Tests seen as crucial for berries
Arkansas Agricultural Experiment Station scientists say current testing regimens may be missing a significant number of disease-causing viruses in berry plants.
“We need to change how we test or index plants because we are missing viruses using the methodologies that are the standard today,” said Ioannis Tzanetakis, pro
fessor of plant virology for the Agricultural Experiment Station, the research arm of the University of Arkansas System Division of Agriculture.
To keep undetected viruses from slipping through, Tzanetakis proposes increasing testing for viruses in berries from the common method of one or two tests to four tests over two years.
Tzanetakis is also director of the Arkansas Clean Plant Center for Berries, one of four centers for berries in the USDA-funded National Clean Plant Network (NCPN) where the virus tests are conducted for nurseries and breeding programs.
The National Clean Plant Network was created to protect U.S. specialty crops — including berries — from the spread of economically harmful plant pests and diseases. It includes scientists, educators, state and federal regulators, nurseries and growers that work together to make sure plant propagation material is clean and available.
The United States ranks second worldwide in berry fruit production, according to FAOSTAT, a collection of databases covering international agricultural statistics for the Food and Agriculture Organization of the United Nations.
According to USDA’s National Agricultural Statistics Service, berry production provides nearly $4 billion to the U.S. economy.
ESSENTIAL TESTS
“This testing is critical to making sure we have healthy plants in the field,” said John Clark, Distinguished Professor and plant breeder for the Agricultural Experiment Station. “Virus testing is key because clean plants are the lifeblood of the berry industry.”
Since 1964, the experiment station has released 21 blackberry varieties and three blueberry varieties. Clark said that Arkansas blackberries are recognized worldwide for quality, productivity and flavor and are grown on every continent except Antarctica.
Clark said breeding programs distribute new fruit varieties by selling tissue cultures from foundation plants maintained by the breeding institutions to licensed propagators. Propagating nurseries use the tissue cultures to grow larger numbers of plants and sell them to other nurseries and commercial growers.
“Viruses have a huge impact on the longevity of plants in the field,” Clark said. “Viral pathogens can lead to reduced plant vigor, lower fruit quality and reduced yields. Most importantly, it’s costly for the growers.
“Everything we release now goes through NCPN,” Clark said.
TESTING THE TESTS
The NCPN center at the Division of Agriculture conducts research and virus tests for berries.
Existing methods involve one or two tests on propagation material over one or two years. Genetic testing using reverse transcriptase polymerase chain reaction, or RT-PCR, is the standard. A more conventional test called biological indexing, used for some clients in other countries, involves inoculating clean plant material with test samples and growing them to see whether symptoms appear.
A project conducted in Tzanetakis’ lab by Agricultural Experiment Station research associate Dan Villamor in collaboration with the Oregon Clean Plant Center in Corvallis compared RTPCR with high-throughput sequencing. HTS is a scientific method that allows researchers to sequence DNA for massive numbers of organism samples quickly, Tzanetakis said. The process enables rapid identification of viruses and other organisms. Also, HTS can identify new viruses, something the RT-PCR cannot do.
The team published a research paper on their results on the American Phytopathological Society Publications website: https://bit.ly/AAES-BerryVirusPaper.
“The introduction of high-throughput sequencing, also known as next-generation sequencing, has altered the landscape of virus detection,” Villamor said in the research paper, for which he is the lead author.
In Villamor’s study, RTPCR and HTS mirrored each other in identifying known virus pathogens in the test samples. But his results showed that 11 viruses, known to be in 16 of the berry samples, went undetected by both testing methods during some seasons.
“We’ve been working on this for five years to make sure we have solid information,” Tzanetakis said. “And this result was surprising.”
Villamor and Tzanetakis conducted the tests on blackberries, raspberries, blueberries and strawberries using plant material with known virus profiles from the USDA National Clonal Germplasm Repository. The samples were infected with known viruses.
“What we found is that things are quite different from what we believed to be happening in virus testing,” Tzanetakis said. “Things that people think are clean may not be. We found that testing once or twice is not enough. You may have to test four times to know what is in there.”
Villamor said more than 50 percent of viruses were going undetected by the conventional method of conducting two tests over two years.
UPPING THE GAME
The unexpected discovery that viruses were going undetected is what led Tzanetakis to his proposal to increase virus testing in berries to four tests over two years.
He also believes that HTS should replace RTPCR in standard testing. Not only can it test more samples more quickly, but it can also find viruses not expected to be present.
“PCR is very sensitive,” Tzanetakis said. “But to get a valid test, you have to understand the virus situation. You must know what you are looking for.”
RT-PCR cannot identify previously unknown viruses and some variants, he said.
“Because viruses mutate, you can miss a new variant because you’re not looking for it,” Tzanetakis said. “In the plant world, mutations are more common than not. Using HTS, you don’t have to know the diversity of the virus. It finds variants and new viruses even if you don’t know it’s there.”
Tzanetakis and his lab team have developed Virfind, a virus detection and discovery software housed at the Arkansas High Performance Computing Center that analyzes the HTS data and identifies unknown viruses and variants.
A research paper describing Virfind can be found online at: https://bit.ly/AAES-VirfindPaper.
In addition to the viruses already known to infect plants in this study, Tzanetakis’ team found six new viruses infecting them.
“This was surprising because those plants were collected from (USDA’s) NCGR and have been studied for decades,” Tzanetakis said.
Once they found the new viruses, they investigated how widespread they are in production fields and nurseries. “One of the new viruses was found in more than 52 percent of more than 600 samples collected from around the country,” Tzanetakis said.
“People have been thinking of HTS as a superior test, and it is,” Tzanetakis said. “But being superior is not the same thing as being perfect. Even if you use RTPCR and HTS, some plants get through with undetected viruses. That’s why we need to increase testing.
“That’s important,” Tzanetakis said, “because if you miss something and propagate a plant millions of times, then you end up with a time bomb on your hands.”
To learn more about Division of Agriculture research, visit the Arkansas Agricultural Experiment Station website: https://aaes.uada.edu/. Follow the agency on Twitter at @ArkAgResearch.
The University of Arkansas System Division of Agriculture offers all its Extension and Research programs and services without discrimination.