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Comparative Evaluation of Diagnostic Methods for Asian Soybean Rust

Wisler, Gail
University of Florida
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End date
  1. Investigators and collaborators will evaluate three diagnostic methods against each other during the 2005 growing season. These evaluations will be performed by NPDN diagnosticians or collaborators in six high-risk states: Mississippi, Kentucky, Tennessee, North Carolina, Georgia, and Florida. The first four states have a large number of soybean-producing acres and, according to the USDA models, are most likely to be among the first states to be affected by the disease in 2005. Florida and Georgia are currently the only states in the continental US that have been confirmed with the disease and as such, will most likely be the lead states in terms of samples. This will ensure that we have first-year data (2005) for this project, regardless of whether all of the other four states confirm soybean rust within their borders.
  2. A bean variety trial will be planted in a previously confirmed county in Florida to provide material for training, observation, and for use in the method comparison study. Samples from this plot will be used in addition to samples submitted through other means, including through the Florida State Department of Agriculture. The addition of multiple hosts will increase the robustness of the method comparison study. This plot will be monitored for both Asian soybean rust and common bean rust and samples will be collected as described in the Methods section. New hosts not previously identified in Florida will be confirmed by the federal APHIS designate. The investigators hope to help fill a void in the literature concerning reports of Asian soybean rust on edible legume varieties. Data collected will be made available in an expedient manner to facilitate scouting preparations across the country.
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NON-TECHNICAL SUMMARY: While several methods exist to diagnose Asian soybean rust, constraints of time, personnel, and finances compel diagnosticians to choose the most efficient and appropriate method. Currently, there is little information to aid in making that decision. The host range of this pathogen on commercial varieties of legumes other than soybeans has been determined partly by artificial inoculation in containment. While this gives producers and others some indication of the host range, the results of natural inoculation in the field is currently unknown in the US. This project examines the methods used to diagnose Asian soybean rust to determine the most efficient and appropriate method. This project also involves the study of hosts and determination of field disease occurrance due to natural inoculum.

APPROACH: Diagnosticians in the participating labs will process 20 samples in triplicate with real-time or traditional PCR protocols and with ELISA protocols. Samples for this experiment will be selected from those submitted to the diagnostic clinics involved. Diagnosticians will process 10 samples that appear to be positive after observation with light microscopy, given that 10 positive samples are received in each lab during the experiment, and 10 that exhibit symptoms not believed to be Asian soybean rust. At the conclusion of the experiment, data will be subjected to the appropriate statistical analysis, to include calculations of coefficients of variation within and between labs. The dependent variables would include 1) reliability of each assay (how often each method gives a false positive/negative), and 2) the time to perform the given assay. A cost-benefit analysis can be done based on the results of 1 and 2. Nineteen commercial varieties of beans, including southern pea, soybean, pea, and azuki, bush, wax, pinto and black turtle beans have been obtained from sources across the US. These beans will be planted in four completely randomized blocks consisting of four two-hundred-foot rows for each variety. The plot will be exposed to the natural inoculum already available at the Pine Acres Research Farm in Citra, FL. The Marion County positive find of Asian soybean rust on kudzu is located in this county, and common bean rust (Uromyces phaseoli) inoculum is also naturally available. This plot was planted the first week of May 2005 and will be scouted every seven to ten days. For each surveillance date, plants will be inspected for signs and symptoms of both Asian soybean rust and common bean rust, which include urediniospores, blisters (premature sori), and pustules (mature sori).

PROGRESS: 2005/08 TO 2006/07
Plant diagnosticians routinely process soybean rust samples with one or more of the following diagnostic protocols: visual assessment using a dissecting microscope, an enzyme-linked immunosorbant assay (ELISA), and pathogen-specific PCR (conventional and real-time). Results of three diagnostic protocols and time requirements for diagnosis were collected for samples with symptoms of soybean rust (presumed positive) and samples without sporulating rust pustules (presumed negative). Data were collected for samples processed in triplicate at six university plant disease diagnostic clinics. The effects of sample treatment and storage on results of two diagnostic protocols also were examined. Visual diagnosis required the least amount of time, whereas ELISA and PCR took approximately six-and-a-half times longer per sample (multiple samples being processed at one time) than a visual diagnosis. ELISA could not detect the pathogen in positive samples after desiccation followed by storage at room temperature and following one or more autoclave treatments for 30 min. However, conventional PCR was capable of detecting P. pachyrhizi-infected plant material following all sample storage regimes. With proper training, diagnosticians are proficient at recognizing soybean rust after observation of pustules and spores using a dissecting microscope. PCR and real-time PCR are useful tools for confirmation of the soybean rust pathogens at the species level, especially in Florida and the Gulf Coast where P. meibomiae is most likely to occur. PCR is also useful for early detection before the development of soybean rust symptoms and diagnostic signs. The ELISA kit that was evaluated is not species-specific and requires too much time to be useful for field employment. We found that the ELISA kit was not as sensitive as PCR when detecting rust on samples that are stored or transported under sub-optimal conditions. Therefore, negative ELISA results of symptomatic tissue should be further investigated using PCR. However, if an ELISA kit were developed that could be read in about 15 min or less, species-specificity would not be necessary in order to be useful for personnel as a rapid confirmation of field observations.

IMPACT: 2005/08 TO 2006/07
Asian soybean rust has the potential to reduce crop yield by as much as 80% if the diseases is not detected early enough to provide disease management. This study emphasized the practicality of the techniques currently in use to detect the pathogen. In addition, six different laboratories were involved, ensuring that the techniques and protocols were followed by at least six different people, to test the validity of published protocols.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Chemical Contaminants
Heavy Metals
Grains, Beans, Legumes