In April 2004, Phytophthora ramorum was detected in two large nurseries in southern California. This was unexpected since P. ramorum had only been found once prior to this detection, in a nursery surrounded by infected California bay trees (Umbellaria californica). With this detection, a cascade of events was set in motion because the nursery in question had delivered more than 1.2 million potentially infected plants to retail and other wholesale nurseries in 47 states. When this occurred, there was no validated molecular diagnostic test and no process in place to develop such tests in a methodical and timely manner. At that time, Mary Palm was the national mycologist in the National Identification Services of the United States Department of Agriculture’s Animal Plant Health Inspection Service, Plant Protection and Quarantine (USDA APHIS PPQ NIS) and Laurene Levy was the director of the National Plant Germplasm and Biotechnology Laboratory in Beltsville, MD (part of the Center for Plant Health Science and Technology, CPHST, within USDA APHIS PPQ). During the plant health emergency that ensued, a number of issues related to diagnostics and surge capacity were discovered. Initially, a confirmatory federal diagnostic required a morphological identification of the oomycete in culture as well as a molecular confirmation. Without a validated molecular diagnostic, a severe backlog of plant samples began to develop since the oomycete culture can take as much as 2 weeks to develop characteristic morphological structures for identification. The culturing process is also labor intensive, requiring a minimum of two separate media for accurate diagnosis. Furthermore, obtaining the oomycete into pure culture was often unsuccessful, even though the samples tested positive for P. ramorum in preliminary molecular tests. A validated molecular assay was essential for rapid and accurate diagnostics. Samples took as long as 6 weeks to go through the system, during which time plants in nurseries were held awaiting either destruction or release, based on the confirmed diagnosis. Through collaboration with University of California scientists, a nested polymerase chain reaction (PCR) protocol that could identify P. ramorum from plant samples was developed. Under Levy’s direction, the CPHST lab took this protocol and developed the first molecular diagnostic for P. ramorum that was validated for regulatory diagnostic purposes. This first diagnostic tool required two different sets of PCR primers and two PCR reactions. The test was deployed to the three West Coast states with the largest influx of regulatory samples within 3 months of the onset of the plant health emergency, and Palm and Levy were part of the team that developed the concept of a potentially actionable suspect sample (PASS) protocol. Sample diagnostic time was reduced to less than 10 days with these changes alone.
Levy’s laboratory was the first federal laboratory to use the new protocols to test plants shipped from the California nursery. Once the infected plants were tracked down, 176 nurseries in 42 states were confirmed to be positive for P. ramorum by the CPHST lab. Since the initial P. ramorum emergency, additional molecular diagnostics have been developed by the CPHST lab, targeting different segments of the pathogen genome and including real-time PCR, further reducing the time necessary for accurate diagnosis. The PPQ Molecular Diagnostics Laboratory established in 2006 and led by Palm became the designated federal laboratory to utilize the improved protocols for confirmatory diagnostics. As a result, typical confirmation of P. ramorum from field sample collection to confirmed diagnosis was reduced to less than 4 days.
As a direct result of this and other plant health emergencies, and of the actions of the National Regulatory Diagnostic Teams that Levy and Palm led, several seminal regulatory diagnostic events occurred:
• The National Plant Diagnostic Network (NPDN) began to develop through NIFA as a means to facilitate surge capacity during plant health emergencies.
• The National Plant Pathogen Laboratory Accreditation Program (NPPLAP) began as a system to deploy validated molecular diagnostics and as a means for laboratories to attain accreditation for use of these diagnostics.
• Proficiency testing programs were developed to ensure network-wide consistency of diagnostic test results in laboratories in PPQ, NPDN, and state departments of agriculture.
• A laboratory-based hands-on training program was developed to enhance the skills of diagnosticians from NPDN, state, and federal laboratories for regulatory plant pathogen identification, detection, and preparedness.
• Potentially Actionable Suspect Sample (PASS) protocols were developed to allow laboratories to triage plant samples that were not suspected to be samples of regulatory importance.
• The national diagnostic system was strengthened to efficiently provide diagnostics for additional plant pathogens of regulatory concern.
The work that these two scientists have done in response to the P. ramorum emergency is just one example of many in which their combined efforts have had a significant positive impact on crop biosecurity and regulatory actions. The development, validation, deployment, training, and use of morphological and molecular diagnostic protocols in preparation and in response to other important regulatory programs include the USDA response to soybean rust, huanglongbing of citrus, daylily rust, gladiolus rust, citrus black spot, and Ralstonia solanacearum Race 3 Biovar 2 and in the successful eradiation of Plum pox virus. What is often not seen is what occurs behind the scenes, as both of these scientists and the work of the talented staff of scientists that they led took steps ensuring that we had the tools, training, and efficient diagnostic systems to respond to any number of significant plant disease events.
