Fungos endofíticos de soja: diversidade e prospecção enzimática

Abstract

Plants are inhabited by a microbiota composed of microorganisms in endophytic conditions and each endophyte-plant relation displays peculiar characteristics that vary according to environmental conditions. Studies related to the identification of endophytic populations associated with plant species allow using them as important tools for obtaining biotechnological products of agricultural and industrial interest. The objective of this study was to isolate and identify endophytic fungi from soybean plants, before and after fungicide application composed by Trifloxystrobin + Prothioconazole in the soybean crops, evaluate the sensitivity of the isolates in vitro against the same fungicide and also evaluate them regarding the biotechnological potential of enzyme production. The soybean plants used were cultivated at the Experimental Nucleus of Agricultural Engineering (NEEA) of the Western Paraná State University, in Cascavel, Paraná, Brazil. The leaves of soybean plants were collected at 48 days after sowing (prior to fungicide application) and at 62 days (14 days after application). The fungi were grouped and classified according to morphological and microscopic aspects, identified at the genera level and analyzed according to indexes such colonization frequency (FC%) and ecological diversity (Simpson (D e 1-D), Shannon-Wiener (H’) and Pielou (e). A total of 259 fungi (122 before the fungicide and 137 after the fungicide application) were obtained, which resulted in the identification of 11 genera, among which Diaporthe and Alternaria were the most abundant with FC of 38.6 and 25.5%, respectively. The frequency and diversity values showed that there was no significant difference between the two harvests carried out, revealing that the attainment of cultivable endophytic fungi from plants treated with chemical pesticides was not affected. The fungi were also tested in vitro for sensitivity to the fungicide at concentrations of 0.2, 2.0 and 3.8 mg L-1 . The results showed that all the fungi evaluated were able to grow in the presence of the fungicide in the solid culture medium, in all of the tested concentrations, with low sensitivity at the concentration of 0.2 mg L-1 . Only four fungi showed sensitivity at the concentration of 2.0 mg L-1 and, even at the highest concentration (3.8 mg L -1 ), the genera Cladosporium, Bipolaris, Cadophora, Rhizoctonia and Alternaria remained showing low sensitivity to the fungicide. The isolated fungi also underwent screening in order to detect the production of cellulase enzymes (CMCases), xylanases, proteases and amylases in solid media and quantify the xylanase activity in submerged cultivation, utilizing straw and sugarcane bagasse as substrate. Assays in solid media showed better results for xylanase with an enzyme index (IE) of 2.14 and for CMCase of 1.92. The submerged cultivation generated better results using 50% straw with 50% bagasse and was able to induce an activity of 383.63 U mL-1 of xylanase. Regarding the kinetic behavior of the crude xylanase produced by the fungus Colletrotrichum boninense, the optimal pH value found was 5.0 and the best temperature range was between 50 and 60 °C. At temperatures of 40 and 50 °C the enzyme preserved 87 and 76% of its activity when incubated for 30 min. The Km and VMax values found against xylan were 99.3 mg mL-1 and 3,333 µmol mL-1 min-1 , respectively.