Isolation and identification of antifungal metabolites from Macfadyena cynanchoides against Aspergillus species

APUD, G.R.; AREDES FERNANDEZ, P.A.; SAMPIETRO, D.A.

PUNTA DEL ESTE

Congreso; 41st World Congress of Vine and Wine; 2018

OIV

The grape black rot disease generated by Aspergillus species,mainly A. niger and A. carbonarius reduce the grape yieldand quality, alter the organoleptic features of wine and contaminate the grapesand wine with ochratoxin A, which exerts adverse health effects in humanand animals. The use of syntheticfungicides remains as the principal wayto control the growth and proliferation of OTA-producing fungi. However,the use of them could lead to environmental pollution, development ofresistance and could have residual effects that are harmful to human and couldproduce alteration in the fermentation or in the final quality of the wine. Theantifungal compounds from plants can be employed as an alternative way tocontrol pathogens owing to they contain wide variety of secondary metaboliteswith antimicrobial properties. In previous assays we demonstrated that thedichlorometane extract of Macfadyenacynanchoides stems showed a significant activity against A. niger and A. carbonarius. Theobjective of this work was to separate and to identify the antifungalmetabolites from Macfadyena cynanchoidesuseful for the control of Aspergillusspecies and additionally determine the joint effect between these compounds andcommercial fungicides. The extract of the plant stem was obtained by macerationwith dichlorometane (fCH2Cl2). The extract compositionwas preliminarly investigated by thin layer chromatography (TLC) and theantifungal constituents were detected by TLC bioautography. The components wereseparated by two sequential chromatography separations in silica gel columns. Theantifungal activity of the pools obtained was tested by dot-blot bioautography assay.The concentration necessary to completely inhibit the Aspergillus growth (MIC) was determined by microdilution assay. The identification of antifungal compoundswas realized in gas chromatography mass spectrometry (GC?MS). The joint effectbetween the plant metabolites and commercial fungicides propiconazole or sodium metabisulfite was evaluated in microdilutionassays by the checkerboard technique. The results obtained with theTLC-bioautography indicated inhibitory activity associated to bands viewedunder UV254nm (Rf=0.70 and Rf=0.63). Dot-blot bioassays of the poolsrecovered from the first separation of constituents in a silica gel columnindicated that pool 6 was the most active on the both Aspergillusstrains. The second separation of constituents from pool 6in a silica gel column yielded a pool 2 which showed antifungal activity in TLCbioautography assay. The GC-MS analysis of pool 2 indicated the presence of lapacholand 1-hydroxy-4-methylanthraquinone. The microdilution assay indicated valuesof MIC= 500 ppm against A. niger and MIC= 1000 ppm against A. carbonarius.The combined effect of pool 2 with sodium metabisulfite or propiconazole wassynergistic and additive respectively, against both Aspergillus strains.We can conclude that our results are the first one indicating thatthe liana M. cynanchoides is a promising source of natural products withantifungal activity on Aspergillus species responsible of grape blackrots. The plant active compounds combined with sodium metabisulfite or propiconazole suggest the possibility of reduce the amount of commercial fungicides for thecontrol of Aspergillus in grapes and wine.