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Portable tests deliver plant disease results in under 30 minutes

North Carolina State University researchers continue to push the field of plant disease diagnosis forward, developing simple-to-run tests capable of delivering results in minutes, sometimes before plants show visible signs of disease.

In an article in the latest issue of the journal Phytopathology, NC State plant pathologist Jean Ristaino and her graduate student, Amanda Mainello-Land, who recently earned her Ph.D., report their success creating tests to tell if rhododendrons have been infected by Phytophthora ramorum, a type of water mold known as an oomycete.

The researchers focused on P. ramorum because it represents a major threat to forest trees and nursery plants such as rhododendrons, camellias and viburnums and impacts forest ecosystems and the commercial industries they support.

The most notorious disease the pathogen causes is sudden oak death, which has killed millions of trees in California and Oregon and spurred state and federal regulations and quarantines in the United States and abroad.

As a Fulbright Fellow on sabbatical in Ireland, Ristaino demonstrated the tests earlier this year to a group of foresters in County Wicklow, where P. ramorum has infected larch trees grown in forest plantations.

"The pathogen has killed millions of planted larch trees in Ireland, a country dependent on forest plantations for wood," Ristaino said.

Tool to aid protectors of forests and plant nurseries

Ristaino said the genetic tests her team developed will give those who manage and protect forests and nurseries the kind of information they need more rapidly to keep the disease from spreading.

Because the nursery plant trade is a major route for movement of P. ramorum from areas in the western United States and a potential route for invasions into forests, nursery surveys are conducted routinely in many states to stop spread, track movement of the pathogen and identify lineages.

Samples are sent for testing at certified labs in the United States and Europe, but most of the samples turn out to be false alarms, said Ristaino, William Neal Reynolds Professor in the Department of Entomology and Plant Pathology and a faculty member with the N.C. Plant Sciences Initiative. Many other Phytophthora species infect forest trees and woody ornamentals.

"The labs are typically using very long, drawn out real-time PCR assays right now that take days to weeks to first collect the sample, send it into the plant disease clinic, run the assay and then get the information back to both state regulators and folks in field," she said.

A simple, inexpensive testing option

Ristaino and her associates compare that to the 20 to 30 minutes it takes to run the tests the team developed.

The tests are called LyoBead LAMP assays because they use LyoBeads and a technology known as loop-mediated isothermal amplification. LAMP employs specially designed primers to recognize segments of DNA characteristic of P. ramorum, and LyoBeads are freeze-dried beads that change colors when exposed to targeted pathogens.

The researchers developed LyoBeads that contain all the reagents needed to run the test in a single tube. The template DNA and buffer is added, and then a small portable heat block, powered by a vehicle's cigarette lighter or solar battery, can be used to run the assays in the field at 65 degrees C for 15 minutes as the DNA amplifies.

Ristaino explained that samples can also be loaded on a microfluidic slide and heated on a smartphone cassette or the heat block. The smartphone camera can be used to photograph the results, or a simple visual color change from violet to blue in the tube will signify positive samples.

Ristaino's team developed four assays: One assay shows whether a sample has been infected with any Phytophthora species known to infect the target hosts, and two others show whether the infecting species is P. ramorum or P. kernoviae, a species of concern in the UK and Europe that hasn't yet shown up in the United States. They have also developed the LAMP test to detect the most common lineage of P. ramorum found in the United States, the NA-1 lineage.

Previously, Ristaino and her associates had developed similar tests for P. infestans, which causes significant potato and tomato disease and was responsible for the historic Irish potato famine.

Ristaino said the LyoBead LAMP assay is scalable to other plant pathogens, as long as specific primers are made to target the pathogen of interest.

Such inexpensive, simple tests can speed diagnosis, reducing laboratory testing or eliminating the need for it, Ristaino said.

"The assay can greatly reduce the workflow in plant diagnostic laboratories since field detection of P. ramorum eliminates the other less important Phytophthora species that might be sent into laboratories," she said.

Detection results can also be linked to public or private databases and used in disease surveillance and risk modeling systems to coordinate containment of the disease.