In searching for evidence in support of this hypothesis or against it, use was made of experiments involving heat inactivation of aster yellows virus in living plants. It had been found that the virus could be destroyed in diseased periwinkle plants and the plants cured by heat treatments at about 104 F. for a certain minimum period (about 12 days), depending on the size of affected plants. If treatments were stopped a bit short of the minimum, the treated plants would recover from the disease and would appear to be cured. Eventually symptoms would reappear, however, and in due course the treated plants become thoroughly diseased.

The time needed for symptoms to reappear in such treated plants varied with the length of treatment. When treatments were relatively short, the time required for reappearance of symptoms was short. When treatments were long but still short of the minimum necessary for cure, the time required for reappearance of symptoms was long. Because cured plants remained healthy indefinitely and no virus could be obtained from them, it was concluded that all of the virus in them had been destroyed by the treatments. Because plants treated for periods too short to cure but long enough to give temporary recovery eventually became as thoroughly diseased as untreated plants, it was concluded that only a portion of the virus present in them at the time of treatment had been destroyed and that the portion not destroyed had multiplied and caused relapse. It was reasoned that, as the amount of virus destroyed would increase and the amount remaining in the plant decrease with length of treatment, it would be expected that the interval preceding relapse would increase with length of treatment, as a small amount of virus remaining in a plant after treatment would require a longer time to reach a given level of concentration than a large amount. If the uninactivated virus in treated plants had not been able to multiply, the plants would not have relapsed.

The heat-treatment experiments with diseased plants suggested that the aster yellows virus in viruliferous insects also might be inactivated by heat. When tests were made it was found that that occurred. Infective insects could be rendered nonviruliferous by heat treatments at about 104 F. for 12 days or longer. Insects rendered nonviruliferous in this way could regain ability to transmit only by being allowed to again feed on a diseased plant. When virus-transmitting insects were heat-treated for periods of 1 to 11 days, they also lost ability to transmit but in these instances the loss was temporary. Such insects always regained ability to transmit without again feeding on a diseased plant but only after a waiting period.

If permanent loss of ability to transmit by infective insects resulted from heat inactivation of all virus in the insects, then the regaining of ability to transmit by insects that were heat-treated for periods only a little shorter than those required to give permanent loss of ability to transmit, and in which we may conclude only a very small amount of uninactivated virus could have remained, must have resulted from virus multiplication in the insects.

The heat-treatment experiments brought strong evidence that the virus of aster yellows multiplied in both the periwinkle plants and in the leafhoppers.

Aster yellows virus has never been transmitted manually by means of plant juices from diseased to healthy plants, but in 1941 L. M. Black showed that it could be transmitted to virus-free aster leafhoppers by needle inoculations with juices from viruliferous leafhoppers. Later he reported in a personal communication that it also could be transmitted to virus-free leafhoppers by needle inoculations with Juices from diseased aster plants.

Dr. Black tried to determine whether or not the virus could be maintained indefinitely by means of serial transfers M insects that were never allowed to feed on a susceptible plant, and thus to obtain further evidence by another experimental method as to whether or rot the aster yellows virus multiplied in its insect vector. Although some evidence was secured that supported the view that it could be so maintained, technical difficulties prevented him from passing the virus serially through a sufficient number of insect colonies to prove conclusively that it could be kept going indefinitely in this way.

In 1951, however, Karl Maramorosch succeeded in passing aster yellows virus serially by needle inoculations through ten colonies of virus-free aster leafhoppers that were never allowed to feed on a susceptible plant. He estimated that the virus present in the leafhopper juice that served as his original inoculum was diluted about 1 to 10,000 at each transfer and that the virus present in the inoculum at the ninth and tenth transfers, if no multiplication had taken place, would have been diluted to 10-11 and 10-40, or many billions of times. But he also showed that the infectivity and presumably the concentration of virus in juices at the ninth transfer was as high as in the juices in the first transfer. He thus confirmed by another method the conclusion that the virus of aster yellows multiplies in the aster leafhopper. That finding is believed to account satisfactorily for the biological relationship long known to exist between aster yellows virus and the aster leafhopper. It also is believed to account for similar relationships between many other plant viruses and their insect vectors.