Ana Niurka Hernández-Lauzardo

Antifungal effects of chitosan with different molecular weights on in vitro development of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill

Determination of the molecular weight of three types of chitosan was carried out by HPSEC-RI. The effect of low, medium and high molecular weight chitosan was evaluated on development of three isolates of Rhizopus stolonifer. Image analysis and electronic microscopy observations were done in spores of this fungus. Germination of R. stolonifer in potato dextrose broth with chitosan was also evaluated. Results pointed out that the low molecular weight chitosan was more effective for inhibition of mycelial growth while the high molecular weight chitosan affected spore shape, sporulation and germination. Studies of scanning and transmission electron microscopy revealed numerous and deeper ridge ornamentations of the chitosan-treated spore.

Current status of action mode and effect of chitosan against phytopathogens fungi

Chitosan is a deacetylated derivative of chitin, consisting mainly of glucosamine units, commercially obtained from crustacean waste. This natural compound is biodegradable and nontoxic and has diverse applications in agriculture, among which highlights the control of fungal diseases in crops of agricultural interest. This review focuses on some basic studies about the mode of action and the effect of chitosan on different phytopathogens fungi. In general, it is known that molecules of this polymer can act on extracellular (plasma membrane) and intracellular level (penetration of chitosan into the fungal cell). The study of the effect of chitosan on different phytopathogens fungi evidence that the response is variable; in some investigations, it was found that the spores are more sensitive than hyphae to the application of chitosan. Even though the progress in understanding the mode of action of this polymer and the various effects that can cause damage are known, it is necessary to carry out more studies about the biological activity of these molecules to propose better control strategies of the phytopathogens fungi.

Influence of chitosan and its derivatives on cell development and physiology of Ustilago maydis

Ustilago maydis, a dimorphic fungus causing corn smut disease, serves as an excellent model to study different aspects of cell development. This study shows the influence of chitosan, oligochitosan and glycol chitosan on cell growth and physiology of U. maydis. These biological macromolecules affected the cell growth of U. maydis. In particular, it was found that chitosan completely inhibited U. maydis growth at 1mg/mL concentration. Microscopic studies revealed swellings on the surface of the cells treated with the polymers, and chitosan caused complete destruction of the membrane and formation of vesicles on the periphery of the cell. Oligochitosan and chitosan caused changes in oxygen consumption, K(+) efflux and H(+)-ATPase activity. Oligochitosan induced a faster consumption of oxygen in the cells, while glycol chitosan provoked slower oxygen consumption. It is noteworthy that chitosan completely inhibited the fungal respiratory activity. The strongest effects were exhibited by chitosan in all evaluated aspects. These findings showed high sensitivity of U. maydis to chitosan and provided evidence for antifungal effects of chitosan derivatives. To our knowledge, this is a first report showing that chitosan and its derivatives affect the cell morphology and physiological processes in U. maydis.

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 F1F0-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 K0.5 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.

Assessment of the effect of chitosan of different molecular weights in controlling Rhizopus rots in tomato fruits

The effect of chitosan was evaluated in three isolates of Rhizopus stolonifer from infected tomatoes that were harvested in different regions of the state of Morelos, México. Changes in the membrane integrity of spores, modifications in pH media and the proteins released were analysed in each isolate against three different molecular weights of chitosan at three fixed concentrations. There was an observed decline in the integrity of spores in the presence of chitosan at all the concentrations evaluated. The protein released was different depending on the isolate, kind of chitosan and used concentration. There was a significant difference in the pH changes on the growth media for all the tested isolates. Chitosan of high molecular weight showed the best results to inhibit the infection caused by R. stolonifer on the tomato fruits. The severity of symptoms of soft rot was not related with the molecular weight of chitosan.