The Pseudogymnoascus sp. is one of the taxa found in harsh environments like the arctic, alpine and Antarctica regions. In Antarctica this species can be found in different habitats such as the lichensphere, marine sediments, soil and associated with macroalgae (DING et al., 2016). Since this species is able to withstand the extreme conditions of the antarctic environment it is possible that it produces specific or new compounds which may be used as source of new antimicrobial products. Extracts from Pseugymnoascus sp. were assayed and showed antifungal and antibacterial activity before (GONÇALVES et al., 2015). These new molecules can be used for example as substitutes for the current chemicals used against phytopathogens such as bacteria of the Xanthomonas species, which cause many plant diseases of economic and social interest in Brazil and other countries. Xanthomonas citri subsp. citri is one of those pathogens, being the causative agent of citrus canker. The present work aimed to purify bioactive intracellular extract from a Pseudogymnoascus spp. isolated from soil under decaying wood at Deception Island, Antarctica. The isolate was cultivated in 180 mL Malt Extract Broth for 20 days at 15 ºC and 150 rpm for secondary metabolite production. The intracellular extract was obtained by cell disruption of the biomass using methanol as solvent. The extract was evaluated in vitro against three Xanthomonas spp, using the resazurin microtiter assay (REMA) for cell viability (SILVA and FERREIRA, 2013), and showed growth inhibition of 89% in Xanthomonas citri subsp. citri (X. citri). The extract was fractionated in two different ways. In the first methodology molecular exclusion chromatography was used, with Sephadex LH-20 as the stationary phase, and gradient elution using methanol, methanol:water (9:1) and water. This step resulted in 4 fractions, partially and fully purified, which were evaluated for inhibitory activity in X. citri. Of these, only one partially purified fraction showed bacterial growth inhibition of 42%. The extract was also subjected to normal phase chromatography, using as stationary phase silica derivatized with cyano groups (CN). This was conducted under gradient elution with dichloromethane:hexane (1:1), methanol:hexane (1:1), methanol, methanol:water (1:1) and water. In this process two purified and three partially purified fractions were obtained. The fractions were tested for antibacterial activity. One of the fractions, which contained only one compound, showed activity against the bacteria with an average of 97% of inhibition. The compound is being analyzed by 1H and 13C NMR and mass spectrometry. From the methodologies used for the purification of the extract, it was possible to verify that for this extract the normal phase chromatography using the silica derivatized with cyano groups was more efficient for the purification of the bioactive compound.

References:

DING et al.. Richness and bioactivity of culturable soil fungi from the Fildes Peninsula, Antarctica. Extremophiles. v. 20, p. 425-435. 2016.
GONÇALVES et al.. Antibacterial, antifungal and antiprotozoal activities of fungal communities present in different substrates from Antarctica. Polar Biology. v. 38, p. 1143-1152. 2015
SILVA IC, FERREIRA H. Drug Sensitivity Assay of Xanthomonas. citri subsp. citri Using REMA Plate Method. Bio-protocol. v. 3, p. 1-4. 2013.