Viral Strike Team Always on Alert
In 2001, a package arrived in the mail, addressed to a little-known team
in a laboratory in a small Ohio town. It contained pieces of sweet corn leaves
infected by a mystery virus. The viral infection showed up as spots and streaks
that ranged in color from pale green to yellow to cream.
Using a tool he helped construct, technician John Abt injects virus into corn kernels via vascular puncture inoculation. The kernels will germinate and grow into an infected plant.
The scientific detectives had little to work with. The leaf bits were drying
up and falling apart, having been taken off fresh corn plants in Georgia fields
several days before. The team had also received a similar package from Arizona,
but those leaves showed different symptoms. To identify the suspected viral
culprits in both these cases, they needed a lot more infected leaves.
Fortunately, this group had the tools and expertise to grow their own supply
of infected plants for study.
The viral strike team, located at the Ohio State University-Ohio Agricultural
Research and Development Center in Wooster, Ohio, serves as the front line for
spotting viral attacks on corn worldwide and on corn and soybeans domestically.
Research associate Dave Fulton (left), of Ohio State University, and research leader Roy Gingery, of ARS, use a scanning electron microscope to help identify pathogens in corn samples from Serbia.
The team is part Agricultural Research Service
(ARS), part Ohio State University (OSU). Roy Gingery heads the ARS half of the
team, which is officially called the Corn and Soybean Research Unit. The other
ARS researchers include plant molecular biologist Peg Redinbaugh, molecular
geneticist Rouf Mian, and plant pathologist Ray Louie, who is officially retired
but works 3 days a week as a research collaborator. Along with their OSU colleagues,
these four study interactions among viruses, plants, and insects to develop
ways to reduce disease losses.
Technician John Abt says they first tried a leaf rub technique
with the mystery virus samples as a way to build up a supply of infected plants.
They rubbed extracts from the infected leaves onto fresh corn leaves growing
in a quarantine greenhouse, but the corn plants didnt get infected.
This didnt particularly surprise Abt or his colleagues; the rubbing technique
doesnt work with most viruses. It may be that this virus needed
to reach vascular cells deep within the plant, and simply rubbing the leaf wasnt
sufficient to let the virus penetrate deep enough to reach those cells,
Another way to create more infected plants for research is to test a variety
of insects to see which ones might transmit the viruses. But there wasnt
enough plant material to feed insects to allow the tests to start.
Technician Tim Mendiola uses "leaf rub inoculation" to transmit bean pod mottle virus to soybeans in a greenhouse at the Ohio Agricultural Research and Development Center in Wooster, Ohio.
Inventing Artificial Insects
Thats when the teams vascular puncture inoculation (VPI) technique
proved instrumental. Louie has been working on this artificial insect since
1991. The initial prototype was based on a simple concept, and it worked, though
it looked pretty primitive. He glued five extremely fine and tiny insect-mounting
pinsknown as minutensto a flattened copper wire, creating
a pin comb. The entire assembly was then inserted into a jewelers engraving
To use VPI, Abt and Louie ground up infected leaf samples to make a liquid
extract, then put a drop of the extract on a presoaked kernel of healthy corn.
Next, they pressed the teeth of the pin comb through the extract into the softened
kernel, infecting the corn embryo with the help of vibrations from the engravers
tool. This enabled them to implant virus into the corn kernel and grow an infected
The VPI method allowed the team to produce enough infected tissue to study
the viruses. It eliminated the need to first find and then mass-rear insect
carriers, and it required only a few leaf samples to work with.
The corn planthopper (Peregrinus maidis) transmits maize mosaic virus.
As a result, the scientists were able to identify what they at first called
the Georgia unknown virus. They later named it maize fine
streak virus. They were surprised to find that the Arizona virus, which
they named maize necrotic streak virus, belonged to the Tombusviridae
family, whose members had been known to attack only broadleaf plants such as
tomatoes and peppernot grain crops like corn. Most tombusviruses are spread
by soil rather than insects, making them less likely to spread far. But
we have to take it seriously, because weve found that if it were to infect
a cornfield, its unlikely the farmer would be able to harvest a single
ear of corn from the field, Redinbaugh says.
She and her team have found one insect carrier of maize fine streak virus,
the black-faced leafhopper. Theyve also mapped the complete genomes of
both of the viruses.
Using VPI, the viral strike team has been able to transfer all major corn viruses
into corn. They have also used it successfully with other crops, including soybean,
wheat, and rice. This technique gives us the option of mailing infected
seeds instead of leaf samples. Seeds withstand shipping better. So far, the
method is working very well, Abt says. He has successfully sent infected
corn kernels to Wisconsin virus researchers.
If you walked in on Abt and Louie at work, youd likely see the old and
new VPI prototypes at work simultaneously. Abt is infecting a tray of corn kernels
with the engraving tool attached to the homemade minuten comb, while Louie uses
a greatly updated version. Short, low sound vibrations have replaced the loud,
high-pitched buzz of the jewelers engraver.
The black-faced leafhopper (Graminella nigrifrons) transmits both maize fine streak virus and maize chlorotic dwarf virus.
Transferring the Technology
The team has a cooperative research and development agreement with Pioneer
Hi-Bred International, Inc., headquartered in Johnston, Iowa, to refine, automate,
and optimize todays VPI technique. Pioneer plans to use VPI in breeding
programs around the world to screen for resistance to important viral diseases.
While VPI is an important contribution to the cause of identifying and fighting
plant viruses, it is by no means the teams only accomplishment.
The scientists supply breeders with viral detection kits. Their corn and soybean
genome maps give breeders a road map with markers and clues to help search their
own proprietary stock of plant germplasm for viral resistance. Besides identifying
new viral diseases and their carriers, the team also seeks to use safe versions
of viruses to transfer plant genes into crops to investigate their functions.
Team members travel to foreign countries occasionally. In 2004, Redinbaugh
and Gingery, accompanied by OSU maize geneticist Rich Pratt, went to Serbia
to help identify a suspected pathogen, perhaps a new virus, attacking corn.
While there, they shared the teams techniques and the VPI method so that
Serb scientists could form their own, similar strike team. USDAs Foreign
Agricultural Service requested their services, and they are still working on
We have to identify the pathogen first and then the vectors or carriers
that spread it. Then well search the corn germplasm collection for genes
resistant to it, Redinbaugh explains.
An important tool for identifying viruses is an electron microscope. The team
also makes use of modern imaging toolssuch as confocal and scanning electron
microscopesat the Wooster facilitys Molecular and Cellular Imaging
Center. Were fortunate that all the team members are located nearby,
Redinbaugh says. Together, we have expertise in several areas, including
cloning, genetics, biology, entomology, and virology.
Answering Viral ThreatsOld and New
Technician Jane Todd uses an aspirator to collect black-faced leafhoppers (Graminella nigrifrons) for transmitting maize fine streak virus.
The team was first formed in the 1960s in response to a dual epidemic of maize
chlorotic dwarf virus (MCDV) and a potyvirus, maize dwarf mosaic virus, that
devastated Ohios cornfields. Today, the Midwest faces potential threats
to soybeans because of several factorsexcessive rainfall, increased insect
numbers from mild winters, widespread use of reduced tillage, less use of crop
rotations, entry of the soybean aphid into the United States, and heightened
threat of exotic viruses as travel and trade increase, including rising imports
of ornamental plants. Ohio, for example, has experienced about every soybean
disease imaginable over the past two wet years, 2003 and 2004. Fortunately,
a dry fall rescued the 2004 soybean harvest, resulting in record yields.
But in recent years, bean pod mottle virus has emerged as a new pathogen of
soybeans in Ohio, along with increased populations of its carrier, the bean
leaf beetle. This virus belongs to an important family of viruses, the Comoviridae,
and can not only lower yields but also reduce seed quality because of discoloration.
Soybean aphid populations have been peaking every other year since they were
first found in the United States in 2001 in the Great Lakes States, so they
may be a problem again in 2005. The researchers are studying the aphids to see
whether theyre transmitting any viruses.
Plant molecular biologist Peg Redinbaugh pollinates corn during experiments to map virus resistance.
Meanwhile, old threats like MCDV cause significant losses in corn in the southeastern
United States. The team, led on this project by OSU entomologist Saskia Hogenhout,
has produced antibodies that have detected several MCDV proteins in infected
plants and insects, including one thought to facilitate transmission. This helper
protein serves as a bridge to bind the virus particles to leafhopper mouthparts.
Characterization of the interaction between the helper protein and the virus
particle will help determine how the virus is transmitted from plant to plant
during epidemics and may suggest ways to disrupt transmission and, ultimately,
Team members Mark Jones, an agronomist, and Bob Anderson, an entomologist (now
retired), were largely responsible for identifying the part of the corn genome
that holds genes for resistance to MCDV and developing genetic markers for these
genes. Redinbaugh says that when she came to Wooster 6 years ago, three accomplishments
helped pave the way for her group to identify the resistance genes in corn:
The team had found a way to transmit the virus with a high success rate; theyd
found corn germplasm with the highest resistance everfrom the Caribbean;
and in collaboration with OSUs Rich Pratt, they had designed a way to
assign scores to indicate the amount of resistance present in crop germplasm.
Now the current team of ARS and OSU researchers is paving the way for future
successes in protecting U.S. crops from viral diseases.By Don
Comis, Agricultural Research Service Information Staff.
This research is part of Plant Diseases, an ARS National Program (#303)
described on the World Wide Web at www.nps.ars.usda.gov.
Roy Gingery, Peg
Redinbaugh, Mark Jones, John Abt, Raymond
Louie, and Rouf Mian are with the USDA-ARS
and Soybean Research Unit, Ohio State University, Ohio Agricultural Research
and Development Center, 1680 Madison Ave., Wooster, OH 44691; phone (330) 263-3838,
fax (330) 263-3841.
"Viral Strike Team Always on Alert" was published in the May
2005 issue of Agricultural Research magazine.