Tracking the Elusive Viroid

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ARS plant pathologist Theodor O. Diener discovered a cell-invading plant pathogen 80 times smaller than a virus-the viroid.

According to accepted scientific dogma, the discovery of the viroid was not supposed to happen.

Finding out what causes potato spindle tuber disease brought about a small revolution in the study, diagnosis, and treatment of viral plant diseases. It also helped change approaches and attitudes in the study of livestock and human diseases.

But as crop diseases go, it wasn't very important. It didn't cost potato farmers millions of dollars in losses or control measures. Of course, if it got into a potato crop, it led to a second-year harvest of spindly, twisted tubers, but that didn't happen often.

Still, the disease made potato breeders nervous. For all they knew, the disease could sweep through their stock, damaging all the breeding potatoes in one year. And they wouldn't know why or be able to do much about it.

And it nagged at plant pathologists. They couldn't figure out what agent caused the disease. After eliminating all the other possibilities, they concluded that it was some kind of virus, even though it didn't behave the way a virus should.

But it wasn't a virus. It was something entirely new.

Potato spindle tuber and at least 15 other crop diseases are caused by viroids, an entity that nobody had ever heard of before 1971, its official date of discovery. Theodor O. Diener, the Agricultural Research Service plant pathologist who discovered the pathogen, named it the “viroid,” because it is “like a virus.”

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VIROIDS!—From Scourge to Boon in the 21st Century?

Like a virus, the viroid invades a cell and hijacks its reproductive mechanisms. It forces the cell to duplicate the viroid's RNA instead of its own. The viroid has no DNA. RNA and DNA are nucleic acids, the molecules of heredity; with the exception of viroids and some viruses, all genes are made of DNA.

The difference between viroids and RNA viruses is that viroids have no protective protein coat. The scientific dogma in 1971 was that an organism with no protein wasn't supposed to be able to replicate itself, even with a host cell's help. And an entity as small as the PSTV (potato spindle tuber viroid)—130,000 daltons—wasn't supposed to be able to infect anything, even a potato.

Until that time, scientists believed that the minimum weight necessary for infectivity was about 1 million daltons. (A dalton, also called an atomic mass unit, equals one-twelfth the mass of a carbon-12 atom.)

Diener wasn't much impressed by scientific dogma. He'd seen it turned upside down too many times. But he was very careful to prove that the viroid really existed. In all, it took him 6 painstaking years.

“Genius,” said Thomas Edison, “is one percent inspiration and ninety-nine percent perspiration.” The perspiration spent in pursuit of the viroid came in years of preliminary work by William B. Raymer, Diener, and many associates.

Raymer, also a plant pathologist, was at the ARS Potato Diseases Investigation Laboratory in Beltsville, Maryland, during the early 1960's. It was Raymer who began the project that eventually led to the discovery of the viroid.

“Perhaps the most important lesson to be gleaned from the discovery of the viroid is the importance of freedom for research scientists to follow leads when they become evident, rather than be tied down by too narrow a position description and predetermined goals.”

Russell L. Steere, ARS botanist (retired), Beltsville, Maryland.

It was Raymer and fellow ARS plant pathologist Muriel O'Brien who came up with a breakthrough in convenience in finding the cause of potato spindle tuber disease—a simple bioassay for the infectious agent.

Since spindle tuber takes a couple of years to show up in potatoes, results of many tests were excruciatingly slow to come. But Raymer and O'Brien found that the unknown pathogen was easily transmitted in tomatoes. Within 2 weeks, a growing tomato plant became dramatically stunted.

Now they could get lots of diseased leaves quickly. High-speed centrifugation, a standard method to purify viruses, would surely turn up the virus, thought Raymer and O'Brien. “Bottling” the virus, capturing it, seemed imminent.

Not exactly. The standard method produced such low amounts of infection that it was clear the cause of potato spindle tuber disease was not a typical virus.

Baffled, Raymer went to Diener, who had recently joined the new Plant Virology Pioneering Laboratory, one of 16 pioneering labs set up by ARS to define the laws and principles of basic problems in agriculture.

Russell L. Steere, botanist and chief of the lab, would later say that “perhaps the most important lesson to be gleaned from the discovery of the viroid is the importance of freedom for research scientists to follow leads when they become evident, rather than be tied down by too narrow a position description and predetermined goals.”

For a year after they teamed up in 1965, Raymer and Diener gradually put to rest the notion that potato spindle tuber disease was caused by a virus. They tried a different form of centrifugation, developed by Myron K. Brakke, an ARS chemist.

This lower leaf of Etrog citron, an indicator host, shows typical symptoms of viroid infection-necrotic cells on the midvein and petiole, where the leaf attaches to the stem.

His density gradient centrifugation technique showed that the pathogen was small and light. So it was unlikely, says Diener, “that the agent was a conventional viral nucleoprotein. It appeared more likely that this material was a free nucleic acid.”

Procedures in enzyme chemistry were next. Diener and Raymer treated extracts of diseased tomato leaves with an enzyme that chews up RNA. With RNA removed from the extracts, the scientists discovered that the treated extract failed to reinfect healthy tomato plants as it had before the enzyme treatment.

RNA in the agent was clearly important. But treatment with enzymes that remove DNA or protein made no difference; neither changed the pathogen's ability to infect tomato plants.

The results told Diener and Raymer that the essential ingredient of the spindle tuber agent was RNA and that it contained no protein.

At this point, in 1966, Raymer left for a job in private industry. Diener spent the next 5 years isolating and characterizing the viroid, verifying his experiments, filling in the holes, preparing to meet the skepticism that generally greets proposals of new, “impossible” concepts.

His concept did meet some resistance, chiefly from animal virologists and medical researchers unfamiliar with his earlier work, but his carefully prepared evidence was overwhelming. And as is often the case, another scientist working on another disease came op with a similar proposal at roughly the same time.

In 1975, Diener was co-recipient of the Alexander von Humboldt Award, which is presented each year for the most significant contribution to agriculture or the agricultural sciences for the past 3 to 5 years.