus Metarhizium anisopliae (Metsch.) Sorokin, 1883, with great concentration and purity of conidia. This method involved the M-61 strain of entomopathogenic fungus in liquid medium of rice, yeast extract, soybean bug extract (Nezara viridula (L., 1758) Hemiptera, Pentatomidae), under six differents concentrations of sugar (0, 2, 4, 6, 8, 10g l-1), and the solid conventional medium of rice grains. The biomasses obtained were filtered and put in an incubator to promote sporulation. The treatments were evaluated through the parameters wet and dry-weight of micelium, number of conidia per gram of substrate, viability and pathogenicity of conidia against the bug. It was observed that the sugar concentration of 2.0g l-1 in extract of N. viridula produced two times more conidia per gram of substrate when compared to 10.0g l-1, and 51 times cheaper than the conventional process of production of fungus. Viability was not affected by the different culture media. Significant differences did not occur in the pathogenicity among culture media and cultivations methods.

Desenvolveram-se um método de cultivo e um meio de cultura para produção massal do fungo Metarhizium anisopliae (Metsch.) Sorokin, 1883, com maior pureza e concentração de conídios. Este método envolveu o cultivo submerso da linhagem M-61 do entomopatógeno em meio líquido de arroz parboilizado, extrato de levedura, extrato do percevejo da soja (Nezara viridula (L., 1758) Hemiptera, Pentatomidae), sob seis diferentes níveis de concentração de açúcar (0, 2, 4, 6, 8, 10g l-1), além do meio convencional sólido de arroz em grão. As biomassas obtidas foram separadas através de tela de nylon (63 mesh) e dispostas em estufa para a esporulação. Os efeitos dos tratamentos foram avaliados pelos parâmetros pesos fresco e seco do micélio, número de conídios por grama de substrato, viabilidade e patogenicidade dos conídios sobre o percevejo. Observou-se que 2.0g l-1 de açúcar em meio de cultura de extrato de N. viridula produziu o dobro do número de conídios por grama de substrato em relação à concentração de 10.0g l-1, a um custo 51 vezes inferior ao obtido no processo convencional de produção do fungo. A viabilidade não foi afetada nos diferentes meios utilizados. Não ocorreram diferenças significativas na patogenicidade em função dos meios de cultura e métodos de cultivo.

Metodologia para produção de Metarhizium anisopliae (METSCH.) sorokin em cultivo submerso: esporulação da biomassa, efeito da concentração de açúcar e custo do inoculant

A method of cultivation and a culture medium were developed aiming at the mass production of fungus Metarhizium anisopliae (Metsch.) Sorokin, 1883, with great concentration and purity of conidia. This method involved the M-61 strain of entomopathogenic fungus in liquid medium of rice, yeast extract, soybean bug extract (Nezara viridula (L., 1758) Hemiptera, Pentatomidae), under six differents concentrations of sugar (0, 2, 4, 6, 8, 10g l-1), and the solid conventional medium of rice grains. The biomasses obtained were filtered and put in an incubator to promote sporulation. The treatments were evaluated through the parameters wet and dry-weight of micelium, number of conidia per gram of substrate, viability and pathogenicity of conidia against the bug. It was observed that the sugar concentration of 2.0g l-1 in extract of N. viridula produced two times more conidia per gram of substrate when compared to 10.0g l-1, and 51 times cheaper than the conventional process of production of fungus. Viability was not affected by the different culture media. Significant differences did not occur in the pathogenicity among culture media and cultivations methods.

Desenvolveram-se um método de cultivo e um meio de cultura para produção massal do fungo Metarhizium anisopliae (Metsch.) Sorokin, 1883, com maior pureza e concentração de conídios. Este método envolveu o cultivo submerso da linhagem M-61 do entomopatógeno em meio líquido de arroz parboilizado, extrato de levedura, extrato do percevejo da soja (Nezara viridula (L., 1758) Hemiptera, Pentatomidae), sob seis diferentes níveis de concentração de açúcar (0, 2, 4, 6, 8, 10g l-1), além do meio convencional sólido de arroz em grão. As biomassas obtidas foram separadas através de tela de nylon (63 mesh) e dispostas em estufa para a esporulação. Os efeitos dos tratamentos foram avaliados pelos parâmetros pesos fresco e seco do micélio, número de conídios por grama de substrato, viabilidade e patogenicidade dos conídios sobre o percevejo. Observou-se que 2.0g l-1 de açúcar em meio de cultura de extrato de N. viridula produziu o dobro do número de conídios por grama de substrato em relação à concentração de 10.0g l-1, a um custo 51 vezes inferior ao obtido no processo convencional de produção do fungo. A viabilidade não foi afetada nos diferentes meios utilizados. Não ocorreram diferenças significativas na patogenicidade em função dos meios de cultura e métodos de cultivo.