Progress 12/15/06 to 12/14/07
Outputs
OUTPUTS: Experiments were conducted to modify and validate the tissue blot immunoassay (TBIA) as a high-throughput, easily processed format for detecting bean or soybean viruses for the Legume Pest Information Platform for Extension and Education (PIPE) for 2007. A supported nitrocellulose membrane gave better results than six other membranes. Interfering green plant residue was removed (decolorizing) by soaking in dilute Triton X-100, and was improved by gentle abrasion. Blocking with a polymer was superior to milk and bovine serum albumin, and was combined with decolorizing. Antibody sources tested were either mouse monoclonal or rabbit polyclonal antibodies for alfalfa mosaic virus (AMV), bean common mosaic virus (BCMV), bean pod mottle virus (BPMV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), peanut mottle virus, peanut stunt virus, soybean mosaic virus (SMV) and tobacco ringspot virus. Optimal antibody concentrations were selected for each virus/antibody
combination to produce intense positive reactions with infected and minimal non-specific reactions with healthy leaf controls. Virus-specific antibody was combined with alkaline phosphatase enzyme-conjugated anti-animal antibody to eliminate an incubation step in processing. Several buffers were tested before selecting one that optimized intensity of positive reactions relative to negative controls. A substrate was selected from among several tested because of its stability during shipment and storage, as well as specificity and reliability. Results with TBIA were verified through tests with leaf tissue known to be infected with SMV or BPMV as confirmed by DAS-ELISA. With SMV there was 100% agreement, with all samples testing positive by ELISA also TBIA positive. In two tests for BPMV, 86% and 96% of ELISA positive field samples were TBIA positive, but other tests were less than 50% reliable. Similar tests were not conducted with other viruses. Prototype kits for TBIA detection of six
legume-infecting viruses were designed. The sampling device was a nitrocellulose membrane covered by a card with 2 sets of 50 holes, designed to test for two viruses in 45 samples collected from sentinel field plots of bean or soybean by a standard PIPE protocol. The kit contained, for each plot, vials of decolorizing/blocking buffer, antibodies, buffer for rinsing and diluting antibodies, and substrate. An instruction sheet with spaces for recording sample data, protocol steps and results was included. Kits were prepared by Agdia and provided below cost to participating laboratories. At the National Plant Diagnostic Network (NPDN) meeting, we had a hands-on TBIA demonstration for diagnosticians. We developed a 45-minute module for a live video-conference for Legume PIPE training, and prepared a Standard Operation Procedure (SOP) for the NPDN diagnosticians website. We spoke to the American Phytopathological Society (APS) diagnosticians group at the national meeting, where updates and
experiences with the method were shared. I also gave presentations and posters at the Potomac Division and national APS and the Bean Improvement Cooperative meetings to scientists, diagnosticians and stakeholders.
PARTICIPANTS: Sue A. Tolin directed the project. The partner organization collaborating on the TBIA research was Agdia, Inc., with Chet Sutula co-directing the project. Bernard Kulemeka was partially funded at Agdia to test TBIA modifications and select antisera, and develop kits. At Virginia Tech, Moss Baldwin was hired for 9 months of 30 hrs per week on this grant for interacting with Agdia on development of the TBIA for legume viruses. She prepared materials for the NPDN demonstration, co-authored the SOP, and prepared images for use in Powerpoint presentations at meetings and training sessions. She attended a training session in Powerpoint to assist in this work. Philip Keating, assigned for 20% of his time, was in charge of maintaining the greenhouse and growing conditions for plants and virus cultures, and assured pesticides were applied to prevent infestations with arthropods that are virus vectors. He also prepared land for testing of beans and soybean for responses to virus,
and assisted Moss Baldwin in field inoculations to grow materials for validation tests with known viruses. Participants of the Legume PIPE steering committee provided advice on sampling and test card formats, and ease of entering data collected in the assays to the NPDN and PIPE websites. Drs. Marie Langham and Howard Schwartz were particularly helpful in their capacity as eastern and western Legume PIPE coordinators. Over 100 state representatives for the Legume PIPE participated in the streaming video TBIA training sessions, hosted by the University of Illinois and Dr. Susan Ratcliffe.
TARGET AUDIENCES: Workshops and experiential learning included and training of NPDN diagnosticians in TBIA for viruses and the Legume PIPE.
Impacts
In our experiments to improve TBIA we combined steps and shortened it to a three step, 3 hour assay. Results compared favorably with ELISA, and had the advantage of no sample grinding and the ability to hold blotted membranes to batch process. Agdia's prototype TBIA kit worked well in tests for 6 viruses. Field-infected plants were, in general, more strongly positive than greenhouse-grown plants, indicating higher virus titer in field-grown plants. In some cases high backgrounds interfered with scoring the results and were associated with the species or cultivar of bean tested or with the failure to follow instructions in the laboratory. Test-wide systemic errors were not noted. Overall, TBIA enabled the Legume Virus PIPE to develop a system for monitoring viruses and built capacity within the plant disease diagnostic network to test large numbers of plants for specific viruses by a simple, high throughput method. Virus detection and incidence data were obtained from
nearly 20,000 plants from 58 soybean and 158 bean sentinel plots sampled twice, and tested for 2-4 viruses per sampling time. Twenty-nine states tested soybean rust sentinel plots for two viruses in two plots, sampled two times. In bean (dry, snap, etc.) 8 states tested for BYMV and CMV, 22 states tested for AMV and BCMV, and 4 states tested for the 4 viruses. TBIA performed well at one or several locations for each of the 6 viruses, giving strong positive results and clear negatives. Samples from soybean in 6 states were positive for BPMV, but none for SMV. Bean samples were positive for AMV in 6 states, for CMV in 3 states, for BCMV group in 3 states, and BYMV in one. The suggestion was made that symptomatic plants be sampled, to help verify the system. Results were discussed on biweekly calls of PIPE participants, when problems with the assay were raised, and attempts made to find solutions to these problems and give advice as protocol clarifications. Very favorable comments were
received from diagnosticians on the protocols and ease of use. The following is an excerpt from an unsolicited comment from a diagnostician: " First, I found the written directions for each step of the test to be concise, clearly written and easy to understand, which made my job easier. The directions for interpretation were clearly written as well. The parts of the kit used for testing were clearly labeled as well. Agdia supplied plenty of reagents to carry out the testing. This was much appreciated, too. Lastly, the actual interpreting of blots was somewhat difficult for me. Some of the blots were clearly a positive or negative while others were a challenge to interpret." Diagnosticians logically believed that pressed leaf samples should look like the controls, which turned out not to be the case. The interpretation problem was in part because the positive and negative controls were microliter spots of re-hydrated sap from freeze-dried leaves, rather than the leaf pressed onto
membranes. Many labs tended to over interpret slightly dark spots as positives in early tests before they had observed a strong positive. Changes in controls and reference images of expected results are needed.
Publications
- Langham, M., Tolin, S., Sutula, C. Schwartz, H., Wisler, G., Karasev, A., Hershman, D., Giesler, L., Golod, J., Ratcliffe, S., Cardwell, K. 2007. Legume/Virus PIPE - A new tool for disease management in legumes. (Abstr.) Phytopathology 97:S61.
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