...From the pages of Agricultural Research magazine
New Test on Tap
Tracking the spread of the plum pox potyvirus in stone
fruit crops could get a lot easier thanks to a new test devised by Agricultural
Research Service scientists.
Plum pox is an aphid-borne disease that causes acidity;
pale, concentric rings; and other defects that diminish the eating quality
of peaches, pears, plums, and other stone fruits. The disease has devastated
the Prunus industry in Europe, and USDA is working hard to prevent
the same thing from happening here. To that end, ARS researchers Bill
Schneider, Reid Frederick, and Vern Damsteegt devised the new test to
expedite year-round monitoring of plum pox, particularly in and around
Pennsylvania's Adams, Cumberland, Franklin, and York countiesthe
only U.S. sites where the disease is known to occur.
Each year, Pennsylvania Department of Agriculture survey crews collect thousands of leaf samples from commercial orchards, nurseries, residential properties, and other sites for analysis in diagnostic laboratories. It's a monumental task, but one that's deemed vital to eradicating plum pox as an economic threat to the rest of the state as well as to the nation's $1.8 billion stone fruit industry.
"What makes this virus so hard to detect is that
it only shows up at certain times of the growing season and in certain
parts of the tree," says Schneider, who, along with Damsteegt and
Frederick, works at ARS' Foreign Disease-Weed Science Research Unit,
in Fort Detrick, Maryland. "We were looking for a faster, more
sensitive method of detecting PPV and chose real-time PCR."
PCR stands for polymerase chain reaction, a chemical procedure
that mass-produces copies of particular DNA fragments or genes so they
can be identified by other means. With real-time PCR, a targeted fragment
becomes detectable virtually the same instant it's mass-produced, or
"Plum pox shows up in spring and tapers off by summer.
So you're limited to a 1- to 2-month window for effective testing,"
says Schneider. "By the time the temperature reaches 84 °F90
°F, the concentration of virus goes down dramatically." When
this happens, he continues, standard tests like the ELISA (Enzyme-Linked
Immunosorbent Assay) can give misleading results, such as false negatives.
Although PCR-based methods are also used, including one called immunocapture
PCR, they're labor intensive and time consuming.
"Immunocapture PCR is technically difficult, and
if you don't do it right, it can lead to false positives," says
Schneider. The ELISA detection of plum pox is based on monoclonal antibodies
binding to the virus's coat proteins, while the PCR methods lock onto
the gene for making the proteins. Both take about a day to run. Schneider
says real-time PCR is faster, simpler, and (unlike ELISA) works well
with samples that have been placed in prolonged cold storagea
feature that's especially convenient when laboratories are pressed for
"From leaf to final results, real-time PCR takes
from 4 to 6 hours, depending on how hard it is to extract viral RNA
from a sample," says Schneider. Also, "Real-time PCR is sensitive
enough that you can pick up the virus in individual aphids, which is
something the other methods don't do."
The added specificity of fluorescent probes used in real-time
PCR also enables it to differentiate plum pox's coat protein gene from
those of other common Prunus-infecting viruses.
"The real-time test is very quantitative," says Schneider, "meaning you can tell how much virus is in a sample," even during hot weather. This information is critical to tracking where, when, and how quickly the virus has spread.By Jan Suszkiw, Agricultural Research Service Information Staff.
This research is part of Crop Production, Product Value,
and Safety, an ARS National Program (#303) described on the World Wide
Web at www.nps.ars.usda.gov.
L. Schneider, Reid
D. Frederick, and Vernon
D. Damsteegt are in the USDA-ARS Foreign
Disease-Weed Science Research Unit, 1301 Ditto Ave., Fort Detrick,
MD 21702; phone (301) 619-7312 [Schneider], fax (301) 619-2880.
"New Test on Tap for Tracking Plum Pox" was published in the October 2003 issue of Agricultural Research magazine.