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Agronomy Day 2009

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Tour B

Utilization of Molecular Detection Techniques to Find Soybean Pathogens

James S. Haudenshield
James S. Haudenshield
Research Plant Pathologist
USDA-Agricultural Research Service, Urbana.
(217) 244-3257
jsh1@illinois.edu
Glen Hartman
Glen Hartman
Research Plant Pathologist
USDA-Agricultural Research Service; and Professor
Department of Crop Sciences, University of Illinois Urbana, Il
(217) 244-3258
ghartman@illinois.edu

Soybeans continue to rise in prominence as a source of feed, food, oil, and renewable energy.  Of many factors impacting yield (including soybean variety, soil fertility, water availability, climatic conditions, and insect pests) microbial pathogens alone can cause losses in excess of 11% of global production.  Roughly 35 fungal, bacterial, protozoal, and viral pathogens are of present economic importance, and individually these may be responsible for losses ranging from slight to complete devastation in a particular field.  Complicating this, a pathogen may be quite destructive one season, but unimportant the next.  Furthermore, some pathogens may require specific environmental conditions to initiate infection of plants, or to spread rapidly leading to an epidemic.  Management of soybean diseases and pests involves many approaches including cultural aspects like crop rotation, tillage practices and cultivar or variety selection. Other management practices include pesticide applications of fungicides or insecticides to the surface of seed or on plant foliage for protection from disease and pest organisms.

Both field diagnosis (for immediate disease control) and research on disease epidimeology and management, inherently require a method of detecting and quantifying the presence of the pathogen responsible.  Traditional methods often require highly skilled diagnosticians, animal-based antibodies, lengthy grow-out or culturing procedures, laboratory equipment and methods unique to each pathogen (nematodes, fungi, etc.), and tissue fixation and microscopic evaluation or even electron microscopic analysis.  Recent advances in molecular biology, however, now permit identification and quantification based on the presence of the pathogen DNA.   The amount of pathogen DNA present is directly proportional to the number of pathogen cells present, which in turn is proportional to the mass of mycleium, the number of fungal spores, bacteria, or protozoa, or the quantity of virus.  Because DNA is fundamental to all life, and yet the primary structure of the DNA is unique to every species, it is possible to employ a streamlined procedure to isolate the DNA from infected plant specimens (or infested soil), and then to utilize a common type of molecular assay (Q-PCR) to determine the precise identity and quantity of pathogen present.  Q-PCR:

  • is highly specific, sensitive, and reproducible.  Single molecule detection is common;
  • is fast: 1.5 hr from reaction assembly through cycling to results for up to 96 samples;
  • requires no plant grow-outs, greenhouse maintenance, inoculations, or microbial culture;
  • is amenable to robotic scaling for mass screenings (used commonly in human disease diagnoses);
  • requires no quarantine precautions in the laboratory or diagnostic facility;
  • assays are already available for pathogens causing many soybean diseases, including:  anthracnose, charcoal rot, sudden death syndrome, soybean rust, phytophthora root and stem rot, brown stem rot, and soybean cyst nematode.

New and/or improved assays are being continually reported.  The Soybean Disease Laboratory at the National Soybean Research Center is a nexus for soybean disease Q-PCR assay development in the United States. In part, this research was supported by the Illinois Soybean Association.

Disease organisms can be founf in different plant tissues

Extract DNA and assay using real-time PCR

change and challenge