STUDIES ON PROPERTIES OP THE CURLY TOP VIRUS ' By C. W. BENNETT 2 Pathologist, Division of Sugar Plant Investigations, Bureau of Plant Industry¡ United States Department of Agriculture INTRODUCTION The determination of properties of the curly top virus is somewhat more difficult than are similar determinations with many other vi- ruses, as the percentage infection from artificial inoculation is too low to afford a critical test for the presence or absence of virus. The unsatisfactory results from artificial inoculations have forced investigators to rely on the natural insect vector, Eutettix tenellus (Baker), for the production of any considerable amount of infection. Experimental work on the properties of the curly top virus was facilitated by the development of a method by Carter (5) ^ for arti- ficially feeding the beet leaf hopper. This method consists in plac- ing a liquid containing the virus in a bag made of animal membrane and allowing nonviruliferous leaf hoppers access to the outside of the bag. The leaf hoppers puncture this membrane and feed sufficiently on the liquid to acquire the virus. In this way the beet leaf hopper may be utilized to transfer virus from the liquid medium to suscep- tible tissue of beet plants. Modifications of Carteras original method of feeding the beet leaf hopper have been devised and used with considerable success in studies on the properties of the virus by those interested in this field of research. Severin and Swezy (14) determined that the virus is filterable, and recently Severin and Freitag (13) published results of further investigations on properties of the virus. In the latter work it was shown that the virus may be recovered from expressed beet juice after a period of aging ranging from 8 to 100 days, the period apparently depending on the previous treatment of the juice and on the conditions under which the virus was preserved. Virus was re- covered from beet juice diluted 1:2,000 and from macerated leaf hoppers diluted 1:22,000. The virus was inactivated by a 10-min- ute exposure at 80^ C., but temperatures considerably below freez- ing apparently had no injurious etfect. No virus was recovered from dried plant tissue nor from dried beet leaf hoppers. Studies on the properties of the curly top virus were started at Riverside, Qalif., the latter part of 1932 and were continued during the following 2 years. In this paper certain methods which have greatly facilitated the experimental work are described and the re- sults of the investigation are presented. METHOD OF MAKING TESTS FOR VIRUS In all of the tests for the presence of active virus in this series of studies, beet leaf hoppers were used as the agents of transfer of virus from the liquid medium to seedling beets. Several types of cages for 1 Received for publication Dec. 4, 1934; issuedApril 1935. 2 The writer is indebted to J. M. Fife, biochemist, Division of Sugar Plant Investigations, Bureau of Plant Industry, for making the hydrogen-ion determinations reported in this paper. 3 Reference is made by number (italic) to Literature Cited, p. 240. Journal of Agricultural Research, Vol. 50, no. 3 Washington, D. C. Feb. 1, 1935 Key no. G-956 (211)] 212 Journal of Agricultural Research Vol. 60, no. 3 feeding leaf hoppers were tried, one of which, designed for feeding the insects with small quantities of exúdate obtained from the beet plant, proved very satisfactory for use with all kinds of virus-containing media. Cages of this type were used for the most part and were constructed as follows: Each feeding cage was made from an ordinary 4-ounce bottle hav- ing a diameter of approximately 2 inches. The bottle was cut into three parts and the top part discarded. The two remaining parts were ground smooth on the cut surfaces. The middle portion, covered at one end with cloth and at the other end with Baudruche capping skin, served as a cage for confining the leaf hoppers. The bottom portion was used as a moist chamber over which the cage was set. Nonviruliferous beet leaf hoppers were placed in the cage through an opening in the cloth cover and subjected to a starvation period of 2 to 18 hours, depending on the temperature. At the expiration of the starva- tion period the cage was invert- ed and drops of the material to be tested for virus content were placed on the capping skin. The cage was then quickly righted, so as not to disturb the drops of medium, and placed over a moist chamber (fig. 1). The cages were placed under lights in a hood at a temperature of 95° to 110° F. and the leaf hoppers allowed a feeding period of 4 to Ü hours. The leaf hoppers were FIGURE I.—Cage usc<i in artificial feeding of Lite beet leaf hopper. then removed and, except as noted, caged singly on seedling beets. The number of plants infected under these conditions was taken as a relative measure of the virus content of the material on which the leaf hoppers had fed. SOURCES OF VIRUS BEET JUICE Juice expressed from sugar beet plants affected with curly top has been used as the source of virus in nearly all of the studies on proper- ties reported up to the present time. This beet juice, when fed directly to the beet leaf hopper, is not an especially favorable liquid from which to obtain virus, since only a small percentage of non- viruliferous beet leaf hoppers become viruliferous as a result of such feeding. Some estimate of the proportion of leaf hoppers that may be expected to acquire virus from beet juice may be obtained from Feb. 1,1935 Studics OU Properties of the Curly Top Virus 213 results selected from one of the experiments of Severin and Freitag (13)j which seems to be representative. Using approximately 33 artificially fed leaf hoppers for inoculating each plant, they obtained 4.6 percent infection from leaf juice, 46.6 percent from root juice, 11.5 percent from leaf juice diluted with 5-percent sugar solution, and 4.3 and 42.1 percent, respectively, from leaf and root juice centrif- ugalized at high speed. These results indicate that even with the higher percentages of infection the actual number of the artificially fed leaf hoppers which picked up virus was small, since it is well known that a single viruliferous leaf hopper is able to transmit disease to a considerable percentage of the plants on which it feeds. The limited capability of the leaf hopper for picking up virus from beet juice is further illustrated by the results shown in table 1, in which less than 4 percent of the total number of leaf hoppers given access to leaf and root juice and caged singly on seedling beets transmitted disease. TABLE 1.—Relative availability to beet leaf hoppers of virus in leaf juice^ rootjuice^ and phloem exúdate of sugar beets affected with curly top Leaf juice Koot juice Phloem exúdate Beet no. Plants Plants Plants Plants Plants Plants ' inoculated infected inoculated infected inoculated infected Number Number Number Number Number Number 1 ... 80 3 80 5 80 26 2 20 0 20 5 3 - _ _- - 20 0 20 Ö 20 7 4 20 0 20 1 20 14 5 20 1 20 0 20 14 Total 160 4 140 6 160 66 Direct utilization of the expressed juice of diseased plants, because of the limited feeding which takes place, the high mortality of the insects, and other circumstances, involves the use of excessively large numbers of beet leaf hoppers to secure results, and the results are often contradictory and difíicult of interpretation. Nevertheless, the method may be used successfully in the study of certain properties of the curly top virus. It is obviously unsuited for studies in which chemical agents on which the leaf hopper will not feed or which are injurious to the leaf hopper are added to the liquid containing the virus. In any type of property studies the method involves the exercise of a great amoimt of patience and effort and necessitates the use of enormous numbers of leaf hoppers. Because of these obvious shortcomings of beet juice as a source from which beet leaf hoppers may acquire virus, the first efforts in this series of property studies were directed towa^rd securing liquids of high virus content more acceptable to the beet leaf hopper than beet juice has proved to be. Attempts were made (1) to obtainthe liquid content of the phloem of the beet and other plants in which there is reason to believe virus occurs in relatively high concentrations and (2) to separate the virus in expressed beet juice from materials that are toxic or unacceptable to the beet leaf hopper. 214 Journal of Agricultural Research Vol. 50, no. 3 PHLOEM EXÚDATE According to the available evidence (2) the curly top virus occurs in the phloem of diseased plants in much greater concentrations than in other tissues. Since it has been shown also that the beet leaf hopper seeks the phloem elements in its feeding, the phloem content of diseased plants should constitute the best possible medium from which to recover virus when the beet leaf hopper is used as the agent of transmission. Many varieties of cucurbits are susceptible to the curly top disease. Since these plants are known to give off considerable quantities of exúdate from the phloem when the stems or petioles are cut, prelimi- nary tests of phloem exúdate as a virus-carrying medium were made with Hubbard squash {Cucúrbita maxima Duchesne) as the source of exúdate.