The discovery was made on a single piece of fruit in an orange tree as part of a routine survey along U.S. Highway 27, a major citrus-hauling corridor, according to a news release.
The USDA's Animal and Plant Health Inspection service issued an emergency action notice for 270 acres of citrus near where the black spot symptoms were found along the Highlands-Polk County line.
The order requires that growers, harvesting crews, haulers and processors or packers follow quarantine procedures.
A delimiting survey of 1,339 acres of citrus located within 9 square miles of the positive tree is underway.
So far, no other positive symptoms have been found.
Polk County, located in central Florida, is part of the larger citrus-producing area called the Ridge.
Citrus black spot is a fungal disease of citrus that causes unsightly lesions on the fruit, making them unmarketable for the fresh market.
In severe cases, it also can affect leaves and cause leaf and fruit drop.
Several commercial fungicides are labeled to control black spot.
Until the Polk County find, black spot had been contained in an area in Collier and Hendry counties.
The disease was first found in the United States in March 2010, when it was confirmed in a grove near Immokalee.
It is found in several other parts of the world, including Argentina, South Africa, Brazil and Australia.
For more information on citrus black spot, visit the University of Florida's citrus black spot website.