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United States Department of Agriculture

Agricultural Research Service

Title: Purification and Characterization of a Prominent Polygalacturonase Isozyme Produced by Phomopsis Cucurbitae in Decayed Muskmelon Fruit

Authors
item Zhang, Jiuxu
item Bruton, Benny
item Biles, Charles - EAST CENTRAL UNIVERSITY

Submitted to: Mycological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 15, 1997
Publication Date: N/A

Interpretive Summary: Economic losses due to postharvest decays are higher than is often recognized. Because of the added costs of harvesting, packing, transporting, and marketing, the value of fresh fruits and vegetables may increase several-fold to the consumer. Latent infections have historically been difficult to control because there is no sign of decay at harvest and consequently the fruit are shipped to terminal markets where the pathogen may cause rapid decay of the produce. Information as to why infections by some fungi remain dormant until after the fruit are harvested is largely unknown. Previous studies demonstrated that the fungus which causes Phomopsis rot of cantaloupe fruit can produce multiple polygalacturonase isozymes. Because polygalacturonase is normally the first enzyme produced by fungi in the decay process, characterization of this enzyme is an important step in understanding latent infections. In the present study, the most prominent polygalacturonase isozyme was identified and purified b ultra filtration, preparative isoelectric focusing, anion exchange and gel filtration chromatography. Studies showed that the isozyme to be highly efficient in maceration of cantaloupe fruit tissue. The isozyme was subsequently sequenced which will allow further investigation of the role of this enzyme in fungal pathogenesis using gene cloning and disruption techniques in our future work.

Technical Abstract: Phomopsis cucurbitae is a latent infecting pathogen that infects unripe muskmelon fruit but causes decay after harvest. This fungus causes severe losses during muskmelon fruit storage and marketing in the U. S., Japan and some Central American countries. Previous studies showed that the fungus produced the cell wall degrading enzyme polygalacturonase (PG) in both culture and muskmelon fruit tissue. The role of P. cucurbitae PG in the fruit decay process and its relating to latent infection is not well understood. A prominent PG isozyme produced by the fungus in decayed fruit was purified to homogeneity by a sequence of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. This isozyme exhibited endo-activity, a molecular weight of 54 kD according to SDS-PAGE, and a pI of 4.2 based on IEF-PAGE. Isozyme activity was optimal at 40-45 deg C and pH 5.0. It had a Km of 44.7 ug/ml and a Vmax of 0.313. The purified isozyme also effectively macerated mature muskmelon fruit tissues. This isozyme was the most prominent of the PG isozymes produced by P. cucurbitae in decaying fruit, and may play an important role in postharvest decay of cantaloupe fruit.

Last Modified: 9/10/2014
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