Location:2010 Annual Report
1a. Objectives (from AD-416)
Objective 1: Elucidate the role of fungal extracellular enzymes as virulence factors important in postharvest decay of fresh fruit including the effect of pathogen modification of the host environment on virulence factors. Objective 2: Evaluate potential control strategies targeting fungal extracellular enzymes to reduce postharvest decay of fresh fruit. Sub-objective 2.A. Optimize the application of recombinant antibodies targeting extracellular enzymes of P. expansum and P. solitum to reduce postharvest decay of pome fruits. Sub-objective 2.B. Determine the effect of antioxidants and their analogs on decay development.
1b. Approach (from AD-416)
To elucidate the role of fungal extracellular enzymes as virulence factors in postharvest decay of apple and pear fruit, we will first compare the ability of several strains of a highly virulent and a weakly virulent pathogen of the same genus to produce extracellular enzymes (such as polygalacturonases and cellulases) during fruit infection and in response to fungal modification of the host/pathogen microenvironment. 'Golden Delicious' apple and 'Anjou' pear fruit will be infected with P. expansum or P. solitum and we will extract, purify, and characterize the extracellular enzymes produced by these organisms using standard procedures. We will develop and utilize recombinant antibodies specific for binding to and inactivating enzymes produced by the pathogens in vivo and in vitro. The antibodies will allow us to determine which specific enzymes play a major role in virulence of Penicillium species on pome fruit. Strategies incorporating recombinant antibodies against virulence factors, antioxidants known to have anti-fungal activity, and mild stress treatments will be tested for their ability to control postharvest decays of pome fruits caused by Penicillium species.
3. Progress Report
Examining fungal virulence factors to develop control strategies for blue mold on pome fruit during postharvest storage. Maintaining fruit quality by preventing decay is critical to prevent postharvest losses. P. expansum and P. solitum are among the most destructive pome fruit pathogens causing blue mold. Polygalacturonases (PGs) are cell wall degrading enzymes that are involved in fungal virulence and facilitate decay. Isolation, purification and biochemical characterization of PGs from P. expansum and P. solitum-decayed pear and apple fruit have been completed. Biochemical characterization of these enzymes and determining their role in fruit decay may provide a foundation for development of novel disease control methods. Synthesis of a recombinant phage display library against P. expansum produced PG and isolation of cDNA clones encoding antibodies against PGs have been achieved. This work will ultimately lead to inhibition studies in the lab and eventually will be tested on apple and pear fruit surfaces. The use of recombinant antibodies to block PG activity may be an effective non-chemical method of preventing postharvest decay caused by Penicillium species on pome fruits.
1. Identification of wild apple germplasm accessions from Kazakhstan for resistance to Penicillium expansum and Colletotrichum acutatum. Maintaining fruit quality by inhibiting decay is crucial to maintain quality and prevent postharvest losses. P. expansum and C. acutatum are the most destructive causal agents of apple fruit decay. Sources of resistance to these fungi are lacking in commercial apple cultivars. However, we have identified several wild apple accessions that are resistant to one or both pathogens. These wild apple fruit will be used to identify the underlying genetic and biochemical resistance mechanisms to fungal decay pathogens. This information will be beneficial to plant breeders and geneticists in transferring resistance to commercial apple cultivars. Ultimately, this knowledge may be applied through genetic manipulation in the host to prevent decay during storage.
2. Isolation and identification of cDNA clones encoding recombinant antibodies against P. expansum PGs. It is necessary to develop alternatives to chemical control for reducing postharvest losses. In cooperation with an ARS scientist at the Appalachian Fruit Research Station, Kearneysville, West Virginia, ARS scientists in Beltsville, Maryland have made significant progress toward construction of a phage library against polygalacturonase from P. expansum, the causal agent of blue mold of apples. We have successfully synthesized the recombinant phage display library, completed 2 rounds of panning, identified several binders, and obtained corresponding cDNA clones. Blocking polygalacturonase activity on the fruit surface using recombinant antibodies against polygalacturonase may lead to a novel control strategy to halt postharvest decay mediated by P. expansum on apple fruit. This control strategy could potentially benefit the postharvest industry by reducing dependency on synthetic fungicides.
Jurick II, W.M., Vico, I., Gaskins, V.L., Garrett, W.M., Whitaker, B.D., Janisiewicz, W.J., Conway, W.S. 2010. Purification and biochemical characterization of polygalacturonase produced by Penicillium expansum during postharvest decay of ‘Anjou’ pear. Phytopathology. 100(1):42-48.