Avaliação do potencial biotecnológico de metabólitos de fungos da rizosfera da laranja no combate ao cancro cítrico
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Data
2021-12-08
Autores
Oliveira, Charles Muller Freire
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Universidade Estadual Paulista (Unesp)
Resumo
O cancro cítrico é uma doença causada pela bactéria Xanthomonas citri subsp. citri que ataca espécies de citrus de importância comercial. Para atenuar perdas, a atual forma de controle da doença envolve pulverização de compostos contendo metais pesados como cobre em sua composição. Tais medidas têm causado grande impacto ambiental devido ao acúmulo destes no solo e por toda a cadeia trófica. Por este motivo, a procura de metabólitos produzidos por micro-organismos tem se tornado crescente nos últimos anos. O objetivo deste trabalho é obter metabólitos obtidos de fungos e/ou extratos fúngicos com potencial atividade contra X. citri subsp. citri. Neste trabalho 16 extratos brutos provenientes do cultivo de fungos isolados da rizosfera da laranja Citrus sinensis foram submetidos a ensaios para avaliação do seu potencial inibitório contra Xanthomonas citri subsp. citri. Os metabolitos foram produzidos em malte 2% em uma temperatura média de 28⁰C à 120rpm por 20 dias. A fase líquida foi extraída usando acetato de etila. Os extratos foram concentrados e dissolvidos em Dimetilsulfóxido (DMSO). O inóculo bacteriano foi preparado utilizando meio NYG à 29⁰C em 200rpm overnight. O bioensaio antibacteriano foi realizado utilizando o ensaio de micro diluição seriada em microplaca de 96 poços, utilizando como indicador resazurina (REMA). A avaliação da atividade inibitória quantitativamente foi realizada utilizando espectrômetro de fluorescência, permitindo verificar a quantidade de células mortas. Dentre os extratos testados 4 apresentaram concentrações inibitórias satisfatória acima de 80%, sendo eles o LMA 1957, LMA 1923, LMA 1844 e LMA 1840. O extrato LMA 1957 apresentou concentrações inibitórias elevadas mesmo nas menores concentrações testadas e, portanto, foi a linhagem escolhida para os testes em planta e purificação onde, após reavaliação de sua atividade inibitória apresentou inibição em concentrações ainda mais baixas acima de 90% na concentração de 0,88µg/mL. Mesmo após o teste em planta em condições diferentes das encontradas no laboratório com análises in vitro e a purificação do extrato os resultados foram considerados satisfatórios. Sendo, no entanto, necessário realizar a identificação destes compostos e análise de toxicidade até a obtenção do resultado final.
Citrus canker is a disease caused by the bacterium Xanthomonas citri subsp. citri that attacks commercially important citrus species. To mitigate losses, the current form of control of the disease involves spraying compounds containing heavy metals in their composition. Such measures have caused great environmental impact due to the accumulation of these metals in the soil and throughout the trophic chain. For this reason, the search for metabolites produced by microorganisms has become increasing in recent years. The objective of this work is to obtain fungi and/or fungal extracts with potential activity against X. citri subsp. citri. In this work 16 crude extracts from fungi isolated from the rhizosphere of Citrus sinensis orange were submitted to assays to evaluate their inhibitory potential against Xanthomonas citri subsp. citri. Metabolites were produced in 2% malt at an average temperature of 28⁰C at 120rpm for 20 days. The liquid phase was extracted using ethyl acetate. The extracts were concentrated and dissolved in dimethyl sulfoxide (DMSO). The bacterial inoculum was prepared using NYG medium at 29⁰C at 200rpm overnight. The antibacterial bioassay was performed using serial micro dilution assay in 96-well microplate using resazurin (REMA) as indicator. The evaluation of the inhibitory activity quantitatively was performed using fluorescence spectrometer, allowing to verify the amount of dead cells. Among the tested extracts, 4 presented satisfactory inhibitory concentrations above 80%, being LMA 1957, LMA 1923, LMA 1844 and LMA 1840. The extract LMA 1957 showed high inhibitory concentrations even at the lowest concentrations tested and, therefore, was the strain chosen for the plant and purification tests where, after reevaluating its inhibitory activity, it showed inhibition at even lower concentrations above 90% at the concentration of 0.88µg/mL. Even after the plant test under different conditions from those found in the laboratory with in vitro analyses and purification of the extract, the results were considered satisfactory. It is, however, necessary to perform the identification of these compounds and toxicity analysis until the final result is obtained.
Citrus canker is a disease caused by the bacterium Xanthomonas citri subsp. citri that attacks commercially important citrus species. To mitigate losses, the current form of control of the disease involves spraying compounds containing heavy metals in their composition. Such measures have caused great environmental impact due to the accumulation of these metals in the soil and throughout the trophic chain. For this reason, the search for metabolites produced by microorganisms has become increasing in recent years. The objective of this work is to obtain fungi and/or fungal extracts with potential activity against X. citri subsp. citri. In this work 16 crude extracts from fungi isolated from the rhizosphere of Citrus sinensis orange were submitted to assays to evaluate their inhibitory potential against Xanthomonas citri subsp. citri. Metabolites were produced in 2% malt at an average temperature of 28⁰C at 120rpm for 20 days. The liquid phase was extracted using ethyl acetate. The extracts were concentrated and dissolved in dimethyl sulfoxide (DMSO). The bacterial inoculum was prepared using NYG medium at 29⁰C at 200rpm overnight. The antibacterial bioassay was performed using serial micro dilution assay in 96-well microplate using resazurin (REMA) as indicator. The evaluation of the inhibitory activity quantitatively was performed using fluorescence spectrometer, allowing to verify the amount of dead cells. Among the tested extracts, 4 presented satisfactory inhibitory concentrations above 80%, being LMA 1957, LMA 1923, LMA 1844 and LMA 1840. The extract LMA 1957 showed high inhibitory concentrations even at the lowest concentrations tested and, therefore, was the strain chosen for the plant and purification tests where, after reevaluating its inhibitory activity, it showed inhibition at even lower concentrations above 90% at the concentration of 0.88µg/mL. Even after the plant test under different conditions from those found in the laboratory with in vitro analyses and purification of the extract, the results were considered satisfactory. It is, however, necessary to perform the identification of these compounds and toxicity analysis until the final result is obtained.
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Palavras-chave
Xanthomonas, REMA, Citrus sinensis, Talaromyces sp, Biotecnologia