The goal of the biocontrol improvement program is to realize the full potential of biological control. This may be accomplished by more extensive strain selection of the same antagonist species, as indicated earlier in the case of M. pulcherrima, by manipulating antagonists and/or their environment, and by applying the antagonists before harvest in addition to one after harvest.
Postharvest application of the antagonists mixture of P. syringae and S. roseus (Janisiewicz and Bors 1995), and C. sake and Pantoea agglomerans (Nunes et al. 2002) improved efficacy of biocontrol of blue mold of apples compared to the individual antagonist. An orchard application of a mixture of A. pullulans with Rhodotorula glutinis was more effective than the individual antagonists, and suppressed apple decays caused by Penicillium spp., B. cinerea, and Pezicula malicorticis after harvest to the same level as the commonly used fungicide Euparen (Leibinger et al. 1997). Nutrient utilization profile of individual antagonists was successfully used to develop antagonist mixtures with a minimum of nutrient overlap between the antagonists and resulted in biocontrol of blue mold of apples that was superior to the individual antagonists (Janisiewicz 1996).
Physiological manipulation has been focused on improving antagonist fitness by growing them under various conditions that improved resistance to desiccation and survival on the fruit. This is of particular importance to antagonists that are applied in the orchard for control of postharvest decays. C. sake cells grown under water stress caused by addition of glucose or glycerol increased after application to apple trees, while those grown on unmodified media did not (Teixido et al. 1998b). This yeast was more water-stress tolerant when grown on a molasses-based medium than on a medium where water activity (aw) was modified by the addition of NaCl (Abadias et al. 2001b). In preparing a freeze-dried formulation, viability of the C. sake cells was best maintained when 10% skim milk was combined with other protectants such as lactose, glucose, fructose, or sucrose (Abadias et al. 2001a). In general, the highest viability of the C. sake cells occurred when the protection and rehydration media were the same.
Control of blue mold of apples was improved by the addition of the amino acids L-asparagine or L-proline to the P. syringae antagonist treatment suspensions (Janisiewicz et al. 1992). These amino acids were selected after screening various C and N sources for their effect on the antagonist and pathogen growth. Both amino acids increased population of the antagonist more than 10-fold in the fruit wounds. The addition of the glucose analog, 2-deoxy-d-glucose, to the antagonistic yeasts S. roseus and C. saitoana significantly improved decay control on apple and citrus, respectively (El-Ghaouth et al. 2000c; Janisiewicz 1994). 2-deoxy-d-glucose can be up taken by the pathogens but it cannot be metabolized as energy source, resulting in reduced pathogen growth, which gives the advantage to the antagonists, and improves biocontrol. Ammonium molybdate stimulated population of the antagonistic yeast C. sake (CPA-1) and improved control of blue mold of apple and pear after harvest (Nunes et al. 2001a). This nutrient also has fungicidal activity and inhibited germination of P. expansum and B. cinerea spores in vitro, and reduced blue mold, gray mold, and Rhizopus stolonifer decay of apple in pre- and postharvest applications (Nunes et al. 2001b).
Use of genetic manipulation to improve BCPD has great potential, but little research has been done in this area. The appearance of decay symptoms on avocado fruit was delayed when the fruit were dipped in a suspension containing a reduced-pathogenicity mutant of the avocado pathogen Colletotrichum gleosporioides (Yakoby et al. 2001). This mutant was generated by restriction enzyme-mediated integration (REMI) transformation, and induced natural resistance of avocado by increasing production of the antifungal diene from 700 to 1,200 mg/g fresh weight 9 days after inoculation. Saccharomyces cerevisiae transformed with a cercopin A-based peptide, that inhibits germination of C. cocodes at 50 mM, was able to inhibit the growth of germinated spores, and inhibited decay development caused by C. cocodes on wounded tomato (Jones and Prusky 2002).
This work demonstrated that microorganisms that colonize fruits can be used as vehicles for providing decay control which may include various biocontrol traits.
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