Effects of chitosan and oligochitosan on development and mitochondrial function of Rhizopus stolonifer.

The antifungal activities of chitosan and oligochitosan have been used to control postharvest decay of the fruits. The effect of chitosan and oligochitosan on mycelium growth, spore germination, and mitochondrial function of Rhizopus stolonifer was evaluated in order to establish a connection between fungus development and the main organelle in charge to provide energy to the cell. The mycelium growth of R. stolonifer was significantly reduced on minimum media amended with chitosan or oligochitosan. The highest antifungal indexes were obtained on media containing chitosan or oligochitosan at 2.0 mg ml(-1). Microscopic observation showed that chitosan and oligochitosan affected the spore germination and hyphae morphology. Both polymers increased oxygen consumption of R. stolonifer. Respiratory activity was restored with NADH in permeabilized treated and untreated cells, and was inhibited with rotenone and flavones. Complex III and IV were inhibited by antimycin A and cyanide, respectively, in treated and untreated cells. Chitosan and oligochitosan increased NADH dehydrogenase activity in isolated mitochondria. However, there were not changes in the cytochrome c oxidase and ATPase activities by effect of these polymers. These results suggest that both chitosan and oligochitosan affect the development of R. stolonifer and might be implicated in the mitochondrial dysfunction.

The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill.

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F(1)F(0)-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K(0.5) for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.

Efecto del quitosano en el desarrollo in vitro de rhizopus stolonifer ehrenb: fr) vuill en dos medios de cultivo / Effect of chitosan on the in vitro developtment of rhizopus stolonifer (ehrenb: fr) vuill on two culture medium

En este trabajo se evaluó el efecto antifúngico del quitosano (0; 0,5; 1,0; 1,5; 2,0 mg mL-1) en el desarrollo in vitro (crecimiento micelial, formación de cuerpos fructíferos, esporulación, germinación y liberación de proteínas) de Rhizopus stolonifer en dos medios de cultivo (papa dextrosa agar y medio mínimo). Los resultados obtenidos demostraron que el quitosano inhibió el crecimiento micelial de R. stolonifer en ambos medios de cultivo. El mayor índice antifúngico se observó en el medio papa dextrosa agar. El quitosano no afectó la formación de los cuerpos fructíferos de R. stolonifer en los medios estudiados. La esporulación y la germinación de las esporas se afectaron en ambos medios de cultivo por efecto de quitosano, siendo más notable en el medio medio mínimo. Se demostró la liberación de proteínas por efecto del quitosano en medio mínimo y caldo papa dextrosa. En general, en este estudio se evidenció el efecto antifúngico del quitosano en el desarrollo in vitro de Rhizopus stolonifer con independencia del medio de cultivo empleado. Sin embargo, en medio medio mínimo podrían observarse mejor los efectos antifúngicos del quitosano.

In this work the antifungal effect of chitosan (0, 0.5, 1.0, 1.5, 2.0 mg mL-1) on the in vitro development (mycelial growth, fruit bodies formation, sporulation, germination and proteins release) of Rhizopus stolonifer on two culture medium (Potato Dextrose Agar and Minimal medium) was evaluated. The obtained results demonstrated that chitosan inhibited the mycelial growth of R. stolonifer in both culture medium. The highest antifungal effect was observed on potato dextrose agar medium. Chitosan was not affected the fruit bodies formation of R. stolonifer on the studied medium. Sporulation and spore germination were affected in both culture mediun by effect of chitosan, it was more noticeable in minimal medium. It was demonstrated proteins release by effect of chitosan in minimal medium and potato dextrose broth medium. In general, in this study it was showed the antifungal effect of chitosan on in vitro development of Rhizopus stolonifer regardless of the culture medium used. However, in minimal medium could be observed best the antifungal effects of chitosan.