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Keeping tabs on plant viruses and viroids.

The tests were positive, but the symptoms weren't.

That's when Bill Lanterman thought of asking USDA for help.

Lanterman, director of the Saanichton Plant Quarantine Station maintained by Agriculture Canada on Vancouver Island, suspected that peaches and Japanese apricots being held in the station might be infected with the devastating plum pox virus (PPV) or another unidentified virus.

Plum pox virus causes peaches, plums, apricots, and several other stone fruit trees to drop their fruit prematurely. If it stays on, the fruit is deformed and deteriorates rapidly. PPV is considered die most serious viral disease of these fruits in Europe, particularly in eastern and Mediterranean regions. And strains of the virus that attack almonds and cherries are spreading. Like most disease-causing viruses, they are carried from infected trees to healthy ones by sucking insects such as aphids.

Lanterman's suspicions were viewed with alarm, because plum pox virus doesn't occur in Canada or the United States. An accidental outbreak would wreak havoc with an industry worth a billion dollars in the United States alone.

The request for help in identification eventually came to plant pathologist Ahmed Hadidi at ARS' National Germplasm Resources Laboratory in Beltsville, Maryland. Hadidi is a world expert on plant viruses and their detection. His specialty: the detection of viral diseases of fruit trees.

He has studied viruses for 33 years and viroids for the last 18. His research has led to the discovery of both a new virus and a new viroid. Viroids are the tiniest disease-causing organism known - about 80 times smaller than the smallest virus.

Detecting Plum Pox Virus

Hadidi enlisted plant pathologist Laurene Levy to help in the search for new and better ways to test for plum pox virus in trees quarantined in the United States.

When Levy and Hadidi tested the Canadian plants for PPV using standard detection methods like ELISA and molecular hybridization, they found that most did indeed react positively.

"In many cases, the problem with detecting PPV is that infected trees often contain very small amounts of the virus," she says, "and it's frequently below the level that current methods can detect. Added to this, the virus can be unevenly distributed throughout the tree."

To overcome these obstacles, Hadidi first analyzed published scientific findings about PPV strains. He found that this virus has unique genetic information at the end of its RNA strand, and this knowledge gave him the key to a solution: He would develop a new diagnostic test around the virus' unique genomic fingerprint.

"To make enough PPV to detect it molecularly, we used a technique developed by Hadidi to detect dapple apple viroid of pome fruit," Levy says. "It's called reverse transcription-polymerase chain reaction, or PCR."

PCR has already proven a useful tool in identifying several viruses, viroids, and mycoplasma-like organisms directly from nucleic acid extracts of infected hosts.

"The technique relies on the virus' unique end, which, like a fingerprint, distinguishes PPV strains from other plant viruses that do not affect stone fruit trees," Levy explains. "These 220 nucleotides are found next to the virus' coat protein gene.

"PCR amplifies this unique genetic end to make additional copies of the specific sequence so as to produce enough material for quick and reliable genetic fingerprinting. Amplification takes only about 3 to 5 hours," she adds.

Next, a small amount of this amplified material is spotted onto a membrane and subjected to a PPV-specific probe - either chemiluminescent (chemical produces light to expose photographic film) or radioactive - to accurately identify it.

"What develops as dark spots on the film is PPV's fingerprint that can be compared to the standard in each test," says Levy. "The more dark spots, the more PPV."

"When we ran the new test on the Canadian plants, only a few - all coming from Asia - tested positive for PPV," Hadidi says. "The other plants were infected with a new virus that we identified and named prunuslatent potyvirus."

"It's the most accurate test to date for pinpointing PPV and for distinguishing it from other viruses," Levy says.

Besides uncovering a new virus, the test solved the mystery of the phantom virus in stone fruit that had eluded identification at the Canadian quarantine station.

"As yet, we have no evidence that prunus-latent produces disease," says Levy, who believes the new test can be used worldwide to identify PPV in plant material.

Adds Hadidi, "This is very important, because we've always believed that PPV originated in Bulgaria. We and the Europeans may have been overlooking the virus from Asia. Now, we can no longer assume plant material from Asia is not infected with the virus."

Hadidi says the potential increase in travel and exchange of goods between the United States and Eastern Europe and the former Soviet Union heightens the risk of PPV striking American orchards.

Hadidi points out, "At present, plants must be grown for weeks or months from quarantined germplasm before seedborne diseases are revealed, whereas the new test screens plants for PPV in a day or two.

"Presently, in most countries, stone fruit seedlings and trees that react positively to PPV antiserum or molecular probes are automatically destroyed," he says. "Some plants, perhaps unnecessarily, if they are not truly infected with that virus."

Detecting Potyviruses

PPV is just one of about nearly 180 viruses - about 36 percent of all known plant viruses - called potyviruses. The name is short for the potato virus Y group.

"Potyviruses make up the largest and most important group of viruses that affect U.S. crops and are responsible for estimated losses of 5 to 20 percent each year," says Roger Lawson, head of ARS' Florist and Nursery Crops Laboratory at Beltsville.

"There are no known incidents of their causing any disease in humans and animals," he adds.

These viruses are carried from plant to plant by aphids when the pests feed on leaves and stems. They also spread during harvesting and are especially devastating to the cut-flower industry.

"Unlike virus-infected food crops that lose their value but can still be used, high-value ornamental crops such as orchids can be totally lost," says Lawson. "In Florida, many commercial gladiolus crops are now almost totally infected by bean yellow mosaic virus, reducing their $13 million annual market value by as much as 15 to 20 percent."

Since 1983, plant pathologist Ramon Jordan has been working in the Florist and Nursery Crops Laboratory, looking at the structure and biochemistry of viruses to better control them. In the course of his research, he and colleague John Hammond developed monoclonal antibody-based technology for a diagnostic kit now used worldwide to detect plant-damaging potyviruses.

Says Jordan, "It will detect at least 55 distinct potyviruses that can lower the market value of vegetables and ornamentals. The kit uses the ARS-patented monoclonal antibody probe to identify the viruses that attack many plant species including beans, wheat, lettuce, and potatoes, as well as lilies, irises, and tulips."

This potyvirus kit is the first capable of detecting such a broad spectrum of plant viruses and is the first marketed under the Technology Transfer Act of 1986. That legislation made it easier for federally funded laboratories to transfer new technologies to industry, by allowing the granting of exclusive licenses to produce or use innovative products, processes, or systems.

In 1988, Agdia, Inc., of Mishawaka, Indiana, was granted such a license to market the ARS-developed potyvirus test kit.

And Now, a New Test To Detect


Although viroids are similar to viruses in the way that they infect their host organism, they have several differences. A typical virus is made up of DNA or its chemical cousin, RNA, coiled inside a protective protein coat that helps the virus enter living cells. But viroids have no coat protein. They're just tiny bits of naked RNA.

Plant pathologist Ed Podleckis has developed a new screening test for detecting viroids that attack pome fruit trees and potatoes.

It's a faster, safer, and reliable new test that uses tissue blot. "Now, apple and pear trees coming into the United States can be tested after being grown in a greenhouse for just 2 months," Podleckis says. "Screening them for apple scar skin viroid takes just about a day or two using our new test.

"This test is 3 to 5 years quicker than the current practice of having to wait for the tree to bear fruit and looking for apple scar skin viroid symptoms like spotting and scarring of fruit to show up. And weather conditions or insect damage or other diseases could mask viroid symptoms."

Hadidi says that Podleckis' test means growers and consumers may not have to wait as long for a new fruit or vegetable variety that owes its flavor, for example, to an imported species. "We've seen prime candidates for new species, such as Chinese pears with superior flavor and market value, that spend years in quarantine."

Explains Podleckis, "A foreign species - in fact, all incoming plants - undergoes quarantine so that no viroids or other disease-causing pathogens accidently enter the country." He spent several months on developing and perfecting a new tissue blot test for detecting apple scar skin viroid.

Apple scar skin viroid infects pome fruit trees (apple, pear, and quince) in China and Japan. It's spread by grafting from cuttings. So Podleckis' apple scar skin viroid test starts with a viroid-free indicator plant to which he grafts a piece of the quarantined plant to be tested.

After the grafted plant grows for a couple of months, he takes a twig or leaf from the plant's new growth and presses it down on a wetted filter so that some of the sap sticks to it.

Podleckis describes this tissue blot test as "sort of a biochemical Dagwood sandwich made up of several layers."

The first layer is the filter and sap that are soaked in a solution containing a probe that specifically binds to any apple scar skin viroid present in the sap spot.

The probe - an RNA mirror image of apple scar skin viroid developed in the National Germplasm Resources lab by Hadidi - is labeled along its length with digoxigenin (DIG). "DIG is a steroid from a plant called Digitalis, a member of the foxglove family," he explains.

The next layer in the sandwich is a solution of molecules with a DIG-binding antibody at one end and the alkaline phosphatase enzyme at the other.

"Capping off the sandwich - like a layer of mustard - is a substrate solution that reacts with the enzyme to produce light in a chemiluminescent reaction. A piece of photographic film is then exposed to the membrane."

The light from the chemiluminescence exposes the film, producing a dark spot wherever apple scar skin viroid is present.

"Using tissue blots," Podleckis says, "eliminates hazardous waste now left from using organic solvents to extract nucleic acids from plant tissues. It also replaces radioactive isotopes with safer chemical probes.

"These nonradioactive probes are every bit as sensitive as the currently used radioactive ones. And they also cost less, can be reused several times, and store well frozen for up to a year."

A similar test, using a different probe, was developed by Podleckis in collaboration with Rosemarie Hammond, a plant pathologist in the Molecular Plant Pathology Laboratory in Beltsville. It is able to detect potato spindle tuber viroid that attacks potato tubers and tomato roots, stems, and leaves.

So far, Podleckis has screened over 200 apple and pear trees for apple scar skin viroid and about 100 potato plants for potato spindle tuber viroid using the new tissue blot technique. He says it has also worked, in lab experiments, for detecting other viroids in other plants.

According to Hadidi, about 25 different viroids are currently known, the first having been discovered in Beltsville in 1971 by ARS plant pathologist Theodor O. Diener. It was potato spindle tuber viroid. [See "Tracking the Elusive Viroid," Agricultural Research, May 1989, pp. 4-7.]

Making Plants Immune to Viruses .

A wide range of ornamental flowers including orchids and gladioulus - as well as some of the country's major economic crops, such as beans, peas, and forage legumes - have long needed effective protection from serious viral enemies. So plant pathologist John Hammond in the ARS Florist and Nursery Crops Laboratory at Beltsville set out to use a new genetic technology - antisense - to build in plant immunity.

"We reversed the coding sequence of a piece of genetic material taken from a plant virus to create an antisense gene," he says. "When we inserted this gene in some experimental tobacco plants, we found it |disarmed' invading plant viruses."

Antisense technology is a form of genetic engineering in which cells are instructed to do the opposite of what one of their genes is telling them to do. Antisense is "not new, but this is the first time it's been used successfully to create virus-resistant plants," Hammond explains.

Plants with the antisense gene will chum out antisense RNA as genetic material that binds to an invading virus' RNA. This binding apparently prevents the virus from reproducing itself in the plant. If the virus can't reproduce and spread, then it can't harm the plant."

He found that the antisense gene that was taken from bean yellow mosaic virus (BYMV) protected the engineered tobacco plants from the virus. Says Hammond, "A desert species of the tobacco plant Nicotiana benthamiana, was used because it's very susceptible to potyviruses."

Hammond worked for about 2 years with plant pathologist Kathryn Kamo and plant geneticist Robert Griesbach on perfecting this technology. He believes that similar techniques should work with related viruses that infect fruit trees. And he estimates that thee antisense gene might be ready in 2 to 5 years for breeders of floral and other crops to use.

Says Hammond, "BYMV-resistant plants would give growers a better chance to increase both yield and quality."

Even more important," adds Lawson, "is the time saved. Antisense gene technology could shorten by generations the time needed to breed resistance into agronomically adapted tree crops. With traditional breeding programs, plants have to be essentially re-bred, and then even more time is needed to screen for resistance."
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Title Annotation:includes article on giving plants viral immunity
Author:Becker, Hank
Publication:Agricultural Research
Date:Jul 1, 1993
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