Purificação parcial de compostos biologicamente ativos a partir de Pycnoporus sanguineus para o controle de ferrugem asiática em soja

Abstract

Induced resistance involves the activation of latent defense mechanisms in plants in response to the treatment with biotic or abiotic agents. The use of crude extracts aimed at inducing resistance mechanisms is an interesting alternative to chemical control, however, in this extracts can occurs besides de presence of inducers, the presence of suppressors. This work objectived a partial purification, through gel filtration chromatography (GFC) and by ammonium sulphate (AS) precipitation, of compounds in basidiocarps crude extracts from Pycnoporus sanguineus efficient to the control of Asian rust in soybean by induction of resistance. Crude extracts from P. sanguineus were submited to gel filtration chromatography (GFC), and five proteins and one carbohydrate peaks were obtained, with molecular weight ranging from 1,82 to 5,18 KDa. It was made the fractionated protein purification from 100 mL of crude extract from P. sanguineus. The fractions obtained were: 0-20%, 20-40%, 40-60%, 60-80% and 80-100% of ammonium sulphate. Incised soybean cotyledons were treated with fractions from GFC and AS precipitation, crude extract 20% (EB 20%), Saccharomyces cerevisiae (25 mg mL-1) and water. After incubation during 20 h, was made biochemical analyses to verify the phytoalexins content and peroxidases, polyphenoloxidases, β-1,3 glucanases and phenylalanine ammonia-lyase activities. The treatments from GFC, protein fractions III (3,44 KDa), IV (2,79 KDa) and V (1,82 KDa), and carbohydrate fraction, fractions 40-60%, 60-80% and 80-100%, obtained by ammonium sulphate precipitation, crude extract (CE) of basidiocarps at 20%, besides control treatments fungicide (tebuconazole, 0,5 g a.i. L-1) and water were used to evaluate the antimicrobial activity and induction of resistance in soybean against P. pacchirhizi in greenhouse. Soybean plants were treated and after three days were inoculated with the pathogen. Samples were collected 0, 1, 3, 6 and 9 days after the treatment for biochemical analyses and the severity was evaluated after 13 days. The data referring to evaluation of antimicrobial activity show that the fractions do not have inhibiting activity of germination of spores. As for severity, the treatments protein fraction III and CE 20% were efficient in the reduction of the number of injuries for cm2. For peroxidase the CE 20% presented tendency in reducing the enzymatic activity. Chitinases and polifenoloxidases did not presented statistical differences between the treatments. For β-1,3 glucanases there was local induction for the treatments with glicide fraction and protein fractions III and V, and fractions 40-60% and 60-80%, while CE 20% presented systemic induction for this enzyme. For phenylalanine ammonia-lyase the purified fractions of P. sanguineus reduced the enzymatic activity, except for CE 20%. It was possible to induce defense mechanisms in soybean against P. pachyrhizi for the application of partially purified fractions from P. sanguineus, indicating its possible use as an alternative method for controling this pathogen