|Pereira, I bastos|
Submitted to: Postharvest Biology and Technology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/10/2007
Publication Date: 2/1/2008
Citation: Janisiewicz, W.J., Pereira, I., Almeida, M.S., Roberts, D.P., Wisniewski, M.E., Kurtenbach, E. 2008. Improved biocontrol of fruit decay fungi with Pichia pastoris recombinant strains expressing Psd1 antifungal peptide. Postharvest Biology and Technology. 47:218-225. Interpretive Summary: Beneficial microbes isolated from the fruit surface have been used commercially for control of fruit decays caused by fungi after harvest for the past ten years. Further expansion of this control method will largely depend on improving the effectiveness of these beneficial microbes. A gene responsible for the production of the antifungal protein in pea seeds was inserted into a genome of the yeast, Picha pastoris, which is used as a model system for expression of foreign genes. The original strain of this yeast is commonly used in animal feed and can also grow well in apple wounds, the point of entry for the fruit decaying fungus. Modified strains of this yeast, containing the pea antifungal protein, controlled blue mold decay more efficiently than the non-modified form of the same yeast on Golden Delicious apple after harvest. Out of 111 modified yeast strains, four repeatedly provided better decay control than the non-modified yeast. We demonstrated that the control of fruit decay can be improved by inserting foreign antifungal genes into yeast. Also, this yeast system could be used as a model system for testing the importance of other genes, isolated from beneficial microbes controlling fruit decay, that are presumably involved in the mechanism responsible for reducing fruit decay. Future work will focus on optimizing conditions for the production of the antifungal protein by yeast in apple wounds.
Technical Abstract: Future expansion of biological control of postharvest diseases will largely depend on improving its effectiveness under an increased range of conditions and expanding the spectrum of activity to new commodities and new diseases. Plasmid pGAPZaC/Psd1, a binary vector containing the cDNA encoding sequence for Psd1 constitutive expression under the control of a GAP promoter, was used to transform the yeast, Pichia pastoris, to evaluate this defensin with regard to enhancing the biocontrol potential of antagonists. Both strains, X-33 and GS 115, were successfully transformed by electroporation and produced the active rPsd1 peptide. The non-transformed strain X-33 grew better than strain GS 115 in Golden Delicious apple wounds and was then chosen as the host for plasmid pGAPZaC/Psd1 in biocontrol tests. The severity and the incidence of blue mold decay caused by Penicillium expansum were significantly reduced on apples treated with pGAPZaC/Psd1/X-33 recombinant when compared to apples inoculated with this fungus alone, or in combination with the non-transformed parental strain X-33 or the pGAPZaC/X33 recombinant containing the empty binary vector. Four selected transformants reduced decay in repeated studies but were effective only when applied at lower (6.3 x 10-5 CFU ml-1) cell concentrations. Realization of the full potential of the Psd1 defensin may be achieved by optimizing its expression and activity on the fruit.