Study of protease inhibitors roles on peanut seed aflatoxin contamination.

MÜLLER, V; ASÍS, R; GIECO, J

Mérida

Congreso; VI Latinamerican Congress of Mycotoxins, II International Symposium on Fungal and Algal Toxins in Industry.; 2010

Sociedad Latinoamericana de Micotoxicología

Background: Aflatoxin contamination is one of the main factors affecting peanut seed quality. One of the strategies to decrease the risk of peanut aflatoxin contamination is the use of genotypes with resistance to Aspergillus infection. In a recent study with the objective to understand the virulence mechanisms of A. flavus and A. parasiticus, we reported that protease production by these fungi is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa (Asis, 2009).Aflatoxin contamination is one of the main factors affecting peanut seed quality. One of the strategies to decrease the risk of peanut aflatoxin contamination is the use of genotypes with resistance to Aspergillus infection. In a recent study with the objective to understand the virulence mechanisms of A. flavus and A. parasiticus, we reported that protease production by these fungi is involved in peanut seed infection and aflatoxin contamination resulting in seed tissue damage, affecting seed viability and facilitating the access of fungi through the testa (Asis, 2009). Aims: In the present work we proposed to evaluate the function of peanut protease inhibitors (PI) during Aspergillus infection and aflatoxin contamination.In the present work we proposed to evaluate the function of peanut protease inhibitors (PI) during Aspergillus infection and aflatoxin contamination. Material and Methods: For this study we used a PI337394 and Florman cultivars previously characterized as resistant and susceptible to Aspergillus infection and aflatoxin contamination in “in vitro” infection assay (Asis 2005). Peanut seeds were infected with A. parasiticus and then fungal colonization, aflatoxin production (Asis, 2005) and PI activity (Sarath, 1989) were determined. We also measured the PI activity in six infected seed cultivars. PI extract of resistant cultivar were incubated with A. parasiticus spores during 24 hs. to evaluated antifungal activity. Also, the resistant cultivar PI were separated and detected by reverse zymography (Le 2004). In parallel an assay were carried out on the polyacrilamide gel to detect an antifungal activity. To evaluate the inhibitor activity of PI on Aspergillus proteases, a zymography (Asis, 2009) of Aspergillus extracellular proteases were incubated with a PI extract of resistant cultivar or a commercial proteases inhibitors mix as a control.For this study we used a PI337394 and Florman cultivars previously characterized as resistant and susceptible to Aspergillus infection and aflatoxin contamination in “in vitro” infection assay (Asis 2005). Peanut seeds were infected with A. parasiticus and then fungal colonization, aflatoxin production (Asis, 2005) and PI activity (Sarath, 1989) were determined. We also measured the PI activity in six infected seed cultivars. PI extract of resistant cultivar were incubated with A. parasiticus spores during 24 hs. to evaluated antifungal activity. Also, the resistant cultivar PI were separated and detected by reverse zymography (Le 2004). In parallel an assay were carried out on the polyacrilamide gel to detect an antifungal activity. To evaluate the inhibitor activity of PI on Aspergillus proteases, a zymography (Asis, 2009) of Aspergillus extracellular proteases were incubated with a PI extract of resistant cultivar or a commercial proteases inhibitors mix as a control. Results and discussion: The results showed a negative correlation between fungal colonization or aflatoxin production and PI activity. Infected seed of resistant cultivar showed an increase of PI activity than uninfected ones, whereas infected seeds of susceptible cultivar showed a decrease of PI activity than the uninfected ones. The PI extract from resistant cultivar exhibited an antifungal activity. The protein separation by electrophoresis showed that PI migrate to the same region where antifungal activity was observed on the gel. Finally, the peanut PI incubated with Aspergillus proteases showed a complete inhibition of proteases activity whereas a partial inhibition was observed by IP commercial mix. This results suggest that PI are related to mechanism of peanut seed to diminish the aflatoxin contamination. We proposed that PI in the resistant cultivar are produced as a response to Aspergillus infection inhibiting the Aspergillus proteases synthesized during seed colonization and probably inhibiting the fungal growth. Actually we are purifying and identifying the proteases inhibitors of peanut seed of resistant cultivar. Thus, protease inhibition during seed infection is shown as an opportunity to reduce aflatoxin contamination.The results showed a negative correlation between fungal colonization or aflatoxin production and PI activity. Infected seed of resistant cultivar showed an increase of PI activity than uninfected ones, whereas infected seeds of susceptible cultivar showed a decrease of PI activity than the uninfected ones. The PI extract from resistant cultivar exhibited an antifungal activity. The protein separation by electrophoresis showed that PI migrate to the same region where antifungal activity was observed on the gel. Finally, the peanut PI incubated with Aspergillus proteases showed a complete inhibition of proteases activity whereas a partial inhibition was observed by IP commercial mix. This results suggest that PI are related to mechanism of peanut seed to diminish the aflatoxin contamination. We proposed that PI in the resistant cultivar are produced as a response to Aspergillus infection inhibiting the Aspergillus proteases synthesized during seed colonization and probably inhibiting the fungal growth. Actually we are purifying and identifying the proteases inhibitors of peanut seed of resistant cultivar. Thus, protease inhibition during seed infection is shown as an opportunity to reduce aflatoxin contamination. Reference: Asis, R., Barrionuevo D. L., Giorda L. M., Nores L. M., and Aldao M. A. 2005 A. Aflatoxin Production in Six Peanut (Arachis hypogaea L.) Genotypes Infected with Aspergillus flavus and A. parasiticus, Isolated from Peanut Production Areas of Cordoba, Argentine. J Agric Food Chem 53 (23): 9274-9280. Asis R., Muller V., Barrionuevo D. L., Araujo S. A and Aldao M. 2009. Analysis of protease activity in Aspergillus flavus and A. parasiticus on peanut seed infection and aflatoxin contamination. European Journal Plant Pathology. 124:391-403. Le, Q. T. and Katunuma, N. 2004. Detection of protease inhibitor by reverse zymography method, performed in tris(hydroxylmethyl)aminomethane-tricine buffer system. Anal Biochem 324:237-240 Sarath, G., Motte, R. S., and Wagner, F. W. 1989. Protease assay methods. In Proteolytic enzimes - a practical approach -, edited by Beynon, R. J., Bond, J.S. IRL: Oxford University Press.