Transmission of Plant Viruses by Cicadellids

Transmission of Plant Viruses by Cicadellids L. M. BLACK Brooklyn Botanic Garden, Brooklyn, New York’ Introduction. . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . , , . . . . . . . . . . . . , . . . . Latent or Incubation Period in the Vector.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission without Evidence of Virus Multiplication in t h e Vector. . . . . . . . . . Transmission with Evidence of Virus Multiplication in the Vector. . . . . . . . . . . . . . Common Relationships between Virus and Leafhopper Vector. . . . . . . . . . . . . . . . . Passage through the Vector Egg.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vector R a n g e . . . . . . . . . . . . . . . . , . . . , . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . Specialization of Virus Varieties in Relation t o Vectors. . . . . , . . . . . . . . . . . . . . . . . Genetics of Vector Ability. . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transmission Process. . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . Effect of Virus on Vector. . . . . . . . . .,.. ..... .,., .... ....... .. .. .. . Transmission of More Than One Virus by a Vector. . . . . . . . . . . . . . . . . . . . , Independence of Each Inoculation by a Cicadellid.. . . . . . . . . . . . . . . . .... ... . Plant Resistance t o Inoculation by Cicadellids. . . . . . . . , . . . . . . . . . . . . . . . . Ecology of Transmission. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Symptoms Ascribed to Insect Toxins.. . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . Conclusion.. . . . . . . . . . . . . . . .. . . .. . . , . . . . . . . . .. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . ,

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Introduction Next t o aphids, cicadellids are the most important vectors of plant viruses. Oman (1949) listed 26 viruses transmitted by 69 species in 7 sub-families of the Cicadellidae.2 Among the viruses transmitted by cicadellids are some of the most important affecting plants, including aster yellows virus, which may cause serious losses in lettuce, endive, carrots, and asters; curly top virus, which once created havoc in the sugar beet fields of the northwestern United States; and viruses causing the important yellows, X disease, and phony diseases of peaches in the United States. The outstanding symptoms produced by cicadellid-transmitted viruses Present address : Department of Botany, University of Illinois, Urbana, Illinois. Among the maladies that may be added to his list are these diseases and the leafhoppers which spread their causal viruses : Western X disease of peaches, Colladonus geminatus (Wolfe, Anthon, and Jones, 1950) ; Australian witches’ broom of alfalfa, OroSius argentatus (Helson, 1951) ; pupation disease of oats, Delphax striatella (Sukhov and Sukhova, 1940) ; and winter wheat mosaic, Deltocephalus striatus (Zazhurilo and Sitnikova, 1941). 69 2

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are varied and include necrosis, as in yellow dwarf of potato and phloem necrosis of elm; vein enlargement and leaf curling, as in curly top; tumors, as in wound tumor disease; chlorotic streaking, as in corn streak; wilt, as in the yellow wilt of sugar beets; and scalding or burning of the leaves, as in Pierce’s disease of grapes. Perhaps the best-known type of virus disease having a cicadellid vector is exemplified by aster yellows. Diseases of this kind exhibit a generalized chlorosis, proliferation of axillary buds, and, in the flowers, virescence, phyllody, and other malformations. All the known vectors of viruses that cause diseases with this syndrome are leafhoppers and it would be surprising to find a virus disease of this type transmitted by an insect other than a cicadellid. Some plant virologists use the term “yellows” in a restricted sense and prefer to apply it only to diseases of this sort; others do not. Some virus diseases whose natural dissemination has been most difficult to discover have been proved to have cicadellid vectors. More than 40 years elapsed between Erwin F. Smith’s demonstration of the infectious nature of peach yellows (Smith, 1891) and Kunkel’s discovery that the causal virus was transmitted by Macropsis trimaculata (Kunkel, 1933). During this time, much effort and many ingenious hypotheses on its dispersal yielded no significant clues. I n a survey whose objective was the discovery of the vectors of phony peach virus, Turner (1952) reported that 22,000 collections of 530 homopterous species were made, including 382 species of Cicadellidae. The vectors proved to be sharpshooters in the sub-family Tett,igellinae, as are the vectors of Pierce’s disease of grapes (Hewitt, Frazier, and Houston, 1942), another virus disease which attacks xylem. I n another instance Baker (1949) reported that 90 species of insects were tested before finding the vector of elm phloem necrosis. In spite of the great difficulty in discovering the vectors of some cicadellid-transmitted viruses, this group may have provided the first demonstration of insect transmission of plant viruses. According to Katsura (1936), the connection between rice stunt and leafhoppers was demonstrated, in unpublished experiments, by a Japanese grower as early as 1884. However, the demonstration that the causal agent of rice stunt was not the insect itself but an autonomous agent carried by the leafhopper was not realized until 1906, and even today, although probably all plant virologists are convinced that this agent is a virus, neither its filterable nature nor its size has been demonstrated. The intrinsic properties of viruses transmitted by leafhoppers are difficult to study. The potato yellow dwarf viruses, which are leafhopper transmitted, produce hundreds of primary lesions in leaves of Nicotiana rustica L. (Black, 1938) and smaller numbers of lesions in other hosts (Hougas, 1951) rubbed with virus-containing solutions under certain conditions. The curly top virus will sometimes produce infections when solutions

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containing i t are pricked into the crowns of young beet plants (Severin, 1924, and others). However, none of the other viruses transmitted by cicadellids has been transferred by inoculating plants with virus-containing solutions from either plants or insects, and many investigators have tried many techniques unsuccessfully. This does not mean that the virus must pass through an insect before it can infect another plant, because transmission from plant to plant is readily accomplished where successful grafts can be made between closely related plants, and it can sometimes be accomplished between distantly related plants by employing dodder (Bennett, 1940; Johnson, 1941). However, these techniques are limited in their usefulness because the virus is necessarily maintained in living cells during the entire procedure of transfer. For example, these methods cannot be employed for determining properties such as the filterability or the size of the virus. For investigations of this sort, two methods have been devised. One consists of feeding the virus in solution t o leafhoppers (Carter, 1927; Severin and Swezy, 1928), and the other of injecting small volumes of virus in solution into leafhoppers (Storey, 1933). Both plants and insects have been used successfully as sources of virus in these techniques. However, since the virus does not induce any symptoms in the leafhoppers, these must subsequently be tested on plants to determine whether or not they have become infective as a result of the treatment. Apparently some viruses can be successfully introduced into leafhoppers by one of these methods but, so far, not by the other, and some can be introduced by both. Recently some of the leafhopper-borne viruses (which are less stable and less concentrated than previously isolated plant viruses) have been identified under the electron microscope. The first leafhopper-borne virus SO identified, potato yellow dwarf virus (Black, Mosley, and Wyckoff, 1948; Brakke, Black, and Wyckoff, 1951), proved to be the largest plant virus known and has the appearance of a small, collapsible organism about 110 mp in diameter. The wound tumor virus, which multiplies in both plants and insects (see below), has recently been shown to have the same form in both plant host and insect vector; whether taken from plants or insects, this virus is a rigid sphere about 80 mp in diameter (Brakke, Vatter, and Black, t o be published). This is the first experimental evidence that indicates the form of the virus in the vector. It is apparent that morphologically different viruses are transmitted by leafhoppers.

Latent or Incubation Period in the Vector Apparently, in the case of most3 viruses transmitted by cicadellids, there is an interval between acquisition of the virus by the insect and its transmission back t o plants. Whenever this period is studied in individual 8 No latent period could be detected in the transmission, by leafhoppers, of the virus of Pierce’s disease of grapes (Severin, 1949).

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insects, it is found to vary greatly in length and it is the minimum such period that is significant. It is usually necessary to test a considerable number of insects, or colonies of insects, to determine the minimum interval with some assurance that further tests will not reduce it. Moreover, recent work by Maramorosch (1950b) demonstrated the importance of making such tests at a temperature that is optimum for transmission of the virus in question. Once the leafhopper transmits virus, it usually retains this ability for life without fresh access to virus from plants. These characteristics, the incubation period and persistence in the vector, led Kunkel (1926) to suggest that the aster yellows virus reproduces in Ma:ros‘eles d.’vtsus.

Transmission without Evidence of Virus Multiplication in the Vector Bennett and Wallace (1938) found that the minimum latent period of the curly top virus in its leafhopper vector, Circulifer tenellus, was 4 hr. and failed t o confirm Severin’s (1931) report of shorter latent periods. As will be seen below, such short periods may indicate that the virus does not multiply in its vector and the periods may be appropriately described as latent periods. Bennett and Wallace (1938) found that, although the curly top virus could be acquired in a 1-min. feeding, individual leafhoppers (not colonies) did not acquire their maximum ability to transmit unless the acquisition feeding lasted for 2 days. Acquisition feedings for longer than 2 days increased the virus concentration in the leafhoppers but did not increase the ability to transmit. Subsequently the ability to transmit and the virus content gradually decreased over periods of 8 to 10 weeks, although the effect on transmission was slight if the original virus content was high. This decrease in transmission ability could not be attributed to the aging of the leafhoppers because infective ability was restored by renewed access to virus from plants. Freitag (1936) also showed gradual loss of ability t o transmit after a single acquisition feeding and restoration of this ability by a second acquisition feeding. Even more convincing is Giddings’ (1950) evidence that eliminates aging as an explanation for the decrease in transmissions. He reared insects on plants infected with curly top virus, strain 2, and then fed them for 16 hr. on a plant infected with strain 3. Fifty percent of the plants infected in the first quarter of the subsequent transfers were infected with strain 3, but only 9 % of those in the last quarter were. A second acquisition feeding on a source of strain 3 significantly increased the proportion of strain 3 infections relative to those of strain 2. These results indicate that the curly top virus either does not multiply or does not multiply sufficiently to maintain itself. In the latter eventuality, it might multiply in local lesions in the leafhopper. Transmission with Evidence of Virus Multiplication in the Vector In most leafhopper-transmitted viruses, the interval between acquisition and transmission of the virus by the vector is longer than with curly top

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virus. The interval varies greatly with individual leahfoppers of a species transmitting a given virus, but even the minimum period may be about 2 weeks. Maramorosch (1950b) studied this period a t different temperatures in a vector of wound tumor virus and, even at optimum temperatures, it was never less than 13 t o 15 days when virus was acquired by feeding. There is much evidence that such intervals represent true incubation periods during which the viruses multiply t o infective concentrations in their vectors. Some virologists have not been convinced, until quite recently, that any plant viruses do multiply in their insect vectors (Bawden, 1950 and earlier editions). Black (19Ej3b) has critically reviewed, in detail, the evidence on multiplication of certain plant viruses in their insect vectors. This evidence is briefly presented below. Rice stunt virus commonly passes from viruliferous females of Nephotettix apicalis var. cincticeps through the eggs t o their progeny but does not pass t o the young by way of the sperms. Starting with a single viruliferous female leafhopper, Fukushi (1940) transferred this insect and each of its progeny, in several generations, to a fresh, healthy rice seedling each day. Progeny were removed from the plant they emerged from before they had any opportunity to feed on it, and daily transfers of the insects were begun before any of them started to infect plants. In this way he insured that they did not pick up virus from plants. In his best experiment Fukushi demonstrated passage of the virus to 82 infective leafhoppers in 6 generations. The experiment lasted 374 days and about 1,200 rice plants were infected. During this time there was no evidence of a decrease either in the percentage of infective leafhoppers or in their infective ability. From Fukushi’s data, Black (1!?53b)has estimated that the initial virus in the starting female in this experiment must have been diluted a t least 1 t o 563,000, and that it probably was actually diluted many times that amount. In a similar investigation, Black (1950) found that the clover club leaf virus was transmitted from viruliferous females of Agallwpsis novella t o their progeny through the egg, and he showed that the virus in a single female was capable of being maintained through 21 generations of progeny, during a period of 5 years, without fresh access to virus from plants. Except for the sampies of progeny that were withdrawn from the line of descent, tested for infectivity, and then discarded, all the insects were maintained during this time on Grimm alfalfa which was shown to be immune from clover club leaf virus. Selection for virus was avoided, and, on the basis of the progeny counted, it was calculated that the quantity of virus in the starting female would have undergone a dilution of 10+ if it had not multiplied. Since the original female could not have contained more than about 10l2virus particles, even on a generous estimate, the experiment

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demonstrated that the virus multiplied in the insect. Transmission of the virus in series, by grafting from plant to plant, showed that it also multiplied in plants. Fukushi found that some females failed to transmit rice stunt virus during their entire life, yet produced viruliferous progeny. In the experiments with clover club leaf virus, the leafhoppers were usually tested on susceptible plants for 9 weeks, yet many that failed to transmit virus to the clover produced infective progeny. Although these insects were not tested on susceptible plants during their entire lives, as were Fukushi's insects, these results support Fukushi's observations. The aster yellows virus undoubtedly multiplies in the insect vector, Maerosteles divisus. Kunkel (1937) showed that infective leafhoppers lost their ability to transmit the virus at 24" C., after they had been kept at 32" C. for 1 or more days. The time the insects remained noninfective was roughly proportional to the time they were held a t the higher temperature, and, if they were kept a t 32" C. long enough, they were rendered permanently noninfective. Moreover, plants infected with this virus could be cured by high-temperature treatments, and Kunkel interpreted his results with the insects on the basis of heat-inactivation of the virus and multiplication a t 24" C. back to an infective concentration in those cases where the insects regained their infective ability. Additional evidence for multiplication of this virus in its vector was obtained by assaying for virus in M . diuisus, in which the virus was undergoing incubation (Black, 1941). Evidence was obtained for a t least a hundredfold increase of the virus during 12 days of the incubation period. Since the insects that were assayed for virus infected none of the susceptible plants they fed on during the incubation period, these insects could not have acquired virus from plants during the period of the assay, and the experiment was interpreted as direct evidence for multiplication of the virus in the insect. It is evident that in such cases the virus multiplies during incubation in the insect, and, indeed, the necessity for such multiplication accounts in part for this period. Kunkel (1948) has indicated that there is a rough correlation between the lengths of the incubation periods of viruses in their leafhopper vectors and in their plant hosts. Maramorosch (1950a) has obtained evidence that, the smaller the dose of aster yellows virus injected into a vector, the longer the incubation period tends to be. It seems appropriate to call these periods incubation periods rather than latent periods. There are, however, indications that other factors besides virus multiplication are involved in the incubation period. The aster yellows virus apparently reaches a maximal concentration in the insect 6 days before the end of the incubation period (Black, 1941). In a third type of experiment Maramorosch (1952a) succeeded in trans-

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mitting the aster yellows virus through ten serial passages from insect to insect without loss of virus concentration. The dilution of starting virus attained in the tenth passage was 10-40. Since the experiment was designed SO that these insects could not acquire virus from plants, it was evident that the virus multiplied in the insect. Thus, three independent experimental approaches adduced evidence for the multiplication of this virus in its vector. In a n experiment similar to Maramorosch’s, Black and Brakke (1952) transmitted wound tumor virus from insect t o insect through seven serial passages and attained an ultimate dilution of 10-18 without decrease in virus concentration in the insects. The insects used as sources of virus had been maintained on immune plants during the experiment, and it was concluded that multiplication in the insect must have occurred. Therefore, there may be two main types of virus transmission by leafhoppers. One type, exemplified by curly top virus, may have a very short incubation period, no multiplication in the vector, and retention of virus proportional t o the amount acquired from plants. The other type, exemplified by the four viruses discussed above, exhibits longer incubation periods in the vector, multiplication in the vector, and retention independent of the amount of virus acquired. Kunkel (personal communication) has recently shown that, in the acquisition of the eastern strain of aster yellows virus by Macrosteles divisus, about 30% of the insects picked up virus in 1 day. In his experiments, retention was apparently not affected by the length of the acquisition feeding. There is no reasonable doubt that the viruses of rice stunt, clover club leaf, aster yellows, and wound tumor multiply in both their insect vectors and plant hosts and that they are, probably, both plant and animal viruses. This fact has an obvious bearing on the classification of viruses, hypotheses of virus reproduction from precursors, and specificity of transmission (Black, 1941). It seems, to the writer, to favor the view that at least some viruses originated by retrograde evolution from more complex parasitic forms (Green, 1935) that were transmitted by insects to the plants they attacked. The common phenomenon of retrograde evolution in parasites might explain the existence of the highly specialized plant-insect relationships of these viruses.

Common Relationships between Virus and Leafhopper Vector The aster yellows virus may be taken as representative of many viruses in this group. In his classical study of aster yellows, Kunkel(1926) showed that the vector could remain inoculative for as long as 100 days after the completion of a 10-day incubation period. He pointed out that the aster yellows virus was not lost during the various molts as the nymphs grew up,

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and that, in nature, it did not pass directly from plant to plant or insect to insect. The virus was not passed from parent vectors to their progeny through the eggs or sperms. In all these respects, the aster yellows virus may be said t o represent the most common relationship between viruses and leafhopper vectors. In its relationship to Macrosteles divisus it is, however, one of the most efficiently transmitted viruses of any that have been studied. Many other viruses are not transmitted as frequently by their vectors. Indeed, the California strain of aster yellows virus itself is not transmitted as efficiently by many of its other vectors.

Passage through the Vector Egg I t has already been indicated that two plant viruses pass from female to progeny through the egg. In these two instances, it has been estimated (Black, l953a) that between 95 and 100% of the eggs of viruliferous females transmit virus, but neither passes to the young by way of the sperm. These two viruses are exceptional, most of those tested having failed to give evidence of transovarian passage. However, recently it was shown (Black, 1953a) that in the case of both wound tumor virus and New Jersey potato yellow dwarf virus, about 1 to 2 % of the young vectors receive virus from their viruliferous parents through the egg. In both rice stunt and clover club leaf viruses there is usually an incubation period in the nymph which has received virus from the egg, but Fukushi (1934) found that insects occasionally transmitted the virus immediately after hatching.

Vector Range Often so many species of insects have been tested without success before finding a leafhopper vector for a virus, that when one was discovered it was regarded as the sole vector. Even today, only one species, Circulifer tenellus, is known to transmit North American curly top virus. This is probably due t o the fact that this species is the only one in the genus that occurs in North America where the virus exists and has been studied. Other species in the genus that live around the eastern Mediterranean might prove to be vectors if tested (Oman, 1949). Severin (1934) reported two species of leafhoppers in the genus Thamnotettiz as vectors of California aster yellows virus. Previously the only known vector had been Macrosteles divisus. Since then, he and his associates have done more than any other workers to break down the concept of specificity in transmission that envisioned a single leafhopper species as the vector of a virus. These workers, according to Freitag, Frazier, and Flock (1952), have since described 23 leafhopper species in several tribes of the Cicadellidae (Oman, 1949) as vectors of California aster yellows

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virus, and 20 species in the family Cicadellidae and 4 in the family Cercopidae as vectors of the virus causing Pierce’s disease of grapes. Apparently only some of these species are important in the spread of the viruses in the field. There is a considerable range in the efficiency of transmission of a given virus by the vectors, and this efficiency is affected by the species of plant host involved (Severin, 194713).

Specialization of Virus Varieties in Relation to Vectors At the same time that the old concept of specificity was being broken down, a new concept of specificity emerged. For it was shown that there exist two kinds of potato yellow dwarf virus specifically transmitted by related leafhoppers (Black, 1944). The New Jersey potato yellow dwarf virus was transmitted by Agallia constricta but not by Aceratagallia sanguinolenta that had fed on the same diseased crimson clover plant; conversely, the New York potato yellow dwarf virus was transmitted from crimson clover by A . sanguinolenta but not by A . constricta. Both were transmitted by Agallia quadripunctata. Such differences in transmission cannot be explained on the basis of such quantitative relationships as different virus concentrations, or different vector efficiencies, but only on a qualitative basis. The relationship of the two viruses was demonstrated by the production, by both viruses, of unique yellow primary lesions on mechanically inoculated leaves of Nicotiana rustica, by cross-protection tests, and by the morphology of the virus particles under the electron microscope (Black, Mosley, and Wyckoff, 1948). There is now evidence for four kinds of curly top virus with different vector and plant host relationships: the type species (U. S.), transmitted by Circulifer tenellus; variety distans (Argentina), not transmitted by C. tenellus but by Agalliana ensigera (Bennett, Carsner, Coons, and Brandes, 1946) ; variety brasiliensis (Brazil), not transmitted by A . ensigera but by Agallia albidula (Bennet and Costa, 1949) ; variety solanacearum (Brazil), not transmitted by A . albidula but by A . Pnsigera and Agallia sticticollis (Costa, 1952). If one lists these curly top viruses and their vectors in a table so that every possible combination is apparent, it becomes obvious that there are many gaps in our knowledge of the virus-vector relationships of this complex. These gaps are difficult to fill because the curly top viruses and vectors occur in certain limited areas of the world, and it is undesirable to risk spreading them to other regions where they might cause significant$ economic losses. Even if this problem, and others of this sort, could be studied in isolation on some appropriate small island, the task would not be an easy one because the host plant adds a third dimension to the investigation since it is known that the plant may significantly affect the trans-

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mitting ability of the vector. Thus, a potential vector might fail to transmit a virus because of the host plant used in a particular test. A similar situation may exist in regard to the yellows types of viruses known in the world today. For example, the relationships of the diseases producing symptoms of big bud-tomato big bud in Australia, the United States, and Puerto Rico; cranberry false blossom in the United States; stolbur of Solanaceae in Russia, Yugoslavia, and Rumania; and little leaf of eggplant in India-are not at all certain. Different vectors have been discovered for these diseases in different countries. To add to the confusion in such cases, the taxonomy of the vector is frequently drastically changed after its vector relationships have been discovered and the insect is restudied. Even when the symptom expression is very similar and the vectors are reported as species of the same genus, as with rice stunt in Japan and the Philippines, one can only be sure that the viruses are similar; they may not be identical, as witness the history of aster yellows in New York and California.

Genetics of Vector Ability Specificity in vector ability may extend to the genes within a single vector species. Storey (1932) demonstrated that, within the species Cicadulina mbila, there were individuals (active) that could, and individuals (inactive) that could not, transmit corn streak virus. Ability to transmit was determined by a single sex-linked dominant gene. Black (1943) showed that Aceratagallia sanguinolenta varies genetically in ability to transmit New York potato yellow dwarf virus. However, in this case, the evidence indicated that multiple factors were involved. In comparable tests, the virus was transmitted by 80 % of the ‘(active” insects, 2 % of the (‘inactive,”and 30 % of the hybrids. The efficiency of transmission by the transmitting insects in the active and inactive groups was significantly different, indicating that some of the wide variation generally observed in vector efficiencies of individuals within a leafhopper species is probably partly genetic. There is evidence that some other leafhoppers also vary genetically in their ability to transmit (Fukushi, 1934; Bennett and Wallace, 1938). Transmission Process The inner stylets of hemipterous insects form two channels. The saliva is believed to be forced down one of these and the plant juices to be sucked up the other. Storey’s three classical papers (1933,1938,1939a) on Cicaduh a mbila and the corn streak virus and his review (193910) have done much to elucidate the process of transmission by leafhoppers and are the basis for much of the information in this section. Storey observed the penetra-

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tion of the leafhopper’s sucking mouth parts (stylets) through a wax membrane into a 10 % sucrose solution. Under these conditions the mandibulary stylets penetrated only a short distance into the membrane and appeared to be wedged apart by the maxillary stylets so that they became anchored in the membrane by their serrated outer surfaces. The maxillary stylets advanced smoothly into the fluid and retired jerkily with rapid sliding movements of the two stylets, one on the other. During these withdrawals, a colorless substance flowed from the tip of the stylets, set to a gel, and was molded internally when the stylets again advanced through it. By repetitions of t*heprocess, a sheath was formed which was made up of two differently staining layers. Storey demonstrated that it required more than 1 min. for the stylets to reach the phloem but, since virus could be acquired in 15 sec., he concluded that it must have been available to the insects in the mesophyll. The corn streak virus was acquired from the chlorotic parts of the leaf but not from the adjacent green areas. Bennett (1934) found that curly top virus was much more readily acquired from the phloem than from other tissues and attributed the proportionately small number of virus acquisitions from parenchyma, t o virus-rich exudate that had escaped from the damaged phloem into such tissues. Bennett and Wallace (1938) believed that such exudate also explained the acquisition of curly top virus by nonvector arthropods that feed on parenchyma only. Sometimes devious methods are necessary for the recovery of virus from certain plants. Giddings (1947) has shown that a vector may acquire virus more readily from dodder that is parasitizing an infected host than it can from the parasitized host itself. Aster yellows virus is difficult to recover from potatoes directly, but infected potato scions may be grafted to Nicotiana rustica and the virus recovered readily from this plant (Younkin, 1943). Moreover, some viruses are not acquired readily by vectors with which they have very intimate relationships. In acquisition feeding periods of 3 to 50 days, only 25 of 1,300 vectors acquired rice stunt virus (Fukushi, 1934). The clover club leaf virus, also, is not as readily acquired by the vector as are many other leafhopper-borne viruses. In some leafhoppers it seems probable that the virus passes, without multiplication, through the different tissues in the body to the salivary glands. I n others, it is certain that, in addition, it must first multiply in one or more sites and may reach a relatively high concentration before the end of the incubation period. Storey also showed that virus acquired by active and inactive races of C. mbila, and by the nonvector Peregrinus maidis, passed through the intestinal tract t o the rectum but was not naturally voided in the feces. It appeared naturally in the blood of active C. mbiEa before the end of the

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incubation period and could be demonstrated in the mid-intestine and salivary glands. Curly top virus occurs in the blood, salivary glands, alimentary tract, and feces of the vector (Bennett and Wallace, 1938). Severin (1947d) reported the presence of the curly top virus in the first three of these sources but he failed to detect it in the ovaries. The curly top virus does not pass through the vector egg, whereas rice stunt and clover club leaf virus do pass through the egg and almost certainly occur in the ovaries. Storey showed that puncturing the mid-intestine of inactive C . mbila rendered them infective, although less efficient than active individuals, thus demonstrating that part of the difficulty in transmission by these individuals resided in the gut wall. A nonvector such as Peregrinus maidis, which transmits a different virus to corn, could not be made to transmit corn streak virus by the same treatment, a fact implying a different order of resistance in this species. That arthropods (leafhoppers, aphids, thrips, and mites) unable to transmit may nevertheless acquire and retain considerable concentrations of curly top virus for 2 to 3 weeks was demonstrated by Bennett and Wallace (1938). The curly top virus is relatively stable and, if it penetrates the gut wall of nonvectors, might be expected to survive, but Maramorosch (195213) has recently reported that the unstable aster yellows virus and the corn stunt virus can be detected in each other’s vectors after an interval that may represent incubation. In these cases some barrier may prevent passage of virus from the blood to the stylet,s. I n contrast to some aphid vectors, the acquisition of virus by leafhoppers is usually little affected by preacquisition fasting (Storey, 1928). However, Bennett and Wallace (1938) found that leafhoppers given a preacquisition fasting of 15 min. to 3 hr. produced more infections with curly top virus than those given either shorter or longer fasting periods. Zazhurilo and Sitnikova (1941) found that, of the first instar nymphs acquiring the virus of winter wheat mosaic, 6.5% picked it up in 1 day, 11.7 -% ’ in 2 days, and 51.4 % in 5 days. There were always some insects that failed t o acquire the virus even with the longest acquisition feeding periods. Insects older than the third instar were unable to acquire virus at all. This is exceptional; usually all stages can acquire virus. Older vectors were also found to be less able to acquire the virus of pupation disease of oats (Sukhov and Sukhova, 1940). There is little reason to question the general assumption that inoculation is accomplished by the sucking mouth parts. Cicadulina mbila with their stylets cut off could not infect plants; others, allowed to contact plants only by passing their stylets through a membrane, could. C . mbila and Circulifer tenellus penetrate through cells to the phloem and C. mbila does not damage cells immediately adjacent t o the stylet sheath. However, not

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all leafhoppers have these feeding characteristics. Some species of Empoasca are very toxic t o plants, a property which might interfere with their role as vectors. It was of considerable interest, therefore, when members of this genus were discovered to transmit a plant virus (Adsuar, 1946; Bird and Adsuar, 1952). Storey found that not all penetrations to the phloem caused infection and that penetrations that did not reach the phloem uniformly failed t o do so. The evidence indicates that the curly top virus must also be introduced into the phloem if infection is to result. Fife and Frampton (1936) obtained experimental evidence that, in seelung the phloem, the stylets of the beet leafhopper follow a gradient of decreasing hydrogen-ion concentration. Elimination of the gradient greatly reduced the chances that the insects would t a p the phloem and reduced the infections in the same proportion. The sheath substances may be the vehicle by which the virus is introduced. However, in Cicadulina mbila a single, 1-hr. inoculation feeding may cause infection, whereas none of several hundred 5-min. inoculation feedings will do so. The fact that many of the 5-min. feedings resulted in sheaths that reached the phloem led Storey (1939) to suspect that another undetected fluid may be the carrier of the virus. The complexity of the salivary glands (Dobroscky, 1931b) would allow for more than a single fluid. Also, Storey observed that particles in suspension a t the stylet tip may pulsate regularly or even be shot away some distance during feeding. I n 1928, Carter obt,ained evidence that viruliferous beet leafhoppers introduced small %mounts of curly top virus into solutions on which they fed by inserting their stylets through a membrane. Storey demonstrated the introduction of corn streak virus into solutions in similar tests only with difficulty. He also showed that on rare occasions the virus was acquired during 24 hr. by insects sucking on the side of a leaf opposite to feeding infective insects. Storey concluded that very small amounts of virus were injected. Curly top virus can move a t the rate of 1 in./min. down a sugar beet petiole after being introduced by infective beet leafhoppers (Bennett, 1934); a similar rapid transport of virus from the inoculation site occurs in corn streak. I t seems probable that multiplication a t the inoculation site is not a prerequisite to transport to other parts of the plant. More recently, the discovery (Hewitt,, Frazier, and Houston, 1942) of the vectors of the virus that causes Pierce's disease of grapes and dwarf of alfalfa has revealed some vectors which acquire virus from, and introduce virus into, the xylem and not into the phloem (Houston, Esau, and Hewitt, 1947). The insects feed on the xylem rather than the phloem, and this virus, significantly, causes symptoms in the xylem and is graft-transmissible

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only when wood is included in the diseased tissue used in grafts. The virus that causes phony peach disease is also restricted to the xylem and is transmitted by insects in the same subfamily, Tettigellinae, to which the important vectors of the virus of alfalfa dwarf belong. The virus of chlorotic streak of sugar cane is transmitted by a leafhopper in the same group (Abbott and Ingram, 1942).

E f e c t of Virus on Vector I n common with other vectors of plant viruses, no cicadellid vector has been found t o be harmed directly by any plant virus it carries. Dobroscky (1931a, b) reported on the anatomy of leafhoppers and cytologically examined viruliferous insects for evidence of infection but failed to find them any different from nonviruliferous insects. Kunkel (1926) and Fukushi (1934) also failed to find any cytological difference. The virus studied by Fukushi produces prominent inclusion bodies in affected plant tissues. Kunkel (1926) and Severin (1947~)found that leafhoppers carrying aster yellows virus lived as long as virus-free leafhoppers.

Transmission of More T h a n One V i r u s by a Vector A single cicadellid species may transmit more than one virus (Oman, 1949). I n fact, a single individual may transmit more than one virus during a period of testing on susceptible healthy plants, as was observed in the case of Agalliopsis novella and the wound tumor and clover club leaf viruses (Black, 1944). Giddings (1950) showed that a single leafhopper, fed on two or more strains of curly top virus, transmitted the different strains indiscriminately, so that test plants are infected with all possible combinations of the strains. Independence of Each Inoculation by a Cicadellid Storey (1938) demonstrated in carefully designed and statistically analyzed experiments, that successful inoculations of corn streak virus by Cicadulina mbila are local and independent. The probability of infection by a group of insects is no more than is to be expected from the sum of the probabilities of infection by individuals within the group. In other words, there is no evidence for more than one sub-infective inoculation interacting to produce infection. There appears to be no real conflict between Storey’s findings and those of Giddings (1946b), indicating that more virus particles must be introduced into resistant beet plants than into susceptible ones t o produce infection. Even in the case of the resistant beets, infection might result from one of many virus particles introduced. None of the data has resolved the question of whether infection results from one or more virus particles introduced by a vector.

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Plant Resistance to Inoculation by Cicadellids Giddings (1937) devised a method of controlled curly top virus inoculations of beets by means of Circulifer tenellus. The method revealed differences in resistance in beet stocks as measured by the percentage of plants infected, severity of symptoms, and length of incubation period in the plant. His laboratory results agreed with those of field tests. Larson (1945), using a similar technique, found varietal differences in the percentage of potato plants infected with potato yellow dwarf. Neither of these techniques allowed for the expression of feeding preferences by the leafhoppers, as did the technique of Wilcox (1951), who was able t o measure the preference of Scleroracus vaccinii, the vector of cranberry false blossom virus, for different cranberry varieties. This preference was related to ability to survive on the variety. His laboratory ratings of known varieties agreed well with the known field resistance or susceptibility to false blossom. The presence of one strain of the curly top virus in the plant being inoculated does not prevent beet leafhoppers from successfully introducing another (Carsner, 1925; Giddings, 1950). Plant resistance may affect transmission by providing a poorer source of virus for the vector. Giddings (1946a) found that leafhoppers that had acquired virus from infected resistant plants produced fewer infections than those that acquired it from infected susceptible plants. Plants infected with mild strains or infected for a long time were also poorer sources of virus.

Ecology of Transmission Perhaps the insect vector whose ecological relationships have been most thoroughly studied is the beet leafhopper, Circulifer tenellus. It is an active flyer, and large numbers can easily be blown long distances by the wind. It does not go into true hibernation during the winter but requires the presence of some food plants to prevent it from dying of starvation. In other words, it cannot carry the virus through the winter without feeding on plants as the clover leafhopper can do with potato yellow dwarf virus (Black, 1937). However, the ability of some individuals of C. tenellus to infect plants 167 days after acquisition (Freitag, 1936) indicates that it can carry virus overwinter. Large populations of the leafhopper may be exterminated by the damp fall and winter climates of the invaded areas. It often multiplies in the spring on a variety of succulent weeds on uncultivated, overgrazed, or abandoned lands, and if these plants dry up after the insects have reached the winged adult stage, the insects take flight. The distance of one spring migratory flight from the San Joaquin Valley into the Sacramento Valley was estimated a t 60 miles. During such flights of

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viruliferous insects, cultivated plants are often invaded by great numbers of leafhoppers, so that serious damage may result. Forecasts of the leafhopper invasions, based on studies of the breeding areas, have been used to reduce losses from the disease (Carter, 1930; Severin, 1933; Piemeisel, Lawson, and Carsner, 1951). The vector of tobacco yellow dwarf is often forced to feed on tobacco due to death by drought of certain weed hosts (Helson, 1942). Ecological conditions, however, may affect not only the vector but the virus within the vector. Kunkel’s (1937) demonstration of the inactivation of aster yellows virus in Macrosteles diuisus a t 32°C. indicates how high summer temperatures may interfere with the spread of this disease. Since the virus is also inactivated in plants by high temperatures, the source of virus for the leafhoppers is probably impoverished by hot summer weather. Maramorosch (1950b), working with wound tumor virus, found that there is an optimum temperature for its incubation in, and transmission by Agallia constricta. This is probably true in other cases. The spread of virus diseases in a field may be obvious from casual observation. Sometimes, when the vector is introducing virus into the field from an outside source, the higher incidence of disease near a margin of the field is striking. Efforts have been made to express spread in mathematical terms. Gregory and Read (1949) found that the cases reported in the literature fit the simple empirical expression log I = a bx as well as any of the more complex formulas that have been advanced. In this expression, I is the number of infective punctures in a given time at a distance x from the source, and a and b are constants for any one given set of field conditions. The spread of aster yellows virus from marginal weeds into cultivated fields is well illustrated by Linn (1940). Severin (1946, 1950) has shown that some vectors of aster yellows virus grow more slowly on healthy plants than on plants with yellows. Some species survive on diseased plants but not on healthy ones. In most cases, however, there is no obvious advantage to growth of the vector on plants infected by the virus they carry, and, in a careful test with the vectors Colladonus geminatus and C . montanus, Severin and Klostermeyer (1950) could find no significant difference in the life histories of either of these leafhoppers on healthy celery and celery with California aster yellows. It has been repeatedly observed by the writer that nymphs of Agalliopsis novella are difficult to maintain on crimson clover infected with the clover club leaf virus which they carry. However, they can readily be reared on healthy crimson clover plants.

+

Symptoms Ascribed to Insect Toxins The feeding of leafhoppers on plants sometimes induces symptoms that resemble those caused by virus diseases, although investigation fails to

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reveal the presence of a virus. Such symptoms are commonly ascribed to insect toxins (Severin, Horn, and Frazier, 1945). The symptoms frequently consist of a vein-clearing that remains localized but simulates quite closely the early vein-clearing due to virus infection (Severin, 1947). With experience, it is usually possible to distinguish one from the other even before enough time has elapsed to determine whether or not systemic infection is going to occur. In rare cases the toxin even produces systemic symptoms closely simulating virus disease. This is so with the systemic disease similar t o aster yellows that is apparently caused by toxic material from Xerophloea vanduzeei (Severin, Horn, and Frazier, 1945). Bennett (1952) reported that certain individuals of Circulifer tenellus produce on young beet seedlings what is apparently a vein-clearing resembling the early symptoms of curly top infection. The writer and others have occasionally observed similar local vein-clearing reactions from the feeding of Macrosteles divisus that may be confused with early aster yellows symptoms. Bennett found that in field collections of C . tenellus the number of individuals that produce such effects rarely exceeded 1 in 200. By selection and breeding he was able to produce a race of C . tenellus in which about 60 % of the individuals caused this reaction. There is a close similarity among many of these toxic vein-clearing reactions produced by the feeding of certain leafhoppers, and, although they may a t first be confused with virus symptoms, they can often, and perhaps always, be distinguished from them on the basis of their symptoms alone. They are attributed to toxins because of a consistent failure to transmit their causal agent in series from plant to plant by any means so far tried. However, because such evidence is negative, it cannot be considered t o eliminate conclusively the possibility that the cause is virus.

Conclusion

It is evident that transmission of plant viruses by cicadellids is a complicated and subtle process about which much is yet to be learned. As indicated in the text, there are probably two major types of transmission by leafhoppers, depending on whether the virus multiplies in the vector or not. At present, the great majority of virus-leafhopper relationships cannot be classified as belonging t o either one or the other of these transmission types. Long incubation periods in the vector may be indicative of multiplication and short ones indicative of its absence. There may always be an element of uncertainty about those viruses which the evidence indicates do not multiply in the vector, because such evidence is, of necessity, negative. REFERENCES Abbott, E. V . , and Ingram, J. W . (1942). Transmission of chlorotic streak of sugar cane by the leaf hopper Draeculacephala portola. Phytopathology 32, 99.

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Adsuar, J. (1946). Transmission of papaya bunchy top by a leaf hopper of the genus Empoasca. Science 103, 316. Baker, W. L. (1949). Studies on the transmission of the virus causing phloem necrosis of American elm, with notes on the biology of its insect vector. J . Econ. Entomol. 42. 729. Bawden, F. C. (1950). Plant viruses and virus diseases. 3rd ed. Chronica Botanica Co. Waltham, Mass. Bennett, C. W. (1934). Plant-tissue relations of the sugar-beet curly-top virus. J. Agr. Research 48, 665. Bennett, C. W. (1940). Acquisition and transmission of viruses by dodder (Cuscuta subinclusa). Phy~opathology 30, 2. Bennett, C. W. (1952). Vein clearing on sugar beet induced by the beet leafhopper. Phytopathology 42, 535. Bennett, C. W., Carsner, E., Coons, G. H., and Brandes, E. W. (1946). The Argentine curly top of sugar beet. J . Agr. Research 72. 19. Bennett, C. W., and Costa, A. S. (1949). The Brazilian curly top of t,omato and tobacco resembling North American and Argentine curly top of sugar beet. J. Agr. Research 78, 675. Bennett, C. W., and Wallace, H. E. (1938). Relations of the curly top virus t o the vector, Eutettix tenellus. J . A ~ TResearch . 68, 31. Bird, J., and Adsuar, J. (1952). Viral nature of papaya bunchy top. J . Agr. Univ. Puerto Rico 36, 5 . Black, L. M. (1937). A study of potato yellow-dwarf in New York. Cornell Univ. Agr. Expt. Sta. Mem. 209, 1 . Black, L. M. (1938). Properties of the potato yellow-dwarf virus. Phytopathology 28. 863. Black, L. M. (1941). Further evidence for multiplication of the aster-yellows virus in the aster leaf hopper. Phytopathology 31, 120. Black, L. M. (1943). Genetic variation in the clover leafhopper’s ability t o transmit potato yellow-dwarf virus. Genetics 28, 200. Black, L. M. (1944). Some viruses transmitted by Agallian leaf-hoppers. Proc. Am. Phil. SOC.88, 132. Black, L. M. (1950). A plant virus that multiplies in its insect vector. Nature 166, 852. Black, L. M. (1953a). Occasional transmission of some plant viruses through the eggs of their insect vectors. Phytopathology 43, 9. Black, L. M. (1953b). Viruses t h a t reproduce in plants and insects. Ann. N . Y . Acad. Sci. 66, 398. Black, L. M., and Brakke, M. K. (1952). Multiplication of wound-tumor virus in an insect vector. Phytopathology 42, 269. Black, L. M., Mosley, V. M., and Wyckoff, R . W. G. (1948). Electron microscopy of potato yellow-dwarf virus. Biochem. et Biophys. Acta 2 , 121. Brakke, M. K., Vatter, A. E., and Black, L. M. (to be published). Brakke, M. K., Black, L. M., and Wyckoff, R. W. G. (1951). The sedimentation rate of potato yellow-dwarf virus. Am. J . Bot. 38, 332. Carsner, E. (1925). Attenuation of the virus of sugar beet curly top. Phytopathology 16, 745. Carter, W. (1927). A technic for use with Homopterous vectors of pIant disease with special reference t o the sugar-beet leaf hopper, Eutettiz tenellus (Baker). J. Agr. Research 34,449. Carter, W. (1928). Transmission of the virus of curly-top of sugar beets through different solutions. Phytopathology 18, 675.

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Carter, W. (1930). Ecological studies of the beet leaf hopper. U . S . Dept. Agr. Tech. Bull. 206, 1. Costa, A.S. (1952). Further studies on tomato curly top in Brazil. Phytopathology 42, 396. Dobroscky, Irene D. (1931a). Studies on cranberry false blossom disease and its insect vector. Contribs. Boyce Thompson Inst. 3, 59. Dobroscky, Irene, D. (1931b). Morphological and cytological studies on the salivary glands and alimentary tract of Cicadula seznotata (Fallen), the carrier of aster yellows virus. Contribs. Boyce Thompson I n s t . 3, 39. Fife, J. M., and Frampton, V. L. (1936). The pH gradient extending from the phloem into the parenchyma of the sugar beet and its relation t o the feeding behavior of Eutettix tenellus. J . Agr. Research 63, 581. Freitag, J. H. (1936). Negative evidence on multiplication of curly-top virus in the beet leafhopper, Eutettiz tenellus. Hilgardia 10, 305. Freitag, J. H., Frazier, N. W., and Flock, R. A. (1952). Six new leafhopper vectors of Pierce’s disease virus. Phytopathology 43,533. Fukushi, T. (1934). Studies on the dwarf disease of rice plant. J . Faculty A g r . Hokkaido I m p . Univ. 37, No.2, 41. Fukushi, T. (1940). Further studies on the dwarf disease of rice plant. J. Faculty Agr. Hokkaido I m p . Univ. 46, No. 3, 85. Giddings, N. J. (1937). A greenhouse method for testing resistance to curly top in sugar beets. Phylopathology 27, 773. Giddings, N. J. (19464. Some factors influencing curly top virus concentration in sugar beets. Phytopathology 36, 38. Giddings, N. J. (194613). Mass action as a factor in curly-top-virus infection of sugar beet. Phytopathology 36, 53. Giddings, N.J. (1947). Dodder a s an aid in testing some plant species for curly-top virus. Phytopalhology 37, 278. Giddings, N.J. (1950). Some interrelationships of virus strains in sugar beet curly top. Phytopathology 40, 377. Green, R. G. (1935). On the nature of filterable viruses. Science 82, 443. Gregory, P. H., and Read, D. R. (1949). The spatial distribution of insect-borne plant-virus diseases. Ann. Applied Biol. 36, 475. Helson, G. A . H. (1942). The leaf hopper Thamnotettix argentata Evans, a vector of tobacco yellow dwarf. J . Council S c i . I n d . Research 16, No. 2, 175. Helson, G. A. 1%. (1951). The transmission of witches’ broom virus disease of lucerne by the common brown leafhopper, Orosius argentatus (Evans). A u s tralian J . Sci. Research B4. No. 2, 115. Hewitt, W. B., Frazier, N. W., and Houston, B. R. (1942). Transmission of Pierce’s disease of grapevines with a leaf hopper. Phytopathology 32, 8. Hougas, K. W. (1951). Factors affecting sap transmission of the potato yellowdwarf virus. Phytopathology 41, 483. Houston, B. R., Esau, Katherine, and Hewilt, W . B. (1947). The mode of vector feeding and the tissues involved in the transmission of Pierce’s disease virus in grape and alfalfa. Pfiytopatho~ogy37, 247. Johnson, F . (1941). Transmission of viruses by the parasitic activities of dodder. Phytopathology 31, 13. Katsura, S. (1936). The stunt disease of Japanese rice, the first plant virosis shown t o be transmitted by a n insect vector. Phytopathology 26, 887. Kunkel, L. 0. (1926). Studies on aster yellows. Am. J . Bat. 13, 646. Kunkel, L. 0. (1933). Insect transmission of peach yellows. Conlribs. Boyce Thompson Znst. 6, 19.

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Kunkel, L. 0 . (1937). Effect of heat on ability of Cicadula sexnotata (Fall.) t o transmit aster yellows. Am. J . Bot. 24, 316. Kunkel, L. 0. (1948). Studies on a new corn virus disease. Arch. ges. Virusjorsch. 4, 24. Larson, R. H. (1945). Resistance in potato varieties t o yellow dwarf. J . Agr. Research 71, 441. Linn, M. B. (1940). The yellows disease of lettuce and endive. Cornell Uniu. Agr. Expl. Sta. Bull. 742, 1. Maramorosch, K . (195Oa). Effect of dosage on length of incubation period of asteryellows virus in its vector. Proc. Soc. Exptl. Biol. M e d . 7 6 , 744. Maramorosch, K. (1950b). Influence of temperature on incubation and transmission of the wound-tumor virus. Phytopathology 40, 1071. Maramorosch, K. (1952a). Direct evidence for the multiplication of aster-yellows virus in its insect vector. Phytopathology 42, 59. Maramorosch, K. (1952b). Studies on the nature of the specific transmission ot aster-yellows and corn-stunt viruses. Phytopathology 42, 663. Oman, P. W. (1949). The nearctic leafhoppers; (Homoptera: Cicadellidae) a generic classification and check list. Entomol. SOC.Wash. Mem. 3. 1. Piemeisel, R . L., Lawson, F. R., and Carsner, E. (1951). Weeds, insects, plant diseases and dust storms. Sci. Monthly 73, No. 2, 124. Severin, H. H. P. (1924). Curly leaf transmission experiments. Phyfopathology 14, 80. Severin, H. H. P. (1931). Modes of curly-top transmission by the beet leaf hopper, Eutettix tenellus (Baker). Hilgardia 6 , 253. Severin, H. H. P. (1933). Field observations on the beet leafhopper, Eutettiz tenellus, in California. Hilgardia 7 , 282. Severin, H. H. P. (1934). Transmission of California aster and celery-yellows virus by three species of leafhoppers. Hilgardia 8 , 339. Severin, H. H. P. (1946). Longevity, or life histories, of leafhopper species on virus-infected and on healthy plants. Hilgardia 17, 121. Severin, H. H. P . (1947a). Plant symptoms induced by feeding of some leafhopper species. Hilgardia 17, 219. Severin, H. H. P. (194715). Newly discovered leafhopper vectors of California aster-yellows virus. Hilgardia 17, 511. Severin, H. H. P . (1947~). Longevity of noninfective and infective leafhoppers on a plant nonsusceptible t o a virus. Hilgardia 17,541. Severin, H. H. P. (1947d). Location of curly-top virus in the beet leafhopper Eutettix tenellus. Hilgardia 17. 545. Severin, H. H. P. (1949). Transmission of the virus of Pierce’s disease of grapevines by leafhoppers. Hilgardia 19, 190. Severin, H. H. P. (1950). Texananus incurvatus. I11 Life history on virus-infected and on healthy plants. Hilgardia 19, 546. Severin, H. H. P., Horn, F. D., and Fraxier, N . F. (1945). Certain symptoms resembling those of curly top or aster yellows, induced by saliva of Xerophloea vanduzeei. Hilgardia 16, 337. Severin, H. H. P., and Klostermeyer, E. C. (1950). Colladonus geminatus and C . montanus, life histories on virus-infected and on healthy plants. Hilgardia 19, 550. Severin, H . H. P., and Swery, Olive. (192b). Filtration experiments on curly top of sugar beets. Phytopathology 18, 681. Smith, E. F. (1891). Additional evidence on the communicability of peach yellows and peach rosette. U . S . Dept. Agr. Veg. Path. Bull. 1, 1.

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Storey, H. H. (1928). Transmission studies on maize streak disease. Ann. A p plied Biol. 16, 1 . Storey, H . H. (1932). The inheritance by a n insect vector of the ability t o transmit a plant virus. Proc. Roy. Sac. (London) Bll2. 46. Storey, H. H. (1933). Investigations of the mechanism of the transmission of plant viruses by insect vectors. 1. PTOC.Roy SOC.(London) B113, 463. Storey, H. H. (1938). Investigations of the mechanism of the transmission of plant viruses by insect vectors. 11. The part played by puncture in transmission. Proc. Roy. SOC.(London) Bl26, 455. Storey, H. H. (1939a). Investigations of the mechanism of the transmission of plant viruses by insect vectors. 111. The insect’s saliva. PTOC.Roy. SOC. (London) Bl27, 526. Storey, H. H. (1939b). Transmission of plant viruses by insects. Botan. Rev. 6. 240. Sukhov, K. S., and Sukhova, M. N. (1940). Interrelations between the virus of a new grain mosaic disease (Zakuklivanie) and its carrier Delphax striatella Fallen. Compt. rend. acad. sci. U.R . 8. S . [n.s.]20, 479; abstracted in Rev. A p p . Mycol. 20, 155. (1941). Turner, W. F. (1952). The role of insect surveys in virus-vector research. Plant Disease Rept.Supp1. 211, 47. Wilcox, R. B. (1951). Tests of cranberry varieties and seedlings for resistance t o the leafhopper vector of false-blossom disease. Phytopathology 41, 722. Wolfe, H. R., Anthon, E. W., and Jones, L. S. (1950). Transmission of western X-disease of peaches by the leafhopper Colladonus geminatus (Van D.). Phytopathology 40, 971. Younkin, S. G. (1943). Purple-top wilt of potatoes caused by the aster yellows virus. A m . Potato J. 20, 177. Zaehurilo, V. K., and Sitnikova, Mme. G. M. (1941). The relation of the virus of winter wheat mosaic t o its vector (Deltocephalus striatus L.). Compt. rend. Pan. Sou. V . I . Lenin acad. agr. sci., Moscow 0 , No. 11, 27-29; abstracted in Rev. A p p . Mycol. 22, 59 (1943).