Fingerprinting a Killer—A Nematode Killer, That Is

A new genetic fingerprinting method is now on hand to identify Pasteuria bacteria that kill soybean cyst nematodes—culprits behind $324 million to $1.4 billion in annual yield losses.

ARS nematologist Greg Noel and colleagues devised the method to help resolve confusion about Pasteuria's taxonomic classification and, in turn, speed efforts to identify strains with the greatest potential as nematode biocontrol agents. (See "Soybean Cyst Nematodes, Look Out!" Agricultural Research, September 1997.)

Conventional methods involve collecting "endospores"—Pasteuria's infectious stage—from nematode specimens so that ribosomal DNA (deoxyribonucleic acid) can be extracted from the bacterium using centrifugation, heat, enzymes, and other chemicals. It's a laborious, time-consuming affair, however, partly because at least 1 million endospores are needed to yield enough DNA to analyze. Contamination by other bacteria is also a problem.

With the new method, which includes use of DNA-multiplying technology called polymerase chain reaction (PCR), "We can extract ribosomal DNA from a single, infected nematode that has several hundred to a few thousand endospores," says Noel, who's in ARS's Soybean/Maize Germplasm, Pathology, and Genetics Research Unit in Urbana, Illinois. His colleagues are Ndeme Atibalentja, at the University of Illinois at Urbana-Champaign, and Aurelio Ciancio, Institute of Plant Protection, Bari, Italy.

For years, says Noel, researchers used morphological, developmental, and pathological characteristics to describe and classify Pasteuria's four known species. Now, comparing differences in the bacteria's ribosomal DNA is deemed more precise.

Although the approach is widely used, Noel felt there had to be an easier way of extracting the DNA from endospore-infected nematodes. Instead of using conventional methods, his group used "glass bead beating." This procedure involves crushing a nematode specimen so that any DNA within it, including that from Pasteuria, is released into solution. Lab-built molecules called primers are then added. They bind with specific fingerprint regions on Pasteuria DNA so it can be multiplied by PCR, cloned, and sequenced for identification.

Noel's group has already used the method to confirm the identity of P. nishizawae in an Illinois soybean plot, marking the first report of this promising biocontrol species in U.S. soils.—By Jan Suszkiw, Agricultural Research Service Information Staff.

Gregory R. Noel is in the USDA-ARS Soybean/Maize Germplasm, Pathology, and Genetics Research Unit, 1102 Goodwin Ave., Urbana, IL 61801; phone (217) 244-3254, fax (217) 333-5251.

"Fingerprinting a Killer—A Nematode Killer, That Is" was published in the March 2004 issue of Agricultural Research magazine.